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Copyright of Journal of Henan Agricultural Sciences is the property of Editorial Board of Journal of Henan Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Edulis Fungi is the property of Acta Edulis Fungi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Hemalin, initially described in Haemaphysalis longicornis, is a protein with anticoagulant activity. We retrieved a gene fragment functionally annotated as hemalin from H. flava salivary gland transcriptomic library, but its full‐length complementary DNA (cDNA) and antithrombogenicity have not been investigated in the species. Here we cloned the full length of hemalin (Hf‐hemalin) by 3′‐end rapid‐amplification of cDNA ends, and the open reading frame (ORF) of Hf‐hemalin was expressed in Escherichia coli. The recombinant protein (rHf‐Hemalin) was tested for antithrombogenicity. The full‐length of Hf‐hemalin was 607 bp with an ORF of423 bp. Protein encoded by Hf‐hemalin was predicted to contain 2 Kunitz domains and a signal peptide. The expression of Hf‐hemalin in salivary glands, midguts and ovaries was higher in the semi‐engorged than the fully engorged. Prokaryotic expression yielded a product of 40 kDa containing a glutathione S‐transferase (GST) tag. Incubation of rHf‐Hemalin with rat plasma significantly extended prothrombin time and activated partial thromboplastin time compared with normal saline and GST controls. Our data demonstrated that Hemalin from H. flava shared a similar primary structure with that from H. longicornis, and was also anticoagulant. Further investigations are needed to test its feasibility to be an antigen candidate for the development of vaccines against ticks. [ABSTRACT FROM AUTHOR]

This paper aims to find out the pathogenesis of konjac soft rot and prevent this disease preferbly, confirm the fun ctions of pectin lyase in pathogenetic process. [ Method] Taking konjac soft rot fungus as material, the important pectinase gene in Konjac soft rot was obtained by PCR cloning technology, and the cloned gene was analyzed by bioinformatics. The prokaryotic expression vector was constructed, and induced by IPTG, and detected by SDS-PAGE electrophoresis. [Result] Konjac soft rot pelD and pelE genes with complete reading frame were obtained. Both genes showed obvious bands at about 45 ku. The enzyme activities of the two genes were significantly increased after induction, and the enzyme activities induced by pelD and pelE were respectively 1. 79 times and 1. 75 times that before induction. [Conclusion] In the study we obtained the konjac pelD gene and pelE gene with normal functions, which laid the foundation for further study of the functions of these two genes. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Henan Agricultural Sciences is the property of Editorial Board of Journal of Henan Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Liver kinase B1 (LKB1) is a member of the serine/threonine kinase family, which plays an indispensable role in the organism of animals. In the current study, the chicken LKB1 protein gene was amplified by PCR based on the primers and cDNA templates. Then, the cloning vector was constructed and the target gene was cloned. After that, the target gene was inserted into the expression vector to construct the recombinant plasmid. The recombinant plasmid was transformed into BL21 (DE3) host cells and the LKB1 recombinant proteins were successfully expressed by using Isopropyl-β-D-thiogalactopyranoside (IPTG). Finally, purified LKB1 proteins were used as antigen and the rabbit-derived antiserums were collected. The antiserum titer determined by ELISA was not less than 1:128000. The results of Western blot suggested that the polyclonal antibody is highly specific to chicken LKB1 protein. Immunofluorescence indicated that the LKB1 protein is mainly expressed in the cytoplasm of liver, heart and hypothalamus cells of chicken. Our study showed that the LKB1 polyclonal antibodies produced by this method are effective and can be used to further study the role of LKB1 in the pathogenesis of chicken disease. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Henan Agricultural Sciences is the property of Editorial Board of Journal of Henan Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Synthetic Cry1Ab/Ac proteins expressed by genetically modified (GM) crops have a high potential to control insect pests without utilizing large amounts of chemical insecticides. Before these crops are used in agriculture, the environmental fate and interactions in the soil must be understood. Stable isotope‐labeled Cry1Ab/Ac protein is a highly useful tool for collecting such data. We developed a protocol to produce 13C/15N single‐labeled Cry proteins. The artificially synthesized gene Cry1Ab/Ac of Bt rice Huahui No. 1, which has been certified by the Chinese government to be safe for human consumption, was subcloned into pUC57, and the expression vector pET‐28a‐CryAb/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM isopropyl thiogalactoside (IPTG) was added to these cells and cultured at 37°C for 4 h to induce the synthesis and formation of inclusion bodies in M9 growth media containing either [U‐13C] glucose (5% 13C‐enriched) or [15N] ammonium chloride (5% 15N‐enriched). Then, Cry inclusion bodies were dissolved in urea and purified by affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blotting. The purities of 13C/15N‐labeled Cry proteins reached 99% with amounts of 12.6 mg/L and 8.8 mg/L, respectively. The δ 13C and ä 15N values of 13C‐labeled Cry protein and 15N‐labeled Cry protein were 3,269‰ and 2,854‰, respectively. A bioassay test revealed that the labeled Cry1Ab/Ac proteins had strong insecticidal activity. The stable isotope‐labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies on Cry proteins in soil and the characteristics of nitrogen (N) and carbon (C) transformations. Our findings may also be employed as a reference for elucidating the environmental behavior and ecological effects of BT plants and expressed products.

[ Objectives] The purpose of this study was to clarify the optimum length of the chromogenic fragments of Cyt-b5 containing Cyt-b5 -like heme/ steroid binding domain (Cyt-b5D) and its adaptability using different prokaryotic expression vectors, and screen out effective visual tag in order to improve the efficiency and convenience of vector construction. [ Methods] The structural domain of Cyt-h5 protein of Chilo suppressalis was analysed and divided into different fragments with different sizes, such as OA, OB, OC, etc. containing Cyt-b5D. These fragments were ligated with prokaryotic expression vectors, including pET-28a ( +), pET-30a ( +) and pET-4la( +),respectively and expressed in Escherichia coli. The color reaction and absorbance of bacterial solution of the screened chromogenic fragments co-expressed with the C. suppressalis RDLJ were detected at different time points. The recombinant proteins expressed by the chromogenic fragments and co-expressed with RDLJ were purified by affinity chromatography and detected by the Western blot. [Results ] The OA fragment was the best chromogenic fragment, which could express the red proteins using three prokaryotic expression vectors. In addition, its color visualization was stable and adaptable when co-expressed with RDLJ. The growth status of chromogenic bacterial solution could he real-time monitored on the basis of its characteristic absorption at 420 nm, whereas the process of protein purification could he estimated according to the red change of the chromatographic column. In addition, the fusion protein could he detected by the Western blot, suggesting that the presence of exogenous chromogenic fragment did not disturb the accurate identification of the target protein. [ Conclusions ] The chromogenic fragment ( OA) could he used as a visual tag in prokaryotic expression system, he able to provide the indication tag for protein expression and purification, and further improve the convenience and timeliness of the experimental operations as described above. [ABSTRACT FROM AUTHOR]

Sanghuangporus baumii, a macrofungus belonging to the Hymenochaetaceae family, has been widely used as a traditional medicine in Asia for centuries. Triterpenoids in S. baumii contribute to the pharmacological activities, however, their content is low in the fruiting body. Phosphomevalonate kinase (PMK) is an important rate-limiting enzyme in the triterpenoids synthesis pathway. In this study, a PMK gene (SbPMK) was cloned and analyzed. Sequence analysis showed that SbPMK has six exons and five introns. The 1,488 bp open reading frame encodes a 489 amino acid polypeptide with a predicted protein molecular weight of 52.47 kDa. The amino acid sequence of SbPMK shares highly similarity with that of PMK sequences from other species, especially in three functional motifs (MI, MII and MIII). The promoter of SbPMK contains typical cis-acting regulatory elements involved in MeJA responsiveness, light responsiveness, anaerobic induction, etc. Furthermore, recombinant SbPMK was overexpressed in Escherichia coli BL21, with a satisfactory solubility. During the fermentation culture of S. baumii, the biomass increased with fermentation and peaked on day 18 (4.80 g/L). The SbPMK expression and the triterpenoids content was peaked on day 6, and the triterpenoids content stabilized at 20.8 mg/g on day 18. Supplemental data for this article is available online at https://doi.org/10.1080/13102818.2021.1938678. [ABSTRACT FROM AUTHOR]

Sanghuangporus baumii (Pilát) (Zhou and Dai), a traditional Chinese medicinal fungus, has been confirmed to produce triterpenoids, which possess a good anti-cancer effect. However, the triterpenoids yield of S. baumii is low and its biosynthetic mechanism is not clearly understood. To investigate the triterpenoids biosynthetic mechanism, the whole cDNA sequence of the HMG-CoA reductase (HMGR) gene was obtained. The open reading frame of HMGR gene cDNA named SbHMGR, comprised 4170 bp, encoding a polypeptide of 1389 amino acids with a predicted protein molecular weight of 147.53 kDa. The HMGR gene includes four exons and three introns. Subsequently, the triterpenoids content was determined at different developmental stages of S. baumii. It showed a dynamic tendency—increased first, then decreased and peaked on 14th day in the mycelium stage. In addition, the changing trend of SbHMGR enzyme activity and transcript level of SbHMGR gene were measured. They were nearly consistent with the triterpenoids content, suggesting that SbHMGR gene may play a key regulatory role in triterpenoids biosynthetic pathway. Similarly, changes in the transcript level of downstream key genes (SbMVD, SbIDI, SbLSD) of SbHMGR gene were also positively correlated with the triterpenoids content, indicating those three genes were also involved in triterpenoids biosynthetic pathway. Moreover, SbHMGR gene had been observed to be expressed in prokaryote. The results will be valuable for elucidation of the biosynthetic pathway of triterpenoids in S. baumii, lay a foundation for genetic modifications to improve triterpenoids yield. [ABSTRACT FROM AUTHOR]

Galactose-binding lectins (GBLs) are a subdivision of jacalin-related lectins and known to perform an essential role in plant protective mechanisms against plant pathogens, fungi, and viruses. A cDNA sequence encoding GBL was cloned and the gene has an open reading frame (ORF) of 657 bp, encoding a protein of 219 amino acids. The protein is predicted to have a molecular weight and isoelectric point (pI) of 23.76 kDa and 6.29, respectively. GBL has a jacalin-type lectin domain and fits into the JRL superfamily. The evolutionary relationship of GBL revealed that the GBL from Morus multicaulis was firmly related to that of Morus rotundibila, Morus alba, Morus notabilis, Artocarpus integer, Artocarpus nitidus and Champedak (cgb). qRT-PCR analysis showed that the gene was expressed in all the tissues tested, leaf, and bud displaying the highest transcript levels. There was a general decrease in the mRNA transcript level under cold and salt stress as compared to the mRNA transcript level of the control. SDS-PAGE and western blot results showed that His-tagged fusion GBL protein was positively expressed in E. coli. In total, our findings disclosed the molecular basis for the signal transduction mechanisms when abiotic stress is induced in a mulberry tree. [ABSTRACT FROM AUTHOR]

Copyright of Forest Research is the property of Forest Research Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective: To express the protein enconded by the Rv 3432 c gene of Mycobacterium tuberculosis ( M . tb ) in vitro by prokaryotic expression, to analyze the structure of the Rv3432c protein by using bioinformatics software, and to explore for new drug targets against M . tb ., Methods: The Rv 3432 c gene was amplified by PCR using the genomic DNA of the inactivated M . tb strain H37Rv as the template and a recombinant plasmid was constructed with the expression vector pET-28a. The expression products were analyzed by SDS-PAGE and purified using affinity chromatography. The biological properties of Rv3432c were analyzed with Protparam, the Pfam online tool, SOMPA, Protscale, TMHMM Signalp 6.0, NetPhos3.1, SUMOsp 2.0, and SWISS-MODEL., Results: pET-28a- Rv 3432 c recombinant plasmid sequencing results were fully consistent with those of the target gene. SDS-PAGE analysis showed that the fusion protein existed in the form of a soluble protein with a relative molecular mass of about 55×10 3 , which matched the expected size. ProtParam analysis showed that the Rv3432c protein was hydrophilic (showing a GRAVY value of －0.079). Rv3432c was a protein with no transmembrane structural domains or signal peptide. The secondary structure of Rv3432c mainly consisted of random coils (39.78%) and α-helix (39.57%) and was relatively loosely structured., Conclusion: We successfully constructed a prokaryotic expression plasmid of the Rv3432c protein and analyzed its structure using bioinformatics, laying the foundation for further research on the role of Rv3432c in the pathogenesis and progression of tuberculosis as well as the identification of new drug targets against M . tb ., Competing Interests: 利益冲突　所有作者均声明不存在利益冲突, (© 2024《四川大学学报（医学版）》编辑部 版权所有Copyright ©2024 Editorial Board of Journal of Sichuan University (Medical Sciences).)

The transcription factor JunB can induce physiological or pathological responses to various stimuli, including immune stimulants and bacteria, and plays an important role in the immune response process. In this study, we identified a JunB family member in Schizothorax prenanti (S. prenanti), which was designated SpJunB. The complete coding sequence (CDS) of SpJunB was 930 bp in length, which was submitted to GenBank (ID: MN215886). SpJunB encodes a putative protein of 309 amino acids, which is highly homologous to JunB of common carp. The SpJunB protein contained a conserved JunB domain, and its 3D structure was also highly similar to (77.61%) the human SpJunB protein. SpJunB was found to be extensively expressed in various tissues, with the highest expression in the spleen. The expression of SpJunB was significantly upregulated after Aeromonas hydrophila (A. hydrophila) challenge. Prokaryotic expression indicated that a 51 kDa recombinant protein was obtained after induction with 1.5 mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h at 37 °C. The expression levels of IL -1β, IL -6 and IL -8 were significantly upregulated (p < 0.01) after treatment of S. prenanti with the SpJunB protein. The activities of SOD, AKP and LZM were also significantly increased (p < 0.01) after the treatment of S. prenanti with the SpJunB protein. Simultaneously, the SpJunB protein reduced the infection rate of A. hydrophila in S. prenanti. In conclusion, SpJunB may improve the immune functions of S. prenanti. It will be beneficial to further study the immune mechanism of JunB in fish. [ABSTRACT FROM AUTHOR]

As a valuable and powerful platform technology, yeast surface display (YSD) has been widely used in various biotechnological fields, but little in agricultural applications. Xylanase can be used as an elicitor in the plant–pathogen interaction system and induce corn defence response. In this study, the YSD technology was used to express the xylanase protein family from Setosphaeria turcica, which is a novel strategy for plant protection. First, five genes encoding xylanase were selected from the S. turcica genome database. The conserved domains of these genes were analyzed, which revealed that these genes were highly conserved. Here, we selected two of the five genes and expressed them by a prokaryotic expression system, but the two xylanase genes were expressed in inclusion bodies and showed no activity. To make them available in the application, we successfully constructed them in a YSD system. The products were detected for enzyme activities and the activities of two xylanases were 16.72 and 19.12 U/mL, respectively. We finally proved that the yeast-displaying xylanase was an efficient strategy to induce corn defence response, as four defence genes in corn, PR-1, PR-4, SOD and CAT were induced after treatment with the yeast displaying xylanases. [ABSTRACT FROM AUTHOR]

Copyright of Xinjiang Agricultural Sciences is the property of Xinjiang Agricultural Sciences Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective * To investigate the activity detection method of α-1,3-mannosyltransferase (ALG3) mutant protein in congenital disorder of glycosylation (ALG3-CDG), and examine the relationship between the activity and the severity of disease. Methods * The mutation sites of the patients in the reports were I69T, W71R, G96R, M157K, R171Q and M209T, respectively. After homologous alignment of the amino acid sequences of Homo sapiens and Saccharomyces cerevisiae (S. cerevisiae) ALG3, six yeast mutant proteins (I70T, Y72R, G98R, L157K, R171Q and M221T) related to ALG3-CDG were designed. These mutants were expressed in Escherichia coli (E. coli). Using E. coli membrane containing wild-type ALG3 or its mutant proteins as catalysts, the relative activities of mutant proteins were calculated by liquid chromatography tandem mass spectrometry based on the catalytic activity of wild type protein. The activities of corresponding mutations in the patients with different survival time were compared. Results * Six ALG3-CDG related S. cerevisiae conserved ALG3 mutant proteins were expressed in E. coli in the comparable protein expression levels with wild type ALG3. The in vitro quantitative activity assay of enzymes showed that compared with the wild type ALG3, the relative activities of the mutant proteins I70T, Y72R, G98R, L157K, R171Q and M221T were 2.8%, 4.9%, 4.5%, 17.2%, 4.8% and 22.3%, respectively. The corresponding mutation activity was significantly higher in the patients with survival longer than 1 year than that in the patients with survival less than 1 year (P=0.002). Conclusion * An in vitro quantitative method is established to measure the enzymatic activities of ALG3-CDG related S. cerevisiae ALG3 mutants, providing the possibility to predict the severity of ALG3-CDG. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Molecular cloning is one of the most fundamental technologies in molecular biology, and has been critical for driving biotechnological advances. In this study, we have developed a novel method for standardized molecular cloning. The cloning technique known as “Nimble Cloning” uses the restriction enzyme, SfiI, in combination with the T5 exonuclease, to linearize the vector and generate 3′-overhangs simultaneously. Both PCR products and plasmids can be used for the cloning reaction in the Nimble Cloning system. The cloning system is highly efficient, suitable for gene expression in both prokaryotic and eukaryotic expression systems, and enables the reuse of DNA fragments or plasmid entry clones. Nimble Cloning is applicable for the cloning of single or multiple fragments, as well as multi-site cloning. Due also to its simplicity and versatility, the cloning method has great potential for the modular assembly of DNA constructs.

Background Glutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the murI gene was shown in Streptococcus mutans, the primary aetiologic agent of human dental caries, studies on S. mutans MurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from the S. mutans UA159 genome. Methods Structure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His6-tagged S. mutans MurI was overexpressed in the expression vector pColdII and further purified using a Ni2+ affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS–PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis–Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures. Results The glutamate racemase gene from S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay, S. mutans MurI displayed unique kinetic parameters (Km, d-Glu→l-Glu = 0.3631 ± 0.3205 mM, Vmax, d-Glu→l-Glu = 0.1963 ± 0.0361 mM min−1, kcat, d-Glu→l-Glu = 0.0306 ± 0.0065 s−1, kcat/Km, d-Glu→l-Glu = 0.0844 ± 0.0128 s−1 mM−1, with d-glutamate as substrate; Km, l-Glu→d-Glu = 0.8077 ± 0.5081 mM, Vmax, l-Glu→d-Glu = 0.2421 ± 0.0418 mM min−1, kcat, l-Glu→d-Glu = 0.0378 ± 0.0056 s−1, kcat/Km, l-Glu→d-Glu = 0.0468 ± 0.0176 s−1 mM−1, with l-glutamate as substrate). S. mutans MurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0. Conclusion The findings of this study provide insight into the structure and biochemical traits of the glutamate racemase in S. mutans and supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.

Abstract Background According to recent studies, the phospholipase A2 receptor 1 (PLA2R1) may be used as a biomarker to diagnose idiopathic membranous nephropathy (iMN). Moreover, the immune-dominant regions of PLA2R1 have been identified. The aim of the present study was to construct a diagnostic antigen based on the immune-dominant region of PLA2R1 and develop a specific serological detection method for PLA2R1 antibodies. Results The tandem multi-epitope diagnostic antigen (designated ‘R101’), which includes aa 39–130 (CysR), aa 238–356 (CTLD1), and aa 1136–1234 (CTLD7) of PLA2R1; thioredoxin at the N-terminus; and a His tag at the C-terminus, was prepared at a concentration of 2.36 mg/mL and purity of 97.32% using Escherichia coli expression and affinity and anion exchange chromatography purification. The integrity and antigenicity of the R101 protein was demonstrated by western blot analysis using anti-Trx, anti-His, and anti-PLA2R1 monoclonal antibodies as the primary antibodies. By analysing 120 positive serum samples identified by biopsy-proven iMN (gold standard) and 240 negative samples identified by an established ELISA based on R101 protein, we concluded that the cut-off value, kappa value, sensitivity, specificity, and agreement rate were 0.305, 0.881, 91.67, 96.25, and 94.72% respectively. The receiver operating characteristic (ROC) curve illustrated that the diagnostic accuracy and practicability of the ELISA was excellent. The area under the curve was 0.986. Conclusions Using prokaryotic expression and chromatography purification, immune-dominant regions of PLA2R1 with excellent antigenicity can be prepared and applied to serological detection of PLA2R1 antibodies.

Abstract Bovine tuberculosis is a disease caused by Mycobacterium bovis (M. bovis) that leads to great economic losses in cattle production. The discovery of a reasonable bioagent to reduce M. bovis infection risk and environment contamination becomes significant and urgent. Previous study reported that human β-defensin-3 (HBD3) participated in Mycobacterial immunity and was recognized as a suitable candidate reagent. However, its minimal inhibitory concentration to M. bovis is not yet reported. In this study, we first purified HBD3 protein by recombinant-DNA technology and prokaryotic expression system. Subsequently, anti-bacterial tests were used to evaluate the basic bioactivity of the protein. Results revealed that recombinant HBD3 (rHBD3) protein inhibits Staphylococcus multiplication but not the host Escherichia coli. The growth curve of M. bovis showed that rHBD3 protein controls the proliferation of M. bovis in 20 μg/ml concentration. In addition, rHBD3 protein-incubated M. bovis exhibited reduced infectivity to alveolar epithelial cells and macrophages. In conclusion, the expression of rHBD3 protein is a potential ideal bio-regent for reducing M. bovis infection.

Abstract Background Mink circovirus (MiCV) is a newly discovered pathogen associated with mink diarrhea. The prevalence and economic importance of this virus remain poorly understood, and no specific serological assay has been developed for the diagnosis of MiCV infection. Results In this study, a recombinant capsid protein antigen expressed in Escherichia coli was utilized to establish an indirect enzyme-linked immunosorbent assay (iELISA). Results revealed that the assay had no cross-reactivity with other related pathogens, and the respective sensitivity and specificity of the proposed iELISA were 92.31% and 91.67% compared with those obtained of Western blot on 138 serum samples from minks. The correlation coefficient between iELISA and Western blot was 0.838 (p > 0.05). iELISA was applied to detect MiCV antibodies in 683 clinical serum samples from different farms from the major mink industry province in China, and 21 of 24 farms with 163 of 683 (23.87%) individuals were tested positive for MiCV antibodies. The positive rates of each of the 21 flocks ranged from 2.33% to 73.68%. Conclusions These results indicated that iELISA was a sensitive and specific method suitable for the large-scale detection of MiCV infections in mink. This study provided an effective method for the serological diagnosis and positive rate investigation of MiCV infection.

Abstract Background Duck enteritis virus (DEV) belongs to the subfamily Alphaherpesvirinae, and information on the DEV UL41 gene is limited. Methods The DEV UL41 gene was cloned into the pET32a(+) vector and expressed in a prokaryotic expression system. Antiserum was raised against a bacterially expressed UL41-His fusion protein for further experiments. Transcription was quantified and UL41 protein expression levels were determined in DEV-infected cells at different time points by RT-qPCR and western blotting, respectively. DEV virions were purified by sucrose gradient centrifugation and analyzed by mass spectrometry to identify protein content. We confirmed the DEV UL41 gene kinetic class using a pharmacological test. IFA was used to analyze the intracellular localization of pUL41. Results The recombinant expression plasmid, pET-32a(+)-UL41, which highly expresses a 76.0 kDa fusion protein, was constructed and expressed in E. coli BL21 (DE3) after induction with 0.2 mM IPTG at 30 °C for 10 h, generating a specific mouse anti-UL41 protein polyclonal antibody. RT-qPCR and western blot analyses revealed that the UL41 transcript number peaked at 36 hpi, and peak protein expression occurred at 48 hpi. The pharmacological test showed that UL41 was a γ2 gene. Mass spectrometry analysis showed that pUL41 was a virion component. IFA results revealed that pUL41 was localized throughout DEV-infected cells but only localized to the cytoplasm of transfected cells. DEV pUL47 translocated pUL41 to the nuclei of DEF cells; this translocation was dependent on predicted pUL47 NLS signals (40–50 aa and 768–777 aa). Conclusions DEV UL41 is a γ2 gene that encodes a virion structural protein, pUL41 localizes throughout DEV-infected cells but only localizes to the cytoplasm of transfected cells. pUL41 cannot autonomously localize to the nucleus, as this nuclear localization is dependent on predicted DEV pUL47 NLS signals (40–50 aa and 768–777 aa).

Saccharum arundinaceus is one of the most important species of sugarcane related species, and with strong resistance. Superoxide dismutases (SODs) play an important role in stress tolerance in plants. In this paper, two novel full-length cDNA sequence of the Cu/Zn SOD gene, denoted as SaSOD-1a and SoSOD1a (GenBank Accession number KJ002569 and KT327179), were isolated from S. arundinaceus and sugarcane, respectively. SaSOD-1a cDNA is 689 bp in length, including 33 bp of 5'-untranslated region (UTR) and 35 bp of the 3'-UTR, and a 621-bp open reading frame (ORF) encoding a 206 amino-acid sequence of the protein which has a conserved domain of superoxide dismutase belonging the SOD family. The SoSOD1a sequence is 690 bp, containing a 600 bp ORF, a 36-bp 5’-UTR and 54-bp 3’-UTR. It encoded the 199 amino-acid sequence of the protein which also has a conserved domain of superoxide dismutase belonging the SOD family. Between these two sequences, there was 97.5% similarity, 17 single nucleotide polymorphism (SNP) sites and 4 small insertion/deletion fragments; 95.5% protein similarity and 10 amino-acid mutation sites. In homologous evolutionary analysis, the Cu/Zn SOD genes from different plant species were rather conservative. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the prokaryotic expression product was a fusion protein. Real-time quantitative polymerase chain reaction analysis demonstrated that the expression profile of SaSOD-1a and SoSOD1a were different under various drought stress duration. It was also suggested that SOD1a has a different drought response mode in sugarcane and the wild species S. arundinaceus.

Ginseng (Panax ginseng C.A. Mey.) is a precious Chinese traditional medicine, for which ginsenosides are the most important medicinal ingredients. Cytochrome P450 enzymes (CYP450) and their primary redox molecular companion NADPH cytochrome P450 reductase (CPR) play a key role in ginsenoside biosynthesis pathway. However, systematic studies of CPR genes in ginseng have not been reported. Numerous studies on ginsenoside synthesis biology still use Arabidopsis CPR (AtCPR1) as a reductase. In this study, we isolated two CPR genes (PgCPR1, PgCPR2) from ginseng adventitious roots. Phylogenetic tree analysis showed that both PgCPR1 and PgCPR2 are grouped in classⅡ of dicotyledonous CPR. Enzyme experiments showed that recombinant proteins PgCPR1, PgCPR2 and AtCPR1 can reduce cytochrome c and ferricyanide with NADPH as the electron donor, and PgCPR1 had the highest enzymatic activities. Quantitative real-time PCR analysis showed that PgCPR1 and PgCPR2 transcripts were detected in all examined tissues of Panax ginseng and both showed higher expression in stem and main root. Expression levels of the PgCPR1 and PgCPR2s were both induced after a methyl jasmonate (MeJA) treatment and its pattern matched with ginsenoside accumulation. The present investigation suggested PgCPR1 and PgCPR2 are associated with the biosynthesis of ginsenoside. This report will assist in future CPR family studies and ultimately improving ginsenoside production through transgenic engineering and synthetic biology.

Terpenoids are one of the main components of plant aromas. In the present study we investigated these compounds in Osmanthus fragrans Lour., which is a fragrant plant widely used for the production of essential oils. Quantitative real-time PCR (qRT-PCR) results of enzymes associated with the 2-C-methylerythritol-4-phosphate pathway confirmed that the TPS is a key enzyme for terpenoid synthesis in O. fragrans. In a series of experiments, we identified the TPS candidate genes in O. fragrans and revealed the underlying catalytic activity and subcellular localisation of the encoded proteins. Because there is no available O. fragrans reference genome, we sequenced and analysed its transcriptome and identified two putative TPS genes, OfTPS1 and OfTPS2. According to qRT-PCR analysis, both genes were most highly expressed at the full-bloom stage, suggesting that OfTPS1 and OfTPS2 contribute to O. fragrans terpenoid synthesis. To verify this hypothesis, we constructed prokaryotic expression vectors to obtain protein. In order to study the function of OfTPS1 and OfTPS2 in the synthesis of monoterpenes, the obtained proteins were reacted with geranyl pyrophosphate. As a result, two kinds of monoterpenes, (E)-β-ocimene and linalool, were detected from reaction products, respectively. In conclusion, OfTPS1 and OfTPS2 are both monoterpene synthases. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The formation of underwater adhesion is a complicated physiological process and many different types of enzymes are found to be essential apart from structural proteins. Previous studies have shown that various tyrosinases were present in marine adhesives, but little information is available about the over-expression and enzymatic characterization of these enzymes. Specifically, this study first identified four significantly up-regulated tyrosinases in the pedal disc of Haliplanella luciae by means of multi-omics technology, and made preliminary bioinformatics predictions. Sequence alignment showed that the Tyr1_Hl contained six conserved His residues that bind to copper ions, of which a tyrosinase with diphenolase activity named as Tyr1_HlΔ, was expressed in Escherichia coli BL21 (DE3) cells and purified by affinity chromatography. Enzymatic characterization showed that the activity of Tyr1_HlΔ was Cu2+ dependent and maximum catalytic activities were in 20 mM Tris–HCl (pH 8.0) at 37 °C. In summary, we identified novel tyrosinases in the pedal disks of sea anemone for the first time and the Tyr1_HlΔ was successfully recombinant expressed. Our study will provide basis for future exploration of bio-adhesion mechanism and design of bio-adhesives derived from sea anemones. [ABSTRACT FROM AUTHOR]

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Actinobacillus pleuropneumoniae (A. pleuropneumoniae/APP) is the pathogen that causes porcine contagious pleuropneumonia. Actinobacillus pleuropneumoniae is divided into 18 serovars, and the cross protection efficacy of epitopes is debatable, which has resulted in the slow development of a vaccine. Consequently, epitope-based vaccines conferring Actinobacillus pleuropneumoniae cross protection have rarely been reported. In this study, B cell epitopes in the head domain of trimeric autotransporter adhesin were predicted, and 6 epitopes were selected. Then, the predicted epitopes (Ba1, Bb5, C1, PH1 and PH2) were connected by linkers to construct a recombinant tandem antigen (rta) gene. The RTA protein encoded by the recombinant rta gene was expressed, and finally the ICR mice were immunized with the RTA protein with or without inactivated Actinobacillus pleuropneumoniae (serovars 1 and 5b) and challenged with Actinobacillus pleuropneumoniae to evaluate the protective effect of the epitope-based vaccine and combined vaccine. The mice in the RTA-immunized group and RTA plus inactivated Actinobacillus pleuropneumoniae vaccine group had a significant improvement in clinical symptoms and a higher level of antibody in the serum than those in the control group. The RTA immune group had a 40% survival rate after Actinobacillus pleuropneumoniae infection, whereas the combination of RTA and inactivated Actinobacillus pleuropneumoniae produced very strong cross immune protection in mice, at least 50% (RTA IB1 + C5) and at most 100% (RTA IB5 + C1), whereas no cross immunoprotection was found in the solo Actinobacillus pleuropneumoniae immune group. Overall, the combination of the RTA protein and inactivated bacteria significantly enhanced the cross protection effects. This implies that RTA protein in combination with a suitable inactivated Actinobacillus pleuropneumoniae strain could be a candidate vaccine for porcine contagious pleuropneumonia. [ABSTRACT FROM AUTHOR]

We optimized a method for the preparation and purification of self-assembled protein nanocontainers EPN, the latest achievement in protein engineering. These nanocontainers are highly stable and provide the possibility of highly specific loading of a protein therapeutic drug. The described technique can be proposed as a tool for production of nanocontainers in a prokaryotic system. The obtained nanocontainers for the delivery of protein preparations can be used in the treatment of chronic, autoimmune, and oncological diseases. [ABSTRACT FROM AUTHOR]

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Thladiantha dubia mosaic virus (ThDMV) from the wild Manchurian tuber gourd (Thladiantha dubia Bunge, Cucurbitaceae), is a novel tentative specie of the genus Potyvirus within the family Potyviridae. It may pose a potential risk to both cultivated crops and plants in the natural ecosystem. In the present study, we developed an ELISA procedure for detection of ThDMV. Coat protein (CP) of ThDMV was successfully expressed in Escherichia coli, and used to immunize rabbit for antiserum after being purified by using Ni-NTA affinity column. Results of indirect ELISA showed that the optimum dilutions of the coating antigen and antiserum were 1:160 (1 μg per well), and 1:2000 respectively. Western blot indicated that antibodies in the antiserum could specifically recognize and bind CP of ThDMV. An ELISA method was developed with the antiserum to detect ThDMV in plant extracts. Samples collected from potato fields in Northeast China and Northwest China were analyzed by ELISA and RT-PCR and both gave consistent results. The ELISA assay developed here has potential to be used as a sensitive, reliable and cost-effective tool for ThDMV detection in epidemiological studies. [ABSTRACT FROM AUTHOR]

Duck Tembusu virus (DTMUV) is a single-stranded, positive-sense RNA arbovirus, belonging to the genus Flavivirus, the family Flaviviridae. As a transmembrane protein, non-structural protein 2A (NS2A) plays an important role in virion assembly, replication complex and antagonizing host immune response. Since NS2A protein contains many hydrophobic amino acids, it is hard to gain the full-length protein of NS2A for prokaryotic expression. Therefore, to make a deep study, prokaryotic expression and polyclonal antibody preparation of truncated DTMUV NS2A was performed. The truncated NS2A gene (178-450 bp) was obtained, and sub-cloned into the prokaryotic vector pGE X-4T-1 (pGE X-4T-1-NS2A178-450bp). Subsequently, the recombinant GST-NS2A60-150aa protein was successfully expressed in E. coli BL21 (DE3) with the induction by 0.3 mmol/l isopropyl ß-D-thiogalactoside (IPTG) for 6 h at 37°C. The GST-NS2A60-150aa protein was extracted from the gel. The BALB/c mice were immunized with the purified recombinant NS2A protein to prepare polyclonal antibodies against the truncated NS2A protein. The titer of the polyclonal antibodies, determined by ELISA analysis, was 1:128,000. The specificity of the polyclonal antibodies (mPAb-DTMUV-NS2A60-150aa) were verified by Western blot analysis. Furthermore, the indirect immunofluorescence (IFA) was performed to explore the subcellular localization of NS2A. NS2A protein was, in the transfected cells, located mainly around nucleus in the endoplasmatic reticulum. Taken together, our study provided a useful tool for the further exploration of the biological functions and molecular mechanism of DTMUV NS2A. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

【Objective】To further study the function of the Orf virus 008 protein,the 008 gene was expressed by prokaryotic expression system and analyzed by bioinformatics.【Method】The 008 gene fragment was amplified by a clinical isolate from Chongqing,and ligated into the pET-28 a(+) vector to construct a recombinant expression plasmid to induce expression of the protein.The 008 protein was analyzed for hydrophobicity using the ProtScale program,the TMHMM server v.2.0 was used to analyze the transmembrane region,the SignalP 5.0 server was used to predict the signal peptide,and the B cell epitope was predicted by Protean.【Result】The ORFV 008 gene can be expressed in E.coli.The molecular weight of the recombinant protein is about 56 kd,which is mainly expressed in the form of inclusion bodies and has specific binding reaction with positive serum,and has good antigenicity.Bioinformatics studies show that the 008 protein belongs to a hydrophobic protein,no transmembrane region and a signal peptide region,and there may be 11 B cell epitope.【Conclusion】The constructed pET-28 a(+)-008 plasmid can be successfully expressed protein 008 under the induction of IPTG,and the protein 008 had good antigenicity [ABSTRACT FROM AUTHOR]

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Copyright of Journal of Zhengzhou University (Natural Science Edition) is the property of Journal of Zhengzhou University (Natural Science Edition) Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

[Objectives] To investigate the expression profile of the saliva protein BphMIF1 gene of the brown planthopper, Nilaparvata lugens, an important insect pest of rice, in response to the feeding activity of this species. [Methods] BphMIF1 was cloned by RT-PCR and sequenced for bioinformatic analysis. The expression levels of BphMIF1 in nymphs (1st-5th instars), adults and different adult body parts, were analyzed with real-time fluorescent quantitative PCR (qPCR), and the prokaryotic expression of BphMIF1 was also quantified. [Results] The BphMIF1 gene was cloned and found to encode 119 amino acids. qPCR results showed that BphMIF1 was rapidly up-regulated in 4th instar nymphs. BphMIF1 was expressed in the head, thorax and abdomen of adults, but was most highly expressed in the abdomen. Prokaryotic expression analysis showed that the BphMIF1 gene was expressed at an induced concentration of 0.5 mmol/L IPTG for 4 h. [Conclusion] The BphMIF1 gene is differentially expressed in different age groups and body parts of N. lugens. These results provide a theoretical basis for the functional study of BphMIF1 in response to the feeding process of N. lugens. [ABSTRACT FROM AUTHOR]

A recombinant gene CCS consisting of three single genes (CAP-ScFv, CPFX-ScFv and SM2-ScFv) were constructed by PCR with three pairs of primers. The length of CCS gene fragment was 2,260 bp. A recombinant plasmid (pGEX-CCS) was obtained in pGEX-6p-1. pGEX-CCS was induced in E.coli BL21(DE3), and the molecular weight of recombinant fusion protein (GST-CCS) was 108.87 kDa. GST-CCS was successfully applied to analysis of CAP-OVA (CAP-ovalbumin conjugate), CPFX-OVA and SM2-OVA simultaneously. The sensitivity of GST-CCS against three veterinary drugs was tested by indirect ELISA at dilution ratio 1:8000. These findings provide a foundation for the construction of fusion genes with linkers and for the potential development of a rapid screening method for the simultaneous detection of veterinary drug residues. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Henan Agricultural Sciences is the property of Editorial Board of Journal of Henan Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)