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A histidine (His)-tag is composed of six His residues and typically exerts little influence on the structure and solubility of expressed recombinant fusion proteins. Purification methods for recombinant proteins containing His-tags are relatively well-established, thus His-tags are widely used in protein recombination technology. We established a one-step enzyme-linked immunosorbent assay (ELISA) for His-tagged recombinant proteins. We analyzed variable heavy and light chains of the anti-His-tag monoclonal antibody 4C9 and used BLAST analyses to determine variable zones in light (VL) and heavy chains (VH). VH, VL, and alkaline phosphatase (ALP) regions were connected via a linker sequence and ligated into the pGEX-4T-1 expression vector. Different recombinant proteins with His tags were used to evaluate and detect ALP-scFv activity. Antigen and anti-His-scFv-ALP concentrations for direct ELISA were optimized using the checkerboard method. ZIKV-NS1, CHIKV-E2, SCRV-N, and other His-tag fusion proteins demonstrated specific reactions with anti-His-scFv-ALP, which were accurate and reproducible when the antigen concentration was 50 µg mL−1 and the antibody concentration was 6.25 µg mL−1. For competitive ELISA, we observed a good linear relationship when coating concentrations of recombinant human anti-Müllerian hormone (hAMH) were between 0.78 and 12.5 µg mL−1. Our direct ELISA method is simple, rapid, and accurate. The scFv antibody can be purified using a prokaryotic expression system, which provides uniform product quality and reduces variations between batches.

In the field of environmental science research, an important research direction is to find a suitable, effective, material and method for desulphurization of fossil fuels and prevent the release of sulfide in fuels into the environment. In this study, a novel catalyst consisting of Pd(CH3COO)2 and MIL-101(Cr) was used to study the sulfur oxidation of organic sulfides, which are difficult to treat, In simulated fuel oil. The optimum reaction conditions were discussed, including the loading of The optimum reaction conditions were discussed, including reaction temperature, the content of Pd in MIL-101(Cr), catalyst dosage, initial BT concentration and cycle times. The BT conversion efficiency of the catalyst in simulated fuel is 88.77%, It shows that the catalyst has good desulfurization performance.

The catalysis of disulphide (SS) bonds is the most important characteristic of protein disulphide isomerase (PDI) family. Catalysis occurs in the endoplasmic reticulum, which contains many proteins, most of which are secretory in nature and that have at least one s-s bond. Protein disulphide isomerase A3 (PDIA3) is a member of the PDI family that acts as a chaperone. PDIA3 is highly expressed in response to cellular stress, and also intercept the apoptotic cellular death related to endoplasmic reticulum (ER) stress, and protein misfolding. PDIA3 expression is elevated in almost 70% of cancers and its expression has been linked with overall low cell invasiveness, survival and metastasis. Viral diseases present a significant public health threat. The presence of PDIA3 on the cell surface helps different viruses to enter the cells and also helps in replication. Therefore, inhibitors of PDIA3 have great potential to interfere with viral infections. In this review, we summarize what is known about the basic structure, functions and role of PDIA3 in viral infections. The review will inspire studies of pathogenic mechanisms and drug targeting to counter viral diseases.

Klebsiella pneumoniae belongs to Enterobacteriaceae, which is the commonest bacterium causing nosocomial respiratory tract infection. It ranks second in bacteremia and urinary tract infection in gram-negative bacteria. Therefore, the rapid and accurate identification of K. pneumoniae was of great significance for the guide of clinical medication, and timely treatment of patients. The purpose of this study was to establish a rapid and sensitive molecular detection method for K. pneumoniae based on loop-mediated isothermal amplification (LAMP) technology. Firstly, local BLAST and NCBI BLAST were used to analyze the genome of K. pneumoniae. According to the principle of interspecific and intraspecific specificity, CelB (GenBank ID 11847805) was selected as the specific gene. Then, the LAMP and PCR identification systems were established with this target gene. Thirty-six clinical isolates of K. pneumoniae and 50 non-K. pneumoniae were used for the specific evaluation, and both LAMP and PCR could specifically distinguish K. pneumoniae from non-K. pneumoniae. A 10-fold series diluted positive plasmids and simulated infected blood samples were used as the templates in the sensitivity assay, and the results showed that the sensitivity could reach 1 copy/reaction. In summary, a rapid, specific, and sensitive LAMP method was established to detect K. pneumoniae in clinics.

With the panel data of 218 prefecture-level cities from 2003 to 2017, this paper empirically tested whether urban environmental legislation realized Porter Hypothesis(PH) in China. After a series of model estimation and robustness tests, the results show that urban environmental legislation increased the number of local green patents, which means that the weak Porter Hypothesis was established. However, the urban environmental legislation did not lead to an increase in green total factor productivity(GTFP). In other words, the strong version of PH did not hold. Further analysis shows that urban environmental legislation led to the decline of GTFP and the increase of green patents in the west of China, but not in the east and central cities. Besides, the legislation did not promote GTFP improvement through green innovation in the short term, which means it did not realize process compensation.

Deoxynivalenol (DON) is a mycotoxin commonly found in cereals and animal feeds, which also is a carcinogen easily soluble in water. So it has drawn considerable interest in food safety. In this study, a specific aptamer named DON-A16 was developed from an immobilized ssDNA library by affinity chromatography column-based the SELEX. The complementary chain competition method for DON detection was established, and its limit of detection (LOD) was 488 ng/mL. Graphene oxide-based fluorescence quenching was developed using DON-A16 with fluorescein (FAM) label; its LOD was 200 ng/mL, and the detection time was 3 h. The recovery rate of fluorescence quenching was also detected in different samples (oat, wheat flour, corn), which showed the commercial DON ELISA kit led to an increase in repeatability, satisfactory recovery and consistency. The three-dimensional structure and the main binding site of of DON-A16 was ultimately obtained by molecular docking simulation.

Background: Shigella is among the most important human pathogenic microorganisms, infecting both humans and nonhuman and causing clinically severe diarrhea. Shigella must be enriched before detection, which is time-consuming. Objectives: To develop a sensitive, rapid, and specific method for Shigella detection. Materials and Methods: Shigella was used as an antigen to generate monoclonal antibodies (mAbs). mAbs were screened via indirect enzyme-linked immunosorbent assay (ELISA) and western blot, and two mAbs were selected. The mAb A3 showed high affinity and specificity and was used to develop immune magnetic beads (IMBs) for Shigella enrichment. An immunocapture (IC)-PCR primer was designed from the ipaH gene, and IC-PCR was developed based on the IMBs and PCR. Results: This system shortened the Shigella detection time to 70 min. The sensitivity of the IC-PCR was 9 colony-forming units.mL-1 in artificial milk. The accuracy of the IC-PCR was confirmed using 46 clinical samples collected from monkeys. The IC-PCR results were consistent with the serological and biochemical assays. Conclusion: The IC-PCR described herein accurately detected Shigella from milk samples, monkeys and can thus be used to complement classical detection methods.

NAC (for NAM, ATAF1-2, and CUC2) proteins are one of the largest transcription factor families in plants. They have various functions and are closely related to developmental processes of fruits. Tomato (Solanum lycopersicum) is a model plant for studies of fruit growth patterns. In this study, the functional characteristics and action mechanisms of a new NAC-type transcription factor, SlNAC3 (SGN-U568609), were examined to determine its role in tomato development and ripening. The SlNAC3 protein was produced by prokaryotic expression and used to immunize New Zealand white rabbits to obtain a specific polyclonal antibody against SlNAC3. By co-immunoprecipitation and MALDI-TOF-MS assays, we showed that there was an interaction between the SlNAC3 protein and Polygalacturonase-2 (PG-2), which is related to the ripening and softening of fruit. Chromatin immunoprecipitation assays revealed the genome of the highly stress-tolerant Solanum pennellii chromosome 10 (sequence ID, HG975449.1), further demonstrating that SlNAC3 is a negative regulator of drought and salinity stress resistance in tomato, consistent with previous reports. These results indicate that SlNAC3 is not only involved in abiotic stress, but also plays a necessary role in mediating tomato ripening.

Background: Zika Virus (ZIKV) is a new type of flaviviruses transmitted by mosquitoes to cause severe diseases including Guillain-Barre syndrome and congenital malformations during recent outbreaks. The early diagnosis of ZIKV infection and efficient vaccines would be effective in controlling epidemics and timely treatment. However, the native nonstructural protein 1 (NS1) mainly acquired from cells infected with ZIKV, which largely limits its application. Objectives: This study aimed to express and purify the recombinant NS1 protein and prepare its antibody that efficiently reacted with the native NS1 protein secreted in the supernatant of ZIKV infected host-cells. Methods: We constructed a prokaryotic expression vector containing the full length of the NS1 gene. Thus, the recombinant NS1 protein was efficiently expressed and purified. The purified NS1 protein was used to prepare an antibody. The Enzyme-linked Immunosorbent Assay (ELISA), Western blot, and Indirect Immunofluorescent Assay (IFA) were used to assess the reactivity and specificity of the antibody. Results: The recombinant NS1 protein from a prokaryotic expression vector had good immunogenicity and the prepared antibody could specifically react with the native NS1 produced in Vero, BHK-21 cells infected with ZIKV. Besides, the ELISA and Western blot showed that the native ZIKV-NS1 protein was secreted extracellularly and could play the role of an early diagnostic biomarker for ZIKV infection. Conclusions: The prepared antibody against the recombinant NS1 protein is a reliable biological tool that enables the rapid and sensitive detection of secreted NS1 from host cells infected with ZIKV. The recombinant NS1 protein and its antibody can contribute to developing vaccines and antibodies targeting the NS1 protein to prevent flavivirus disease progression.

Salmonella, a common foodborne pathogen, causes many cases of foodborne illness and poses a threat to public health worldwide. Immunological detection systems can be combined with nanoparticles to develop sensitive and portable detection technologies for timely screening of Salmonella infections. Here, we developed an antibody-probe-based immuno-N-hydroxysuccinimide (NHS) bead (AIB) system to detect Salmonella. After adding the antibody probe, Salmonella accumulated in the samples on the surfaces of the immuno-NHS beads (INBs), forming a sandwich structure (INB&ndash, Salmonella&ndash, probes). We demonstrated the utility of our AIB diagnostic system for detecting Salmonella in water, milk, and eggs, with a sensitivity of 9 CFU mL&minus, 1 in less than 50 min. The AIB diagnostic system exhibits highly specific detection and no cross-reaction with other similar microbial strains. With no specialized equipment or technical requirements, the AIB diagnostic method can be used for visual, rapid, and point-of-care detection of Salmonella.

Background: Klebsiella pneumoniae is an important human pathogen that causes severe diseases including urinary tract infection, pneumonia, and bacteremia. However, a rapid and sensitive detection method remains to be developed. Objectives: This study aimed to develop a rapid, real-time, and visual detection method for K. pneumoniae. This is a primary screening method to improve the diagnosis of K. pneumoniae infection and save much precious time for clinical practice. Methods: Klebsiella pneumoniae was used as an antigen to produce a monoclonal antibodies (mAb). The mAb 1E6 recognizing outer membrane protein C on the surface of K. pneumoniae was screened by an indirect enzyme-linked immunosorbent assay (ELISA), Western Blot, and mass spectrometry assay, and then conjugated with the protein A/G coated magnetic beads to generate the immune magnetic beads (IMBs). Thereafter, the IMBs were used to capture K. pneumoniae, and the complex (beads-mAb-K. pneumoniae) was used for loop-mediated isothermal amplification (LAMP) assay. Results: We developed a rapid, real-time, and visual detection method employing an immunocapture loop-mediated isothermal amplification (IC-LAMP). The sensitivity of the IC-LAMP was 4 CFU mL-1. The process of K. pneumoniae detection lasted ~ 60 minutes and had no cross-reaction to other microbial strains. Besides, the accuracy of IC-LAMP was further verified by examining 39 clinical isolates. Conclusions: Without the need for enrichment of bacteria and extraction of its genome, the IC-LAMP developed here could be used as a primary screening method supplementary to traditional detection methods to improve the diagnosis of K. pneumoniae infection for clinical practice.

Background: The outbreak of Zika virus (ZIKV) in many countries caused alarming numbers of babies born with microcephalus and severe neurologic disorders, however, the methods for the detection of the ZIKV are limited at present. Objectives: The aim of this study was to produce polyclonal antibody against full length envelop protein of ZIKV (ZIKV-E protein) in prokaryotic system and to evaluate its efficacy to capture ZIKV virion. Methods: The recombinant full length ZIKV-E protein was purified and then used as an immunogen to vaccinate BALB/c mice to produce polyclonal antibody. Protein A/G coated magnetic beads were coated with the polyclonal antibody to capture the ZIKV virion in the culture medium of Vero cells infected with ZIKV. The products of immunoprecipitation were further used for Western Blot, Loop-mediated isothermal amplification (LAMP), and PCR assay. Results: Western Blot and indirect immunofluorescence analysis showed that the mouse polyclonal antibody could react specifically with the native E protein in Vero cells infected with ZIKV. The results of Western Blot, Immunocaptured-LAMP (IC-LAMP), and Immunocaptured-PCR (IC-PCR) showed that polyclonal antibody of ZIKV-E recombinant protein can capture ZIKV virion specifically, however, the polyclonal antibody against the ZIKV-NS1, Shigella, and Salmonella without such a function. Conclusions: These findings may provide the basis for the development of the rapid diagnostic kits such as, IC-PCR, IC-LAMP, and sandwich ELISA. The serum virion capture activity elicited by prokaryotic expressed recombinant ZIKV-E protein may also provide a basis for further preparation of neutralizing antibody and protein subunit vaccine candidate against ZIKV.

Background: Brucella spp. are Gram-negative bacteria that cause a zoonotic disease called brucellosis in humans as well as many animals. Brucella suis (B.suis) is one of the greatest threats to the human health and food safety. Studying macrophage and B. suis interaction is critical for understanding the chronic infection mechanism. However, the interaction mechanisms, especially for molecular events triggered by B. suis infected macrophage, such as biological pathways, are still obscure. Objectives: We will use gene set enrichment analysis (GSEA) to microarray in an attempt to find critical pathways in the interaction of macrophage and B. suis. Methods: We applied a standardized microarray preprocessing and GSEA to 2 independent macrophage and B. suis interaction studies including smooth virulent B. suis strain 1330 (S1330) data sets and rough attenuated B. suis strain VTRS1 (VTRS1) data sets. Integrative analysis was used to find critical pathways for 2 independent macrophage and B. suis interaction data sets. Results: The results demonstrated that for S1330 data sets, 8 and 13 common up- and down- regulated pathways were found in 4 interaction stages including 4h, 8h, 24h, and 48h post macrophage infected S1330 B. suis, and for VTRS1 data sets, we found 30 and 19 common up- and down- regulated pathways. Comparing the results of S1330 and VTRS1 data sets, 6 and 8 common up- and down- regulated pathways were identified. Conclusions: The study of macrophage and B. suis interaction through pathway analysis highlighted genes weakly connected to the phenotype, and discovered common critical pathways in the process of macrophages and different phenotypes of B. suis interaction. The identified pathways will shed light on the understanding of the functional events within macrophage post infected B. suis.

Shigella is an important human food-borne zoonosis bacterial pathogen, and can cause clinically severe diarrhea. There is an urgent need to develop a specific, sensitive, and rapid methodology for detection of this pathogen. In this study, loop-mediated isothermal amplification (LAMP) combined with magnetic immunocapture assay (IC-LAMP) was first developed for the detection of Shigella in pure culture, artificial milk, and clinical stool samples. This method exhibited a detection limit of 8.7 CFU/mL. Compared with polymerase chain reaction, IC-LAMP is sensitive, specific, and reliable for monitoring Shigella. Additionally, IC-LAMP is more convenient, efficient, and rapid than ordinary LAMP, as it is more efficiently enriches pathogen cells without extraction of genomic DNA. Under isothermal conditions, the amplification curves and the green fluorescence were detected within 30 min in the presence of genomic DNA template. The overall analysis time was approximately 1 h, including the enrichment and lysis of the bacterial cells, a significantly short detection time. Therefore, the IC-LAMP methodology described here is potentially useful for the efficient detection of Shigella in various samples.

Background: Rabies is a zoonotic infectious disease that infects the human and animal central nervous system. Worldwide, especially in low-income countries, this disease is still a burden for public health. Among the rabies virus proteins, phosphoprotein plays a very important role in viral infection, and this research found that immunization of rabies virus vaccines could widely induce antibody responses against phosphoprotein, therefore rabies virus phosphoprotein may be a useful target for development of rapid and low cost serological diagnosis test, and therapeutic drugs. Objectives: The aim of this study was to prepare anti-rabies virus phosphoprotein monoclonal antibody, which is used for rapid detection and diagnosis of rabies virus infection. Methods: The phosphoprotein gene was amplified by the polymerase chain reaction (PCR), and sub-cloned in a his-tagged prokaryotic expression vector to generate a recombinant plasmid named pET-32a-RABV-P. Recombinant RABV-P was induced by isopropyl-β-D-Thiogalactopyranoside (IPTG), and then purified by the Ni-NTA purification system. Immunization of BALB/c mice with the recombinant protein was performed, and the spleen cells of the immunized mice and SP2/0 myeloma cells were fused together to obtain the monoclonal cell strains, and then identification of the characteristics of the antibody by the enzyme linked immunosorbent assay (ELISA), western blotting, and indirect immunofluorescence assay was done. Results: The prokaryotic expression vector of pET-32a-RABV-P was successfully constructed. The fusion protein was expressed in Escherichia coli Rosetta and purified. After cell fusion, a hybridoma cell line 1A4 was successfully obtained. The antibody titer of the anti-RABV-P ascites reached 256,000. The results of western blotting and indirect immunofluorescence assay indicated that 1A4 hybridoma cell line was able to produce specific antibodies against rabies virus phosphoprotein. Conclusions: Recombinant rabies virus phosphoprotein could be successfully expressed in E. coli. A specific monoclonal antibody against rabies virus phosphoprotein has been successfully prepared.

Background and Aims Aluminium (Al) toxicity is a limiting factor for plant growth and crop production in acidic soils. Citrate exudation and activation of the plasma membrane H+-ATPase are involved in soybean responses to Al stress. Auxin has crucial functions in plant growth and stress responses. However, little is known about possible interactions between auxin and citrate exudation under Al stress. In this study, we elucidated the regulatory roles of IAA in Al-induced citrate exudation in soybean roots. Methods We measured IAA content, Al concentration, citrate exudation, plasma membrane H+-ATPase activity, expression of the relevant genes and phosphorylation of the plasma membrane H+-ATPase by integrating physiological characterization and molecular analysis using hydroponically grown soybean. Key Results The concentration of IAA was increased by 25 and 50 μm Al, but decreased to the control level at 200 μm Al. External addition of 50 μm IAA to the root medium containing 25, 50 or 200 μm Al decreased root Al concentration and stimulated Al-induced citrate exudation and the plasma membrane H+-ATPase activity. Reverse transcription-PCR analysis showed that exogenous IAA enhanced the expression of citrate exudation transporter (GmMATE) but not the plasma membrane H+-ATPase gene. The western blot results suggested that IAA enhanced phosphorylation of the plasma membrane H+-ATPase under Al stress. Conclusions Auxin enhanced Al-induced citrate exudation through upregulation of GmMATE and an increase in phosphorylation of the plasma membrane H+-ATPase in soybean roots.

Background Herpes B virus (BV) is a zoonotic disease caused by double-stranded enveloped DNA virus with cercopithecidae as its natural host. The mortality rate of infected people could be up to 70% with fatal encephalitis and encephalomyelitis. Up to now, there are no effective treatments for BV infection. Among the various proteins encoded by monkey B virus, gD, a conserved structural protein, harbors important application value for serological diagnosis of frequent variations of the monkey B virus. Objectives This study aimed to expressed the gD protein of BV in Escherichia coli by a recombinant vector, and prepare specific monoclonal antibodies against gD of BV to pave the way for effective and quick diagnosis reagent research. Materials and Methods The gD gene of BV was optimized by OptimWiz to improve codon usage bias and synthesis, and the recombinant plasmid, pET32a/gD, was constructed and expressed in E. coli Rosetta (DE3). The expressed fusion protein, His-gD, was purified and the BALB/c mice were immunized by this protein. Spleen cells from the immunized mice and SP2/0 myeloma cells were fused together, and the monoclonal cell strains were obtained by indirect enzyme-linked immunosorbent assay (ELISA) screening, followed by preparation of monoclonal antibody ascetic fluid. Results The optimized gD protein was highly expressed in E. coli and successfully purified. Five monoclonal antibodies (mAbs) against BV were obtained and named as 4E3, 3F8, 3E7, 1H3 and 4B6, and with ascetic fluid titers of 2 × 106, 2 × 105, 2 × 105, 2 × 103 and 2 × 102, respectively. The 1H3 and 4E3 belonged to the IgG2b subclass, while 3E7, 3F8 and 4B6 belonged to the IgG1 subclass. Conclusions The cell lines obtained in this work secreted potent, stable and specific anti-BV mAbs, which were suitable for the development of herpes B virus diagnosis reagents.

Annexins have been suggested to play pivotal roles in stress resistance and plant development. However, related studies on fruit-bearing plants, especially on fruit development, are very limited. In the present study, we provide a comprehensive overview of the annexin family in tomato, describing the gene structure, promoter cis-regulatory elements, organ expression profile, and gene expression patterns under hormone and stress treatments. Bioinformatic analysis revealed that the nine tomato annexins were structurally different from their animal counterparts, but highly conserved annexin domains were still found in most of them. Cis-regulatory element prediction showed that there were important elements in the 2kb upstream promoter regions, including stress- and hormone-responsive-related elements. The expression patterns of these genes were investigated, and the results revealed that they were regulated under developmental processes and environmental stimuli. Among them, AnnSl1.1 and AnnSl2 were highly expressed in most of the tested organs. Genes preferentially or specifically expressed in organs, such as stigma or ovary (AnnSl6), stamen (AnnSl8), and fruit pericarp (AnnSl1.2 and AnnSl9), were identified. Some annexin genes were induced by plant hormones including abscisic acid (AnnSl3, AnnSl6, AnnSl8, and AnnSl9) and gibberellic acid (AnnSl1.1, AnnSl1.2, AnnSl4, and AnnSl7). Most of these annexin genes were induced by salt, drought, wounding, and heat or cold stresses. The present study provides significant information for understanding the diverse roles of annexins in tomato growth and development.

Plant microRNAs (miRNAs) are vital components of the translation control system that regulates plant development and reproduction. The biological function of sly-miR156 was investigated by over-expression in tomato plants. Transgenic tomato plants exhibited a drastically altered phenotype, with reduced height, smaller but more numerous leaves, and smaller fruit. The inflorescence structure of sly-miR156 over-expressing plants phenocopied the sft mutant. The putative targets of sly-miR156 were identified by data base search and included six SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box transcription factor genes. Their expression patterns were then determined in 35S-miR156a and wild type tomato plants. These target genes, as well as the tomato FLOWERING LOCUS T (FT) ortholog SFT, were significantly down-regulated in sly-miR156 over-expressing plants. These studies reveal novel phenotypes regulated by miR156.

BACKGROUND: Rabies virus (RABV) is one of the old deadly zoonotic viruses. It attacks the central nervous system and causes acute encephalitis in humans and animals. Host factors are known to be essential for virus infection and replication in cells. The identification of the key host factors required for RABV infection may provide important information on RABV replication and may provide new potential targets for RABV drug discovery. OBJECTIVES: This study aimed to investigate the change in the subcellular distribution and expression of the host protein Prefoldin subunit 1 (PFDN1) in RABV-infected cells and the viral expression of plasmids in the transfected cells. MATERIALS AND METHODS: Mouse Neuro-2a (N2a) cells were infected by RABV or transfected with the plasmids of the nucleoprotein (N) and/or phosphoprotein (P) gene of RABV. The subcellular distribution of PFDN1 was analyzed by confocal microscopy, and the transcription levels of PFDN1 in the N and/or P gene of the RABV-transfected or RABV-infected N2a cells were assessed via real-time quantitative polymerase chain reaction. RESULTS: Confocal microscopy showed that PFDN1 was colocalized with the N protein of RABV in the infected N2a cells and was mainly recruited to the characteristic Negri-Body-Like (NBL) structures in the cytoplasm, as well as the cotransfection of the N and P genes of RABV. The transcription of PFDN1 in the RABV-infected N2a cells was upregulated, whereas the transfection of the N and/or P genes did not result in the upregulation of PFDN1. CONCLUSIONS: The results of this work demonstrated that the subcellular distribution of PFDN1 was altered in the RABV-infected N2a cells and colocalized with the N protein of RABV in the NBL structures.

Background: Rabies virus (RABV) is a deadly neurotropic virus that causes the disease of rabies in humans and animals. L protein is one of the large structural protein of rabies virus, which displays multiple enzymatic activities, and is required for viral transcription and replication. Objectives: A truncated L protein of Rabies virus is being cloned, expressed and purified to produce relevant polyclonal antibody. Materials and Methods: The gene fragment of L protein of RABV was subcloned into prokaryotic expression vector pBackground: Rabies virus (RABV) is a deadly neurotropic virus that causes the disease of rabies in humans and animals. L protein is one of the large structural protein of rabies virus, which displays multiple enzymatic activities, and is required for viral transcription and replication. Objectives: A truncated L protein of Rabies virus is being cloned, expressed and purified to produce relevant polyclonal antibody. Materials and Methods: The gene fragment of L protein of RABV was subcloned into prokaryotic expression vector pET-28a and transformed into E. coli Rosetta DE3 host strain. The recombinant L protein of RABV was expressed and characterized by SDS-PAGE and western blot analysis using anti-his tag antibody. Mice were immunized with the purified recombinant L protein, the reaction of the anti-serum was checked by immunofluorescence and dot-blot, respectively. Results: The results of PCR and sequencing confirmed that the fragment of L gene of RABV was successfully cloned into the expression vector. The expression of recombinant L protein fragment induced by IPTG was confirmed by the band of 43 kDa in SDS-PAGE and western blot. The antiserum of purified L protein immunized mice was reacted with RABV infected N2a cells and suckling mouse brain tissue lysates. Conclusions: Our data showed that the recombinant L protein produced by pET-28a vector was very successful, and the purified L protein could efficiently induce the antibody response in mice. The antiserum could recognize the virus in RABV infected cells and tissue very well.ET-28a and transformed into E.coli Rosetta DE3 host strain. The recombinant L protein of RABV was expressed and characterized by SDS-PAGE and western blot analysis using anti-his tag antibody. Mice were immunized with the purified recombinant L protein, the reaction of the anti-serum was checked by immunofluorescence and dot-blot, respectively. Results: The results of PCR and sequencing confirmed that the fragment of L gene of RABV was successfully cloned into the expression vector. The expression of recombinant L protein fragment induced by IPTG was confirmed by the band of 43 kDa in SDS-PAGE and western blot. The antiserum of purified L protein immunized mice was reacted with RABV infected N2a cells and suckling mouse brain tissue lysates. Conclusions: Our data showed that the recombinant L protein produced by pET-28a vector was very successful, and the purified L protein could efficiently induce the antibody response in mice. The antiserum could recognize the virus in RABV infected cells and tissue very well.

Wild species often show more tolerance to environmental stress factors than their cultivated counterparts. An early responsive-to-dehydration gene was cloned from a drought- and salt-tolerant wild tomato Solanum pennellii (SpERD15). SpERD15 transcript accumulated differentially in different organs, and was remarkably induced by dehydration, salinity, cold and treatment with plant growth regulators. The protein encoded by SpERD15 was predominantly localized in the nucleus. Interestingly, we found that the majority of the transgenic tobacco plants were co-suppressed along with the overexpressing line. Overexpressing plants manifested stress tolerance accompanied by the accumulation of more soluble sugars and proline, and limited lipid peroxidation compared with co-suppression lines, which were more sensitive than the wild type. The differential contents of these compatible solutes in different transgenic lines were related to the changes in the expression of the genes involved in the production of some important osmolytes (P5CS and Sucrose synthase). Reduced lipid peroxidation over a broad range of stress factors was in agreement with increased expression of stress-responsive genes (ADH and GAPDH). Overexpression of SpERD15 increased the efficiency of PSII (F(v)/F(m)) in transgenic tobacco plants by maintaining PSII quinone acceptors in a partially oxidized form. The results show that SpERD15 augments stress tolerance by enhancing the efficiency of PSII through the protection of cellular membranes, as conferred by the accumulation of compatible solutes and limited lipid peroxidation.