Search

A DNA-binding protein (DBP) [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05), indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (p<0.01). The transcriptional level of dbp-ko-Bacmid early gene lef-3, ie-1, dnapol, late gene vp39 and very late gene p10 were statistically significantly lower than dbp-re-Bacmid and wtBacmid (p<0.01). The results presented are based on Western blot analysis, which indicated that the lack of dbp gene would lead to low expressions of lef3, vp39, and p10. In conclusion, dbp was not only essential for early viral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle. [ABSTRACT FROM AUTHOR]

Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people’s interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs. [ABSTRACT FROM AUTHOR]

To better understand the molecular mechanism underlying of diapause in Antheraea pernyi (A.pernyi), we cloned a novel diapause-associated protein 3 (DAP3) gene from A.pernyi by reverse transcription-polymerase chain reaction (RT-PCR) and studied the biological functions. Sequence analysis revealed that this gene encodes 171 amino acids and has a conserved domain of Copper/Zinc superoxide dismutase (Cu/Zn-SOD). Western blot and qRT-PCR results showed that DAP3 was mainly expressed in the pupal stage, and gradually decreased as diapause development. DAP3 was also expressed in 1st and 5th instar larvae of A.pernyi. In tissues of 5th instar larvae of A.pernyi, DAP3 was mainly expressed in the epidermis, followed by the head, hemolymph and fat body. To identify the SOD activity of DAP3, we constructed a prokaryotic expression vector by inserting the coding region sequence into plasmid pET-28a (+) and obtained the purified rHIS-DAP3 fusion protein by Ni-NTA affinitive column. Importantly, we found the SOD activity of DAP3 fusion protein was approximately 0.6674 U/µg. To further confirm the SOD activity of DAP3 in vivo, we induced the oxidative stress model of pupae by UV irradiation. The results showed that both the mRNA and protein level of DAP3 significantly increased by UV irradiation. Furthermore, the SOD activity of the total lysate of pupae increased significantly at 10 min post UV irradiation and transiently returned to normal level afterwards. These results suggested that DAP3 might be a novel protein with SOD activity getting involved in regulation of diapause in A.pernyi. [ABSTRACT FROM AUTHOR]

To better understand the molecular mechanism underlying of diapause in Antheraea pernyi (A.pernyi), we cloned a novel diapause-associated protein 3 (DAP3) gene from A.pernyi by reverse transcription-polymerase chain reaction (RT-PCR) and studied the biological functions. Sequence analysis revealed that this gene encodes 171 amino acids and has a conserved domain of Copper/Zinc superoxide dismutase (Cu/Zn-SOD). Western blot and qRT-PCR results showed that DAP3 was mainly expressed in the pupal stage, and gradually decreased as diapause development. DAP3 was also expressed in 1st and 5th instar larvae of A.pernyi. In tissues of 5th instar larvae of A.pernyi, DAP3 was mainly expressed in the epidermis, followed by the head, hemolymph and fat body. To identify the SOD activity of DAP3, we constructed a prokaryotic expression vector by inserting the coding region sequence into plasmid pET-28a (+) and obtained the purified rHIS-DAP3 fusion protein by Ni-NTA affinitive column. Importantly, we found the SOD activity of DAP3 fusion protein was approximately 0.6674 U/µg. To further confirm the SOD activity of DAP3 in vivo, we induced the oxidative stress model of pupae by UV irradiation. The results showed that both the mRNA and protein level of DAP3 significantly increased by UV irradiation. Furthermore, the SOD activity of the total lysate of pupae increased significantly at 10 min post UV irradiation and transiently returned to normal level afterwards. These results suggested that DAP3 might be a novel protein with SOD activity getting involved in regulation of diapause in A.pernyi. [ABSTRACT FROM AUTHOR]