Identification Of Structural Proteins Of Bombyx Mori Parvo-like Virus (China Zhenjiang Isolate)

Bombyx mori parvo-like virus (BmPLV) causes flacherie diseases of silkworm and impairs silk production. The infection of Bombyx mori larvae with BmPLV leads to thorax transparency and diarrhea. Since the symptoms caused by this virus are very close to those caused by Bombyx mori densovirus type I (termed BmDNV-1 Ina isolate), so Bombyx mori parvo-like virus was initially called Bombyx mori densovirus type II which was the major pathogens that cuts the production of cocoon. The most notable characteristic in the genome of BmPLV-Z is that it has 2 sets of different single-stranded linear DNA molecular Viral DNA 1(VD1) and Viral DNA 2 (VD2), with the sequence which is able to encode DNA polymerase by itself. The structural proteins of virus determine the nature and composition of viral capsid and the viral infection characteristics of particles, and the nature and composition of BmPLV-Z structural proteins were not to be determined yet.In our study, firstly we cloned vp gene into a prokaryon expression vector pMAL-c2x to construct a recombinant plasmid pMAL-c2x-vp, and was expressed in E. coli TB1. Protein was purified and injected into rabbit. The antibodies were purified with protein A affinity column. Secondly, the virions were purified by cesium chloride density gradient centrifugation from the feces of Bombyx mori larvae infected with BmPLV-Z and analyzed by SDS-PAGE, Western-blotting and MALDI-TOF/MS. Lastly, the vp was expressed in Sf9. We mainly completed the following work:Prokaryotic Expression of Viral Protein and Preparation of AntibodyThe vp gene was cloned into a prokaryon expression vector pMAL-c2x to construct a recombinant plasmid pMAL-c2x-vp, expressed under the induction of IPTG and identified by SDS-PAGE and mass spectrum. The molecular mass of expressed protein is a about 97 kDa. Protein was purified and injected into rabbit. The antibodies were purified with protein A affinity column.Identification of Structural Proteins of VirionsBmPLV-Z virions were purified from the feces of infected Bombyx mori larvae by cesium chloride density gradient centrifugation and examined under electron microscope. Proteins of the virions were separated by SDS-PAGE and identified by mass spectrometry. The isolated proteins were verified by Western blotting using polyclonal antibody against virion and recombinant viral protein (VP) expressed in E. coli respectively. The results showed that the viral polypeptides were mainly composed of 7 proteins named P1 to P7. Western-blotting with anti-virion serum indicated that structural proteins P5, P6 and P7 had molecular weight around 50, 55 and 130 kDa, respectively, were the major immunogens of virion. Among them, P5 and P6 were coded by VD1-ORF3 , P7 was encoded by VD2-ORF1. The Construction of Eukaryotic Expression vector of vpThe vp fragment was inserted into pFastBacHTb, within E. coli DH10Bac, the vp gene was transposed into the bacmid. The recombinant bacmid DNA was isolated, purified and transfected into Sf9 cells to yield the recombinant AcNPV carrying the vp gene. Cells were harvested 48 h post-infection, then scraped into the medium. There were tow proteins detected by Western blot.