Purification of Chinese Sacbrood Virus (CSBV), Gene Cloning and Prokaryotic Expression of its Structural Protein VP1

• Published in: Molecular biotechnology 2018-12, Vol.60 (12), p.901-911
• Main Authors: Wu, Pengjie, Yu, Huimin, Xu, Jin, Wu, Jiangli, Getachew, Awraris, Tu, Yangyang, Guo, Zhanbao, Jin, Hongyan, Xu, Shufa
• Format: Article
• Language: English
• Subjects: Amino acids; Animals; Bees - virology; Binding sites; Biochemistry; Biological effects; Biological Techniques; Biotechnology; Capsid Proteins - genetics; Cell Biology; Chemistry; Chemistry and Materials Science; Cloning; Cloning, Molecular; Disulfide bonds; E coli; Escherichia coli - genetics; Gene expression; Helices; Human Genetics; Larva - virology; Larvae; Molecular weight; Original Paper; Phylogeny; Protein Science; Protein structure; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA Viruses - genetics; RNA Viruses - isolation & purification; RNA, Viral - analysis; RNA, Viral - genetics; Secondary structure; Sequence Analysis, RNA; Viruses; VP1 protein
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-018-0121-4

The aim of this study was to purify the Chinese Sacbrood Virus Beijing Miyun (BJMY-CSBV) from infected Apis cerana larvae, clone structural protein gene VP1 (named BJMY-CSBV-VP1), and investigate its biological information. The result indicated that the capsid of CSBV is of spherical shape. Gene clone experiment showed that the BJMY-CSBV-VP1 gene sequence comprised 945 bp, encoding 315 amino acids with relative molecular weight of 35.59 kDa and isoelectric point 9.38 pI. Phylogenetic analysis of amino acid sequences showed that the BJMY-CSBV-VP1 and LNDD_2015 were grouped together. Protein secondary structure prediction showed that the gene contained two α-helices, thirteen β-folds, six polypeptide binding sites, and no disulfide bridge. Simultaneously, the BJMY-CSBV-VP1 was ligated to the expression vector pET32a(+) and then transformed into the Escherichia coli BL21 (DE3) for prokaryotic expression. The optimal expression experiment revealed that the protein was found in the inclusion body. The recombinant protein was successfully purified by washing buffer combined with supersonic fragmentation. In this study, we obtained the purified BJMY-CSBV particles, cloned BJMY-CSBV-VP1 gene, investigated the detailed information of the gene by analyzing the sequence, and obtained the purified recombinant protein, which could help for further understanding of the function of the structural protein gene VP1.