Research On Prokaryotic Expression System Of Porcine Circovirus Virus Capsid Protein

Porcine circovirus (PCV) is the smallest virus that have been found. The porcine circovirus disease caused by PCV was recognized as an important disease to the global pig industry, it is also a member of the three big epidemic disease which cause closely attention by China’s pig industry. To be a new vaccines, subunit vaccines has many advantages compared to traditional vaccines. The capsid protein encoded by ORF2 gene is the major structural protein of the virus, it has immunogenicity and to be considered as a major target of subunit vaccines research. The insect expression system is widely used to produce subunit vaccines in domestic and overseas market, the operation of the system is troublesome and highly cost. In order to achieve efficient expression of the capsid protein in Escherichia coli and Bacillus subtilis, lay an important foundation for the development of subunit vaccines, prokaryotic expression system was used as the research target in this study. The main subjects of the experiment are as follows:(1 Construction of E.coli expression vector and induction of the target protein expression;(2)Construction of Bacillus subtilis exocrine expression system induced by IPTG and xylose respectively, induction and detection of target protein. Detection the mRNA of ORF2 gene in Bacillus subtilis exocrine expressed system through relative quantitative fluorescence PCR;(3) Building the Bacillus subtilis intracellular expression system and induced the expression of interest protein.Since the N-terminal nuclear localization sequence of ORF2 gene is rich in continuous rare codons, the expression of full-length ORF2 gene is difficult in E. coli. In this paper, we constructed a truncated gene expression vector, also we optimized the full-length gene expression vector. After induced, the target protein was detected by SDS-PAGE and Western-blot.Full-length and truncated ORF2 gene ware cloned on exocrine Bacillus subtilis expression vector induced by IPTG and xylose. Transforming the vector into Bacillus subtilis 168 and WB600, we failed to detect the expression of interest protein according to SDS-PAGE and Western-blot after the induction.Taking Bacillus subtilis ribosomal protein L7Ae as a reference gene, detected the mRNA of full-length and truncated ORF2 gene in Bacillus subtilis exocrine expressed system through relative quantitative fluorescence PCR, calculated the relative expression of ORF2 gene according to the method of the Ct value comparison (2-ΔΔCt), understand the transcription of ORF2 gene in Bacillus subtilis exocrine expression system. The reasons that can not be expressed in the system was further studied which can be used to guide subsequent improved expression.Based on starting plasmid pHT43, by removing the signal peptide sequence and transforming its cloning restriction sites, the Bacillus subtilis expression systems was constructed. The original ORF2 gene is difficult to express in Bacillus subtilis intracellular expression system. After the optimization, truncated ORF2 gene can be expressed under induction in Bacillus subtilis intracellular expression system, and we can detect the protein by Western-blot.