Expression Of Bovine Papillomavirus 13 L1 Gene

Bovine papillomavirus (BPV) can cause bovine papillomatosis (BP), which is chronic hyperplastic disease. BPV genome includes three regions:early transcription region (E), late transcription region (L) and non coding region (NCR). L region codes the capsid protein, which can induce the body to produce neutralizing antibody and stimulate protective immunity, and also is the main target antigen for preventive immunity. At present, the immune of BPV is virus-like particle (VLP) preventive vaccination established on the basis of L1 gene. Although VLP has good effects, it is expensive.In Part 1, the specific primers were designed according to the published sequence of BPV13, and LI gene fragment was amplified by PCR. After being excised by BamH. I and Hind?, the fragment was inserted into the prepared vector pET28a to construct the recombinant expression plasmid pET28a-L1. The recombinant plasmid pET28a-L1 was identified by restriction enzyme digestion and sequencing and used to transform into E.coli BL21 (DE3) cells. The expression of pET28a-L1 was induced by IPTG after screening the best concentration and time. The expressed protein was purified by Ni-NTA His-Bind Resin and identified by SDS-PAGE. The results showed that L1 gene was exactly inserted into the prokaryotic expression vector pET28a, after being induction, the target protein containing His tag was successfully expressed by bacteria which included recombinant plasmid pET28a-L1; SDS-PAGE electrophoresis results showed that the molecular mass of the protein was about 60 kDa, consistent with the expected size; after ultrasound treatment, SDS-PAGE electrophoresis results showed that the protein existed in the inclusion body; the target protein that verified by Western blotting was a fusion protein with His tag.In Part 2, the purified fusion protein was used to immune white rabbit by subcutaneous multi-point injection to prepare antibody of rabbit anti BPV13-L1 polyclonal antibody. Indirect ELISA method was used to detect the antibody titer, the results showed that the antibody titer was more than 1:25600. Western blotting analysis results showed that the polyclonal antibody has strong specificity.In Part 3, LI gene was amplified through PCR. After excised by Xho I and BamH I, the fragment was inserted into the eukaryotic expressing vector pEGFP-N1 to construct the recombinant plasmid. The recombinant plasmid was transfectd into NIH3T3 cells and the expression of L1 protein was detected by fluorescence microscope, qRT-PCR and Western blotting. The results showed that the recombined eukaryotic expressing plasmid was constructed successfully, and L1 protein was expressed, which lay the foundation for the development of BPV 13 vaccine.