Stable Expression And Antigenicity Identification Of PCV 2 Cap In Drosophila S2 Cell Lines

Porcine circovirus（PCV）can be divided into porcine circovirus type 1（PCV 1）and porcine circovirus type 2（PCV 2）.PCV 2 has pathogenicity,is the main cause of postweaning multisystemic wasting syndrome and other circovirus-related diseases.Since the first discovery of the virus in Canada in 1991,the virus and its complications have been spread all over the world,and becoming one of the major diseases that endangering the pig industry.The virus can be replicated in the host’s immune system cells,so the development of attenuated or inactivated vaccines is difficult.The existing vaccine has the problems of high preparation cost,short effect time and biological safety,so the popularization and the effective use of the vaccine are seriously restricted.Therefore,the development of new subunit vaccine based on genetic engineering technology is the hot research direction of vaccine research.Analysis of the PCV 2 gene sequence revealed that the genome has two major open reading frames（ORFs）.The Cap protein encoded of ORF2 was the main constituent unit of porcine circovirus capsid protein and was the main immunoprotective antigen of PCV 2.The Cap protein encoded of ORF2 can induce the body to produce specific immune response,which is ideal target gene for the development of new genetically engineered subunit vaccine.In order to obtain the expression vector capable of carrying the target gene,in this study,PCV 2 cap gene was amplified by PCR firstly,and then it was attached to the Drosophila S2cell expressing vector p MT/Bip/V5-His A under the effect of T4 DNA ligase enzyme to construct a recombinant pMT-Cap plasmid.The recombinant plasmid was co-transfected with the pCoHygro plasmid and then stained the appropriate concentration of antibiotic Hygromycin B to screen out the cell lines that containing the target gene and stable inheritance.The stable cell lines were cultured by adding the 500μM CuSO_4,and translate exogenous gene synthesis PCV 2 Cap protein.Under the induction of Bip secretion signal peptide in pMT/Bip/V5-His A vector,the target protein can be secreted into the culture supernatant.In the test,different induction times（0 h,24 h,48 h,72 h,96 h）were set to induce expression and the supernatant was collected for Western Blot detection.The results showed that PCV 2 Cap protein could be expressed correctly in Drosophila S2 cells,and the expression level of Cap protein was the highest at about 72 h after induction.At this point,a sufficient amount of Cap protein expressed in the cultured stable cell line was collected at72 h.First of all,the expression of PCV 2 Cap protein was detected by Western Blot method.Then the expression of PCV 2 Cap gene was detected by indirect immunofluorescence experiments,it was find that there were red fluorescence in the stable cell The results showed that PCV 2 Cap gene was expressed in Drosophila S2 cells.The PCV 2 Cap protein was assembled into porcine circovirus type 2 virus-like particles（PCV 2 VLPs）in Drosophila S2 cells to form a white regular circular with a diameter about 17 nm under the transmission electron microscopy.The antigenicity of the purified protein was determined by indirect ELISA method after centrifugation by sucrose density,the results showed that the protein antigenicity of the bottom of the ultracentrifugation tube was higher,which indicated that the purification effect was good.In order to detect whether PCV 2 VLPs could induce immune response in mice,PCV 2VLPs and GEL adjuvant were mixed in 15%ratio and then immunizing mice.The blood samples from mice were collected at different time（7 d,14 d,21 d,28 d,35 d）.The specific IgG antibody production in serum was detected by ELESA.It was found that VLPs was assembled by PCV 2 Cap protein could induce the humoral immunity of mice and produce specific antibodies,and the antibody level reached the highest level at 28 days after immunization.It was proved that the protein has a good antigenicity.There is potential for further development of the vaccine.This experiment provides a basis for the development of PCV 2 gene engineering subunit vaccine.