Preparation Of Polyclonal Antibodies Against VP1 Protein Of Chicken Anemia Virus And Development Of Stable Cell Lines With Co-expression Of VP1 And VP2 Genes

Chicken infectious anemia(CIA)is an immunosuppressive disease caused by chicken anemia virus(CAV).CAV genome mainly encodes three proteins:VP1,VP2 and VP3.Among them,VP1 is the capsid protein and key immune protein of CAV,and VP2 is the regulatory protein which can assist VP 1 to form a correct conformation.They can coordinate with each other to induce neutralization antibodies.In order to increase the expression of VP1 in the prokaryotic expression system,the specific antibodies were prepared based on the truncated expression recombinant plasmid of VP1 was constructed and proteins were purified.Another,VP1 and VP2 genes were inserted into the double promoter vector to screen the cell lines stably expressing VP1 and VP2.The results can lay a foundation for the further study of the pathogenic mechanism of CAV and the development of CAV vaccine.(1)VP1 truncated prokaryotic expression and its polyclonal antibody preparationAccording to the antigenicity of VP1 protein by DNASTAR software,according to the existence of two main epitope aggregation regions,the truncated expression gene regions:N-terminal 16-615bp(600bp)and 616-1350bp(735bp)were determined.Two pairs of specific primers were designed to amplify the gene fragments by PCR and ligated to the prokaryotic expression vector pET32a(+).The recombinant plasmids were identified by PCR detection,double endonuclease digestion and sequencing.Two recombinant strains(pET32a-VP 1-600 and pET32a-VP 1-735)were induced by IPTG at room temperature and low temperature,respectively and then their expression forms were detected.The results showed that two prokaryotic expression plasmids pET32a-VP 1-600 and pET32a-VP1-735 were constructed.The fusion proteins of VP1-600 and VP1-735 induced by IPTG were mainly expressed as inclusion bodies.The proteins were dissolved in 8M urea,purified by nickel affinity chromatography and checked with running SDS-PAGE.The results showed that VP 1-600 and VP 1-735 fusion proteins with high purity were obtained,which could be used as immunogens.The purified proteins were emulsified with Freund's adjuvant respectively and immunized the New Zealand white rabbits by subcutaneous multi-point injection.After four immunizations,blood samples were collected to prepare antisera.The antibody level was detected by indirect ELISA,and the immune reaction between polyantibodies and VP 1-600 and VP 1-735 proteins was detected by Western Blotting method.The results showed 1:204,800 ELISA titers anti-VP1-600 and 1:819,200 anti-VP1-735 proteins rabbit polyclonal antibodies were obtained,respectively.The antibodies could effectively recognize the corresponding target proteins by Western Blotting detection,which indicates the prepared polyclonal antibodies could be used for the detection of VP1 proteins.(2)Construction of stable cell line with co-expression of VP1 and VP2 genesBased on the specific overlap primers for cloning of VP1 and VP2 designed,the full-length genes of VP1 and VP2 were amplified by PCR and liked to the eukaryotic expression vector pCB15 with double promoters.The construct was transformed into E.coli DH5? competent cells.The positive clones were identified by PCR amplification,restriction endonuclease digestion and sequencing.The results showed that VP2 and VP1 were successfully inserted into the same pCB15 vector.The constructed recombinant plasmid was transfected into 293T cells by liposome.The resistant cells were screened with 1.5 ?g/mL puromycin.The gene expression and protein expression of VP1 and VP2 in the selected cells were detected by PCR and Western Blotting.Finally,VP1 and VP2 genes and their proteins were detected in the cells after puromycin screening.These indicate a stably cell line co-expressing VP1 and VP2 were successfully obtained.In summary,two recombinant plasmids for truncated prokaryotic expression of VP1 were constructed.The fusion proteins were obtained by IPTG induction,and two kinds of rabbit anti-VP 1 polyclonal antibodies were prepared.After VP1 and VP2 genes were cloned into double promoter vector pCB15,cell lines stably co-expressing VP1 and VP2 were successfully obtained by puromycin screening.Our results can lay a foundation for further induction of neutralization antibody production and development of vaccine based on CAV VP1 and VP2 co-expression.