Expression And Immunogenicity Analysis Of Feline Immunodeficiency Virus P24 Protein

Feline immunodeficiency virus?FIV?and human immunodeficiency virus are both retroviridae,members of the genus lentivirus,which cause predominantly immunodeficiency diseases in cats.Individuals infected with feline immunodeficiency virus may have no symptoms for several years.In the early stages of their illness,they may be predominantly fever,diarrhea,swollen lymph nodes,neutropenia in leukocytes,oral inflammation,rhinitis,loss of appetite,and emaciation.FIV infection occurs all over the world,since its first discovery in 1985,prevalent in the United States,Japan,Canada and other countries.FIV has a strong mutational abilities,strains isolated in different regions are different.In this experiment,the p24 fragment of FIV gag gene was cloned from the DNA extracted from the positive whole blood of FIV,the protein was prokaryotic and eukaryotic expressed and finally the monoclonal antibody was prepared,lays a foundation for the establishment of FIV serological testing methods.The experimental results are as follows:According to the GenBank accession:NC₀01482.1,a pair of primers of p24 protein coding region was designed.Total RNA was extracted from a serologically positive cat blood sample and reverse transcribed into cDNA,then the p24 gene was amplified by PCR and identified by nucleic acid electrophoresis.A target fragment of 622 bp was successfully obtained and encoded 207 amino acids.The homology reached 98%after sequencing.The target fragment was subcloned and identificated.After identification,the target fragment with the sticky end was ligated into the expression vector pET-28a???,and the recombinant prokaryotic expression plasmid pET-28a-p24 was successfully constructed.The recombinant plasmids were transformed into E.coli BL21?DE3?.The recombinant protein were expressed in inclusion bodies by SDS-PAGE and the conclusion of Western-blot is that the recombinant protein could react with Anti-His tag mouse monoclonal antibody proved that the recombinant p24 protein was well expressed.The recombinant vector for prokaryotic expression was massive induced,prokaryotic expressed p24 protein was purified by gel electrophoresis.The purified p24 protein was injected into mice intraperitoneally after mixing with an equal amount of Freund's complete adjuvant.After three additional boosts were performed with Freund's incomplete adjuvant to mix the purified protein,after a total of four injections the polyclonal antibodies were obtained with high specificity.Polyclonal antibody's titer was measured by indirect ELISA and the results showed that titer reached 1:51200.Indirect immunofluorescence results showed that the polyclonal antibody has good specificity.In order to be able to attach the target fragment to the pCMV-HA eukaryotic expression vector,a set of primers with different cleavage sites needs to be designed.After subclone the correct target fragment with different cleavage sites was ligated into pCMV-HA vector to construct p24 gene eukaryotic expression vector pCMV-HA-p24.The recombinant plasmids were transfected into HEK-293T cells by liposome-mediated method.The expression vector without the target fragment transfected 293T cells served as a control group,and Western-blot was used to detect the expression of p24 protein.At this part of the experiment,pCMV-HA-p24 eukaryotic expression vector was successfully constructed and the correct recombinant plasmid was transfected into HEK293T cells by liposome.The results of immunofluorescence showed that most cells in the selected HEK-293T-p24 cell line could display green fluorescence.HEK-293T cells as a control showed no fluorescence,indicating that p24 protein was successfully and efficiently expressed in the HEK-293T-p24 cell line.The characteristic of eukaryotic expression is that once successfully expressed,the protein has specific biological activity,so the eukaryotic p24 protein will be used as a test protein for the detection of murine polyclonal antibodies.