Preparation And Preliminary Application Of PFV Whole Virus Particle And Its IN, LP And Tas Protein Antiserum

Foamy viruses are the only members of the genus Spumavirus,Spumaretroviruses,one of two families of the Retroviridae.As indicated by their names,FVs possess highly fusogenic activities which induce infected cells in vitro to form foam-like multinucleated cells.But FVs in hosts could establish lifelong persistent infections in the absence of any pathogenicity.Otherwise,FVs have a long genome and a wide host range.Therefore,FVs are ideal materials for constructing gene transfer vector.When working on the PFV gene transfer vector,it is required to detect the efficiency of its infection on protein expression level.So in this study,genetic engineering cloning,expression and purification of the IN,LP and Tas proteins of PFV,and preparation of the IN antiserum,LP antiserum and Tas antiserum were carried on to support the qualitative and quantitative detection of the PFV gene transfer vector on protein level.This research also could help to the further studies about the structural protein and related function mechanism of PFV.Firstly,the antigenic epitope of IN,LP and Tas proteins were analyzed using the DNAStar in this study.Result showed that these protein sequences which contained potential antigenic epitope,could be designated as antigen to prepared and gained antiserums.Secondly,the pET-32a-IN,pET-32a-LP,pET₃2a-tas expression vectors were constructed,and then which were transfered into E.coli BL21?DE3?,E.coli BL21?DE3?plysS,E.coli Transetta?DE3?for expression.After induced by IPTG,the IN,LP and Tas fusion proteins were identified by SDS-PAGE.Meanwhile,the dissolubility of these proteins were analyzed.Thirdly,after denaturing in urea,the IN,LP and Tas fusion proteins were separated by SDS-PAGE and isolated by cutting the gel slices that contained the right bands.Then these gel slices were grinded with PBS and used to immunize the New Zealand rabbits to prepare the antiserum against IN,LP and Tas proteins,respectively.At the same time,two other rabbits were immunized the concentrated PFV virus particles.The obtained antiserums were analyzed by ELISA?Enzyme-linked immunosorbent assay?and Western blot imprinting method.Finally,the PFV proteins expression in infected cells and in the eukaryotic expression vector transfected cells were detected in protein levels with these antiserums.The main results obtained were as follows:1.The antigenic epitope analyses of IN,LP and Tas proteins showed that the 20-28 aa,127-137 aa,273-287 aa sections of IN protein,the 64-67 aa,93-100 aa sections of LP protein,the 45-56 aa,118-128 aa,176-205 aa,247-269 aa sections of Tas protein contained potential antigenic epitope.2.The 1092 bp,381 bp,903 bp fragments of IN,LP,tas genes were obtained by PCR.The fragments were connected to pET-3 2a?+?,and then the sequencing analyses by Clustal Omega program showed that the amplified sequences were identical to the sequence of pHSRV13 published in Genbank.It meant that the recombinant prokaryotic expression vectors with the fused His-Tag were constructed successfully.3.The recombinant vectors were transfered into E.coli and the expression was induced by IPTG.The IN,LP,Tas fusion proteins band with relative molecular weight of 58 KD,36 KD,54 KD were identified by SDS-PAGE,which was consistent with the expectation.Moreover,these poroteins were expressed in the form of inclusion body under the different conditions.4.The inclusion bodys were denatured in urea and then used to immunize the New Zealand rabbits.At the same time,the antigenic proteins were purified by His-Bind???Quick 900 Cartridges purification systerm.ELISA showed the titer of the IN antiserum,LP antiserum and Tas antiserum might achieve 1:2 048 000.Western blot showed these antiserums could interact with the expressed IN,LP,Tas proteins specifically.5.The PFV whole virus antiserum was obtained through immunized rabbits with PFV virus particles,Western blot showed that the antiserum could specifically bind to PFV structural proteins.6.The immunoglobulin IgG of these antiserums were separated and purificated.Result showed that the IgG concentration of IN antiserum,LP antiserum,Tas antiserum and PFV virus particles antiserum were 2.517?g/?L,1.860?g/?L,2.515?g/?L,2.666?g/?L.7.In BHK-21 cell infected with PFV,the Pol/IN,Env/LP,Bet/Tas proteins could be detected with IN antiserum,LP antiserum,Tas antiserum,respectively.In HT1080 cell transfected with the eukaryotic expression vector of pCI-Env,pCI-Tas,respectively,the Env/LP,Tas proteins also could be detected with corresponding antiserums.These results showed that these antiserums had good immunoreactivities which confirmed that these antiserums reacted specifically to specific proteins.These antiserums provided a base for the diagnosis of PFV gene delivery system and for the studies about the structural protein and related functional mechanism of PFV.