Screening and immunogenicity analysis of candidate antigenic proteins for ixodes subunit vaccine
The aim of this study is to screen the best candidate antigenic proteins for ixodes subunit vaccine.In this study,the target genes in PET-28a-GSTK1,PET-28a-GSTE3,PET-28a-Fe1,PET-28a-Fe2 and PET-28a-CPL plasmids which had been constructed previously were detected by PCR.The expression of five recombinant proteins(rGSTK1,rGSTE3,rFe1,rFe2 and rCPL)was detec-ted by SDS-PAGE after IPTG induction,and the four recombinant proteins(rGSTK1,rFe1,rFe2 and rCPL)were purified by His-tagged protein purification kit,and the protein concentrations were determined by BCA protein quantification kit.The reactivity of the four recombinant proteins was determined by Western blot,and the potency of the polyclonal antibodies and the pattern of antibody elongation after immunization were determined by indirect Elisa.The results showed that the five target gene bands were correct and did not lose.SDS-PAGE showed that except for rG-STE3,the other four proteins(rGSTK1,rFe1,rFe2 and rCPL)were stably expressed in the form of inclusion bodies with the sizes of 28,25,26,27 kDa,respectively.His label was used to purify the protein and determine the protein concentration.The results showed that the target protein with high purity could be obtained by elution with 250 mmol/L imidazole,and the protein concen-trations were 2.633,2.532,2.582 and 2.506 g/L,respectively.The purified recombinant protein was used to immunize BALB/c mice to prepare polyclonal antibodies.Indirect ELISA results showed that the titers of the four recombinant proteins(rGSTK1,rFe1,rFe2 and rCPL)were as high as 1∶819 200,1∶819 200,respectively 1∶1 638 400 and 1∶409 600,indicating that the immunoge-nicity of the four recombinant proteins was good,rFe2 had the best immunogenicity.The antibody produced by the four recombinant eggs reached the peak at 21 days after triple immunization and gradually decreased,but the antibody level at 63 days after triple immunization was still at a high level,with rFe2 having the highest antibody level at 1∶819 200;RGSTK1 came second at 819 200.Western blot results showed that all the four recombinant proteins could react specifically with its polyclonal antibody.rFe2 showed the strongest reactivity,followed by rGSTK1.In this study,the stability,high expression,antibody level after immunization and reactivity with polyclonal antibod-ies of the recombinant proteins were used as indicators to screen rFe2 as the best candidate anti-genic protein,and rGSTK1 had similar effect and the combination of the two proteins can also be considered,providing an experimental basis for the subsequent development of anti-tick vaccine.