Expression Of Broiler Glutathione S-transferase A3 Protein And Preparation Of Its Polyclonal Antibody

Glutathione S-transferase A3(GSTA3),as a member of the α family of the GSTs superfamily,has a detoxification function and participates in the regulation of anti-oxidative stress.It is the most efficient steroid isomerization known to humans.Enzymes also play a very important role in the treatment of tibial dyschondroplasia in broilers.This study constructed a pGEX-6P-1-Gsta3 prokaryotic expression vector by cloning the genes encoding the GSTA3 protein.The fusion protein obtained by inducing expression was immunized with New Zealand white rabbits to prepare polyclonal antibodies.It lays the foundation for the study of the function and structure of GSTA3 protein.In this experiment,total RNA was extracted from chicken tissue and reverse transcribed into c DNA.Specific primers were designed to amplify chicken Gsta3 gene using c DNA as a template by PCR technology,and the Gsta3 target fragment was ligated to T Vector p MD19(Simple)vector to construct Recombinant plasmid p MD19-T-Gsta3;In this experiment,pGEX-6P-1 plasmid and p Cold-TF plasmid were used to construct two prokaryotic expression vectors pGEX-6P-1-Gsta3 and p Cold-TF-Gsta3 with different tags.The results were verified by sequencing.Recombinant plasmid was transformed into E.coli BL21 strain,and the fusion protein was induced by IPTG.The fusion proteins purified by affinity chromatography were named GST-GSTA3,His-GSTA3;the purified GST-GSTA3 protein is combined with E.coli BL21 strain.The purified GST-GSTA3 protein was fully emulsified with Freund’s adjuvant and used as an immunogen.Female New Zealand white rabbits were immunized with multiple subcutaneous injections on the back to obtain GSTA3 polyclonal antibodies.His-GSTA3 protein is used as the coating antigen in the indirect ELISA method.After the optimal coating concentration is determined by the checkerboard titration method,the polyclonal antibody against chicken GSTA3 protein is detected and screened.Western blot and indirect immunofluorescence test(IFA)determine the specificity of the antibody to the GSTA3 protein.After testing,the antibody titer was 1:102400,and the ra bbit anti-chicken GSTA3 polyclonal antibody was successfully prepared;Western blot confirmed that the prepared rabbit anti-chicken GSTA3 polyclonal antibody had good reactivity to the GSTA3 protein.IFA shows that the antiserum can specifically recognize the natural GSTA3 protein in chicken fibroblasts.