Recombinant human TGF-β1 prokaryotic expression and polyclonal antibody preparation

Purpose To clone human TGF-β1 gene and prokaryotic expression of TGF-β1 protein through the expression of this gene,and then to get the rabbit-anti-human TGF-β1 polyclonal antibody from the immune rabbit with this protein.Methods Using RT-PCR technique to amplified human TGF-β1 gene sequence.Then the target gene was cloned into a prokaryotic expression vector pET-28a by using the recombinant DNA technique.After induced by IPTG,the protein TGF-β1 was expressed in E.coli BL21(DE3).The protein testified its antigenicity by Western-blot.Next,we immunized New Zealand white rabbits with the obtained recombinant protein as antigen.We collected the immune sera of rabbit.Then purified antibody by ammonium sulfate fractionationthen,the indirect Enzyme-linked immunosorbent assay was used to detect the polyclonal antibodies titer.Western-blot techniques were used to identify antibody specificity.Results The encoding sequence and expression vector of TGF-β1 was obtained while the interest protein was mainly expressed in the inclusion body.Western-blot detection of the target protein displays antigenicity.The purified rabbit anti-human polyclonal antibodies titer is 1∶ 10 000.Conclusion The rabbit anti-human TGF-β1 polyclonal antibodies titer is higher and has a better specificity.