Prokaryotic Plasmid Construction And Expression Of DOCK8 Core Functional Region And Preparation Of Polyclonal Antibody

Objective: Prokaryotic plasmid DHR2,the core domain of DOCK8(dedicator of cytokinesis 8),is constructed and transformed by Escherichia coli to induce the target protein,so as to explore the best induction conditions for the target protein.Through the preparation of polyclonal antibody against DOCK8,we preliminarily analyze whether the target protein has corresponding immunogenicity,which provides a basis for further exploring the immune function and molecular mechanism of DHR2,the core functional area of DOCK8,and the construction of anti-caries vaccine.Methods: This study consists of two parts Part I: Construction of prokaryotic expression plasmid p ET28a-DOCK8/DHR21.Total RNA was extracted from SD rat kidney,specific primers were designed,and DHR2 coding gene,the core functional area of DOCK8,was obtained by reverse transcription PCR in two steps.2.The intermediate vector p MD-DOCK8/DHR2 plasmid was constructed by T-A cloning.3.The positive plasmid with correct identified sequence was recombined with p ET28a(+)expression vector by directional cloning to obtain recombinant plasmid p ET28a-DOCK8/DHR2;Part II: Protein induction and purification of p ET28a-DOCK8/DHR2 prokaryotic expression plasmid and preparation of its polyclonal antibody1.The recombinant plasmid p ET28a-DOCK8/DHR2 was transformed into E.coli Rosetta(DE3)and IPTG was added to induce the expression of the target protein.2.DOCK8/DHR2 protein was purified by Ni-NTA affinity chromatography,and the purified protein was identified by Western blot.3.The purified DCOK8/DHR2 protein was used as antigen to immunize New Zealand white rabbits to prepare polyclonal antibody.4.Indirect ELISA was used to determine the titer of rabbit anti-DOCK8/DHR2 serum.5.Western blot was used to detect the specificity of rabbit anti-DOCK8/DHR2 serum.Results:1.DOCK8/DHR2 gene fragment was successfully amplified,and recombinant cloning vector p MD-DOCK8/DHR2 and recombinant expression plasmid p ET28a-DOCK8/DHR2 were constructed.2.The recombinant expression plasmid p ET28a-DOCK8/DHR2 was digested with Eco RI and Hind III to obtain a 1.2kb fragment,which is the same size as the target gene fragment.After sequencing and comparison analysis,the target gene sequence has 100% homology and no gene mutation.3.After being induced by different concentrations of inducer IPTG,they all expressed efficiently in the form of inclusion bodies.SDS-PAGE analysis showed that p ET28a-DOCK8/DHR2 was expressed at 54 k Da after induction,and the optimal final concentration of IPTG was 0.5m M.The optimal induction conditions were optimized at different temperatures and times,and the best induction effect was found at 37? for 4h.4.The recombinant protein was purified by Ni2+ affinity chromatography.The protein expressed by p ET28a-DOCK8/DHR2 was identified by Western blot.It was found that the protein expressed by p ET28a-DOCK8/DHR2 could bind specifically to anti-His monoclonal antibody,indicating that the induced protein was the recombinant protein.5.The anti-DOCK8/DHR2 polyclonal antibody was successfully prepared,and the antibody titer determined by ELISA could reach 1: 1024000.6.Western blot showed that the anti-DOCK8/DHR2 polyclonal antibody had good specificity.Conclusion:1.In this study,recombinant plasmid p ET28a-DOCK8/DHR2 was successfully constructed.2.The recombinant plasmid p ET28a-DOCK8/DHR2 was transformed into Rosetta(DE3)competent cells and induced to express by IPTG.The recombinant DOCK8/DHR2 proteins were successfully obtained and all of them existed in the form of inclusion bodies.3.New Zealand white rabbits were immunized with DOCK8/DHR2 protein to prepare polyclonal antibody.The titer of the antibody is as high as 1: 1024000,and the polyclonal antibody can specifically bind to DOCK8/DHR2 protein.