ORP6的原核表达、鉴定、纯化及多克隆抗体的制备

Recently, ORP6 highly express in brain and skeletal muscle were reported in some articles. However, there is no further investigation on why ORP6 highly express in these two tissues. For a better study on the cellular function of ORP6, we constructed the recombinant plasmid, pGEX-4T-1-ORP6m by cloning technology, purified the fusion protein expressed by Rossetta, E.coli., immunized New Zealand White rabbits to prepare Anti-ORP6 polyclone antibody. Firstly, we amplified human ORP6(ORP6m) gene sequence which is encoding amino acid (872-934). And then the gene sequence is inserted into prokaryotic expression vector pGEX-4T-1 following by transforming into RosettaTM（DE3）expressing the fusion protein GST-ORP6m by IPTG induction. Using the GST-tag in the fusion protein to affinity purify, the purity of the antigen protein is 90%. The purified antigen is emulsified with Freund's adjuvant completely before immunizing New Zealand White rabbits. We purified antibody from the anti-serum, finally, we prepare Anti-ORP6 polyclone antibody. Using Western blot to detect the specificity of the antibody. The result showed the specific binding activity of the antibody against endogenous and exogenous ORP6. To prepare the polyclonal antibody is to lay the foundation for further ORP6 functional research.