Prokaryotic Expression And Preparation Of Antibody Of Spider Dragline Like Gene

Because of spider drag silk have excellent mechanical properties it could be increasingly used as material in medical、building and so on. But it is hard to get the natural spider silk. Using prokaryotic bioreactor is effective method to get a large of artificial spider silk. We constructed prokaryotic expression plasmids pGEX-S and pGEX-2S which were expressed in BL21, the recombinant protein from the higher expression cell was used as antigen and immunized mice to preparing anti-serum. Though this research we explored and established suitable prokaryotic expression system of recombinant spider silk protein and provided material for following detected. The results are as follows:1. Spider dragline silk protein gene synthesis:We synthesized spider dragline silk protein gene monomer artificially according to the published cDNA fragments of Nephila clavata spider, then constructed the diploid (2S).2.Construction of the expression vector:We linked gene monomer (S) into cloning vector pUC57, then got the recombinant plasmid pUC57-S,we synthesized the dimer (2S) after enzyme digestion, ligation and transformation; S and2S were respectively connected with the expression vector pGEX-2T. Finally we obtained plasmid pGEX-S and pGEX-2S which can be used for prokaryotic expression.3.Expression and purification the recombinant protein in prokaryotes:We transformed the recombinant plasmid pGEX-S and pGEX-2S into BL21cells, then explored the expressional condition arid found the best concentration of IPTG was0.8mmol/1,as well as the time was4h to induce expression. The expression of S-GST was obviously higher than2S-GST by Western Blot. Recombinant protein S-GST was purified by affinity choromatography and had preparing polyclonal antibody4.Polyclonal antibody of dragline silk protein gene(S) preparation:Polyclonal antibody against S was generated by immunizing CD I mouse with purified recombinant protein S-GST. ELISA results indicated that two mice anti-serum concentration was high,it can be used for subsequent study.