Prokaryotic Expression, Purification And Preparation Of Antibody Of Spider Dragline Silk Protein Dimmer

In order to prepare anti-serum contains spider dragline silk protein for in vitro detection, a prokaryotic expression plasmid 2S-pET-52b (+) was constructed and then expressed in BL21-pLysS cells for producing of 2S recombinant protein which was used as antigen after purificated by the means of affinity choromatography. The results are showed below:(1) Construction of expression vector 2S-pET-52b (+):recombinant plasmid 2S-pET-52b (+) was made up by two fragments:2S and pET-52b (+).2S was designed according to the core sequence of the dimerization spider dragline silk gene and obtained through PCR used 2S-UHS-3.1 as template, the PCR product was then conjuncted with linear vector pEasy-3.1 to construct recombinant plasmid 2S-pEasy-3.1.2S fragment through digestion of 2S-pEasy-3.1 was fused before the tag of 6xHis in pET-52b(+) to generate the expression vector 2S-pET-52b (+)-.(2) Prokaryotic expression and purification of recombinant protein:recombinant plasmid 2S-pET-52b-(+) was transformed into BL21-pLysS cells, and then screened for the most suitable strain and expressional conditions include concentration, temperature and time of IPTG induction. Recombinant protein was purified by the means of His-tag-specific affinity choromatography after expressed.(3) Polyelonal antibody preparation:polyclonal antibody against 2S was generated by immunizing New Zealand rabbits with purified recombinant protein 2S-His. Elisa results indicated that when diluted anti-serum abtained to 1:25,600, the prepared polyclonal antibody had better immunoreactivity.In conclusion, this research not only laid basis for in vitro spider dragline silk protein detection, but also provides a new approach for specific expression of spider dragline silk protein in the hair.