Prkaryotic Expression's System Construction Of Interleukin-17A To Mice

ObjectiveINTERLEUKIN-17(IL-17) is the proinflammatory cytokine which was newly found in recent years and has extensive biological activity, hence, it maybe one of the important factors of cause and development to some diseases. IL-17 is one protein which is generated by T cell and makes an effect through conveying to the receptor of some cells. The IL-17 to human is the dimeric peptides homologous secreted by activatory CD4+ memory T cells. But IL-17 to small mouse contains leader signal sequence which consists of 21 amino acid residue and the total length is 158 amino acid residue. The amino acid residue sequence of IL-17 to human has 62.5% homology with small mouse while 58% with big mouse. The experiments from home and abroad corroborate that IL-17 has high conveying in many types of nervous system disease. IL-17 to patients with multiple sclerosis(MS) has a higher convening and it is also related some to the severity of disease. IL-17A is the most important IL-17 family members, involved in immune-mediated inflammatory injury, the systemic inflammatory response play an important role in use.If the specific antibody to IL-17A is created, and corroborated in experiments has outstanding treatment effect, it will not only provide a new therapeutic schedule to multiple sclerosis in clinic but also realize its extensive use in the nervous system disease.MethodsFirstly, this subject designs the primer of IL-17A to small mouse according to the genome sequence of small mouse in Genbank. By PCR method, the genetic fragment of IL-17A to small mouse will be amplified from genome of small mouse through hifi enzyme and the length is 631bp. It is added A in the end, then clone the target fragment genetic to T cloning vector pMD19-T according the right reading frame, then gaine the recon of pMD19-T(+) which contains IL-17A genetic fragment. Transform the competent cell(DH5a) of colon bacillus, and do the appraisement of Double Digests and order-checking to masculine recon which is preliminary screened by penbritin. Do Double Digests treatment to masculine recon whose appraisement of Double Digests is correct and order-checking result has same height as the IL-17A gene order to small mouse in Genbank. Then connect it to expression vector pET32a(+) which was same treated by Double Digests and gain the reforming expression vector pET32a(+)/IL-17A to small mouse, transform the expression bacteria of colon bacillus BL21(DE3),37℃,1mM IPTG,200rpm, introduce and reform expression of protein.ResultsAfter detecting in SDS PAGE and Western blotting, it will show that fused protein expression with 53kDa around molecular weight has the same size as expectations (52.8kDa) and most expression protein can exist in soluble type.ConclusionThe results of experimental indicates that dna recombinant plasmid pET32a(+)/IL-17A to small mouse can successfully express the target protein in the expression bacteria of colon bacillus BL21(DE3) and the fused protein which is gained in experiments has same size as expectations.