神经菌毛素1b结构域的克隆及表达

摘要

Objective:To develop recombinant human neuropilin 1 b domain(HuNRP1b).Methods:cDNA of HuNRP1b was amplified by PCR technique from pGEM-NRP1 cloning vector and then subcloned into plasmid pColdTF.After screening and sequencing,the constructed recombinant plasmid pCold-HuNRP1b was transformed into competent cell E.coli BL21 (DE3) and expressed by the induction of IPTG.Results:Enzyme digestion and DNA sequencing results showed that pCold-HuNRP1b was successfully constructed.SDS-PAGE analysis showed that the expressed product was a fusion protein with a molecular weight of 90 kDa.Conclusion:Recombinant protein HuNRP1b was successfully constructed,which has laid a foundation for the production of recombinant HuNRP1b and development of monoclonal antibodies against HuNRP1b.%目的:制备重组入神经菌毛素1(NRP1)b结构域蛋白.方法:采用PCR技术,从pGEM-NRP1克隆载体中扩增人NRP1 b结构域(HuNRP1b)cDNA,并将其克隆入pColdTF,构建重组原核表达质粒pCold-HuNRP1b并转化大肠杆菌BL21 (DE3)感受态,制备重组大肠杆菌BL21(DE3) (pCold-HuNRP1b),低温条件下用IPTG诱导重组菌的表达,制备重组蛋白TF-HuNRP1b.结果:酶切、测序结果表明pCold-HuNRP1b构建成功,经SDS-PAGE分析,重组质粒转化表达菌后重组蛋白TF-HuNRP1b获得表达,融合蛋白相对分子质量约90× 103.结论:获得原核表达的重组HuNRP1b蛋白,为该蛋白的规模化生产及其单克隆抗体的制备提供了有效的生物材料.