【摘要】 目的:利用PCR产物靶向克隆法构建人癌-睾丸抗原NY-ESO-1的原核表达载体。方法:从人新鲜的食管癌组织中提取总RNA,通过RT-PCR技术扩增NY-ES0-1基因3′端的340bp片段,采用靶向克隆法将目的基因插入到原核表达载体pET-15b中,得到重组表达质粒pET-15b-NY-ESO-1,经过筛选,挑选出阳性克隆进行XhoⅠ和BamHⅠ双酶切图谱分析、PCR检测和扩增产物核苷酸序列分析等,鉴定所构建的原核表达载体。结果:扩增得到NY-ESO-1基因片段,经测序证实与GenBank公布的序列一致,重组的质粒经过PCR,酶切鉴定获得了一个大小为340bp的特征性片段,证明重组质粒中已成功的插入了目的基因NY-ESO-1。结论:靶向克隆法可快速、高效地构建人NY-ESO-1基因的原核表达载体,为制备pET-15b-NY-ESO-1融合蛋白奠定了基础。更多还原

【Abstract】 Objective: To construct the prokaryotic expression vector of human cancer-testis gene NY-ESO-1 by the method of LP Recco PCR Cloning.Methods: Total RNA was extracted from esophageal carcinoma tissue,the primer containing special enzyme was designed,NY-ESO-1 fragment was amplified by RT-PCR,and then was cloned into the expression vector pET-15b by LP Recco PCR Cloning.The recombinants plasmid pET-15b-NY-ESO-1 were identified by restriction endonucleases digest analysis and sequence analysis.Results: The sequence of NY-ESO-1 segment was amplified and identical with that published in GenBank.The target gene NY-ESO-1 was successfully cloned into pET-15b prokaryotic expression vector.Conclusion: LP Recco PCR Cloning can efficiently and conveniently construct high quality prokaryotic expression vector of human NY-ESO-1 gene,which provide the expression vector to produce pET-15b-NY-ESO-1 fusion protein.更多还原