摘要
目的构建原核表达载体pGEX-4T含人可溶性白细胞分化抗原40配体(soluble cluster of differentiation antigen 40 ligand,sCD40L)重组质粒。方法通过反转录-聚合酶链反应扩增,获得人sCD40L的基因片段;将其连接入T载体,用5-溴-4-氯-3-吲哚-β-D半乳糖苷与异丙基-β-D-硫代半乳糖苷筛选阳性克隆,鉴定正确后经BamHⅠ和XhoⅠ双酶切,再与pGEX-4T原核表达载体连接,最后经酶切及测序鉴定。结果以L02细胞的互补DNA为模板,扩增出474 bp的目的基因;将目的片段与T载体连接并进行蓝白筛选得到阳性克隆;构建pGEX-4T-sCD40L重组质粒,经转化和筛选获得重组菌,经酶切、基因测序证实序列正确。结论成功构建pGEX-4T-sCD40L原核表达载体,为进一步探讨sCD40L在肝癌诊断中的作用奠定基础。
Objective To construct containing human soluble cluster of differentiation antigen 40 ligand(sCD40L) recombinant plasmid pGEX-4T-sCD40 L.Methods The gene fragment of human leukocyte sCD40 L was amplified by reverse transeription-polymerase chain reaction(RTPCR).The fragment was ligated into T vector and ligated with 5-bromo-4-chloro-3-indolyl-β-D-galactoside(X-gal) and isopropyl-β-D-thiogalaetopyranoside(IPTG).The positive clones were screened by IPTG.The positive clones were digested with BamH Ⅰ and Xho Ⅰ,then ligated with pGEX-4T prokaryotic expression vector,and identified by restriction enzyme digestion and sequencing.Results The target DNA fragment of 474 bp was amplified by the complementary DNA(cDNA)of L02 cells.The recombinant plasmid pGEX-4T-sCD40 L was constructed by ligating the target fragment with T vector and screening for blue-white.The recombinants were obtained by transformation and screening,and the sequences were confirmed by restriction enzyme digestion and gene sequencing.Conclusion The prokaryotic expression vector pGEX-4T-sCD40 L has been successfully constructed,which lays a foundation for further study on the role of sCD40 L in the diagnosis of hepatocellular carcinoma(HCC).
出处
《转化医学杂志》
2017年第1期12-15,共4页
Translational Medicine Journal
基金
国家自然科学基金资助项目(81361120401)
首都卫生发展科研专项项目(首发2014-1-1151)
关键词
可溶性白细胞分化抗原40配体
原核表达载体
融合蛋白
Soluble cluster of differentiation antigen 40 ligand(sCD40L)
Prokaryotic expression vector
Fusion protein
