摘要
目的:克隆出人T-钙粘蛋白基因,并应用pEGFP-N1构建其重组真核表达载体。方法:Trizol液氮研磨法提取出人子宫平滑肌组织总RNA。设计一对分别含有EcoRⅠ、BamHⅠ酶切位点的人T-钙粘蛋白基因上下游引物。利用Pfx DNA聚合酶高保真RT-PCR扩增cDNA,并将其连接到pEGFP-N1的多克隆位点,然后通过卡那霉素抗性筛选、双酶切及PCR鉴定,选取鉴定正确的克隆测序。结果:提取的人子宫平滑肌组织总RNA可清楚看到28S、18S和5S rRNA带形。反转录出的cDNA为2.142kb,将其与pEGFP-N1均进行EcoRⅠ和BamHⅠ酶切,之后用T4连接酶将二者进行连接,产物电泳可见在7 500bp附近有一条带,即为pEGFP-N1/T-cadherin,双酶切与测序结果表明目的基因序列克隆正确。结论:重组真核表达载体pEGFP-N1/T-cadherin构建成功,为下一步研究人T-钙粘蛋白基因在黑色素瘤中的作用奠定基础。
Objective： To clone human T- cadherin gene, and construct its recombinant eukaryotic exprression vector with pEGFP- N1. Method：Extract the total RNA from uterine smooth muscle tissue by grinding in liquid nitrogen Trizol. Design a couple of upstream and downstream primer of human T - cadherin gene which contain EcoR I and BarnH I restriction sites respectively, eDNA is extended byPfx DNA polymerase high fidelity RT - PCR , and connect it into multiple cloning sites of pEGFP - N1 , then choose the correct cloning se- quence by kanamycin resistance selection, and identification by double digestion and PCR and sequenced. Result：We can clearly see the band shape of 28S, 18S and 5S rRNA from the extracted total RNA of Human uterine smooth muscle tissue. Reverse transcription cDNA is 2. 142kb. Digest both cDNA and pEGFP - NI by EcoR I and BamH I , then connect them by using T4 ligase, electrophoresis of the product show a band near the 7 500bp, which is pEGFP - N1/T - cadherin. Double digested and sequencing results show that the se- quence of the target gene cloning is correct. Conclusion： The expression vector pEGFP - N1/T - cadherin is constructed succesfully , which lay foundation for the further study on whether the T - cadherin will play an important role in melanoma.
出处
《生物技术》
CAS
CSCD
北大核心
2013年第2期9-13,共5页
Biotechnology
基金
河北省科学技术研究与发展计划项目("T钙粘蛋白在恶性黑素瘤中表达及基因转染研究"
09276101D-14)资助
