摘要
本实验克隆并构建了携带红荧光报告基因的Nudel真核表达载体,为后续研究Nudel基因的功能打下基础。查阅Gene card可知,Nudel基因在肺组织中的RPKM值为7.97,本研究以人肺癌细胞A549细胞为材料,提取总RNA并反转录出cDNA,再以cDNA为模板扩增Nudel基因;将目的基因与pMD18-T载体连接,测序鉴定并命名为pT-Nudel;然后再将Nudel基因亚克隆至含红荧光报告基因的载体pDsRed-C1,构建的表达载体命名为pRed-Nudel;提取去内毒素的超纯质粒,转染COS7细胞,检测Nudel的表达情况以及抑癌基因Pten对Nudel表达的影响。结果表明,克隆的Nudel基因经测序鉴定正确;将Nudel基因亚克隆至红荧光表达载体pDsRed-C1,成功构建pRed-Nudel,并在COS7中成功表达。同时观察到与突变型的PtenC124S相比,野生型的Pten有抑制Nudel表达的可能,需要后续的实验进一步验证。
In this study, Nudel gene was cloned and used to construct a eukaryotic expression vector har-boring a red fluorescent gene. According to the Gene card data, the RPKM value of Nudel is 7.97 in lung tissue, so the total RNA was extracted from the lung cancer cells A549. The cDNA was made from the RNA and used as template to amplify Nudel gene. Nudel gene was cloned into pMD18-T vector and named as pT-Nudel after being confirmed by sequencing. Then this gene was subcloned to pDsRed-C1 vector which harbors a red fluorescent gene, to achieve the expression vector pRed-Nudel. Ultrapure plasmids pRed-Nudel free of endotoxin were isolated and used to transfect COS7 cells. The expression of Nudel and the impact of tumor suppressor gene Pten on the expression of Nudel were investigated. The results showed that the Nudel gene was successfully cloned and the expression vector pRed-Nudel was successfully constructed by subcloning Nudel gene into the expression vector pDsRed-C1. Nudel was expressed in COS7 cells. Different from the mutated PtenC124S, the wild type Pten restrained the expression of Nudel possibly, which needs further verification.
出处
《微生物前沿》
2018年第4期149-155,共7页
Advances in Microbiology
基金
湖北省自然科学基金资助项目(2015CFB490)
全国大学生创新创业大赛项目(201710488013)的资助。
