目的 构建真核表达质粒pcDNA6.0-TNFR2196MET和pcDNA6.0-TNFR2196ARG.方法 人工合成TNFR2196MET目的 片段,与pMD18-T simple 载体连接形成克隆载体,再通过设计突变引物,PCR定点突变扩增出含有突变位点的质粒,转化到肠埃希菌感受态细胞JM109中,通过氨苄青霉素抗药性筛选出成功转化的阳性克隆,并进行扩增.抽提质粒进行测序鉴定,得到pMD18-T simple-TNFR2196ARG.双酶切pMD18-T simple-TN-FR2196MET、pMD18-T simple-TNFR2196ARG及pcDNA6.0,将TNFR2196MET、TNFR2196ARG插入pcDNA6.0线性质粒,即构建成pcDNA6.0-TNFR2196MET和pcDNA6.0-TNFR2196ARG真核表达质粒,转化到Ecoli感受态中,筛选阳性克隆行菌液PCR和双酶切及测序鉴定.结果 酶切鉴定和测序分析验证TNFR2基因196MET和196ARG表达载体构建成功.结论 成功构建表达载体为下一步心血管疾病研究奠定了实验基础.%Objective To construct the eukaryotic expression plasmid, pcDNA6.0-TNFR2196MET and pcDNA6.0-TNFR2196ARG Methods TNFR2196MET target fragment was artificially synthesized and was linked to pMD18-T simple for forming cloning vectors. This was followed by amplification of plasmids with point mutation for further transforma -tion into JM109, the competent E. Coll cells, via polymerase chain reaction on the basis of mutation primers designed previously. The positive clones via transformation were screened based on ampicillin resistance and were subjected to amplification. The pMD18-T simple-TNFR2196ARG was obtained by sequencing of the extracted plasmids. The plasmids, pMD18-T simple-TNFR2196MET, pMD18-T simple-TNFR2196ARG; and pcDNA6.0, underwent double enzyme digestion. Both TNFR2196MET and TNFR2196ARG were then inserted into pcDNA6.0, the linear plasmid, for construction of eukaryotic expression vectors, pcDNA6.0-TNFR2196MET and pcDNA6.0-TNFR2196ARg;, which were subsequently transformed into competent E. Coli followed by screening the positive clones as confirmed by polymerase chain reaction, double enzyme digestion and sequencing test. Results TNFR2 gene expression plasmids, pcDNA6.0-TNFR2196MET and pcDNA6.0-TN-FR2196ARG, were successfully constructed, as confirmed by DNA sequencing and enzyme digestion analysis. Conclusion The successful construction of pcDNA6.0-TNFR2196MET and pcDNA6.0-TNFR2196ARG; expression plasmids has provided foundation for further research on cardiovascular diseases.
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