RegⅣ基因缺失CRD结构域片段获得及其表达载体的构建与转染-东南大学学报医学版

RegⅣ基因缺失CRD结构域片段获得及其表达载体的构建与转染

单位：1. 东南大学基础医学院病理学与病理生理学系, 江苏南京 210009;
2. 南京中医药大学病理学教研室, 江苏南京 210046

分类号：Q786; Q781; Q782

出版年·卷·期（页码）：2009·28·第三期（166-170）

摘要：

目的: 探讨应用基因重叠延伸拼接PCR (SOE-PCR)技术构建Reg Ⅳ基因缺失CRD 结构域表达载体,并转染Reg Ⅳ低表达结直肠癌细胞系。方法: 应用SOE-PCR法获得Reg Ⅳ缺失CRD 结构域片段,构建真核表达载体并转染Reg Ⅳ低表达结直肠癌LoVo细胞,G418筛选,RT-PCR检测鉴定。结果: 经酶切鉴定筛选出的重组体测序结果与目的序列完全一致,重组载体构建成功,经pcDNA3.1-Reg Ⅳ-domain△转染的LoVo细胞Reg Ⅳ-domain △呈阳性表达。结论: 利用SOE-PCR技术可成功构建Reg Ⅳ基因缺失CRD 结构域的表达载体。

Objective To construct the expression vector of Reg Ⅳ gene deleting the CRD domain and transfect it into human colon cancer LoVo cell line which is negative for Reg Ⅳ gene expression.Methods The fragment of Reg Ⅳ gene deleting the CRD domain was obtained by SOE-PCR.Recombinant plasmids of Reg Ⅳ were constructed and tranfected into human colon cancer LoVo cell line.Selection for transfected cells was carried out in a medium containing G418 and identified by RT-PCR.Results The sequence of the recombinant vector digested by enzyme was completely consistent with the target sequence and the recombinant vector was successfully constructed.Conclusion The expression vector of Reg Ⅳ gene deleting the CRD domain can be successfully constructed by SOE-PCR.

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