Reg Ⅳ基因缺失CRD结构域片段获得及其表达载体的构建与转染  

Construction of the expression vector of Reg Ⅳ gene deleting the CRD domain and its transfection in colorectal cancer cell lines

作　　者：郭英[1,2] 徐佳佳[1] 商延芳[1] 李楠[1] 高峰[1] 黄培林[1] 

机构地区：[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

出　　处：《东南大学学报（医学版）》2009年第3期166-170,共5页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目(30400534)

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-dom ain△转染的LoVo细胞Reg Ⅳ-dom ain△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 SOEPCR.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 SOEPCR.

关 键 词：REG Ⅳ 碳水化合物识别域 结直肠癌 基因转染 基因重叠延伸拼接PCR 

分 类 号：Q786[生物学—分子生物学] Q781