小鼠PMX—IRES—Wnt5a逆转录病毒载体的构建及在3T3细胞中的表达(1)
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[摘要] 目的 构建小鼠Wnt—5a逆转录病毒载体，观察Wnt—5a在3T3细胞的表达情况。 方法 采用同源重组技术将小鼠Wnt—5a插入逆转录病毒载体PMX—IRES—GFP，构建PMX—IRES—Wnt5a重组质粒，利用脂质体转染3T3细胞，RT—PCR检测Wnt—5a mRNA表达情况，Western—Bloting检测Wnt—5a蛋白表达情况。 结果 经限制性内切酶证实成功构建了携带Wnt—5a基因的逆转录病毒载体PMX—IRES—Wnt5a，RT—PCR检测结果显示Wnt—5a在转染的3T3细胞的表达明显增高。 结论 成功构建Wnt—5a逆转录病毒载体，并能在3T3细胞成功表达Wnt—5a。
[关键词] Wnt—5a；逆转录病毒载体
[中图分类号] R782 [文献标识码] A [文章编号] 1673—9701(2012)25—0007—02
Construction of mouse PMX—IRES—Wnt5a retroviral expression vector and expression of Wnt—5a in NIH3T3 cells
YAN Chun1 LIU Caixia2 YAN Shaorong1 LI Qiang1 LIN Sen1 WU Wenbing1 WU Lifeng1 CHEN Jiong3
1. Department of Clinical Laboratory，the People’s Hospital of Rui’an City in Zhejiang Province，Rui’an 325200，China；
2. Department of Clinical Laboratory，the Second Affiliated Hospital of Wenzhou Medical College，Wenzhou 325027，China；3. Department of Burn，the People’s Hospital of Rui’an City in Zhejiang Province，Rui’an 325200，China
[Abstract] Objective To construct the retroviral expression vector of PMX—IRES—Wnt5a，transfect into 3T3 cells，and observe the expression of Wnt—5a. Methods Gene recombinant technology was employed to clone mouse Wnt—5a gene to the retroviral expression vector of PMX—IRES—GFP， to obtain the recombined plasmid PMX—IRES—Wnt5a，transfect into 3T3 cells by Lipofectamine 2000. The result of transfection was analyzed by RT—PCR and Western—Blotting. Results Identification by enzyme digestion confirmed successful construction of the retroviral expression vector of PMX—IRES—Wnt5a ......
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