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反义及正义α－平滑肌肌动蛋白逆转录病毒载体的构建与表达
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发布时间：2007-01-16
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[摘要] 目的：将人的α－平滑肌肌动蛋白（α－SMA）基因，与载体pLXSN质粒连接，重组反义和正义逆转录病毒载体，评价含α－SMA的逆转录病毒的转染能力，为瘢痕的基因治疗提供一种新的手段。方法：含人类α－SMA cDNA目的片段的pBS/SK质粒和载体pLXSN质粒连接分别酶切、再平末端化、酶连接反应，重组反义和正义α－SMA cDNA的质粒，构建逆转录病毒载体，并用DOTAP转染培养细胞，RT－PCR鉴定。结果：NIH3T3细胞被反义和正义重组逆转录病毒感染且携带目的基因的病毒基因组正确整合到NIH3T3细胞基因橹校籶LXSN/as－SMA感染 NIH3T3细胞后胞体逐渐失去典型的梭形，突起变短。与之相反，pLXSN/s－SMA感染NIH3T3细胞后细胞突起长，与相邻突起开始形成连接。辅助病毒检测没有发现抗性克隆出现。结论：反义和正义α－SMA重组逆转录病毒可以高效感染包装细胞，目的基因正确整合到细胞基因组中。
[关键词]逆转录病毒；载体；α－平滑肌肌动蛋白；基因
[中图分类号] R619.6 [文献标识码]A [文章编号]1008－6455（2003）03－0229－03
Construction and expression of the α－SMA recombinant sense and antisense retrovirus vector
WANG Zhen－xiang, WU Jun, YI Shao－xuan and Li Shi－rong
(Department of Plastic Surgery Southwest Hospital the Third Military Medical University, Chongqing 400038, China)
Abstract: Objective Using the gene of α－smooth muscle actin(α－SMA) and PLXSN plasmid，recombinant sense and antisense retrovirus vector were constructed. It was analysed whether they were easy to transfect into the target cells. Methods A recombined sense and antisense α－SMA cDNA was obtained using gene recombination technique to clone the－SMA cDNA to vector, which a gene of－smooth muscle actin(α－SMA) and PLXSN plasmid were cut respectively and link into sense, antisense recombinants, and then transfected into PA317 and NIH3T3 cells by DOTAP method. Results Recombined sense and antisense －SMA cDNA carried by retrovirus can integrate into NIH3T3 cell correctly. Using both of Recombined retrovirus transfected NIH3T3 cells, it were observed that their cell morphology were changed and two kinds of recombinants were test out. Conclusion：Recombined sense and antisense －SMA cDNA were carried by retrovirus effectively and can integrate into PA317 and NIH3T3 cell correctly.
Key words: retrovirus; vector;α－smooth muscle actin; gene
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