构建人csp-B核基质结合区克隆及其介导的反转录载体  

Cloning of human csp-B matrix attachment region sequence and construction of its retrovirus vector

作　　者：昝玉玺[1] 王俐[1] 张俊河[1] 王天云[1] 

机构地区：[1]新乡医学院生物化学与分子生物学教研室,河南省新乡市453003

出　　处：《中国组织工程研究与临床康复》2010年第11期1948-1950,共3页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30470030);河南省自然科学基金(0511042300);河南省科技攻关项目(0624410041)~~

摘　　要：背景:核基质结合区是染色质被限制酶消化后仍附着在核基质上的DNA序列。大量实验表明,核基质结合区可以作为DNA复制的起始点或调控基因的转录,构建核基质结合区表达载体能提高外源基因表达水平,增强外源基因表达的稳定性及提高转化细胞稳定株的频率等。目的:通过克隆人基因组不同的核基质结合区片段,构建核基质结合区介导的包含氯霉素乙酰转移酶(CAT)报告基因的反转录病毒载体pLXSN-MAR,以探索核基质结合区对基因表达的影响。方法:开放性实验于2007-01/12在新乡医学院生物化学与分子生物学实验室及分子研究室完成。自行构建含氯霉素乙酰转移酶报告基因的质粒PLXSN-CAT载体。TaqDNA聚合酶、T4DNA连接酶、DNAMarker、限制性内切酶BamHⅠ、凝胶纯化试剂盒、质粒提取试剂盒均购于大连TaKaRa公司;引物由上海生工生物工程技术服务有限公司合成。以人基因组DNA为模板,采用聚合酶链反应扩增csp-B核基质结合区序列,克隆入反转录载体PLXSN-CAT中,经限制性内切酶酶切及DNA序列分析鉴定目的基因。结果与结论:聚合酶链反应扩增的特异性片段长度为931bp,以此构建的重组质粒命名为PLXSN-CAT-MAR,经BamHⅠ酶切后显示5.9kb和931bp左右的两条片段,测序结果与Gen-bank中的人csp-B核基质结合区(Genbank序列号M62716)序列一致,证明人核基质结合区片段已成功克隆到了反转录载体PLXSN-CAT中。提示成功构建了PLXSN-CAT-MAR反转录表达载体。BACKGROUND： Matrix attachment region （MAR） are DNA elements that bound to the nuclear matrices after chromatin digested with restriction endonuclease. Plenty of studies have shown that MAR considered as initial point of DNA replication or transcription of regulatory gene. Thereby, construction of MAR expression vector can elevate the overall level of transgene expression, enhance stability of exogenous gene, as well as increase frequency of stable transfectant cells. OBJECTIVE： To construction pLXSN-CAT recombinant retrovirus vector that containing chloramphenicol acetyltransferase （CAT） via cloning MAR sequence of human, and to explore the influence of MAR on the gene expression. METHODS： An open experiment was performed at the Department of Biochemistry and Molecular Biology, Xinxiang Medical College from September 2007 to December 2007. The PLXSN-CAT vector of CAT was constructed by the laboratory. TaqDNA polymerase, T4 DNA ligase, DNA Marker, restriction enzyme BamHⅠ, agarose gel DNA purification kit, as well as plasmid purification kit were purchased fromTakara Biotechnology （Dalian） Co., Ltd. The sequence of csp-B MAR was amplified by polymerase chain reaction （PCR） method applied to human DNA. The fragment was inserted into retrovirus vector PLXSN-CAT plasmid. The recombinant plasmid was verified by double digestion and DNA sequencing. RESULTS AND CONCLUSION： The length of specific fragment applied by PCR was 931 bp, and the recombinant plasmid PLXSN-CAT-MAR presented two bands： 5.9 kb and 931 bp using respective restriction enzymes BamHⅠ. The sequence of MAR was confirmed by blasting to Genbank （serial numobr： M62716）. It suggested that MAR had been cloned into PLXSN-CATR vector correctly. The recombinant retrovirus vector PLXSN-MAR was successfully constructed.

关 键 词：核基质结合区 csp-B 基因克隆 反转录载体 载体构建 

分 类 号：R318[医药卫生—生物医学工程]