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催乳素调控序列荧光素酶报告基因的构建 被引量：1

Construction of a luciferase report vector with prolatin regulating sequence
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摘要 目的通过将人催乳素(hPRL)启动子上游的调控基因(包括启动子,-3518 bp^+15 bp)插入荧光素酶报告基因pGL3-Basic中,构建成含催乳素调控序列的荧光素酶报告基因(PRL3400-pGL3-Basic),用于催乳素在淋巴细胞中的表达调控研究。方法将含hPRL启动子的PGEM-PRL3400,及荧光素酶报告基因pGL3-Basic转染大肠肝菌(JM109)后扩增,提取并纯化PGEM-PRL3400和pGL3-Basic;分别以SacⅠ、BamHI酶切PGEM-PRL3400,SacⅠ、BglⅡ酶切pGL3-Basic;电泳并回收PRL3400片段和pGL3-Basic酶切大片段,在T4 DNA连接酶的作用下,将PRL3400片段插入荧光素酶报告基因pGL3-Basic中。结果通过酶切及基因测序的方法证实所构建质粒含有pGL3-Basic全序列及催乳素启动子上游调控序列,并且PRL3400片段调控序列的插入位置与方向正确。结论该荧光素酶报告基因构建成功。 Objective To analyse the mechanisms of PRL gene expression in lymphocytes, this study take an upstream of hPRL regulating sequence （include promoter, - 3518 bp ＋ 15bp） to build in luciferase report vector： pGL3 - Basic. Methods E. coli JM109 was transformed with PGEM- PRL3400（include upstream of hPRL regulating sequence- 3518 bp- ＋ 15bp） and pGL3 - Basic, then cultured JM109 in LB, extracted vectors and purified DNA. PGEM - PRL3400 was digested by restriction enzymes （Sac Ⅰ and BamH Ⅰ ） as well as pGL3 - Basic was digested by Sac Ⅰ and Bgl Ⅱ. The corresponding production of the lysis reaction was separated by gel electrophoresis, thus PRL3400 and pGL3 - Basic fragments were obtained. PRL3400 and pGL3 - Basic fragment were linked by T4 DNA ligase. Results The recombination plasmid was tested by gel electrophoresis and ,sequence analysis, it was proved that the plasmid include pGL3 - Basic DNA sequence and PRL regulating sequence （include promoter, - 3518 bp- ＋ 15bp） ; the sites and direction of insertion was corrected. Conclusion The combination plasmid PRL3400 - pGL3 - Basic is successfully constructed.
作者 郭继强 郝文慧 王辉
机构地区 新乡医学院免疫学研究中心
出处 《新乡医学院学报》 CAS 2006年第5期452-455,共4页 Journal of Xinxiang Medical University
基金 河南省医药卫生创新人才项目(编号:20011013)
关键词 催乳素 荧光素酶报告基因 基因调控 JURKAT细胞 prolactin luciferase report vector regulation of gene expression Jurkat cell
分类号 R392.11 [医药卫生—免疫学]
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参考文献7

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二级参考文献11

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共引文献5

同被引文献11

  • 1孙翔,孙清河,王辉.催乳素对人B淋巴细胞增殖功能的影响[J].新乡医学院学报,2006,23(2):130-131. 被引量：4
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新乡医学院学报
2006年 第5期

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