获得重构基因的简捷方法——重叠延伸PCR

doi:卫生部临床学科重点科研项目；吉林省科技厅

获得重构基因的简捷方法——重叠延伸PCR

刘玲丽¹，谭岩^1*，刘力华¹，张琨²，时阳¹，许淑芬¹，方艳秋¹，段秀梅¹，姜艳芳¹，王晓祺¹

1.吉林大学第一医院中心实验室，吉林长春130021;2.吉林省疾病预防控制中心,吉林长春130061

收稿日期:2003-11-25 修回日期:1900-01-01 出版日期:2004-09-28 发布日期:2004-09-28
通讯作者: 谭岩

Splicing overlap extension by PCR：a convenientmethod to obtain recombinant gene

LIU Ling-li¹, TAN Yan^1*, LIU Li-hua¹, ZHANG Kun², SHI Yang¹, XU Shu-fen¹, FANG Yan-qiu¹, DUAN Xiu-mei¹, JIANG Yan-fang¹, WANG Xiao-qi¹

1. Department of Central Laboratory, First Hospital, Jilin University, Changchun 130021,China;2. Center of Disease Control and Prevention of Jilin Province, Changchun 130061,China

Received:2003-11-25 Revised:1900-01-01 Online:2004-09-28 Published:2004-09-28
Contact: TAN Yan

摘要/Abstract

摘要： 目的：构建IFN-β信号肽序列与白细胞介素18（IL-18）成熟肽的重组嵌合分子。方法：从BALB/c鼠基因组DNA扩增IFN-β信号肽序列；由小鼠Pro-IL-18真核表达质粒获得IL-18成熟肽序列；以非对称PCR技术得到IFN-β信号肽编码区的反义链和IL-18成熟肽正链；采用重叠延伸PCR(SOE-PCR)产生具有IFN-β信号肽与IL-18成熟肽的嵌合编码序列；定向连方式构建嵌合IL-18的表达质粒并测序。结果：连续胶PAGE表明非对称PCR可分别扩增出两个靶序列的特异性单链，大小符合预期。IFN-β信号肽对称PCR产物与IL-18非对称PCR产物混合作为模板，进行重叠延伸；电泳分析可见清晰的嵌合片段；测序显示重组质粒的表达框由两个设计的靶序列构成，且无移码。结论：重叠延伸PCR是一种获得重构基因的简捷方法。

关键词: 重叠延伸PCR, 非对称PCR

Abstract: Objective To construct a hybrid molecule composed of IFN-β signal sequence and IL-18 mature sequence. Methods Firstly, through routine PCR, IFN-β signal sequence was amplified from BALB/c mouse genome DNA while IL-18 mature sequence from a recombinant plasmid containing mouse Pro-IL-18 sequence. Secondly, the sense strand of IL-18 mature sequence and the anti-sense strand of IFN-β　signal sequence were obtained by asymmetric PCR. Thirdly, a hybrid IL-18 containing IFN-β signal sequence and IL-18 mature sequence was obtained through splicing overlap extension by PCR. At last, the hybrid IL-18 was bonded to pCDNA3 expression vector by the way of orientation linkage and sequenced. Results Continuing PAGE showed the specific single sequence of the two target products could be amplified by asymmetric PCR, and the size was accord with expectation. The best way to obtain hybrid molecule was the mixing of IFN-β signal sequence and IL-18 mature sequence to overlap extention PCR. Squencing proved that expression box of recombinant plasmid was composed of the expected target sequence. And there was no base malposition. Conclusion Splicing overlap extension by PCR is a convenient method to obtain recombinant gene.

Key words: overlap extention PCR, asymmetric PCR

中图分类号:

Q784

刘玲丽，谭岩，刘力华，张琨，时阳，许淑芬，方艳秋，段秀梅，姜艳芳，王晓祺. 获得重构基因的简捷方法——重叠延伸PCR[J]. J4, 2004, 30(5): 713-716.

LIU Ling-li, TAN Yan, LIU Li-hua, ZHANG Kun, SHI Yang, XU Shu-fen, FANG Yan-qiu, DUAN Xiu-mei, JIANG Yan-fang, WANG Xiao-qi. Splicing overlap extension by PCR：a convenientmethod to obtain recombinant gene[J]. J4, 2004, 30(5): 713-716.

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