人白细胞介素12两个亚基基因p35、p40拼接的新方法

doi:吉林省科技厅科技发展计划项目资助课题（20

人白细胞介素12两个亚基基因p35、p40拼接的新方法

杜珍武^1,2,齐熙明³,王茜¹,吴晓冬¹,杨绍娟¹,张桂珍¹

(1.吉林大学中日联谊医院中心研究室，吉林长春 130033; 2.吉林大学药学院基因工程教研室, 吉林长春 130021;3.河北省秦皇岛市第一医院中心实验室，河北秦皇岛 066000)

收稿日期:2008-12-03 修回日期:1900-01-01 出版日期:2009-03-28 发布日期:2009-08-14
通讯作者: 张桂珍

A new method for linking two subunits gene p35 and p40 of IL-12

DU Zhen-wu^1,2,QI Xi-ming³,WANG Qian¹,WU Xiao-dong¹,YANG Shao-juan¹,ZHANG Gui-zhen¹

(1.Central Laboratory,China-Japan Union Hospital,Jilin University,Changchun 130033,China;2.School of Pharmacy,Jilin University,Changchun 130021,China;3.Central Laboratory,First Hospital of Qinhuangdao City，Qinhuangdao 066000,China)

Received:2008-12-03 Revised:1900-01-01 Online:2009-03-28 Published:2009-08-14
Contact: ZHANG Gui-zhen

摘要/Abstract

摘要： 目的: 探讨人白细胞介素12(IL-12)两个亚基基因通过连接肽基因进行拼接的新方法。为进一步研究IL-12基因的生物学功能以及应用提供实验基础。方法：采用两个牛弹性蛋白基序（10个氨基酸）的基因序列为两个亚基基因拼接的连接肽基因。根据人IL-12 p35、p40两个亚基基因片段以及连接肽基因核苷酸序列设计引物，其中p40基因下游引物包括部分连接肽基因序列以及Kpn Ⅰ 酶切位点，p35基因上游引物包括部分连接肽基因序列以及Kpn Ⅰ 酶切位点。通过聚合酶链反应(PCR)方法分别获得人IL-12 p35与p40基因片段。利用Kpn Ⅰ内切酶对p35与p40基因的PCR产物进行酶切，酶切产物回收后利用T4 DNA连接酶进行连接，应用p40基因上游引物与p35基因下游引物对连接产物进行PCR扩增。利用Kpn Ⅰ内切酶对连接产物进行酶切鉴定。将扩增的连接产物克隆入T载体，挑选阳性克隆并进行酶切鉴定，酶切鉴定正确的克隆进行拼接产物的核苷酸序列测序。结果：通过PCR分别获得约1 000 bp大小的p40基因片段与600 bp大小的p35基因片段，两个基因酶切片段连接后PCR扩增获得1 600 bp左右的拼接产物，采用Kpn I 内切酶对拼接产物进行酶切后获得约1 000和600 bp大小的基因片段，分别与p40和p35基因片段大小一致。拼接产物的T载体经酶切鉴定可见1 600 bp 大小的酶切产物，拼接产物测序结果与GenBank核酸数据库上的p40基因、p35基因以及连接肽基因核苷酸序列完全一致。结论：利用该基因分子拼接技术成功地将人白介素12两个亚基基因通过连接肽基因进行拼接。建立了一种简单、方便、有效进行人白细胞介素12两个亚基基因拼接的新方法，为进一步研究IL-12的生物学功能以及基因治疗提供实验基础。

关键词: 聚合酶链反应, 基因拼接

Abstract: Abstract:Objective To discuss a new method for linking two subunit genes p35 and p40 of IL-12 by a linkage peptide,so that offer a experimental base for studying the biological function and application of IL-12. Methods Two　 bovine elastin motifs (ten amino acid) genes were used as linkage peptide for linking two subunits gene. The primers for explanding p35 and p40 genes of IL-12 were designed according to the gene nucleotide sequences of p35 and p40 genes and linkage peptide.The part linkage peptide gene sequences and Kpn Ⅰ digestion site were arranged separately to antisense primer of p40 gene and sense primer of p35 gene.The p35 and p40 genes were explanded by PCR.The PCR products of p35 and p40 genes were digested with Kpn Ⅰ restriction endonuclease.The two digestion products were ligated by T4DNA ligase after two digestion products were purified.The ligated products were explanded by PCR with sense primer of p40 gene and antisense primer of p35 gene.The PCR products were identified by restriction endonuclease digestion with Kpn Ⅰ.The PCR products were cloned into T clone vector,and the positive clones were picked out,then the T clone vector was identified by restriction endonuclease digestion.The nucleotide sequences of ligated products were judged by sequencing analysis. Results The p40 gene product that nucleotide number was 1 000 bp and the p35 gene product that nucleotide number was 600 bp were obtained by PCR.The ligated products of two subunits of IL-12 that nucleotide number was 1 600 bp were obtained.Two gene segments were obtained by digesting the ligated products with Kpn Ⅰ endonuclease.The sequencing analysis results showed that the sequence of ligated products was same as the gene nucleotide sequences of p35 and p40 genes and linkage peptide reported in GenBank.Conclusion The two subunits of IL-12 are successfully linked with linkage peptide by using this method.A new,simple,convenient and effective method for linking two subunits of IL-12 is found,and the paper offers a experimental base for farther study on the biological function and application of IL-12.

Key words: polymerase chain reaction, gene linking the paper offers

中图分类号:

杜珍武,齐熙明,王茜,吴晓冬,杨绍娟,张桂珍. 人白细胞介素12两个亚基基因p35、p40拼接的新方法[J]. J4, 2009, 35(2): 309-313.

DU Zhen-wu,QI Xi-ming,WANG Qian,WU Xiao-dong,YANG Shao-juan,ZHANG Gui-zhen. A new method for linking two subunits gene p35 and p40 of IL-12[J]. J4, 2009, 35(2): 309-313.

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