小鼠Tim-３真核表达载体pTARGET-Tim-3的构建

山东大学学报(医学版) ›› 2010, Vol. 48 ›› Issue (11): 37-40.

小鼠Tim-３真核表达载体pTARGET-Tim-3的构建

孟静1，刘晓立2，郭春3，马春红3

山东大学 1.第二医院检验科，济南 250033； 2.第二医院肾移植科，济南 250033；
3.医学院免疫学研究所，济南 250012

收稿日期:2010-07-09 出版日期:2010-11-16 发布日期:2010-11-16
通讯作者: 马春红(1970- )，女，博导，主要从事肿瘤免疫研究。E-mail:machunhong@sdu.edu.cn
作者简介:孟静(1973- ),女，硕士，主管技师，主要从事分子免疫和体液免疫的研究。 E-mail:mengjing189@126.com
基金资助:
山东省科技厅攻关计划资助项目（2007GG30002028）;山东省卫生厅青年基金资助项目（2007QZ011）。

Construction of the mouse T cell immunoglobulin mucin 3(Tim-3) eukaryotic expression vector

MENG Jing1， LIU Xiao-li2， GUO Chun3， MA Chun-hong3

1. Clinical Laboratory； 2. Department of Kidney Transplantation, The Second Hospital of Shandong University, Jinan 250033, China；
3. Institute of Immunology, School of Medicine, Shandong University, Jinan 250012, China

Received:2010-07-09 Online:2010-11-16 Published:2010-11-16

摘要/Abstract

摘要：

目的构建小鼠Tim-3真核表达载体,并在黑色素瘤细胞系B16中表达小鼠TIM-3。方法以脾细胞RNA为模板，逆转录PCR扩增鼠Tim-3编码区基因,T-A克隆至真核表达载体pTARGET,构建重组质粒pTARGET-Tim-3,采用脂质体转染法转染黑色素瘤细胞系B16细胞,以逆转录PCR和Western blot验证Tim-3在B16细胞中的表达。结果利用酶切和测序的方法，筛选、鉴定pTARGET-Tim-3真核表达载体,转染黑色素瘤细胞系B16细胞,经逆转录PCR和Western blot证实TIM-3高效表达。结论成功构建小鼠Tim-3真核表达载体, Tim-3在转染的小鼠B16细胞系中高表达。

关键词: 基因；克隆，分子；DNA，重组；真核表达；小鼠

Abstract:

Objective To construct an eukaryotic expression vector of murine T cell immunoglobulin mucin 3(Tim-3), and express Tim-3 in the melanoma cell line B16. Methods The murine Tim-3 coding region was amplified by reverse transcription PCR （RT-PCR）, using RNA from spleen cells as a template. The Tim-3 cDNA was cloned into the eukaryotic expression vector pTARGET by T-A cloning. The recombinant vector was transfected into the melanoma cell line B16 by Lipofectamine. Tim-3 expression was analyzed by RT-PCR and Western blot. Results The murine Tim-3 expression vector was identified by enzyme digestion and sequencing. RT-PCR and Western blot results showed specific Tim-3 expression in B16 cells transfected with the recombinant vector. Conclusion The Tim-3 coding region was successfully cloned into the eukaryotic expression vector and highly expressed in the melanoma cell line B16.

Key words: Genes; Cloning， molecular； DNA， recombinant； Eukaryotic expression; Mice

中图分类号:

R392.12

孟静1，刘晓立2，郭春3，马春红3 . 小鼠Tim-３真核表达载体pTARGET-Tim-3的构建[J]. 山东大学学报(医学版), 2010, 48(11): 37-40.

MENG Jing1，LIU Xiao-li2，GUO Chun3，MA Chun-hong3. Construction of the mouse T cell immunoglobulin mucin 3(Tim-3) eukaryotic expression vector[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2010, 48(11): 37-40.

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