粉尘螨变应原第1组分原核表达质粒pCold-TF-Der f 1构建及鉴定

中国媒介生物学及控制杂志 ›› 2012, Vol. 23 ›› Issue (4): 289-291.

粉尘螨变应原第1组分原核表达质粒pCold-TF-Der f 1构建及鉴定

杨李, 周鹰, 王运刚, 马桂芳, 孙金霞, 崔玉宝

盐城卫生职业技术学院医学检验技术教研室, 江苏盐城 224006

收稿日期:2012-02-20 出版日期:2012-08-20 发布日期:2012-08-20
通讯作者: 崔玉宝,Email: ybcui1975@hotmail.com
基金资助:
国家自然科学基金(30660166,NSFC81001330);盐城市科技发展计划项目(YK2008079);江苏省卫生厅招标课题(Z200914);江苏省卫生职业技术教育研究立项课题(J200907);江苏省“六大人才高峰”第六批项目

Construction and identification of prokaryotic expression plasmid pCold-TF-Der f 1 for the dust mite allergen Der f 1

YANG Li, ZHOU Ying, WANG Yun-gang, MA Gui-fang, SUN Jin-xia, CUI Yu-bao

Department of Clinical Laboratory, Yancheng Health Vocational & Technical College, Yancheng 224006, Jiangsu Province, China

Received:2012-02-20 Online:2012-08-20 Published:2012-08-20
Supported by:
Supported by the National Natural Science Foundation of China(No. 30660166, NSFC81001330), Scientific Development Plan of Yancheng City (No. YK2008079), Jiangsu Provincial Health Department (No. Z200914), Jiangsu Provincial Health Department(No. J200907), and“Six Adults Just”High Peak, Personal Department of Jiangsu Province

摘要/Abstract

摘要： 目的建立粉尘螨变应原第1组分全长基因原核表达质粒pCold-TF-Der f 1。方法以质粒pET-28a(+)-Der f 1为模板扩增目的基因Der f 1,克隆至pCold-TF DNA载体,转化大肠埃希菌BL21,IPTG诱导表达并用SDS-PAGE(十二烷基磺酸钠-聚丙烯酰胺凝胶电泳)和蛋白印迹法(Western blotting,WB)验证产物。结果 PCR扩增获得Der f 1编码全长基因,成功构建表达质粒pCold-TF-Der f 1,SDS-PAGE和WB验证表明该质粒在大肠埃希菌中正常表达,且基本为可溶性表达。结论成功建立尘螨变应原原核表达质粒pCold-TF-Der f 1,并成功实现其原核表达,为进一步生产基因工程变应原提供基础依据。

关键词: 尘螨变应原, 基因重组, 原核表达

Abstract: Objective To construct prokaryotic expression plasmid pCold - TF - Der f 1 for the dust mite allergen Der f 1. Methods The Der f 1 gene in full length was amplified from pET-28a(+)-Der f 1 plasmid and cloned to the vector pCold-TF- DNA, with the recombinants then transferred into Escherichia coli BL21. The genetically engineered bacteria with pCold-TF-Der f 1 plasmids were induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blotting. Results The Der f 1 gene was acquired by PCR, with pCold-TF-Der f 1 successfully constructed, and expressed in E. coli. which was indicated SDS-PAGE and western blotting. Conclusion The prokaryotic expression plasmid pCold-TF-Der f 1, which contain Der f 1 gene fragment of Dermatophagoides farinae, can be successfully constructed with its expression in E. coli BL21 accomplished.

Key words: Dust mite allergen, Gene recombination, Prokaryotic expression

中图分类号:

R384.4

杨李, 周鹰, 王运刚, 马桂芳, 孙金霞, 崔玉宝. 粉尘螨变应原第1组分原核表达质粒pCold-TF-Der f 1构建及鉴定[J]. 中国媒介生物学及控制杂志, 2012, 23(4): 289-291.

YANG Li, ZHOU Ying, WANG Yun-gang, MA Gui-fang, SUN Jin-xia, CUI Yu-bao. Construction and identification of prokaryotic expression plasmid pCold-TF-Der f 1 for the dust mite allergen Der f 1[J]. Chines Journal of Vector Biology and Control, 2012, 23(4): 289-291.

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