奶牛黏附分子MadCam-1基因的克隆及原核表达

›› 2011, Vol. 38 ›› Issue (8): 131-134.

奶牛黏附分子MadCam-1基因的克隆及原核表达

刘文博¹, 陈媛媛¹, 孙玲², 李德朋¹, 付云贺¹, 张乃生¹, 郭梦尧¹, 杨正涛¹

1. 吉林大学畜牧兽医学院,吉林长春 130062;2. 吉林大学白求恩学院病原生物学实验室,吉林长春 130021

收稿日期:1900-01-01 修回日期:2011-05-05 出版日期:2011-08-20 发布日期:2011-08-20
通讯作者: 杨正涛

Cloning and Prokaryotic Expression of MadCam-1 Gene from the Dairy Cows

LIU Wen-bo¹, CHEN Yuan-yuan¹, SUN Ling², LI De-peng¹, FU Yun-he¹, ZHANG Nai-sheng¹, GUO Meng-yao¹, YANG Zheng-tao¹

1. College of Animal Science and Veterinary Medicine,Jilin University, Changchun 130062,China;2. Department of Pathogenobiolog,Norman Bethune College of Medicine,Jilin University, Changchun 130021,China

Received:1900-01-01 Revised:2011-05-05 Online:2011-08-20 Published:2011-08-20

摘要/Abstract

摘要： 原核表达MadCam-1基因、纯化MadCam-1黏附蛋白,并制备其多克隆抗体,为进一步功能研究奠定基础。根据MadCam-1的已知基因序列设计并合成1对引物,应用PCR技术扩增MadCam-1基因片段插入到原核表达载体 pGEX-4T-1中,构建重组表达质粒 pGEX-4T-1-MadCam-1,转化入E.coliBL21(DE3),经IPTG诱导,对其产物进行SDS-PAGE分析和Western blotting鉴定,以纯化后的融合蛋白为抗原,免疫家兔,获得抗血清。结果表明,重组表达质粒pGEX-4T-1-MadCam-1经PCR及双酶切鉴定证明构建正确,测序结果与GenBank中登录的基因序列同源性为100%。表达产物经Western blotting鉴定,结果证实成功表达了分子质量约为50 ku 的蛋白质。纯化的蛋白质免疫家兔后,经ELISA检测多克隆抗体效价为1∶32000。因此,成功获得了奶牛 MadCam-1多克隆抗体,为进一步研究奶牛黏附分子 MadCam-1的功能奠定了基础。

关键词: MadCam-1; 克隆; 表达; 纯化; 多克隆抗体

Abstract: To construct expression vertor of MadCam-1 gene, to expression and purify recombination protein and to prepare polyclonal antibody, which have laid a foundation of studying its function. A pair of primers was designed according to the sequence of MadCam-1 from GenBank. The MadCam-1 gene was amplified by PCR. The expression vector pGEX-4T-1-MadCam-1 was reconstructed and transformed into E.coli BL21,then it was induced by IPTG.The expressed product was identified by SDS-PAGE and Western blotting. Rabbits were immunized with the purified MadCam-1 to generate antisera. The results showed that the homology of 100% of the cloned MadCam-1 gene to that reported in GenBank. The recombinant plasmid pGEX-4T-1-MadCam-1 was constructed successfully. The expression product was identified by Western blotting. The results showed that the molecular weight of recombinant protein pGEX-4T-1-MadCam-1 was 50 ku.Then rabbits were injected with purified protein to induce immunoreactions and the potency of the antibody was as high as 1∶32000. Therefore,the polyclonal antibody of MadCam-1 was induced.The results of this study lay a foundation for further study of the function of dairy cows MadCam-1.

Key words: MadCam-1; cloning; expression; purification; polyclonal antibody

中图分类号:

刘文博;陈媛媛;孙玲;李德朋;付云贺;张乃生;郭梦尧;杨正涛. 奶牛黏附分子MadCam-1基因的克隆及原核表达[J]. , 2011, 38(8): 131-134.

LIU Wen-bo;CHEN Yuan-yuan;SUN Ling;LI De-peng;FU Yun-he;ZHANG Nai-sheng;GUO Meng-yao;YANG Zheng-tao. Cloning and Prokaryotic Expression of MadCam-1 Gene from the Dairy Cows[J]. China Animal Husbandry & Veterinary Medicine, 2011, 38(8): 131-134.

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