猪唾液酸黏附素氨基端结构域基因的克隆表达及其多克隆抗体制备

中国畜牧兽医

猪唾液酸黏附素氨基端结构域基因的克隆表达及其多克隆抗体制备

张继希¹, 张志远², 张宜娜¹, 周永辉¹, 张祥¹, 郝一妹¹, 张元元¹, 夏平安¹, 崔保安¹

1. 河南农业大学牧医工程学院,河南郑州 450002;
2. 中国动物疫病预防控制中心,北京 100125

收稿日期:2012-03-09 出版日期:2012-09-20 发布日期:2012-09-18
通讯作者: 夏平安(1964－),男,湖北人,教授,硕士生导师,研究方向:动物疫病分子病原学及分子免疫学。
作者简介:张继希(1986－),男,河南人,硕士生,研究方向:动物病原分子生物学与免疫学。
基金资助:
河南省重点攻关项目(92102110018)。

Cloning and Expression of the Amino Structure Domain Gene of Sialoadhesin from Swine and Preparation of Polyclonal Antibody against it

ZHANG Ji-xi¹, ZHANG Zhi-yuan², ZHANG Yi-na¹, ZHOU Yong-hui¹, ZHANG Xiang¹, HAO Yi-mei¹, ZHANG Yuan-yuan¹, XIA Ping-an¹, CUI Bao-an¹

1. College of Animal Science and Veterinary Medicine,Henan Agricultural University, Zhengzhou 450002,China;
2. China Animal Disease Control Center,Beijing 100125,China

Received:2012-03-09 Online:2012-09-20 Published:2012-09-18

摘要/Abstract

摘要： 本试验旨在克隆出猪唾液酸黏附素的近氨基端结构域基因,进而构建原核表达载体并诱导表达重组蛋白,以制备多克隆抗体。试验中应用RT-PCR技术从健康仔猪肺巨噬细胞中克隆出猪唾液酸黏附素的近氨基端结构域的cDNA序列,并将其亚克隆到原核表达载体pET-32a中,构建重组表达质粒pET-Sn150,成功表达了分子质量大小约为43.1 ku的γ亚单位重组蛋白。将重组蛋白pET-Sn150免疫小鼠,制备获得鼠抗pET-Sn150重组蛋白多克隆抗体,将得到的重组蛋白多克隆抗体采用间接 ELISA 和Western blotting试验方法检测,ELISA测定多克隆抗体的血清效价为1∶12800;Western blotting试验结果证明,制备的鼠抗pET-Sn150多克隆抗体可以与重组蛋白进行特异性结合,从而证明重组蛋白具有较好的免疫原性。本试验克隆出猪唾液酸黏附素的近氨基端结构域基因并成功表达,为进一步研究其结构与功能奠定了基础。

关键词: 唾液酸黏附素; 克隆; 原核表达; 多克隆抗体

Abstract: The pig sialoadhesin near the amino structure domain cDNA sequence which was cloned from the 90-day age lung health piglets macrophages with RT-PCR was subcloned into a prokaryotic expression vector pET-32a, to construct a recombinant expression plasmid pET-Sn150 which was successfully used to express a gamma recombinant protein subunits whose molecular weight was about 43 ku. By using the recombinant protein pET-Sn150 to immune the mouse, the mice sialoadhesin resistance gamma-irradiation His recombinant protein polyclonal antibody which could be tested by Western blotting and indirect ELISA could be achieved. And the result of indirect ELISA showed that the titer of the polyclonal antibody was 1∶12800. The specific binding of the mice sialoadhesin resistance gamma-irradiation His recombinant protein polyclonal antibody with the restructuring γ and unit protein revealed by the result of Western blotting proved that the recombinant protein had good immunogenicity, and that layed the foundation for a further study of its construction and function as the pig sialoadhesin near the amino structure domain gene was successfully cloned and expressed.

Key words: sialoadhesin; gene clone; prokaryotic expression; polyclonal antibody 

中图分类号:

Q786

张继希, 张志远, 张宜娜, 周永辉, 张祥, 郝一妹, 张元元, 夏平安, 崔保安. 猪唾液酸黏附素氨基端结构域基因的克隆表达及其多克隆抗体制备[J]. 中国畜牧兽医.

ZHANG Ji-xi, ZHANG Zhi-yuan, ZHANG Yi-na, ZHOU Yong-hui, ZHANG Xiang, HAO Yi-mei, ZHANG Yuan-yuan, XIA Ping-an, CUI Bao-an. Cloning and Expression of the Amino Structure Domain Gene of Sialoadhesin from Swine and Preparation of Polyclonal Antibody against it[J]. .

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