猪繁殖与呼吸综合征病毒GP5a多克隆抗血清的制备

doi:10.13523/j.cb.20150806

中国生物工程杂志

2015, Vol. 35

Issue (8): 38-43 DOI: 10.13523/j.cb.20150806

研究报告

猪繁殖与呼吸综合征病毒GP5a多克隆抗血清的制备

王青, 徐彦召, 魏晓晓, 王秋霞, 杭柏林, 孙亚伟, 王飞飞, 胡建和

河南科技学院新乡 453003

Preparation of Polyclonal Antiserums of GP5a Protein of Porcine Reproductive and Respiratory Syndrome Virus

WANG Qing, XU Yan-zhao, WEI Xiao-xiao, WANG Qiu-xia, HANG Bo-lin, SUN Ya-wei, WANG Fei-fei, HU Jian-he

Henan Institute of Science and Technology, Xinxiang 453003, China

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摘要：

构建XX2012株猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV) GP5a蛋白基因的真核表达载体,并制备出GP5a蛋白的多克隆抗血清。以XX2012株PRRSV GP5a蛋白的基因序列为扩增模板,设计并合成一对特异性扩增引物,通过RT-PCR扩增得到该病毒株的GP5a蛋白全长基因片段,将其定向克隆到真核表达载体pCAGG中,构建含有GP5a蛋白基因的重组表达载体pCAGGS-GP5a,并将获得的pCAGGS-GP5a载体采用基因免疫的方式免疫BALB/c小鼠制备GP5a蛋白的多克隆抗体。结果显示,成功克隆出了XX2012株PRRSV GP5a蛋白全长基因,片段大小为156 bp;构建的pCAGGS-GP5a载体经PCR、双酶切、测序鉴定均无误;通过三次基因免疫小鼠成功制备出了GP5a蛋白的多克隆抗体,间接免疫荧光试验证实制备出的多克隆抗体能特异性识别病毒感染后的细胞。由此获得了PRRSV GP5a 蛋白基因的真核表达载体,制备出了GP5a蛋白的特异性多克隆抗血清,为今后开展GP5a蛋白的亚细胞定位及相关功能的研究奠定了基础。

关键词： 猪繁殖与呼吸综合征病毒; GP5a蛋白; 真核表达; 基因免疫

Abstract:

According to the sequences of GP5a gene of XX2012 strain HP-PRRSV, a pair of specificity primers were designed synthesis. The complete gene fragment of GP5a protein was amplified by RT-PCR, and the eukaryotic expression vector pCAGGS-GP5a which containing GP5a protein gene was constructed. Polyclonal antiserums of GP5a protein were prepared by gene immunization pCAGGS-GP5a vector in BALB/c mice. The results showed that the sequences of GP5a protein gene were consistent with predicted size, 156 base pairs. PCR, digestion and sequencing results showed that the recombinant plasmid pCAGGS-GP5a was correct. After the mice were immuned with pCAGGS-GP5a three times, the separated serums of these mice contain the polyclonal antibody of GP5a protein, and the results of indirect immuno-fluorescence assay (IFA) showed that the polyclonal antiserums can specifically recognize cells infected by the virus. In summary, the recombinant plasmid pCAGGS-GP5a containing GP5a protein gene was constructed, and the polyclonal antibody of GP5a protein was prepared, which laid the foundation for the further study of subcellular localization and relevant function of GP5a protein.

Key words: Eukaryotic expression Porcine reproductive and respiratory syndrome virus (PRRSV) GP5a protein Gene immunization

收稿日期: 2015-03-06 出版日期: 2015-08-25

ZTFLH:

Q789

基金资助:

国家自然科学基金青年科学基金项目(31402208)、河南省教育厅科学技术研究重点项目指导计划(14B230003)资助项目

通讯作者: 徐彦召 E-mail: haoxyz@163.com

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引用本文:

王青, 徐彦召, 魏晓晓, 王秋霞, 杭柏林, 孙亚伟, 王飞飞, 胡建和. 猪繁殖与呼吸综合征病毒GP5a多克隆抗血清的制备[J]. 中国生物工程杂志, 2015, 35(8): 38-43.

WANG Qing, XU Yan-zhao, WEI Xiao-xiao, WANG Qiu-xia, HANG Bo-lin, SUN Ya-wei, WANG Fei-fei, HU Jian-he . Preparation of Polyclonal Antiserums of GP5a Protein of Porcine Reproductive and Respiratory Syndrome Virus. China Biotechnology, 2015, 35(8): 38-43.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150806 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I8/38

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