猪繁殖与呼吸综合征病毒GP5蛋白的原核表达及多克隆抗体制备
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Prokaryotic Expression of GP5 Protein of PRRSV and Preparation of Its Polyclonal Antibody
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    为体外表达猪繁殖与呼吸综合征病毒(PRRSV)糖基化囊膜蛋白GP5,以及制备其多克隆抗体,以PRRSV PC疫苗株RNA为模板,应用RT-PCR扩增GP5基因,并克隆至原核表达载体pET-32a(+),构建重组表达载体pET-32a-GP5。经过酶切和测序鉴定后,将阳性重组质粒转化到大肠杆菌BL21(DE3)感受态细胞中,加入IPTG低温诱导表达。使用亲和层析法纯化PRRSV GP5蛋白。然后将纯化的蛋白免疫北京大耳白兔制备多克隆抗体,并进行酶联免疫吸附试验(ELISA)和间接免疫荧光分析(IFA)。结果显示,表达的PRRSV GP5原核蛋白以可溶性和包涵体两种形式存在,相对分子质量大约30 kDa;制备的GP5蛋白多克隆抗体ELISA效价可达1:64 000;IFA检测显示,制备的多克隆抗体能够识别PRRSV。重组PRRSV GP5蛋白及其多克隆抗体的成功制备为PRRSV血清学检测方法的建立提供了良好的生物学材料。

    Abstract:

    In order to express the glycosylated envelope protein GP5 of porcine reproductive and respiratory syndrome virus(PRRSV)in vitro,and to prepare its polyclonal antibody,GP5 gene was amplified by RT-PCR by taking the RNA of PRRSV PC vaccine strain as a template,then cloned into the prokaryotic expression vector pet-32a(+)to construct the recombinant expression vector of pET-32a-GP5. After enzyme digestion and sequencing analysis,the positive recombinant plasmid was transformed into E. coli BL21(DE3)cells,and induced by IPTG at low temperature. The PRRSV GP5 protein was purified by affinity chromatography,and then was vaccinated into Beijing white rabbit to prepare polyclonal antibody,then enzyme linked immunosorbent assay(ELISA)and indirect immunofluorescence analysis(IFA)were carried out. The results showed that the expressed GP5 prokaryotic protein was available in soluble supernatant and inclusion body,with a molecular weight of approximate 30 kDa;the ELISA titer of the polyclonal antibody against GP5 protein was up to 1:64 000,and it was shown that PRRSV could react with the polyclonal antibody. In conclusion,good biological materials were provided for establishing serological detection methods for PRRSV through successful preparation of recombinant PRRSV GP5 protein and its polyclonal antibody.

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乌吉斯古楞,刘亭岐,巨敏莹,张斌,韩宇,刘建奇,宋庆庆,关平原.猪繁殖与呼吸综合征病毒GP5蛋白的原核表达及多克隆抗体制备[J].《中国动物检疫》编辑部,2020,37(10):121-126.

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  • 在线发布日期: 2020-09-29
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