為體外表達豬繁殖與呼吸綜合征病毒（PRRSV）糖基化囊膜蛋白GP5，以及制備其多克隆抗體，以PRRSV PC疫苗株RNA為模板，應(yīng)用RT-PCR擴增GP5基因，并克隆至原核表達載體pET-32a（+），構(gòu)建重組表達載體pET-32a-GP5。經(jīng)過酶切和測序鑒定后，將陽性重組質(zhì)粒轉(zhuǎn)化到大腸桿菌BL21（DE3）感受態(tài)細胞中，加入IPTG低溫誘導(dǎo)表達。使用親和層析法純化PRRSV GP5蛋白。然后將純化的蛋白免疫北京大耳白兔制備多克隆抗體，并進行酶聯(lián)免疫吸附試驗（ELISA）和間接免疫熒光分析（IFA）。結(jié)果顯示，表達的PRRSV GP5原核蛋白以可溶性和包涵體兩種形式存在，相對分子質(zhì)量大約30 kDa；制備的GP5蛋白多克隆抗體ELISA效價可達1:64 000；IFA檢測顯示，制備的多克隆抗體能夠識別PRRSV。重組PRRSV GP5蛋白及其多克隆抗體的成功制備為PRRSV血清學(xué)檢測方法的建立提供了良好的生物學(xué)材料。
Prokaryotic Expression of GP5 Protein of PRRSV and Preparation of Its Polyclonal Antibody
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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