科研動態(tài)

為體外表達牛源多殺性巴氏桿菌（Pasteurella multocida）PlpE基因及制備抗PlpE蛋白多克隆抗體，根據(jù)PlpE基因核酸序列設(shè)計了1對特異性引物，通過PCR擴增PlpE基因，構(gòu)建重組質(zhì)粒pET-32a-PlpE；搖菌提取質(zhì)粒，進行雙酶切鑒定，鑒定正確后將重組質(zhì)粒pET-32a-PlpE轉(zhuǎn)化至BL21（DE3）感受態(tài)細胞，加入IPTG進行誘導(dǎo)，對誘導(dǎo)蛋白進行SDS-PAGE和Western Blot鑒定；使用純化蛋白PlpE免疫北京大耳白兔，制備多克隆抗體，并進行間接ELISA和Western Blot分析。結(jié)果顯示，擴增的PlpE基因大小為1 050 bp，在原核細胞中誘導(dǎo)表達的PlpE蛋白約56 kDa。間接ELISA結(jié)果顯示，所制備多克隆抗體效價可達1:512 000；Western Blot結(jié)果顯示，所制備多克隆抗體反應(yīng)性良好。PlpE蛋白的成功表達和多克隆抗體的成功制備，為下一步多殺性巴氏桿菌診斷方法的建立和疫苗研發(fā)奠定了基礎(chǔ)。

Prokaryotic Expression of PlpE Protein of Bovine Pasteurella multocida and Preparation of Polyclonal Antibodies

In order to express the PlpE gene of bovine Pasteurella multocida in vitro and prepare corresponding polyclonal antibodies，a pair of specific primers were designed according to the nucleic acid sequence of the gene，then pET-32a-PlpE，the recombinant plasmid，was constructed through PCR amplification，after plasmid extraction and identification by double enzyme digestion，the recombinant plasmid was transformed into BL21（DE3）cells and the bacteria was induced by IPTG and the induced protein was identified by SDS-PAGE and Western Blot；Beijing White Rabbits were vaccinated with purified PlpE protein to prepare polyclonal antibodies，which were analyzed by indirect ELISA and Western Blot. The results showed that the amplified PlpE gene was 1 050 bp，and the induced PlpE protein was about 56 kDa. It was shown that，by indirect ELISA，the titer of the polyclonal antibodies could be up to 1:512 000；and by Western Blot，the antibody could react well with the positive serum. It was concluded that a foundation was laid for the establishment of the method for identifying Pasteurella multocida and development of relevant vaccines in the future.

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