【摘要】：【目的】構(gòu)建綿羊肺炎支原體(MO)p74基因重組單核細(xì)胞增生李斯特菌(LM)并分析其免疫原性,為研發(fā)MO重組活疫苗奠定基礎(chǔ)�！痉椒ā坎捎肞CR方法從LM-SB5株基因組中擴(kuò)增出rli60基因的上、下游同源片段a和b,從質(zhì)粒pET-32a(+)-p74中擴(kuò)增出MOp74目的基因片段,利用重疊延伸PCR方法(SOE-PCR)將擴(kuò)增的3個(gè)片段融合成ap74b基因片段,克隆入pMD19-T載體中進(jìn)行測(cè)序鑒定。將ap74b片段從pMD19-T載體上切下亞克隆入pKSV7穿梭載體,構(gòu)建pKSV7-ap74b重組穿梭質(zhì)粒,電轉(zhuǎn)化至LM-SB5株感受態(tài)細(xì)胞,用氯霉素和高溫雙重壓力篩選得到重組菌LM-Δrli60-ap74b。并通過SDS-PAGE及Western blot分析重組菌P74蛋白的表達(dá)情況,通過小鼠免疫試驗(yàn)檢測(cè)其免疫原性�！窘Y(jié)果】成功擴(kuò)增出了rli60基因的上下游同源片段a、b及p74目的片段,采用SOEPCR方法獲得融合片段ap74b,并成功亞克隆于穿梭質(zhì)粒pKSV7中。對(duì)重組菌PCR鑒定結(jié)果表明,外源基因MO p74定點(diǎn)整合于LM基因組中。體外穩(wěn)定性檢驗(yàn)表明,p74基因能在LM基因組中穩(wěn)定存在。SDS-PAGE結(jié)果表明,重組菌LM-Δrli60-ap74b可表達(dá)蛋白分子質(zhì)量約為17ku重組蛋白;Western blot分析表明,表達(dá)的重組蛋白能與MO陽性血清發(fā)生特異性免疫反應(yīng)。小鼠免疫試驗(yàn)表明,重組菌LM-Δrli60-ap74b菌液可誘導(dǎo)機(jī)體產(chǎn)生抗MO特異性抗體,證實(shí)該重組菌具有一定的免疫原性,重組菌能誘導(dǎo)機(jī)體產(chǎn)生1∶8~1∶16的抗體效價(jià)�！窘Y(jié)論】獲得了具有免疫原性的綿羊肺炎支原體p74基因重組單核細(xì)胞增生李斯特菌。
[Abstract]:The expression of P74 protein was analyzed by SDS-PAGE and Western blot, and the immunogenicity of P74 protein was detected by mouse immunoassay. [results] the upstream and downstream homologous fragments of rli60 gene AGB and p74 were successfully amplified. The fusion fragment ap74b, was obtained by SOEPCR and subcloned into shuttle plasmid pKSV7. The results of PCR identification showed that the foreign gene MO p74 was integrated into the LM genome. The results of in vitro stability test showed that the p74 gene was stable in the LM genome. SDS-PAGE showed that the recombinant strain LM- 螖 rli60-ap74b could express the protein with molecular weight about the same as that of the 17ku recombinant protein. The expressed recombinant protein could react specifically with MO positive serum.
【作者單位】： 石河子大學(xué)動(dòng)物科技學(xué)院;阿克蘇地區(qū)動(dòng)物疫病控制診斷中心;中國農(nóng)業(yè)科學(xué)院蘭州獸醫(yī)研究所;
【基金】：國家自然科學(xué)基金項(xiàng)目(31360596) 國家國際科技合作專項(xiàng)(2014DFR31310)
【分類號(hào)】：S852.4
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本文編號(hào)：2245805