本文選題：綿羊肺炎支原體 + 單增李斯特菌��； 參考：《西南農(nóng)業(yè)學(xué)報(bào)》2017年09期

【摘要】：【目的】本文研發(fā)了MO重組活疫苗�！痉椒ā坷肞CR技術(shù)從LM-SB5株基因組中擴(kuò)增出rli60基因上下游同源片段a、b,從pMD19-T-meAg15中擴(kuò)增出MO meAg15目的片段,采用重疊延伸PCR(SOE-PCR)技術(shù)將擴(kuò)增得到的3個(gè)基因片段融合成ameAg15b,將其克隆測(cè)序鑒定。將ameAg15b目的基因亞克隆到pKSV7穿梭載體,構(gòu)建pKSV7-ameAg15b重組穿梭質(zhì)粒,將該重組穿梭質(zhì)粒電轉(zhuǎn)化至LM-SB5感受態(tài)細(xì)胞,在氯霉素、高溫雙重壓力篩選重組菌LM-Δrli60-ameAg15b�！窘Y(jié)果】重組菌PCR鑒定表明,外源基因meAg15定點(diǎn)整合于LM基因組中。遺傳穩(wěn)定性試驗(yàn)表明,meAg15基因可以在LM基因組中穩(wěn)定存在。SDS-PAGE結(jié)果表明,重組菌LM-Δrli60-ameAg15b可表達(dá)相對(duì)分子量為14.5 kDa的重組蛋白;Western blot分析說明,表達(dá)的重組蛋白能與MO陽(yáng)性血清發(fā)生特異性免疫反應(yīng)�！窘Y(jié)論】小鼠免疫試驗(yàn)表明,重組菌LM-Δrli60-ameAg15b菌體可誘導(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。
[Abstract]:[methods] the upstream and downstream homologous fragment of rli60 gene was amplified from the genome of LM-SB5 strain, and the meAg15 fragment of MO was amplified from pMD19-T-meAg15. The three amplified gene fragments were fused to ameAg15b by overlapping extension PCR- SOE-PCR technique, and their cloning and sequencing were identified. The ameAg15b target gene was subcloned into pKSV7 shuttle vector, and the recombinant shuttle plasmid pKSV7-ameAg15b was constructed. The recombinant shuttle plasmid was electrotransformed into LM-SB5 competent cells. The recombinant strain LM- 螖 rliame60-Ag15b. was screened under chloramphenicol and high temperature double pressure. The foreign gene meAg15 was integrated into LM genome. The results of genetic stability test showed that the stably exists of the gene in LM genome. SDS-PAGE showed that the recombinant strain LM- 螖 rli60-ameAg15b could express the recombinant protein with relative molecular weight of 14.5 kDa. [conclusion] mice immunological test showed that the recombinant strain LM- 螖 rli60-ameAg15b could induce the production of anti-MO specific antibody, which proved that the recombinant strain had certain immunogenicity.
【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(guó)農(nóng)業(yè)科學(xué)院蘭州獸醫(yī)研究所;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(31360596) 國(guó)家國(guó)際科技合作專項(xiàng)(2014DFR31310) 兵團(tuán)中青年科技創(chuàng)新領(lǐng)軍人才計(jì)劃(2016BC001)
【分類號(hào)】：S852.4

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