發(fā)布時(shí)間：2018-06-03 15:58

  本文選題：伽氏疏螺旋體尚志株 + P　； 參考：《中國(guó)畜牧獸醫(yī)》2017年01期

【摘要】：為獲得伽氏疏螺旋體尚志株(B.garinii SZ)的P66蛋白,本研究從培養(yǎng)的螺旋體菌液中提取總RNA,反轉(zhuǎn)錄合成第一鏈cDNA,利用PCR擴(kuò)增P66基因。將目的片段連接表達(dá)載體pET-30a(+),并轉(zhuǎn)化大腸桿菌表達(dá)菌BL21(DE3),經(jīng)PCR、雙酶切及測(cè)序驗(yàn)證正確后,進(jìn)行IPTG誘導(dǎo)表達(dá)和純化,然后將純化的融合蛋白免疫新西蘭大白兔,制備多克隆抗體。SDS-PAGE結(jié)果顯示,獲得約70ku的表達(dá)產(chǎn)物;Western blotting分析表明,表達(dá)產(chǎn)物與抗His標(biāo)簽的小鼠單克隆抗體、螺旋體鼠源陽(yáng)性血清、兔抗P66多克隆抗體均能發(fā)生反應(yīng),獲得的多克隆抗體也可識(shí)別天然蛋白。本試驗(yàn)成功表達(dá)了B.garinii SZ株P(guān)66蛋白,并制備了多克隆抗體,為后續(xù)B.garinii SZ株P(guān)66蛋白的功能研究奠定了基礎(chǔ)。
[Abstract]:In order to obtain the P66 protein of B. garinii SZ, the total RNAs were extracted from the cultured spirochetes, and the first strand cDNAwas synthesized by reverse transcription. The P66 gene was amplified by PCR. The target fragment was ligated into the expression vector pET-30a (pET-30a) and transformed into E. coli expression strain BL21DDE3. The recombinant protein was induced and purified by IPTG after PCR, double enzyme digestion and sequencing. Then the purified fusion protein was immunized with New Zealand white rabbit. The results of preparation of polyclonal antibody. SDS-PAGE showed that the expressed product of about 70ku could react with mouse monoclonal antibody, spirulina positive serum and rabbit anti-P66 polyclonal antibody. The obtained polyclonal antibodies can also recognize natural proteins. In this experiment, the P66 protein of B.garinii SZ strain was successfully expressed, and the polyclonal antibody was prepared, which laid a foundation for the functional study of P66 protein of B.garinii SZ strain.
【作者單位】： 中國(guó)農(nóng)業(yè)科學(xué)院蘭州獸醫(yī)研究所家畜疫病病原生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室甘肅省動(dòng)物寄生蟲(chóng)病重點(diǎn)實(shí)驗(yàn)室;江蘇省動(dòng)物重要疫病與人獸共患病防控協(xié)同創(chuàng)新中心;
【基金】：農(nóng)業(yè)科技創(chuàng)新工程(ASTIP) 國(guó)家肉牛牦牛產(chǎn)業(yè)技術(shù)體系(NBCIS CARS-38)
【分類(lèi)號(hào)】：S852.6

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1 于培發(fā);伽氏疏螺旋體P66基因的特征與抗原性研究[D];中國(guó)農(nóng)業(yè)科學(xué)院;2016年

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本文編號(hào)：1973356