本文選題：無乳鏈球菌 + 菌毛島嶼��； 參考：《中國病原生物學(xué)雜志》2017年07期

【摘要】：目的構(gòu)建無乳鏈球菌菌毛島嶼輔助蛋白AP1、菌毛骨架蛋白BP主要抗原域串聯(lián)表達(dá)重組載體,對(duì)表達(dá)產(chǎn)物進(jìn)行抗原性分析及鑒定。方法利用DNAstar Protean功能篩選AP1和BP主要抗原結(jié)構(gòu)域,引入15位柔性多肽,通過重疊延伸PCR技術(shù)串聯(lián)融合AP1、BP主要抗原決定簇基因區(qū),PCR擴(kuò)增串聯(lián)體基因片段,克隆至pET-30a(+)載體后轉(zhuǎn)化BL21(DE3)細(xì)胞,表達(dá)的目的蛋白經(jīng)親和層析純化后進(jìn)行Western blot鑒定。結(jié)果 PCR擴(kuò)增出321bp的AP1和660bp的BP主要抗原域,經(jīng)重疊延伸PCR獲得966bp的AP1、BP串聯(lián)體基因片段,DNA測(cè)序表明無堿基缺失、突變和移碼。構(gòu)建的重組表達(dá)載體經(jīng)IPTG誘導(dǎo)能可溶性表達(dá)分子質(zhì)量約45ku的目的蛋白,純化后重組蛋白的純度≥95%,Western blot分析表明,重組融合蛋白能被兔源抗無乳鏈球菌陽性血清識(shí)別。結(jié)論重疊延伸PCR獲得AP1、BP主要抗原域串聯(lián)體,構(gòu)建的pET-30a(+)/AP1+BP重組表達(dá)載體能以包涵體形式表達(dá)目的蛋白,表達(dá)產(chǎn)物具有良好的反應(yīng)原性,為研究基于AP1、BP蛋白的致病機(jī)制及其免疫活性奠定了實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Objective to construct the recombinant vector expressing the main antigen domain of Streptococcus lactis tuber island helper protein AP1 and its skeleton protein BP in tandem, and to analyze and identify the antigenicity of the expressed product. Methods the main antigenic domains of AP1 and BP were screened by DNAstar Protean function, and 15 position flexible polypeptides were introduced. The tandem gene fragments were amplified by tandem fusion of AP1GBP major antigen-determinant gene region by overlapping extension PCR technique. The target protein was cloned into pET-30a () vector and transformed into BL21DE-3 cells. The expressed protein was purified by affinity chromatography and identified by Western blot. Results the main antigenic domains of 321bp AP1 and 660bp BP were amplified by PCR. The sequence of 966bp AP1 BP tandem gene fragment obtained by overlapping extension PCR showed that there was no base deletion, mutation and frameshift. The recombinant expression vector was induced by IPTG to express the target protein with molecular weight of about 45ku. The purity of purified recombinant protein was 鈮，

本文編號(hào)：1802164