本文選題：糞腸球菌 + Ace　； 參考：《微生物學(xué)雜志》2008年03期

【摘要】：構(gòu)建含糞腸球菌表面蛋白Ace保守序列A的重組載體,在大腸埃希菌M15中進(jìn)行誘導(dǎo)表達(dá)、純化表達(dá)產(chǎn)物,研究其黏附活性。以糞腸球菌標(biāo)準(zhǔn)株JH2-2為模板進(jìn)行PCR擴(kuò)增ace基因保守序列A,構(gòu)建重組質(zhì)粒pQE30/Ace,轉(zhuǎn)化至表達(dá)宿主菌M15中進(jìn)行誘導(dǎo)表達(dá),利用SDS-PAGE和Western blot進(jìn)行分析和鑒定表達(dá)結(jié)果,Ni-NTA親和層析柱純化重組蛋白,同時(shí)用純化的Ace重組蛋白免疫新西蘭兔,用所得相應(yīng)的抗血清分別進(jìn)行黏附和黏附抑制實(shí)驗(yàn)。結(jié)果可見,構(gòu)建的重組質(zhì)粒經(jīng)酶切鑒定和測(cè)序鑒定證明其中插入片段為ace基因保守序列A,測(cè)序結(jié)果與Genbank上登錄序列完全一致;SDS-PAGE分析顯示,重組工程菌表達(dá)了一相對(duì)分子質(zhì)量(Mr)約為37 ku的目的蛋白條帶,Western blot檢測(cè)其能與6×His單克隆抗體發(fā)生特異性反應(yīng)。糞腸球菌JH2-2能夠黏附于膠原蛋白Ⅰ表面;抗Ace多克隆抗體可抑制46℃培養(yǎng)的JH2-2對(duì)膠原蛋白Ⅰ的黏附,這種抑制作用與其稀釋度呈負(fù)相關(guān)。構(gòu)建的原核表達(dá)載體PQE30/Ace在E.coli M15中成功地表達(dá),純化的重組Ace蛋白具有膠原黏附活性。
[Abstract]:A recombinant vector containing conserved sequence A of Enterococcus faecalis surface protein (Ace) was constructed and expressed in Escherichia coli M15. The expression product was purified and its adhesion activity was studied. The conserved sequence of ace gene was amplified by PCR using the standard strain of Enterococcus faecalis JH2-2 as template. The recombinant plasmid pQE30 / Acewas constructed and transformed into the expression host strain M15 for induction expression. The recombinant protein was purified by Ni-NTA affinity chromatography column with SDS-PAGE and Western blot. New Zealand rabbits were immunized with the purified Ace recombinant protein. The adhesion and adhesion inhibition tests were carried out with the corresponding antiserum. The results showed that the constructed recombinant plasmid was identified by restriction endonuclease digestion and sequencing, and the inserted fragment was conserved by ace gene. The results of sequencing were consistent with those of Genbank and the results of SDS-PAGE analysis showed that the recombinant plasmid was a conserved sequence of ace gene. The recombinant engineering strain expressed a target protein band of about 37 ku, which could react with 6 脳 His monoclonal antibody by Western blot. Enterococcus faecalis JH2-2 could adhere to the surface of collagen 鈪，

本文編號(hào)：1791816