本文選題：腸致病性大腸埃希菌 + 緊密素��； 參考：《中國(guó)病原生物學(xué)雜志》2017年10期

【摘要】：目的制備EPEC粘附素IntiminC280抗血清,檢測(cè)其中和EPEC對(duì)Hep-2細(xì)胞粘附作用。方法用基因克隆技術(shù)構(gòu)建IntiminC280表達(dá)載體pET32(α)-intiminC280,轉(zhuǎn)化表達(dá)宿主菌BL21,用IPTG誘導(dǎo)His-IntiminC280融合蛋白的表達(dá),SDS-PAGE分析其表達(dá)形式;用NI柱親和層析法純化融合蛋白,并輔以佐劑免疫家兔,4次免疫后采血,分離血清,對(duì)流免疫電泳法檢測(cè)抗血清效價(jià);以該抗血清作為一抗,采用Western blot檢測(cè)EPEC粘附素Intimin的表達(dá);用不同稀釋度的抗血清中和EPEC,顯微鏡下觀察細(xì)菌被中和后對(duì)Hep-2細(xì)胞的粘附情況。結(jié)果成功構(gòu)建BL21/pET32(α)-intiminC280原核表達(dá)系統(tǒng),表達(dá)蛋白的分子質(zhì)量單位為50ku,與預(yù)期相符。用純化的His-IntiminC280免疫家兔獲得高效價(jià)抗血清,16倍稀釋后仍能有效抑制EPEC對(duì)Hep-2細(xì)胞的粘附。結(jié)論 IntiminC280抗血清具有抑制是EPEC粘附Hep-2細(xì)胞作用,可作為EPEC疫苗研發(fā)的備選抗原。
[Abstract]:Objective to prepare the antiserum of EPEC adhesion hormone IntiminC280 and to detect the adhesion of EPEC to Hep-2 cells. Methods IntiminC280 expression vector pET32 (偽 -intiminC280) was constructed by gene cloning technique and transformed into host strain BL21. The expression of His-IntiminC280 fusion protein was analyzed by SDS-PAGE induced by IPTG, and the fusion protein was purified by NI affinity chromatography. After the rabbits were immunized with adjuvant for 4 times, the serum was isolated, the titer of antiserum was detected by convection immunoelectrophoresis, and the expression of imtimin was detected by Western blot. The adherence of bacteria to Hep-2 cells was observed under microscope with different dilution of antiserum neutralization and EPECs. Results the prokaryotic expression system of BL21 / pET32 (偽 -intimin C280) was successfully constructed. The molecular weight unit of the expressed protein was 50ku. 16 times dilution of purified His-Intimin C280 immunized rabbits with high titer antiserum could effectively inhibit the adhesion of EPEC to Hep-2 cells. Conclusion the inhibition of Intimin C280 antiserum is the adhesion of EPEC to Hep-2 cells and can be used as an alternative antigen for the development of EPEC vaccine.
【作者單位】： 川北醫(yī)學(xué)院附屬醫(yī)院兒科;川北醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)院病原生物學(xué)與免疫學(xué)實(shí)驗(yàn)教學(xué)中心;
【基金】：四川省教育廳項(xiàng)目(No.15ZA0205)
【分類號(hào)】：R392

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