本文選題：腸致病性大腸桿菌　切入點(diǎn)：緊密素　出處：《川北醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:制備EPEC粘附素IntiminC300抗血清,通過體外細(xì)胞實(shí)驗觀察該抗血清能否影響EPEC對Hep-2細(xì)胞的粘附,以確定IntiminC300是否可作為EPEC感染的保護(hù)性抗原。方法:用酶切和PCR方法鑒定IntiminC300表達(dá)質(zhì)粒pET32a-intiminC300,把質(zhì)粒轉(zhuǎn)化表達(dá)宿主菌BL21,構(gòu)建IntiminC300原核表達(dá)系統(tǒng);用IPTG誘導(dǎo)His標(biāo)簽-IntiminC300融合蛋白的表達(dá),SDS-PAGE分析其表達(dá)形式;用NI柱親和層析方法純化融合蛋白,用該純化蛋白作為抗原輔以佐劑免疫家兔,4次免疫后,心臟采血收集抗血清;對流免疫電泳檢測抗血清的效價,并用該抗血清作為一抗,用Westen Blotting方法檢測了EPEC粘附素IntiminC300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Hep-2細(xì)胞的粘附情況。結(jié)果:1.成功構(gòu)建了BL21/pET32a-intiminC300原核表達(dá)系統(tǒng),并用SDS-PAGE檢測到His-IntiminC300融合蛋白既有可溶性表達(dá)形式又有包涵體表達(dá)形式,但以包涵體表達(dá)形式為主;2.通過His標(biāo)簽蛋白純化試劑盒(包涵體蛋白),得到純化蛋白。3.成功制得高效價抗血清(對流免疫電泳檢測效價達(dá)1:32)。4.體外中和實(shí)驗觀察到該抗血清在稀釋倍數(shù)16以內(nèi)皆能有效抑制EPEC對Hep-2細(xì)胞的粘附。結(jié)論:EPEC粘附素IntiminC300不僅具有良好的免疫原性,且其抗體能有效阻止細(xì)菌對上皮細(xì)胞的粘附,可以預(yù)防EPEC的感染,因此IntiminC300是EPEC重要保護(hù)性抗原,可作為EPEC疫苗研究的理想備選抗原。
[Abstract]:Aim: to prepare IntiminC300 antiserum of EPEC adhesion hormone, and to observe whether the antiserum can affect the adhesion of EPEC to Hep-2 cells by cell experiment in vitro. Methods: the IntiminC300 expression plasmid pET32a-intiminC300 was identified by enzyme digestion and PCR. The plasmid was transformed into the host strain BL21 to construct the IntiminC300 prokaryotic expression system. IPTG induced expression of His tagged -Intimin C300 fusion protein was analyzed by SDS-PAGE, and the fusion protein was purified by NI column affinity chromatography. The purified protein was used as antigen and adjuvant to immunize rabbits for 4 times. The titer of antiserum was detected by convection immunoelectrophoresis, and the expression of EPEC adhesin IntiminC300 was detected by Westen Blotting method. The adhesion of bacteria to Hep-2 cells after neutralization was observed under microscope with different dilution antiserum neutralization. Results: 1. The prokaryotic expression system of BL21/pET32a-intiminC300 was successfully constructed. The His-IntiminC300 fusion protein was detected by SDS-PAGE in both soluble expression and inclusion body expression. The purified protein was obtained by His tag protein purification kit. 3. The high titer antiserum was prepared successfully (the titer of Convective immunoelectrophoresis was 1: 32. 4). The neutralization experiment in vitro showed that. The antiserum can effectively inhibit the adhesion of EPEC to Hep-2 cells within the dilution multiple of 16. Conclusion the antiserum IntiminC300 has good immunogenicity. Its antibody can effectively prevent bacterial adhesion to epithelial cells and prevent the infection of EPEC. Therefore, IntiminC300 is an important protective antigen for EPEC and can be used as an ideal alternative antigen for the study of EPEC vaccine.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 楊健;王朝莉;馮麗;;Intimin及其受體Tir在致病性大腸桿菌致細(xì)胞線粒體功能障礙中的作用[J];中國生物制品學(xué)雜志;2010年12期

相關(guān)碩士學(xué)位論文 前3條

1 郭永明;EPEC粘附相關(guān)分子抗血清制備及其中和能力探討[D];川北醫(yī)學(xué)院;2015年

2 李英;致病性大腸桿菌IntiminC280、BfpA和EspA原核表達(dá)載體的構(gòu)建及蛋白的表達(dá)與純化[D];川北醫(yī)學(xué)院;2012年

3 李程;腸致病性大腸桿菌eae基因的克隆及其原核表達(dá)[D];長春理工大學(xué);2010年

，

本文編號：1627665