【摘要】： 研究目的:用基因工程技術(shù)克隆和表達(dá)幽門螺桿菌(Helicobacter pylori,H.pylori)的hpaA基因,以期獲得重組蛋白HpaA,并鑒定其抗原性和免疫原性,為進(jìn)一步制備預(yù)防H.pylori感染的HpaA制劑提供了材料。采用鎳柱層析和切膠純化兩種方法提取可溶性rHpaA,并比較其效果,為hpaA表達(dá)中的包涵體純化提供一條經(jīng)濟(jì)便捷的方法。 方法:用PCR方法從H.pylori DNA中擴(kuò)增hpaA基因片段。插入原核表達(dá)載體pQE30,轉(zhuǎn)化受體菌DH5α,經(jīng)克隆及序列分析正確后在大腸桿菌中進(jìn)行表達(dá)。SDS-PAGE分析表達(dá)蛋白的分子量和表達(dá)形式,經(jīng)鎳柱層析純化后Western blot證實(shí)其抗原性;免疫小鼠后ELISA檢測(cè)血清特異性抗體鑒定其免疫原性。將SDS-PAGE電泳后的表達(dá)蛋白用4M的乙酸鈉顯色指示切膠,所切膠塊在透析袋中經(jīng)電場(chǎng)作用,提取可溶性目的蛋白,Western blot鑒定其抗原性;同時(shí)以間接ELISA法比較分別以切膠純化法和鎳柱層析法所得表達(dá)蛋白與抗體結(jié)合的滴度。 結(jié)果:重組表達(dá)質(zhì)粒pQE30-hpaA構(gòu)建成功,插入的基因片段全長(zhǎng)782bp,與基因文庫(kù)中的hpaA基因同源性達(dá)97.0%。SDS-PAGE顯示表達(dá)產(chǎn)物相對(duì)分子量為30kD,表達(dá)量占全菌總量的32.3%,主要以包涵體形式表達(dá),具有良好的抗原性和免疫原性,鎳柱層析純化后純度達(dá)97%。重組蛋白包涵體經(jīng)切膠純化后可得到純度93.9%的可溶性重組HpaA,Western blot鑒定切膠純化法提取的可溶性rHpaA也具有良好的抗原性,經(jīng)ELISA法比較,其抗體結(jié)合滴度與鎳柱層析法提取的rHpaA相近。 結(jié)論:H.pylori hpaA基因片段克隆表達(dá)成功,獲得高純度可溶性的重組HpaA蛋白,具有良好的抗原性和免疫原性,為進(jìn)一步制備預(yù)防H.pylori感染的HpaA制劑提供了材料。并證實(shí)了切膠純化包涵體蛋白的方法切實(shí)可行,與傳統(tǒng)方法比較,簡(jiǎn)單經(jīng)濟(jì)實(shí)用,為大量制備可溶性HpaA提供了新的方法。
[Abstract]:Objective: to clone and express the hpaA gene of Helicobacter pylori (H.pylori) by genetic engineering technique, to obtain the recombinant protein HpaA, and to identify its antigenicity and immunogenicity. Soluble rHPAA was extracted by nickel column chromatography and gel cut purification, and its effect was compared, which provided an economical and convenient method for the purification of inclusion body in hpaA expression. Methods: hpaA gene fragments were amplified from H. pylori DNA by PCR. PQE30 was inserted into the prokaryotic expression vector pQE30 and transformed into the receptor strain DH5 偽. After cloning and sequence analysis, the protein was expressed in E. coli. The molecular weight and expression form of the expressed protein were analyzed by SDS-PAGE. The antigenicity of the expressed protein was confirmed by Western blot after purification by nickel column chromatography. The immunogenicity of mice was determined by Elisa after immunization. SDS-PAGE was used to show the antigenicity of the expressed protein by 4M sodium acetate. The gel was extracted from the dialyzed bag by electric field, and the antigenicity of the protein was identified by Western blot. At the same time, the titers of the expressed protein combined with antibody were compared by indirect Elisa and nickel column chromatography. Results: the recombinant expression plasmid pQE30-hpaA was successfully constructed, and the inserted gene fragment was 782bp. the homology with the hpaA gene in the gene library was 97.0.SDS-PAGE, which showed that the relative molecular weight of the expressed product was 30kD. the expression amount accounted for 32.3kD. it was mainly expressed in the form of inclusion body. It has good antigenicity and immunogenicity, and its purity is 97% after purification by nickel column chromatography. The soluble rHpaA extracted from recombinant protein inclusion body was purified by gelling gel. The soluble rHpaA extracted by gumming and purification method also had good antigenicity. The titer of antibody binding of recombinant protein inclusion body was similar to that of rHpaA extracted by nickel column chromatography. Conclusion the hpaA gene fragment of H. pylori was cloned and expressed successfully, and a high purity and soluble recombinant HPA protein was obtained. It has good antigenicity and immunogenicity, which provides a material for the further preparation of H.pylori infection prevention preparation. Compared with the traditional method, the method is simple, economical and practical. It provides a new method for the preparation of soluble HpaA.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R378.2

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 李艷青;幽門螺桿菌烷基過(guò)氧化氫還原酶基因克隆、表達(dá)及多克隆抗體制備[D];鄭州大學(xué);2009年

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本文編號(hào)：2121145