幽门螺杆菌重组HpaA蛋白的表达及其包涵体纯化方法的探讨
发布时间：2018-07-14 08:59
【摘要】： 研究目的:用基因工程技术克隆和表达幽门螺杆菌(Helicobacter pylori,H.pylori)的hpaA基因,以期获得重组蛋白HpaA,并鉴定其抗原性和免疫原性,为进一步制备预防H.pylori感染的HpaA制剂提供了材料。采用镍柱层析和切胶纯化两种方法提取可溶性rHpaA,并比较其效果,为hpaA表达中的包涵体纯化提供一条经济便捷的方法。 方法:用PCR方法从H.pylori DNA中扩增hpaA基因片段。插入原核表达载体pQE30,转化受体菌DH5α,经克隆及序列分析正确后在大肠杆菌中进行表达。SDS-PAGE分析表达蛋白的分子量和表达形式,经镍柱层析纯化后Western blot证实其抗原性;免疫小鼠后ELISA检测血清特异性抗体鉴定其免疫原性。将SDS-PAGE电泳后的表达蛋白用4M的乙酸钠显色指示切胶,所切胶块在透析袋中经电场作用,提取可溶性目的蛋白,Western blot鉴定其抗原性;同时以间接ELISA法比较分别以切胶纯化法和镍柱层析法所得表达蛋白与抗体结合的滴度。 结果:重组表达质粒pQE30-hpaA构建成功,插入的基因片段全长782bp,与基因文库中的hpaA基因同源性达97.0%。SDS-PAGE显示表达产物相对分子量为30kD,表达量占全菌总量的32.3%,主要以包涵体形式表达,具有良好的抗原性和免疫原性,镍柱层析纯化后纯度达97%。重组蛋白包涵体经切胶纯化后可得到纯度93.9%的可溶性重组HpaA,Western blot鉴定切胶纯化法提取的可溶性rHpaA也具有良好的抗原性,经ELISA法比较,其抗体结合滴度与镍柱层析法提取的rHpaA相近。 结论:H.pylori hpaA基因片段克隆表达成功,获得高纯度可溶性的重组HpaA蛋白,具有良好的抗原性和免疫原性,为进一步制备预防H.pylori感染的HpaA制剂提供了材料。并证实了切胶纯化包涵体蛋白的方法切实可行,与传统方法比较,简单经济实用,为大量制备可溶性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 伪. 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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378.2
本文编号：2121145
[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 伪. 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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378.2
【引证文献】
相关硕士学位论文 前1条
1 李艳青;幽门螺杆菌烷基过氧化氢还原酶基因克隆、表达及多克隆抗体制备[D];郑州大学;2009年
，本文编号：2121145
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