衣原体噬菌体Vp1蛋白的表达、提纯及杂交瘤细胞的培养
发布时间：2018-08-20 15:31
【摘要】： 衣原体(Chlamydia)已越来越成为人类疾病的重要病原体,曾引起全世界范围内沙眼的广泛流行,造成严重的危害,迄今仍是一些发展中国家致盲的首要原因。噬菌体(bacteriophage;phage)是一群能特异地感染宿主细菌,可将其裂解的病毒,它不仅在分子生物学研究中起重要作用,作为抗菌剂治疗细菌感染正日益受到重视。衣原体噬菌体是微病毒,目前只是在肺炎衣原体、流产衣原体、鱼类衣原体等数种衣原体中发现有噬菌体的存在,研究显示组成衣原体噬菌体壳的衣壳蛋白主要成分是Vp1、Vp2和Vp3,其中Vp1是最主要的结构蛋白,在噬菌体对衣原体的黏附和植入中可能有重要作用。同时该蛋白高度保守而特异,因而是在其它衣原体种—尤其是沙眼衣原体寻找衣原体噬菌体的良好标志物。本课题旨在研究分离和纯化后的Vp1蛋白的免疫原性,探索制备Vp1蛋白的单克隆抗体,为进一步研究其单克隆抗体的临床意义及Vp1蛋白与衣原体作用的相关机制奠定基础,并通过纯化的Vp1蛋白和由此得到的Vp1单抗进行临床筛查,试图发现沙眼衣原体噬菌体的存在。 目的:表达、提纯衣原体噬菌体Vp1蛋白,免疫小鼠,制备杂交瘤细胞。 方法:培养带有vp1-pET-30a(+)重组质粒的大肠杆菌BL21,通过IPTG进行诱导表达,进行超声波碎菌后,用6M脲溶解。将离心、重悬后的蛋白经0.45μm孔径滤器过滤后溶液清亮,然后经过含有镍离子的透析柱透析,用bindingbuffer、washingbuffer依次清洗透析柱,洗脱杂蛋白,最后用stritebuffer将结合在镍离子上的Vp1蛋白洗脱下来,复性后经SDS-PAGE电泳鉴定。 将复性后的Vp1蛋白进行蛋白定量,将约70μg蛋白注射于4周龄的BALB/C小鼠的腹腔内进行免疫,一月后取其内眦静脉血,将其作为一抗,以羊抗小鼠IgG为二抗,进行Western-blot。取追加免疫三天后的小鼠的脾细胞,与骨髓瘤细胞以10:1的数量混合后,进行细胞融合,将融合后的细胞加到含有小鼠腹腔巨噬细胞的96孔板内,在37℃含二氧化碳的温箱中孵育。取经过HAT、HT筛选后杂交瘤细胞的上清液进行ELISA,检测是否含有特异性抗体。 结果:带有vp1-pET-30a(+)重组质粒菌BL21经IPTG诱导表达后,进行SDS-PAGE电泳和Western Blot,均显示从细菌裂解物离心沉淀中能获得约70kD的衣原体GPIC噬菌体衣壳蛋白Vp1,提纯后在SDS-PAGE上的可显示单一的条带,说明提纯后的Vp1蛋白纯度较高。Western-blot在70KD大小处有清晰的目的条带,这说明免疫后的小鼠血清中含有抗Vp1蛋白的抗体。取加强免疫后的小鼠的脾脏,与骨髓瘤细胞进行融合,形成了杂交瘤细胞,并且能够分泌一定的抗体。 结论:1、含有vp1-pET30a(+)重组质粒的大肠杆菌经诱导后能够表达衣原体噬菌体Vp1衣壳蛋白。2、Vp1蛋白分离纯化成功。3、纯化后的Vp1蛋白具有免疫原性,用Vp1蛋白免疫小鼠后小鼠血清中能够产生针对Vp1蛋白的抗体。4、免疫小鼠的脾细胞与骨髓瘤细胞能够进行细胞融合,融合后的杂交瘤细胞既能够进行体外培养,又能够分泌Vp1的特异性抗体。
[Abstract]:Chlamydia has become an increasingly important pathogen of human diseases, causing widespread epidemic of trachoma worldwide, causing serious harm, and is still the primary cause of blindness in some developing countries. Chlamydia phages are microviruses. At present, only a few kinds of Chlamydia pneumoniae, Chlamydia abortion, Chlamydia fishes and other kinds of Chlamydia have been found in the presence of phages. Studies have shown that the main capsid proteins that make up the phage shell of Chlamydia pneumoniae are Chlamydia. The main components are Vp1, Vp2 and Vp3, among which Vp1 is the most important structural protein and may play an important role in the adhesion and implantation of phages to Chlamydia. At the same time, the protein is highly conserved and specific, so it is a good marker for other Chlamydia species, especially Chlamydia trachomatis, to find the phages of Chlamydia. The immunogenicity of purified Vp1 protein and the preparation of monoclonal antibodies against Vp1 protein were explored to lay a foundation for further study of the clinical significance of the monoclonal antibody and the mechanism of the interaction between Vp1 protein and Chlamydia. The purified Vp1 protein and the resulting Vp1 monoclonal antibody were screened for Chlamydia trachomatis phages. Exist.
Objective: to express and purify Chlamydia phage Vp1 protein, immunize mice and prepare hybridoma cells.
METHODS: E. coli BL21 with recombinant plasmid vp1-pET-30a (+) was cultured and induced to express by IPTG. After ultrasonic fragmentation, it was dissolved by 6M urea. The Vp1 protein bound to nickel ion was eluted by stritebuffer and renatured and identified by SDS-PAGE electrophoresis.
The refolded Vp1 protein was quantified and injected into the abdominal cavity of 4-week-old BALB/C mice for immunization. One month later, the blood from the inner canthus vein was taken as an antibody. The sheep anti-mouse IgG was used as a second antibody and Western-blot was performed. The spleen cells of the three-day-old mice were taken and mixed with the myeloma cells in the amount of 10:1. After fusion, the fused cells were added to 96-well plate containing mouse peritoneal macrophages and incubated in a temperature incubator containing carbon dioxide at 37 C. The supernatant of hybridoma cells screened by HAT and HT was taken for ELISA to detect the presence of specific antibodies.
Results: The recombinant plasmid BL21 with vp1-pET-30a(+) was induced by IPTG and expressed by SDS-PAGE electrophoresis and Western Blot. The results showed that about 70 kD of Chlamydia GPIC phage capsid protein Vp1 could be obtained from the centrifugal precipitation of bacterial lysates, and a single band could be displayed on SDS-PAGE after purification. Western blot showed a clear target band at 70 KD, indicating that the serum of immunized mice contained antibodies against Vp1 protein.
Conclusion: 1. E. coli containing vp1-pET30a (+) recombinant plasmid can express Chlamydia phage Vp1 capsid protein. 2. Vp1 protein was isolated and purified successfully. 3. The purified Vp1 protein has immunogenicity. The antibody against Vp1 protein can be produced in the serum of mice immunized with Vp1 protein. Tumor cells can fuse with each other. The fused hybridoma cells can not only be cultured in vitro, but also secrete specific antibodies against Vp1.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2194131
[Abstract]:Chlamydia has become an increasingly important pathogen of human diseases, causing widespread epidemic of trachoma worldwide, causing serious harm, and is still the primary cause of blindness in some developing countries. Chlamydia phages are microviruses. At present, only a few kinds of Chlamydia pneumoniae, Chlamydia abortion, Chlamydia fishes and other kinds of Chlamydia have been found in the presence of phages. Studies have shown that the main capsid proteins that make up the phage shell of Chlamydia pneumoniae are Chlamydia. The main components are Vp1, Vp2 and Vp3, among which Vp1 is the most important structural protein and may play an important role in the adhesion and implantation of phages to Chlamydia. At the same time, the protein is highly conserved and specific, so it is a good marker for other Chlamydia species, especially Chlamydia trachomatis, to find the phages of Chlamydia. The immunogenicity of purified Vp1 protein and the preparation of monoclonal antibodies against Vp1 protein were explored to lay a foundation for further study of the clinical significance of the monoclonal antibody and the mechanism of the interaction between Vp1 protein and Chlamydia. The purified Vp1 protein and the resulting Vp1 monoclonal antibody were screened for Chlamydia trachomatis phages. Exist.
Objective: to express and purify Chlamydia phage Vp1 protein, immunize mice and prepare hybridoma cells.
METHODS: E. coli BL21 with recombinant plasmid vp1-pET-30a (+) was cultured and induced to express by IPTG. After ultrasonic fragmentation, it was dissolved by 6M urea. The Vp1 protein bound to nickel ion was eluted by stritebuffer and renatured and identified by SDS-PAGE electrophoresis.
The refolded Vp1 protein was quantified and injected into the abdominal cavity of 4-week-old BALB/C mice for immunization. One month later, the blood from the inner canthus vein was taken as an antibody. The sheep anti-mouse IgG was used as a second antibody and Western-blot was performed. The spleen cells of the three-day-old mice were taken and mixed with the myeloma cells in the amount of 10:1. After fusion, the fused cells were added to 96-well plate containing mouse peritoneal macrophages and incubated in a temperature incubator containing carbon dioxide at 37 C. The supernatant of hybridoma cells screened by HAT and HT was taken for ELISA to detect the presence of specific antibodies.
Results: The recombinant plasmid BL21 with vp1-pET-30a(+) was induced by IPTG and expressed by SDS-PAGE electrophoresis and Western Blot. The results showed that about 70 kD of Chlamydia GPIC phage capsid protein Vp1 could be obtained from the centrifugal precipitation of bacterial lysates, and a single band could be displayed on SDS-PAGE after purification. Western blot showed a clear target band at 70 KD, indicating that the serum of immunized mice contained antibodies against Vp1 protein.
Conclusion: 1. E. coli containing vp1-pET30a (+) recombinant plasmid can express Chlamydia phage Vp1 capsid protein. 2. Vp1 protein was isolated and purified successfully. 3. The purified Vp1 protein has immunogenicity. The antibody against Vp1 protein can be produced in the serum of mice immunized with Vp1 protein. Tumor cells can fuse with each other. The fused hybridoma cells can not only be cultured in vitro, but also secrete specific antibodies against Vp1.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
【参考文献】
相关期刊论文 前3条
1 张爱华;余模松;;噬菌体抗体库技术[J];国际生物制品学杂志;2006年01期
2 向代军,姚学军,刘汉燕;超声处理对大肠杆菌HCV-NS_3融合蛋白纯化的影响[J];数理医药学杂志;2002年06期
3 龚向东,叶顺章,张君炎,张国成,邵长庚,梁国钧,姜文华,夏强,王全佩;1991～2001年我国性病流行病学分析[J];中华皮肤科杂志;2002年03期
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