【摘要】： 衣原體(Chlamydia)已越來(lái)越成為人類(lèi)疾病的重要病原體,曾引起全世界范圍內(nèi)沙眼的廣泛流行,造成嚴(yán)重的危害,迄今仍是一些發(fā)展中國(guó)家致盲的首要原因。噬菌體(bacteriophage;phage)是一群能特異地感染宿主細(xì)菌,可將其裂解的病毒,它不僅在分子生物學(xué)研究中起重要作用,作為抗菌劑治療細(xì)菌感染正日益受到重視。衣原體噬菌體是微病毒,目前只是在肺炎衣原體、流產(chǎn)衣原體、魚(yú)類(lèi)衣原體等數(shù)種衣原體中發(fā)現(xiàn)有噬菌體的存在,研究顯示組成衣原體噬菌體殼的衣殼蛋白主要成分是Vp1、Vp2和Vp3,其中Vp1是最主要的結(jié)構(gòu)蛋白,在噬菌體對(duì)衣原體的黏附和植入中可能有重要作用。同時(shí)該蛋白高度保守而特異,因而是在其它衣原體種—尤其是沙眼衣原體尋找衣原體噬菌體的良好標(biāo)志物。本課題旨在研究分離和純化后的Vp1蛋白的免疫原性,探索制備Vp1蛋白的單克隆抗體,為進(jìn)一步研究其單克隆抗體的臨床意義及Vp1蛋白與衣原體作用的相關(guān)機(jī)制奠定基礎(chǔ),并通過(guò)純化的Vp1蛋白和由此得到的Vp1單抗進(jìn)行臨床篩查,試圖發(fā)現(xiàn)沙眼衣原體噬菌體的存在。 目的:表達(dá)、提純衣原體噬菌體Vp1蛋白,免疫小鼠,制備雜交瘤細(xì)胞。 方法:培養(yǎng)帶有vp1-pET-30a(+)重組質(zhì)粒的大腸桿菌BL21,通過(guò)IPTG進(jìn)行誘導(dǎo)表達(dá),進(jìn)行超聲波碎菌后,用6M脲溶解。將離心、重懸后的蛋白經(jīng)0.45μm孔徑濾器過(guò)濾后溶液清亮,然后經(jīng)過(guò)含有鎳離子的透析柱透析,用bindingbuffer、washingbuffer依次清洗透析柱,洗脫雜蛋白,最后用stritebuffer將結(jié)合在鎳離子上的Vp1蛋白洗脫下來(lái),復(fù)性后經(jīng)SDS-PAGE電泳鑒定。 將復(fù)性后的Vp1蛋白進(jìn)行蛋白定量,將約70μg蛋白注射于4周齡的BALB/C小鼠的腹腔內(nèi)進(jìn)行免疫,一月后取其內(nèi)眥靜脈血,將其作為一抗,以羊抗小鼠IgG為二抗,進(jìn)行Western-blot。取追加免疫三天后的小鼠的脾細(xì)胞,與骨髓瘤細(xì)胞以10:1的數(shù)量混合后,進(jìn)行細(xì)胞融合,將融合后的細(xì)胞加到含有小鼠腹腔巨噬細(xì)胞的96孔板內(nèi),在37℃含二氧化碳的溫箱中孵育。取經(jīng)過(guò)HAT、HT篩選后雜交瘤細(xì)胞的上清液進(jìn)行ELISA,檢測(cè)是否含有特異性抗體。 結(jié)果:帶有vp1-pET-30a(+)重組質(zhì)粒菌BL21經(jīng)IPTG誘導(dǎo)表達(dá)后,進(jìn)行SDS-PAGE電泳和Western Blot,均顯示從細(xì)菌裂解物離心沉淀中能獲得約70kD的衣原體GPIC噬菌體衣殼蛋白Vp1,提純后在SDS-PAGE上的可顯示單一的條帶,說(shuō)明提純后的Vp1蛋白純度較高。Western-blot在70KD大小處有清晰的目的條帶,這說(shuō)明免疫后的小鼠血清中含有抗Vp1蛋白的抗體。取加強(qiáng)免疫后的小鼠的脾臟,與骨髓瘤細(xì)胞進(jìn)行融合,形成了雜交瘤細(xì)胞,并且能夠分泌一定的抗體。 結(jié)論:1、含有vp1-pET30a(+)重組質(zhì)粒的大腸桿菌經(jīng)誘導(dǎo)后能夠表達(dá)衣原體噬菌體Vp1衣殼蛋白。2、Vp1蛋白分離純化成功。3、純化后的Vp1蛋白具有免疫原性,用Vp1蛋白免疫小鼠后小鼠血清中能夠產(chǎn)生針對(duì)Vp1蛋白的抗體。4、免疫小鼠的脾細(xì)胞與骨髓瘤細(xì)胞能夠進(jìn)行細(xì)胞融合,融合后的雜交瘤細(xì)胞既能夠進(jìn)行體外培養(yǎng),又能夠分泌Vp1的特異性抗體。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

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本文編號(hào)：2194131