隐孢子虫特异性人源抗体Fab片段的制备和研究

发布时间：2018-10-23 11:09

【摘要】： 隐孢子虫属于顶复门胞内寄生机会致病性原虫，可感染人和多种动物，引起以腹泻为主要症状的隐孢子虫病，是危害全球的公共卫生问题，至今仍无有效的控制和治疗手段。大量研究表明，高滴度牛初乳(HBC)、高滴度牛初乳免疫球蛋白(HBCIg)、一系列针对隐孢子虫表面致病相关蛋白的特异性多克隆抗体及鼠源单克隆抗体等均可在动物或人体内产生抗隐孢子虫作用，证明了被动免疫治疗隐孢子虫感染的有效性，故本研究拟进一步研制隐孢子虫特异性人源抗体。首先从奶牛粪便中分离、鉴定、纯化获得微小隐孢子虫卵囊，感染SCID小鼠保种；以微小隐孢子虫卵囊免疫志愿者，获得含有较高滴度抗微小隐孢子虫IgG抗体的外周血，从中分离淋巴细胞，利用工程抗体技术构建非依赖性克隆滴度为8×10~6的人抗隐孢子虫免疫球蛋白G基因文库；选择位于隐孢子虫胞外发育阶段虫体表面和顶端、直接参与虫体对宿主细胞的吸附和入侵过程的糖蛋白gp40／15作为抗体库筛选的抗原靶的，应用DNA重组技术将扩增得到的Cpgp40／15基因克隆入原核表达载体pET19b中，转化宿主菌、诱导表达重组蛋白，纯化得到可被含有抗微小隐孢子虫特异性抗体的血清识别、具有特异免疫原性的重组抗原gp40／15，每100ml培养菌液中可获得高纯度重组蛋白3.6mg；采用克隆印迹法和ELISA对抗体库进行多轮筛选，共筛选克隆数约为1.01×10~6个，经ELISA、IFA等方法反复验证，最终获得两个阳性克隆，分别命名为Agp40—1和Agp40—2；Agp40-1的轻链可变区近似系为A20，基因同源性为95％；重链可变区近似系为VH3-30，基因同源性为92％；Agp40-2的轻链可变区近似系为O12，基因同源性为97％；重链可变区近似系为VH3-30，基因同源性为92％；分别对两个阳性克隆进行大量表达，并以金属螯合亲和层析法进行纯化，得到重轻链结构完整、纯度较高的抗体Fab片段，得量为1mg／L培养液；经ELSIA、斑点印迹、免疫印迹、IFA等检测证实两个纯化Fab片段均可特异性识别天然及重组微小隐孢子虫表面抗原；Biacore检测分析表明，Agp40-1的解离常数为5.25×10~(-9)，，Agp40-2的解离常数为1.47×10~(-9)，均具有较高的亲和力；体外功能检测中，两个纯化Fab片段对微小隐孢子虫卵囊感染宿主细胞(Caco-2)具有一定的抑制作用，对微小隐孢子虫子孢子附着宿主细胞(Caco-2)具有一定的阻断作用。上述人源性抗隐孢子虫工程抗体IgG Fab片段的制备和研究工作，为进一步发展被动免疫策略治疗和预防隐孢子虫感染提供了理论和实践依据。
[Abstract]:Cryptosporidium belongs to opportunistic protozoa, which can infect human and many animals and cause Cryptosporidiosis with diarrhea as its main symptom. Cryptosporidium is a global public health problem, and there are no effective control and treatment methods. A great deal of research has shown that High titer bovine colostrum (HBC), high titer bovine colostrum immunoglobulin (HBCIg), a series of specific polyclonal antibodies against Cryptosporidium surface pathogenicity related proteins, and murine monoclonal antibodies can produce anti-Cryptosporidium effects in animals or human beings. It is proved that passive immunotherapy is effective in the treatment of Cryptosporidium infection, so this study intends to further develop the specific human antibody against Cryptosporidium. Firstly, Cryptosporidium microspore oocysts were isolated from cow feces, identified, purified and preserved in SCID mice. The peripheral blood containing higher titer of IgG antibody against Cryptosporidium microsporidium was obtained by immunizing volunteers with Cryptosporidium microsporidium oocysts. The G gene library of human Cryptosporidium immunoglobulin (Ig) was constructed by using engineering antibody technique, and was selected to be located on the surface and apical of Cryptosporidium during the extracellular development stage of Cryptosporidium, and the immunoglobulin G gene library of Cryptosporidium was constructed by using engineering antibody technique. Glycoprotein gp40/15, which is directly involved in the adsorption and invasion of host cells, was used as the antigen target of antibody library screening. The amplified Cpgp40/15 gene was cloned into the prokaryotic expression vector pET19b by DNA recombination technique and transformed into host strain. The recombinant protein was induced to express and purified to be recognized by sera containing specific antibodies against Cryptosporidium minimus. The recombinant antigen gp40/15, with specific immunogenicity could obtain high purity recombinant protein 3.6 mg per 100ml culture solution. The antibody library was screened by clone blot and ELISA. The total number of clones screened was about 1.01 脳 106. After repeated verification by ELISA,IFA and other methods, two positive clones were obtained, named Agp40-1 and Agp40-2;, respectively. The light chain variable region approximate line of Agp40-1 is A20, the gene homology is 95%, the heavy chain variable region approximate line is VH3-30, gene similarity 92Agp40-2, the light chain variable region approximate line is O12, and the gene homology is 97%. The similarity of heavy chain variable region was that the homology of VH3-30, gene was 92.The two positive clones were respectively expressed in large quantities and purified by metal chelating affinity chromatography to obtain Fab fragments with complete heavy and light chain structure and high purity. ELSIA, dot blot, Western blot, IFA and so on confirmed that two purified Fab fragments could specifically recognize natural and recombinant surface antigens of Cryptosporidium minimus. Biacore analysis showed that the dissociation constants of Agp40-1 and Agp40-2 were 5.25 脳 10 ~ (-9) and 1.47 脳 10 ~ (-9), respectively. It can block the attachment of Cryptosporidium microspore spores to host cells (Caco-2). The preparation and study of the IgG Fab fragment of human engineering antibody against Cryptosporidium provided a theoretical and practical basis for the further development of passive immunization strategy for the treatment and prevention of Cryptosporidium infection.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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