呼吸道合胞病毒治疗性单克隆抗体的制备及其免疫保护作用研究

发布时间：2018-01-13 06:14

本文关键词：呼吸道合胞病毒治疗性单克隆抗体的制备及其免疫保护作用研究　出处：《安徽医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)广泛分布于世界各地,是导致婴幼儿严重下呼吸道感染最重要的病毒病原。病毒感染机体后的免疫保护机制尚未明确,也无特异性防治方法。本研究尝试利用杂交瘤技术,制备具有中和活性的单克隆抗体,并开展RSV免疫治疗研究,以期为研制具有自主知识产权的RSV治疗性单克隆抗体奠定基础。方法:用RSV活病毒通过滴鼻免疫小鼠,取免疫小鼠的脾脏与骨髓瘤细胞进行细胞融合,通过有限稀释法亚克隆和间接ELISA进行筛选,获得稳定分泌抗RSV单克隆抗体(Monoclonal antibody, McAb)的杂交瘤细胞株。将杂交瘤细胞注入小鼠腹腔诱生腹水,利用G蛋白亲和层析法从腹水中纯化抗体。使用SDS-PAGE检测纯化后抗体的纯度,间接ELISA、间接免疫荧光(indirect immunoflurescence, IIF)和Western blot方法分析抗体对RSV融合蛋白(fusion protein, F)的特异性结合能力,间接ELISA、非竞争ELISA及竞争ELISA分别测定抗体的亚型、亲和常数和抗原识别表位,为了更好的开展抗体生物活性研究,我们还建立了免疫酶法(immunoenzyne, IE)分析RSV病毒滴度的方法,应用IE法体外蚀斑减少中和实验,确定该株单抗的中和活性,并计算中和效价,通过融合抑制实验确定该株单抗是否具有融合抑制活性。用Trizol法从杂交瘤细胞株中提取总RNA,应用鼠源抗体重链和轻链通用引物,通过PCR扩增抗体的轻链和重链可变区基因,并克隆入pGEM-T载体中,进行核酸序列分析,并与现有的RSV单克隆抗体进行同源性比对。利用RSV感染的动物模型,进一步分析单克隆抗体的体内中和活性。结果:获得1株稳定分泌抗RSV F蛋白的杂交瘤细胞株F8,纯化后抗体的纯度达到95%以上。该单抗能够识别F蛋白单体,抗体亚型为IgG1,亲和常数(Ka)为6.8×108 L/mol,抗原识别表位位于F蛋白的抗原表位区205-222位氨基酸。该株单抗可以在体外抑制RSV对HEp-2细胞的感染,具有中和活性,同时具有融合抑制活性。扩增出抗体轻链和重链可变区基因,并克隆入pGEM-T载体中。测序结果显示重链可变区基因序列全长371 bp,编码124个氨基酸;轻链可变区基因序列全长323 bp,编码107个氨基酸,在GeneBank对氨基酸序列进行比对分析,两者均符合小鼠IgG可变区基因的特征。与美国专利网上提交的具有中和活性的RSV鼠源单抗的重链轻链同源性分别为95.37%与75.4%。抗体体内保护实验显示,该抗体可降低感染RSV小鼠的肺脏病毒滴度。结论:制备了能特异性识别RSV F蛋白的单克隆抗体F8,完成了纯化和性质鉴定,扩增出抗体轻链和重链可变区基因,与已知的具有中和活性的RSV单抗的可变区基因有较高的同源性,可以确定所得到的序列为RSV抗体轻链和重链可变区序列,而非其他基因的序列。以IE法为基础的RSV病毒滴度分析方法显示F8在体内体外具有中和活性和融合抑制活性,为RSV感染的免疫治疗和抗体人源化改造奠定了坚实基础。
[Abstract]:Objective: human respiratory syncytial virus (Human Respiratory Syncytial Virus, RSV) is widely distributed in the world, is the leading cause of infant severe viral pathogens of lower respiratory tract infection. The most important viral infection immune protective mechanism of body is not clear, nor specific control methods. This study uses the hybridoma technique, preparation of monoclonal antibody with neutralizing activity, and carry out the research of RSV immune therapy, in order to provide the basis for the development of therapeutic monoclonal antibody RSV with independent intellectual property rights.
Methods: RSV live virus by intranasal immunization, spleen and bone marrow cells from the immunized mice were fused, screened by limited dilution. Cloning and indirect ELISA, stably secreting monoclonal antibody against RSV (Monoclonal antibody McAb) hybridoma cells. The hybridoma cells are injected into the abdominal cavity of mice induced ascites, purified from ascites by affinity chromatography using SDS-PAGE G protein. The purity of the purified antibody detection, indirect ELISA, indirect immunofluorescence (indirect immunoflurescence IIF) analysis of antibody against RSV fusion protein and Western blot method (fusion protein F) the specific binding ability, indirect ELISA, subtype of non competition ELISA and ELISA were determined by competitive antibody, affinity constant and antigen recognition epitopes, in order to carry out the biological activity of antibody, we also established ELISA (immunoenzyne IE), RSV analysis method of virus titer, the application of IE method in vitro plaque reduction neutralization test, determine the neutralizing activity of mAb, and calculate the neutralization titer, through fusion inhibition experiment to determine the McAb with fusion inhibitory activity. Total RNA was extracted from hybridoma cell lines by Trizol method and the application of mouse antibody the heavy chain and light chain primer, light chain and heavy chain antibody variable region gene was amplified by PCR, and cloned into the pGEM-T vector, nucleic acid sequence analysis and homology comparison with RSV monoclonal antibody. The existing animal model of RSV infection, further analysis of in vivo neutralizing monoclonal antibodies.
Results: 1 strains of stable secretion of anti RSV protein F hybridoma cell strain F8, purified antibody purity is more than 95%. The monoclonal antibody to F protein identification monomer, antibody subtype IgG1, affinity constant (Ka) of 6.8 * 108 L/mol antigen epitopes in F protein epitopes 205-222 amino acids. The McAb can inhibit RSV in vitro in HEp-2 cells infected with neutralizing activity, also has the fusion inhibitory activity. Amplified gene antibody light chain and heavy chain variable region, and cloned into pGEM-T vector. The sequencing result showed that the gene sequence of heavy chain variable region was 371 BP, encoding 124 amino acid; light chain variable region gene sequence length of 323 BP, encoding 107 amino acids, the amino acid sequences of GeneBank were compared and analyzed, both in line with the characteristics of IgG gene in mice. The variable region of RSV rats with neutralizing activity of the source file with the patent Online The heavy chain light chain homology of the monoclonal antibody was 95.37% and the 75.4%. antibody in vivo protection experiment showed that the antibody could reduce the titer of lung virus in RSV mice.
Conclusion: the preparation of the monoclonal antibody against F8 F protein specifically recognizes the RSV, completed the identification and purification of amplified gene, antibody light chain and heavy chain variable regions, have high homology with known variable region gene with neutralizing activity of RSV monoclonal antibody, sequence can be determined the sequence of light chain RSV antibody and VH gene sequence, rather than the other. The virus titer RSV based on the IE method of analysis showed that the F8 with neutralizing activity and fusion inhibitory activity in vitro and in vivo, laid a solid foundation for immunotherapy and humanized antibody RSV infection.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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