本文關(guān)鍵詞：呼吸道合胞病毒治療性單克隆抗體的制備及其免疫保護(hù)作用研究　出處：《安徽醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人呼吸道合胞病毒 F蛋白 單克隆抗體 中和活性 融合抑制活性

【摘要】： 目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)廣泛分布于世界各地,是導(dǎo)致嬰幼兒嚴(yán)重下呼吸道感染最重要的病毒病原。病毒感染機(jī)體后的免疫保護(hù)機(jī)制尚未明確,也無特異性防治方法。本研究嘗試?yán)秒s交瘤技術(shù),制備具有中和活性的單克隆抗體,并開展RSV免疫治療研究,以期為研制具有自主知識(shí)產(chǎn)權(quán)的RSV治療性單克隆抗體奠定基礎(chǔ)。 方法:用RSV活病毒通過滴鼻免疫小鼠,取免疫小鼠的脾臟與骨髓瘤細(xì)胞進(jìn)行細(xì)胞融合,通過有限稀釋法亞克隆和間接ELISA進(jìn)行篩選,獲得穩(wěn)定分泌抗RSV單克隆抗體(Monoclonal antibody, McAb)的雜交瘤細(xì)胞株。將雜交瘤細(xì)胞注入小鼠腹腔誘生腹水,利用G蛋白親和層析法從腹水中純化抗體。使用SDS-PAGE檢測純化后抗體的純度,間接ELISA、間接免疫熒光(indirect immunoflurescence, IIF)和Western blot方法分析抗體對(duì)RSV融合蛋白(fusion protein, F)的特異性結(jié)合能力,間接ELISA、非競爭ELISA及競爭ELISA分別測定抗體的亞型、親和常數(shù)和抗原識(shí)別表位,為了更好的開展抗體生物活性研究,我們還建立了免疫酶法(immunoenzyne, IE)分析RSV病毒滴度的方法,應(yīng)用IE法體外蝕斑減少中和實(shí)驗(yàn),確定該株單抗的中和活性,并計(jì)算中和效價(jià),通過融合抑制實(shí)驗(yàn)確定該株單抗是否具有融合抑制活性。用Trizol法從雜交瘤細(xì)胞株中提取總RNA,應(yīng)用鼠源抗體重鏈和輕鏈通用引物,通過PCR擴(kuò)增抗體的輕鏈和重鏈可變區(qū)基因,并克隆入pGEM-T載體中,進(jìn)行核酸序列分析,并與現(xiàn)有的RSV單克隆抗體進(jìn)行同源性比對(duì)。利用RSV感染的動(dòng)物模型,進(jìn)一步分析單克隆抗體的體內(nèi)中和活性。 結(jié)果:獲得1株穩(wěn)定分泌抗RSV F蛋白的雜交瘤細(xì)胞株F8,純化后抗體的純度達(dá)到95%以上。該單抗能夠識(shí)別F蛋白單體,抗體亞型為IgG1,親和常數(shù)(Ka)為6.8×108 L/mol,抗原識(shí)別表位位于F蛋白的抗原表位區(qū)205-222位氨基酸。該株單抗可以在體外抑制RSV對(duì)HEp-2細(xì)胞的感染,具有中和活性,同時(shí)具有融合抑制活性。擴(kuò)增出抗體輕鏈和重鏈可變區(qū)基因,并克隆入pGEM-T載體中。測序結(jié)果顯示重鏈可變區(qū)基因序列全長371 bp,編碼124個(gè)氨基酸;輕鏈可變區(qū)基因序列全長323 bp,編碼107個(gè)氨基酸,在GeneBank對(duì)氨基酸序列進(jìn)行比對(duì)分析,兩者均符合小鼠IgG可變區(qū)基因的特征。與美國專利網(wǎng)上提交的具有中和活性的RSV鼠源單抗的重鏈輕鏈同源性分別為95.37%與75.4%�？贵w體內(nèi)保護(hù)實(shí)驗(yàn)顯示,該抗體可降低感染RSV小鼠的肺臟病毒滴度。 結(jié)論:制備了能特異性識(shí)別RSV F蛋白的單克隆抗體F8,完成了純化和性質(zhì)鑒定,擴(kuò)增出抗體輕鏈和重鏈可變區(qū)基因,與已知的具有中和活性的RSV單抗的可變區(qū)基因有較高的同源性,可以確定所得到的序列為RSV抗體輕鏈和重鏈可變區(qū)序列,而非其他基因的序列。以IE法為基礎(chǔ)的RSV病毒滴度分析方法顯示F8在體內(nèi)體外具有中和活性和融合抑制活性,為RSV感染的免疫治療和抗體人源化改造奠定了堅(jiān)實(shí)基礎(chǔ)。
[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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 陳慧中;流行性喘憋性肺炎的研究進(jìn)展[J];臨床兒科雜志;2005年09期

2 董繼華,盧銀平,江漢珍,田順新;炭凝集試驗(yàn)快速檢測病毒的研究[J];細(xì)胞與分子免疫學(xué)雜志;2001年05期

3 池永斌,呂建新,陸永綏;微量酶聯(lián)夾心雜交法定量檢測TNF-α mRNA[J];中國病理生理雜志;2003年09期

4 鄧潔;錢淵;朱汝南;王芳;趙林清;;2000年冬-2006年春北京地區(qū)急性呼吸道感染患兒中呼吸道合胞病毒的監(jiān)測[J];中華兒科雜志;2006年12期

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