抗SARS相关冠状病毒重组纤突蛋白、核衣壳蛋白单克隆抗体的制备和应用研究

发布时间：2018-05-29 04:33

本文选题：严重急性呼吸综合征相关冠状病毒 + 纤突蛋白　；参考：《天津医科大学》2006年硕士论文

【摘要】：严重急性呼吸综合征(severe acute respiratory syndrome,SARS)又称为传染性非典型肺炎,是一种新出现的烈性传染病。世界卫生组织(WHO)已确认该病的病原体为一种新的冠状病毒,命名为SARS冠状病毒(SARS-CoV)。SARS疫病全球暴发给国际社会造成了极大的恐慌,给世界各国的卫生事业以及经济发展带来了严重后果。但是SARS在临床上鉴别诊断比较困难,因此建立安全、特异、敏感的SARS-CoV抗原检测方法对SARS的早期诊断、预防和治疗具有重要意义。本研究旨在制备抗基因工程表达的纤突蛋白(S)、核衣壳蛋白(N)的单克隆抗体(mAb),初步建立早期检测SARS-CoV抗原的双抗体夹心ELISA方法。我们以基因工程表达的S、N蛋白为免疫原,采用常规杂交瘤技术分别制备分泌抗S、N蛋白的mAb杂交瘤细胞株;对其进行鉴定,包括细胞克隆株稳定性和染色体分析,检测杂交瘤细胞培养上清及腹水中mAb效价、免疫球蛋白(Ig)分型,特异性分析,中和试验,以及相关表位分析。以抗S蛋白、N蛋白抗体分别建立双抗体夹心ELISA法检测SARS-CoV抗原,对180份健康人血清、临床诊断为SARS的12份抗体阴性血清和12份抗体阳性血清进行检测。结果如下:成功制备出了3株分泌抗S蛋白mAb(B8G2,B10F6,C10G10)和2株分泌抗N蛋白mAb(F2G6G3,G5H12G12)的杂交瘤细胞株;杂交瘤细胞染色体计数接近两种亲本细胞染色体数目的总和;细胞培养上清和小鼠诱生腹水中mAb效价高;3株抗S蛋白mAb均为IgG1(κ),而抗N蛋白mAb G5H12G12为IgG2a(κ)、F2G6G3为IgG1(κ);单抗特
[Abstract]:Severe acute respiratory syndrome (SARS), also known as infectious atypical pneumonia, is a new severe infectious disease. The World Health Organization (WHO) has identified the pathogen of the disease as a new coronavirus named SARS coronavirus SARS-CoV. The global outbreak of SARS has caused great panic in the international community. For the world's health care and economic development has brought serious consequences. But the differential diagnosis of SARS is difficult in clinic, so it is important to establish a safe, specific and sensitive detection method of SARS-CoV antigen for early diagnosis, prevention and treatment of SARS. The aim of this study was to prepare a monoclonal antibody (mAba) against the genetically expressed plasminogen, nucleocapsid protein N, and to establish a sandwich ELISA method for the early detection of SARS-CoV antigens. The mAb hybridoma cell lines secreting anti-SfN protein were prepared by conventional hybridoma technique and identified, including cell clone stability and chromosome analysis. The mAb titers, immunoglobulin titers, specificity analysis, neutralization test and related epitope analysis were detected in the supernatant and ascites of hybridoma cells. Double antibody sandwich ELISA method was established to detect SARS-CoV antigen with anti S protein N protein antibody, and 12 antibody negative sera and 12 antibody positive sera were detected in 180 healthy people's sera. The results are as follows: three hybridoma cell lines secreting anti-S protein mAbB8G2OB10F6C10G10) and two hybridoma cell lines secreting anti-N protein mAb-F2G6G3 G5H12G12) were successfully prepared, and the chromosome number of hybridoma cells was close to the sum of the chromosomes of the two parents. The mAb titers of the supernatants of cell culture and the ascites of mice were all IgG1 (kappa) and IgG2a (魏 F2G6G3) for anti-S protein mAb, and IgG1 (kappa; monoclonal antibody) for anti-N protein mAb G5H12G12.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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