抗人类疱疹病毒6型南京株E5单克隆抗体的制备、鉴定及初步应用
发布时间：2018-09-13 13:54
【摘要】： 人类疱疹病毒6型(Human herpesvirus 6,HHV-6)是一类嗜人类淋巴细胞的双链DNA病毒,于1986年由美国癌症中心的Salahuddin等首先从淋巴增生及AIDS患者外周血单个核细胞中分离到,与人类疱疹病毒7型(HHV-7)及人巨细胞病毒(CMV)同属于疱疹病毒β亚科。目前认为HHV-6是婴幼儿急疹(ES)的病因,另外还与神经胶质瘤、脑膜脑炎、AIDS、慢性疲劳综合症、器官移植后感染、多发性硬化症等多种疾病有关,但其致病机理目前尚未清楚。 HHV-6基因组为一线性双链DNA分子,大小约160～170Kbp,结构上可分为3个部分,即序列独特区(UL区),左右末端各有一个正向重复序列(DRL和DRR区),且有119个开放读码框(ORF)。根据其基因结构差异和抗原性可分为HHV-6A、B两种亚型,GS、U1102是HHV-6A亚型代表株,而Z29是B亚型代表株,且序列的同源性在96%以上,HHV-6广泛存在于健康成人及儿童外周血淋巴细胞和唾液中,其原发感染一般发生在6个月～2岁的婴幼儿,高峰期在6～9个月,其后在体内建立持续性感染,并长期呈潜伏感染。 我室于1994年在国内首次分离鉴定出HHV-6南京地方株8株,并详细研究了南京株E5(CN5)的病毒学、免疫学、生物学特性及病毒形态学超微结构,为本次研究奠定了良好的基础。 本研究采用蔗糖密度梯度离心法纯化的南京株HHV-6 E5病毒抗原免疫8周龄、雌性BALB/c小鼠,采用常规方法融合,间接ELISA方法筛选,并经3次有限稀释法亚克隆后,获得3株能持续稳定分泌特异性抗HHV-6抗体的杂交瘤细胞株,分别命名为JA9、JYE7、JYE8。将获得的3株杂交瘤细胞株传代扩增,注射到用Pristane预刺激的BALB/c小鼠体内制备腹水,并进行初步纯化。单抗Ig亚类鉴定表明:JA9为IgG1,κ亚型、JYE7和JYE8为IgM,λ亚型;腹水效价分别为1:0.8X10~5、1:0.256X10~5、1:0.128 X10~5;杂交瘤细胞染色体计数显示3株细胞染色体数均约95条以上;间接免疫荧光实验显示:3株单抗均能与HHV-6 E5感染的CBMCs呈阳性反应,而与未感染CBMCs呈阴性反应;其中JA9单抗Western-blot实验结果进一步表明其可与HHV-6 E5约75ku大小的病毒蛋白结合。 本研究收集口腔肿瘤(口腔鳞癌及其癌前病变)、脑肿瘤(神经胶质瘤及脑膜瘤)、婴幼儿急疹、免疫抑制剂使用者(肾移植及肾病综合症)及健康人群唾液标本分别为41份、40份、36份、37份、40份,共194份。采用巢式PCR及ELISA法检测唾液标本中HHV-6的阳性率,结果患者的HHV-6阳性检出率均高于健康人群且有统计学意义。 本研究在自分离鉴定的HHV-6南京株E5的基础上,成功制备并初步鉴定出3株抗人类疱疹病毒6型南京株E5的单克隆抗体,将为HHV-6的进一步研究奠定基础,并为临床诊断提供了可能。
[Abstract]:Human herpesvirus type 6 (Human herpesvirus 6 HHV-6) is a human lymphocytic double-stranded DNA virus. It was first isolated from peripheral blood mononuclear cells (PBMC) of patients with lymphoproliferative and AIDS by Salahuddin et al of the American Cancer Center in 1986. Human herpesvirus 7 (HHV-7) and human cytomegalovirus (CMV) belong to herpesvirus 尾 subfamily. At present, it is believed that HHV-6 is the etiology of acute rash (ES) in infants and young children. In addition, it is related to glioma, meningitis, chronic fatigue syndrome, infection after organ transplantation, multiple sclerosis and other diseases. However, the pathogenetic mechanism of HHV-6 is not clear. The HHV-6 genome is a linear double-stranded DNA molecule with a size of about 160 ~ 170kbp. it can be divided into three parts structurally. That is, sequence unique region (UL region), with a forward repeat sequence (DRL and DRR region) at the left and right ends, and 119 open reading frame (ORF). According to the difference of gene structure and antigenicity, the two subtypes of HHV-6A,B, GSH U1102, are the representative strains of HHV-6A subtype, while Z29 is the representative strain of subtype B, and the homology of HHV-6 is more than 96% in peripheral blood lymphocytes and saliva of healthy adults and children. The primary infection usually occurred in children aged 6 months to 2 years old, the peak period was 6 ~ 9 months, then persistent infection was established in the body, and long term latent infection occurred. Eight local strains of HHV-6 were first isolated and identified in our laboratory in 1994, and the virology, immunology, biological characteristics and ultrastructure of the virus were studied in detail, which laid a good foundation for this study. In this study, HHV-6 E5 virus antigen of Nanjing strain purified by sucrose density gradient centrifugation was immunized with 8-week-old female BALB/c mice. It was screened by conventional fusion method, indirect ELISA method, and subcloned by three times limited dilution method. Three hybridoma cell lines, named JA9,JYE7,JYE8., have been obtained, which can continuously secrete specific anti HHV-6 antibodies. The three hybridoma cell lines were amplified and injected into BALB/c mice prestimulated with Pristane to prepare ascites for preliminary purification. The McAb Ig subclass identification showed that IgG1, 魏 subtype JYE7 and JYE8 were IgM, 位 subtypes, ascites titers were 1: 0.8X105: 1: 0. 256X105: 1: 0. 128X105.The chromosome count of hybridoma cells showed that the chromosome number of the three cell lines was above 95. Indirect immunofluorescence assay showed that all of the McAbs could react positively with CBMCs infected with HHV-6 E5, but negative with uninfected CBMCs, and JA9 McAb Western-blot further showed that the McAb could bind to the viral protein of HHV-6 E5 about the size of 75ku. In this study, oral tumor (oral squamous cell carcinoma and its precancerous lesion), brain tumor (glioma and meningioma), infantile rash were collected. The saliva specimens of immunosuppressant users (kidney transplantation and nephrotic syndrome) and healthy population were 41, 40, 36, 37, 37, 40, respectively. Nested PCR and ELISA were used to detect the positive rate of HHV-6 in saliva specimens. The results showed that the positive rate of HHV-6 in patients was higher than that in healthy subjects. On the basis of self-isolation and identification of HHV-6 Nanjing strain E5, three monoclonal antibodies against human herpesvirus type 6 strain E5 were successfully prepared and preliminarily identified, which will lay a foundation for further study of HHV-6. It also provides the possibility for clinical diagnosis.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
[Abstract]:Human herpesvirus type 6 (Human herpesvirus 6 HHV-6) is a human lymphocytic double-stranded DNA virus. It was first isolated from peripheral blood mononuclear cells (PBMC) of patients with lymphoproliferative and AIDS by Salahuddin et al of the American Cancer Center in 1986. Human herpesvirus 7 (HHV-7) and human cytomegalovirus (CMV) belong to herpesvirus 尾 subfamily. At present, it is believed that HHV-6 is the etiology of acute rash (ES) in infants and young children. In addition, it is related to glioma, meningitis, chronic fatigue syndrome, infection after organ transplantation, multiple sclerosis and other diseases. However, the pathogenetic mechanism of HHV-6 is not clear. The HHV-6 genome is a linear double-stranded DNA molecule with a size of about 160 ~ 170kbp. it can be divided into three parts structurally. That is, sequence unique region (UL region), with a forward repeat sequence (DRL and DRR region) at the left and right ends, and 119 open reading frame (ORF). According to the difference of gene structure and antigenicity, the two subtypes of HHV-6A,B, GSH U1102, are the representative strains of HHV-6A subtype, while Z29 is the representative strain of subtype B, and the homology of HHV-6 is more than 96% in peripheral blood lymphocytes and saliva of healthy adults and children. The primary infection usually occurred in children aged 6 months to 2 years old, the peak period was 6 ~ 9 months, then persistent infection was established in the body, and long term latent infection occurred. Eight local strains of HHV-6 were first isolated and identified in our laboratory in 1994, and the virology, immunology, biological characteristics and ultrastructure of the virus were studied in detail, which laid a good foundation for this study. In this study, HHV-6 E5 virus antigen of Nanjing strain purified by sucrose density gradient centrifugation was immunized with 8-week-old female BALB/c mice. It was screened by conventional fusion method, indirect ELISA method, and subcloned by three times limited dilution method. Three hybridoma cell lines, named JA9,JYE7,JYE8., have been obtained, which can continuously secrete specific anti HHV-6 antibodies. The three hybridoma cell lines were amplified and injected into BALB/c mice prestimulated with Pristane to prepare ascites for preliminary purification. The McAb Ig subclass identification showed that IgG1, 魏 subtype JYE7 and JYE8 were IgM, 位 subtypes, ascites titers were 1: 0.8X105: 1: 0. 256X105: 1: 0. 128X105.The chromosome count of hybridoma cells showed that the chromosome number of the three cell lines was above 95. Indirect immunofluorescence assay showed that all of the McAbs could react positively with CBMCs infected with HHV-6 E5, but negative with uninfected CBMCs, and JA9 McAb Western-blot further showed that the McAb could bind to the viral protein of HHV-6 E5 about the size of 75ku. In this study, oral tumor (oral squamous cell carcinoma and its precancerous lesion), brain tumor (glioma and meningioma), infantile rash were collected. The saliva specimens of immunosuppressant users (kidney transplantation and nephrotic syndrome) and healthy population were 41, 40, 36, 37, 37, 40, respectively. Nested PCR and ELISA were used to detect the positive rate of HHV-6 in saliva specimens. The results showed that the positive rate of HHV-6 in patients was higher than that in healthy subjects. On the basis of self-isolation and identification of HHV-6 Nanjing strain E5, three monoclonal antibodies against human herpesvirus type 6 strain E5 were successfully prepared and preliminarily identified, which will lay a foundation for further study of HHV-6. It also provides the possibility for clinical diagnosis.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
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