抗人類皰疹病毒6型南京株E5單克隆抗體的制備、鑒定及初步應用
發(fā)布時間：2018-09-13 13:54
【摘要】： 人類皰疹病毒6型(Human herpesvirus 6,HHV-6)是一類嗜人類淋巴細胞的雙鏈DNA病毒,于1986年由美國癌癥中心的Salahuddin等首先從淋巴增生及AIDS患者外周血單個核細胞中分離到,與人類皰疹病毒7型(HHV-7)及人巨細胞病毒(CMV)同屬于皰疹病毒β亞科。目前認為HHV-6是嬰幼兒急疹(ES)的病因,另外還與神經膠質瘤、腦膜腦炎、AIDS、慢性疲勞綜合癥、器官移植后感染、多發(fā)性硬化癥等多種疾病有關,但其致病機理目前尚未清楚。 HHV-6基因組為一線性雙鏈DNA分子,大小約160～170Kbp,結構上可分為3個部分,即序列獨特區(qū)(UL區(qū)),左右末端各有一個正向重復序列(DRL和DRR區(qū)),且有119個開放讀碼框(ORF)。根據(jù)其基因結構差異和抗原性可分為HHV-6A、B兩種亞型,GS、U1102是HHV-6A亞型代表株,而Z29是B亞型代表株,且序列的同源性在96%以上,HHV-6廣泛存在于健康成人及兒童外周血淋巴細胞和唾液中,其原發(fā)感染一般發(fā)生在6個月～2歲的嬰幼兒,高峰期在6～9個月,其后在體內建立持續(xù)性感染,并長期呈潛伏感染。 我室于1994年在國內首次分離鑒定出HHV-6南京地方株8株,并詳細研究了南京株E5(CN5)的病毒學、免疫學、生物學特性及病毒形態(tài)學超微結構,為本次研究奠定了良好的基礎。 本研究采用蔗糖密度梯度離心法純化的南京株HHV-6 E5病毒抗原免疫8周齡、雌性BALB/c小鼠,采用常規(guī)方法融合,間接ELISA方法篩選,并經3次有限稀釋法亞克隆后,獲得3株能持續(xù)穩(wěn)定分泌特異性抗HHV-6抗體的雜交瘤細胞株,分別命名為JA9、JYE7、JYE8。將獲得的3株雜交瘤細胞株傳代擴增,注射到用Pristane預刺激的BALB/c小鼠體內制備腹水,并進行初步純化。單抗Ig亞類鑒定表明:JA9為IgG1,κ亞型、JYE7和JYE8為IgM,λ亞型;腹水效價分別為1:0.8X10~5、1:0.256X10~5、1:0.128 X10~5;雜交瘤細胞染色體計數(shù)顯示3株細胞染色體數(shù)均約95條以上;間接免疫熒光實驗顯示:3株單抗均能與HHV-6 E5感染的CBMCs呈陽性反應,而與未感染CBMCs呈陰性反應;其中JA9單抗Western-blot實驗結果進一步表明其可與HHV-6 E5約75ku大小的病毒蛋白結合。 本研究收集口腔腫瘤(口腔鱗癌及其癌前病變)、腦腫瘤(神經膠質瘤及腦膜瘤)、嬰幼兒急疹、免疫抑制劑使用者(腎移植及腎病綜合癥)及健康人群唾液標本分別為41份、40份、36份、37份、40份,共194份。采用巢式PCR及ELISA法檢測唾液標本中HHV-6的陽性率,結果患者的HHV-6陽性檢出率均高于健康人群且有統(tǒng)計學意義。 本研究在自分離鑒定的HHV-6南京株E5的基礎上,成功制備并初步鑒定出3株抗人類皰疹病毒6型南京株E5的單克隆抗體,將為HHV-6的進一步研究奠定基礎,并為臨床診斷提供了可能。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
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