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
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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。
Human Herpesvirus 6(HHV-6)is a double-stranded DNA virus with preferential tropism for human lymphocytes,and was originally isolated from peripheral boold mononuclear cells(PBMCs)of patients with lymphoproliferative disease and AIDS,and isβ-herpesvirus subfamily with human cytomegalovirus(CMV)and Human Herpesvirus 7(HHV-7).At present,HHV-6 has been suggested to be a cause of Exanthema Subitum(ES),and may be associated with neurospongioma, meningocerebritis,AIDS,chronic fatigue syndrome,organ transplantations and multiple sclerosis(MS),et al,but mechanisms of its pathopoiesis are unknowed.
HHV-6 contains a liner double-standed DNA genome of approximately 160 Kbp to 170Kbp with 119 open reading frames(ORF), and can be comprised of three domains of UL,DRL and DRR.HHV-6 is classified into two variants by difference of genetic structure and antigenicity:HHV- 6A and HHV- 6B.GS and U1102 strains are A variant,and Z29 strain is B variant respectively representative,and over 96%sequences of two variants are similar.HHV-6 resides in PBMCs and saliva of healthy adults and children widesprendly.HHV-6 primary infection hppens in six-month to two-year old infants generally,and following establishes a long-term latent infection.
The HHV-6 Nanjing local strain E5(CN5)was isolated from PBMCs of a child with ES disease and grew in CBMCs or JJhan cells in our lab in 1996.HHV-6 E5 has been reseached detailly in virulogy, immunology,bionomics,morphology and ultrastructure of virus to provid conditions for further study.
In the study,we purified the HHV-6 E5 virus by two-step sucrose gradient centrifugation.Then the purifed virus was immunized to BALB/c mouse,and the spleen cells of the immunized mouse were fsed with SP2/0 myeloma cells.The positive clones which produced McAbs against HHV-6 were screened by ELISA.As a result,clones named as JA9,JYE7,JYE8 secreting specific McAbs against HH-6 were obtained.Among these,JA9 was of the IgG1,JYE7 and JYE8 were the IgM.Ascitic titres of the antibodies were 1:0.8X10~5,1:0.256X10~5, 1:0.128X10~5,when tested by ELISA respectively.Westen-blot showed that JA9 was bound to 75kD of protein of HHV-6.The results of three McAbs detecting HHV-6-infected CBMCs were positive,but uninfected CBMCs were negative by indirect immunofluorescence.
In the study,saliva samples of patients with oral tumour(oral squamous cell carcinoma and precancerous change),brain tumor (neurospongioma and meningothelioma),ES,immunosuppressive patients(renal transplant and hydropigenous nephritis)and healthy populations were 41、40、36、37 and 40 respectively,total 194.The positive results of these saliva samples were statistical significance by nested PCR and ELISA.The McAbs against HHV-6 may be useful tool for further studying and clinic laboratory detection of HH-6.
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。
Human Herpesvirus 6(HHV-6)is a double-stranded DNA virus with preferential tropism for human lymphocytes,and was originally isolated from peripheral boold mononuclear cells(PBMCs)of patients with lymphoproliferative disease and AIDS,and isβ-herpesvirus subfamily with human cytomegalovirus(CMV)and Human Herpesvirus 7(HHV-7).At present,HHV-6 has been suggested to be a cause of Exanthema Subitum(ES),and may be associated with neurospongioma, meningocerebritis,AIDS,chronic fatigue syndrome,organ transplantations and multiple sclerosis(MS),et al,but mechanisms of its pathopoiesis are unknowed.
HHV-6 contains a liner double-standed DNA genome of approximately 160 Kbp to 170Kbp with 119 open reading frames(ORF), and can be comprised of three domains of UL,DRL and DRR.HHV-6 is classified into two variants by difference of genetic structure and antigenicity:HHV- 6A and HHV- 6B.GS and U1102 strains are A variant,and Z29 strain is B variant respectively representative,and over 96%sequences of two variants are similar.HHV-6 resides in PBMCs and saliva of healthy adults and children widesprendly.HHV-6 primary infection hppens in six-month to two-year old infants generally,and following establishes a long-term latent infection.
The HHV-6 Nanjing local strain E5(CN5)was isolated from PBMCs of a child with ES disease and grew in CBMCs or JJhan cells in our lab in 1996.HHV-6 E5 has been reseached detailly in virulogy, immunology,bionomics,morphology and ultrastructure of virus to provid conditions for further study.
In the study,we purified the HHV-6 E5 virus by two-step sucrose gradient centrifugation.Then the purifed virus was immunized to BALB/c mouse,and the spleen cells of the immunized mouse were fsed with SP2/0 myeloma cells.The positive clones which produced McAbs against HHV-6 were screened by ELISA.As a result,clones named as JA9,JYE7,JYE8 secreting specific McAbs against HH-6 were obtained.Among these,JA9 was of the IgG1,JYE7 and JYE8 were the IgM.Ascitic titres of the antibodies were 1:0.8X10~5,1:0.256X10~5, 1:0.128X10~5,when tested by ELISA respectively.Westen-blot showed that JA9 was bound to 75kD of protein of HHV-6.The results of three McAbs detecting HHV-6-infected CBMCs were positive,but uninfected CBMCs were negative by indirect immunofluorescence.
In the study,saliva samples of patients with oral tumour(oral squamous cell carcinoma and precancerous change),brain tumor (neurospongioma and meningothelioma),ES,immunosuppressive patients(renal transplant and hydropigenous nephritis)and healthy populations were 41、40、36、37 and 40 respectively,total 194.The positive results of these saliva samples were statistical significance by nested PCR and ELISA.The McAbs against HHV-6 may be useful tool for further studying and clinic laboratory detection of HH-6.
引文
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