CN1238499C - 艾滋病病毒0型株的一种抗体及其生产细胞系与应用 - Google Patents
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Abstract
本发明公开了艾滋病病毒O型株的一个抗体及其生产细胞系与应用，本发明的目的是提供能与艾滋病病毒O型株专一结合的单克隆抗体，该抗体具有对表位DE，特别是表位ELDEWA的特异性。能够分泌与艾滋病病毒O型株专一结合的单克隆抗体的小鼠杂交瘤细胞系14D9，已于2002年06月27日保藏于中国微生物菌种保藏管理委员会普通微生物中心。本发明的单克隆抗体在鉴别、诊断HIV-1病毒O型株、制备用于HIV-1O型毒株感染被动免疫药物中将得到广泛应用。以本发明单克隆抗体为活性成分的药物可望在治疗艾滋病中得到应用。
Description
技术领域
本发明涉及艾滋病病毒O型株的一个抗体及其生产细胞系与应用。
背景技术
获得性免疫缺陷综合症，又称艾滋病，是由人类免疫缺陷病毒I型(HIV-1)引起的一种免疫性疾病，目前尚无彻底治愈的有效药物，也没有疫苗可以预防。免疫缺陷病毒的高变异性一直是HIV疫苗和药物设计中的难题。
最近国外抗艾滋病药物临床研究结果证明，在被动免疫治疗中，组合使用针对HIV-1膜蛋白gp160(膜蛋白gp120及跨膜蛋白gp41的前体蛋白)上的几个特定中和表位的中和抗体(其中包括ELDKWA表位特异性的单克隆抗体2F5)，能抑制HIV-1病毒的粘膜传染以及母婴传播，并能清除血液中的HIV-1病毒(Nature Medicine 1999，5：204；Nature Medicine 2000，6：200；Nature Medicine 1999，5：211)；人免疫缺陷病毒(HIV-1)跨膜蛋白gp41上的主要中和表位ELDKWA的表位特异性单克隆抗体能够体外抑制多种HIV-1病毒株感染靶细胞(J.Virology 1993，67：6642；AIDS Res.Human Retroviruses 1994，10：1651；AIDS 1996，10：587)。研究证明，HIV-1能够通过中和表位的限制性变异逃脱中和抗体的中和作用(Immunity 10，431-438，1999)。ELDKWA是公认的重要的HIV-1中和表位，该表位的保守性很强，但在D或K位点的某些限制性变异，能够使病毒逃脱单抗的中和作用。
发明内容
本发明的发明人经研究发现了艾滋病病毒O型株的一个新的免疫学表位，该表位包括氮末端为天门冬氨酸，碳末端为谷氨酸的两个相邻氨基酸残基。与该两个氨基酸残基相连的，通常氮末端连接的氨基酸残基为亮氨酸，碳末端连接的氨基酸残基为色氨酸，这时该免疫学表位的氨基酸残基序列为LDEW，在该序列的基础上，通常氮端连接的氨基酸残基为谷氨酸，碳端连接的氨基酸残基为丙氨酸，这时该免疫学表位的氨基酸残基序列为ELDEWA。
发明人通过对HIV数据库(http：//hiv-web.lanl.gov)的检索发现，所有HIV-1病毒O型株的跨膜蛋白gp41均带有ELDEWA表位(如表1所示)，而其它HIV-1病毒亚型不带有此表位，进一步证实ELDEWA是HIV-1病毒O型株的特有表位，可用于HIV-1病毒O型株鉴别和诊断的靶位点，也可以作为治疗药物的靶位点。
表1：HIV-1病毒O型株带有特征性的ELDEWA表位
| 登录号 | 序列 | 氨基酸残基的序号 | |
| 1 | AF009033 | LELDEWAS | 668-675 |
| 2 | AF407418 | LELDEWAS | 675-682 |
| 3 | AJ302646 | LELDEWAS | 680-687 |
| 4 | AJ302647 | LELDEWAS | 669-676 |
| 5 | L20571 | LELDEWAS | 674-681 |
| 6 | L20587 | LELDEWAS | 660-667 |
| 7 | NC002787 | LELDEWAS | 680-687 |
| 8 | U82990 | LELDEWAS | 658-665 |
| 9 | U82991 | LELDEWAS | 670-677 |
| 10 | U82992 | LELDEWAS | 668-675 |
| 11 | U82993 | LELDEWAS | 669-676 |
| 12 | X96522 | LELDEWAS | 674-681 |
| 13 | X96526 | LELDEWAS | 677-684 |
本发明的目的是提供能与艾滋病病毒O型株专一结合的单克隆抗体，该抗体具有对表位DE，特别是表位ELDEWA的特异性。
能够分泌与艾滋病病毒O型株专一结合的单克隆抗体的小鼠杂交瘤细胞系14D9，已于2002年06月27日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)，保藏号为CGMCC №0753。
小鼠杂交瘤细胞系14D9产生的单克隆抗体14D9能够特异性识别HIV-1病毒O型株跨膜蛋白gp41特有的ELDEWA-表位，而不识别其它HIV-1病毒亚型所携带ELDKWA-、ELNKWA-和ELEKWA-表位。
本发明的单克隆抗体在鉴别、诊断HIV-1病毒O型株、制备用于HIV-1 O型毒株感染被动免疫药物中将得到广泛应用。以本发明单克隆抗体为活性成份的药物可望在治疗艾滋病中得到应用。
下面结合具体实施例对本发明做进一步说明。
附图说明
图1为本发明单克隆抗体与带有不同表位的重组、可溶性gp41融合蛋白反应后的电泳图。
具体实施方式
实施例1、鼠类来源的杂交瘤细胞系14D9的制备过程：
1、人工合成一条含有HIV-1 O型株跨膜蛋白gp41特有的ELDEWA-表位的表位多肽，该表位多肽含有4次重复的ELDEWA-免疫学表位，其氨基酸残基序列为：CELDEWAGELDEWAGELDEWAGELDEWA；
2、利用MBS(m-maleimidobenzoyl-N-hydroxy succinimide ester)将上述表位多肽与载体蛋白BSA耦联；
3、将上述耦联物与福氏佐剂混合(两种物质的重量比为，耦联物∶福氏佐剂＝1∶1)免疫Balb/c小鼠，每两周免疫一次。首次使用完全福氏佐剂，以后使用不完全福氏佐剂。每次免疫抗原剂量为：含10微克表位多肽的耦联物/次/只，共免疫3次；
4、将免疫后的小鼠脾细胞与小鼠骨髓瘤细胞采用常规细胞融合技术融合，并制备杂交瘤，从杂交瘤细胞中筛选出能分泌ELDEWA-表位特异性单克隆抗体的杂交瘤；
5、克隆杂交瘤，获得能分泌ELDEWA-表位特异性单克隆抗体的克隆杂交瘤细胞系14D9。
实施例2、本发明抗体对HIV-1病毒O型株ELDEWA-表位的特异性识别
将杂交瘤细胞系14D9分泌的单克隆抗体与带有不同表位的重组、可溶性gp41融合蛋白进行温浴反应，结果如图1所示，从图中的结果可以看出，杂交瘤细胞系14D9分泌的单克隆抗体对HIV-1病毒O型株ELDEWA-表位具有特异性识别，图中，A：分子量蛋白；B：本发明单克隆抗体识别带有ELDEWA表位的rsgp41(GST-rsgp41ELDEWA)〔42KD〕；C：本发明单克隆抗体不识别带有ELDKWA表位的rsgp41(GST-rsgp41ELDKWA)；D：本发明单克隆抗体不识别GST；E：本发明单克隆抗体识别含有ELDEWA表位的GST-ELDEWA(26KD)。
Claims (3)
1、一种鼠类来源的杂交瘤细胞系14D9，其特征在于所述细胞系的保藏号为CGMCC№0753。
2、权利要求1所述的杂交瘤细胞系14D9生产的单克隆抗体。
3、权利要求2所述的单克隆抗体在体外鉴别HIV-1病毒O型株中的应用。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021253714A CN1238499C (zh) | 2002-07-29 | 2002-07-29 | 艾滋病病毒0型株的一种抗体及其生产细胞系与应用 |
| PCT/CN2002/000551 WO2004011633A1 (fr) | 2002-07-29 | 2002-08-09 | Anticorps dirige contre la souche de type o du vih, lignee cellulaire d'hybridomes produisant cet anticorps et utilisations correspondantes |
| AU2002325466A AU2002325466A1 (en) | 2002-07-29 | 2002-08-09 | The antibody directed against the hiv strain type o, the hybridoma cell line producing it and the uses thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021253714A CN1238499C (zh) | 2002-07-29 | 2002-07-29 | 艾滋病病毒0型株的一种抗体及其生产细胞系与应用 |
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| Publication Number | Publication Date |
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| CN1472313A CN1472313A (zh) | 2004-02-04 |
| CN1238499C true CN1238499C (zh) | 2006-01-25 |
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| CN (1) | CN1238499C (zh) |
| AU (1) | AU2002325466A1 (zh) |
| WO (1) | WO2004011633A1 (zh) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5459060A (en) * | 1989-08-24 | 1995-10-17 | Bioclonetics Incorporated | Human monoclonal antibodies directed against the transmembrane glycoprotein (gp41) of human immunodeficiency virus-1 (HIV-1) |
| DE19809785C2 (de) * | 1998-03-08 | 2000-02-10 | Wolfgang Bergter | Radioimmunpharmakon zur Behandlung der HIV-1-Infektion |
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- 2002-08-09 AU AU2002325466A patent/AU2002325466A1/en not_active Abandoned
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| Publication number | Publication date |
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| AU2002325466A1 (en) | 2004-02-16 |
| WO2004011633A1 (fr) | 2004-02-05 |
| AU2002325466A8 (en) | 2004-02-16 |
| CN1472313A (zh) | 2004-02-04 |
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