中国临床康复
中國臨床康複
중국림상강복
CHINESE JOURNAL OF CLINICAL REHABILITATION
2005年
19期
213-215
,共3页
李妍妍%杨笛%刘剑南%卞智萍%徐晋丹%陈相健%顾春荣%张寄南
李妍妍%楊笛%劉劍南%卞智萍%徐晉丹%陳相健%顧春榮%張寄南
리연연%양적%류검남%변지평%서진단%진상건%고춘영%장기남
抗体,单克隆%基因%序列分析%聚合酶链反应
抗體,單剋隆%基因%序列分析%聚閤酶鏈反應
항체,단극륭%기인%서렬분석%취합매련반응
背景:为将鼠抗人cTnI单克隆抗体应用人体进行体内诊断或作为治疗心肌损伤的药物载体,必须降低鼠源性抗体的免疫源性,克服其人抗鼠抗体反应,需要对其进行人源化改造.目的:克隆鼠抗人cTnI单克隆抗体Fab段基因并进行序列分析.设计:单一样本研究.单位:一所医科大学附属医院的心血管病研究所.材料:本实验于2003-01/2004-05在南京医科大学第一附属医院心血管病研究所完成.分泌鼠抗人cTnI单克隆抗体的杂交瘤细胞株(JS200202)由南京医科大学第一附属医院心血管病研究所提供.方法:设计扩增鼠IgG重链Fd段及κ轻链引物,从分泌cTnI单抗的杂交瘤细胞中提取总RNA,RT-PCR扩增,对扩增产物进行分子克隆、测序及序列分析.主要观察指标:重链Fd段和κ轻链基因序列及其所属亚型.结果:重链和轻链引物分别扩增出一约700 bp和800 bpDNA片段.经序列分析,与已发表的鼠IgG基因序列对比,其核苷酸及其所推导的氨基酸序列符合鼠IgG1Fab段特征.在GenBank登录,登录号为AY484430(重链),AY484431(轻链);氨基酸序列登录号为AAR83243(重链),AAR83244(轻链).结论:本实验室获得了完整的鼠源性抗人cTnI单克隆抗体Fab段基因,为鼠抗人cTnI单克隆抗体的人源化改造奠定了基础.
揹景:為將鼠抗人cTnI單剋隆抗體應用人體進行體內診斷或作為治療心肌損傷的藥物載體,必鬚降低鼠源性抗體的免疫源性,剋服其人抗鼠抗體反應,需要對其進行人源化改造.目的:剋隆鼠抗人cTnI單剋隆抗體Fab段基因併進行序列分析.設計:單一樣本研究.單位:一所醫科大學附屬醫院的心血管病研究所.材料:本實驗于2003-01/2004-05在南京醫科大學第一附屬醫院心血管病研究所完成.分泌鼠抗人cTnI單剋隆抗體的雜交瘤細胞株(JS200202)由南京醫科大學第一附屬醫院心血管病研究所提供.方法:設計擴增鼠IgG重鏈Fd段及κ輕鏈引物,從分泌cTnI單抗的雜交瘤細胞中提取總RNA,RT-PCR擴增,對擴增產物進行分子剋隆、測序及序列分析.主要觀察指標:重鏈Fd段和κ輕鏈基因序列及其所屬亞型.結果:重鏈和輕鏈引物分彆擴增齣一約700 bp和800 bpDNA片段.經序列分析,與已髮錶的鼠IgG基因序列對比,其覈苷痠及其所推導的氨基痠序列符閤鼠IgG1Fab段特徵.在GenBank登錄,登錄號為AY484430(重鏈),AY484431(輕鏈);氨基痠序列登錄號為AAR83243(重鏈),AAR83244(輕鏈).結論:本實驗室穫得瞭完整的鼠源性抗人cTnI單剋隆抗體Fab段基因,為鼠抗人cTnI單剋隆抗體的人源化改造奠定瞭基礎.
배경:위장서항인cTnI단극륭항체응용인체진행체내진단혹작위치료심기손상적약물재체,필수강저서원성항체적면역원성,극복기인항서항체반응,수요대기진행인원화개조.목적:극륭서항인cTnI단극륭항체Fab단기인병진행서렬분석.설계:단일양본연구.단위:일소의과대학부속의원적심혈관병연구소.재료:본실험우2003-01/2004-05재남경의과대학제일부속의원심혈관병연구소완성.분비서항인cTnI단극륭항체적잡교류세포주(JS200202)유남경의과대학제일부속의원심혈관병연구소제공.방법:설계확증서IgG중련Fd단급κ경련인물,종분비cTnI단항적잡교류세포중제취총RNA,RT-PCR확증,대확증산물진행분자극륭、측서급서렬분석.주요관찰지표:중련Fd단화κ경련기인서렬급기소속아형.결과:중련화경련인물분별확증출일약700 bp화800 bpDNA편단.경서렬분석,여이발표적서IgG기인서렬대비,기핵감산급기소추도적안기산서렬부합서IgG1Fab단특정.재GenBank등록,등록호위AY484430(중련),AY484431(경련);안기산서렬등록호위AAR83243(중련),AAR83244(경련).결론:본실험실획득료완정적서원성항인cTnI단극륭항체Fab단기인,위서항인cTnI단극륭항체적인원화개조전정료기출.
BACKGROUND: To apply mouse anti-human cTnI monoclonal antibody as the drug vector in the treatment and diagnosis of myocardial injury, it is important to degrade the immunity of murine antibody and overcome human anti-mouse reaction. Humanization has been applied as an attempt to resolve this problem.OBJECTIVE: To clone murine anti-cTnI Fab fragment and analyse the nucleotide and deduced amino acid sequences.DESIGN: Single sample study.SETTING: An institute of cardiovascular disease under a medical university-affiliated hospitalMATERIALS: The study was conducted in the Institute of Cardiovascular Diseases, First Affiliated Hospital of Nanjing Medical University from January 2003 to May 2004. The hybridoma cell line JS200202 which secrets the anti-cTnI monoclonal antibody was provided by Institute of Cardiovascular Disease, First Affiliated Hospital of Nanjing Medical University.METHODS: IgG heavy chain primers and κ light chain primers of amplified mouse were designed. Total RNA was extracted from hybridoma cells which secrete cTnI. Reverse transcription polymerase chain reaction(RT-PCR) was amplified. Cloning and subsequent sequence analysis of the Fab fragment was performed. The deduced amino acid sequence was compared and analysed with previously published sequences.MAIN OUTCOME MEASURES: Heavy chain Fd segment and κ light chain gene sequence and its subgroups.RESULTS: A band of approximate 700 and 800 base pairs were amplified using IgG heavy chain primers and κ light chain primers respectively. Sequence analysis indicated that the deduced amino acid sequences were in consistent with the characterization of the amino acid in the murine IgGl Fab fragment(GenBank accession NO AY484430, AY484431; Protein Bank accession NO AAR83243, AAR83244).CONCLUSION: A complete murine anti-cTnI Fab fragment was obtained in this study, which may provide basis for the production of the chimeric anti-cTnI antibody.
