摘要
目的 :探讨Kinectin的抗原性 ,为下一步研究Kinectin在白塞氏病中的意义奠定基础。方法 :从人Hep2细胞抽提RNA ,利用RT PCR扩增能覆盖Kinectin全长的 3个片段 ,其位置分别相当于氨基酸 2 2～ 5 10 (kin 5 ) ,5 0 3～ 994 (kin M)和 92 0～1346 (kin 3)。将扩增的PCR片段克隆到pET4 2 a(+)载体中 ,诱导表达的融合蛋白用白塞氏病病人血清进行Westernblot分析。结果 :构建的 3个表达Kinectin部分片段载体 (pET kin 5、pET kin M和pET Kin 3)具有正确的阅读框架 ,DNA测序符合率为99 % ,能在大肠杆菌中表达相应的肽段。 8份阳性血清中 ,用Westernblot分析发现 ,6份血清与kin M反应 ,5份血清与kin 3反应 ,只有一份血清与kin 5有反应。结论 :成功地构建表达覆盖Kinectin全长的 3个载体 ,并初步发现Kinectin的抗原性可能主要位于其中间段和羧基端。
Objective:Have disclosed that the Behcet's patient have autoantibodies to 160 kD protein which is subsequently identified as protein kinectin by gene library screening using a Behcet's patient serum(BD4).To investigate the antigeneic location of kinectin.Methods:Purified RNA from Hep2 line culture and amplified three kinectin fragment by RT PCR using three pairs of primers(kin 3,base sequence 920～ 1 346;kin M,503～994;kin 5,22～510),which combine to cover nearly the full sequence of kinectin molecules listed in Genbank(Z22551);then cloned the three fragments into pET 42a(+) vector and expressed in BL21(DE3) E.coli.The whole cell lysate was put onto SDS PAGE and subsequently transferred to a nitrocellous membrane,then detected for the antibody of Behcet's patient serum by ECL system.Results:Three PCR fragment posed a 99% correspondent rate with kinectin sequence.All of the expression vectors has a correct readframe and expressed three fusion peptides of molecular weight 89(kin 5)?89(kin M) and 82 kD(kin 3) respectivey by Western blot analysis.Of eight patients,6 patients serum reacted to kin M,5 to kin 3 and 1 to kin 5;none of ten normal controls reacted to all the three fusion fragments.Conclusion:Three PCR fragment of kinectin covering sequence 133 to 4 107(aa22～1 346) have been successfully cloned into pET 42a(+) vector and expressed in BL21(DE3) E.coli.The preliminary analysis demonstrates that the antigeneic region of kinectin is mainly located in the middle and carboxyl terminal portion.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2003年第4期282-285,共4页
Chinese Journal of Immunology
基金
国家自然科学基金 ( 3 0 2 712 2 5 )
上海市高等学校青年科学基金资助项目 (No .0 1QN75 )
关键词
白塞氏病
抗原
重组
表达
抗原性
分析
免疫印迹
Behcet's disease
Kinectin
Antigeneic analysis
Immunoblot
Prokaryotic expression
