白塞氏病相关抗原的重组表达及抗原性初步分析①
冯修高 陈顺乐② 陆 瑜 叶 萍 沈 南 叶 霜 耿文勤 俞 钱 捷
(上海市仁济医院风湿病科,上海200001)
中国图书分类号 R59312 文献标识码 A 文章编号 10002484 X( 2003) 0420282204
[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RT2PCR扩增能覆盖Kinectin全长的3个片段,其位置分别相当于氨基酸22～510(kin25),503～994(kin2M)和920～
1 346(kin23).将扩增的PCR片段克隆到p ET422a(+)载体中,诱导表达的融合蛋白用白塞氏病病人血清进行Western blot分
析.结果:构建的3个表达Kinectin部分片段载体(p ETPkin25,p ETPkin2M和p ETPKin23)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Western blot分析发现,6份血清与kin2M反应,5份血清与kin23反
应,只有一份血清与kin25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.
[关键词] 白塞氏病; Kinectin ;抗原分析;免疫印迹;原核表达
Cloning and expression of Behcet'disease2related autoantigen and preliminary anal2
ysis of its antigeneicity
FENGXiu2Gao,CHENShun2Le,LUYuetal.DepartmentofRheumatology,RenjiHospital,Shanghai200001,China
[Abstract] 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 RT2PCR using three pairs of primers(kin23 , base sequence
920～1 346 ;kin2M ,503～994 ; kin25 ,22～510),which combine to cover nearly the full sequence of kinectin molecules listed in Genbank
(Z22551);then cloned the three fragments into p ET242a(+)vector and expressed in BL21(DE3)E. coli. The whole cell lysate was put onto
SDS2PAGE 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(kin25),89(kin2M)and 82 kD(kin23)respectivey by Western blot analysis. Of eight pa2

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