中华皮肤科杂志 ›› 2006, Vol. 39 ›› Issue (1): 38-40.

• 论著 • 上一篇    下一篇

In-cell PCR技术扩增白癜风自身抗体基因

田艳丽, 李春英, 王刚, 高天文, 卢涛, 李淼   

  1. 第四军医大学西京医院全军皮肤性病中心 西安 710032
  • 收稿日期:2005-02-03 发布日期:2006-01-15
  • 基金资助:
    国家自然科学基金资助项目(30300307)

In-cell assembly of single-chain Fv from peripheral blood lymphocytes from vitiligo patients

TIAN Yan-li, LI Chun-ying, WANG Gang, GAO Tian-wen, LU Tao, LI Miao   

  1. Centre of Dermatology of Chinese PLA, Xijing Hospital, the Fourth Military Medical University, Xi'an 710032, China
  • Received:2005-02-03 Published:2006-01-15

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摘要： 目的 从白癜风患者外周血B淋巴细胞中扩增获得原始亲本配对的抗体基因.方法 以正常人的黑素细胞为抗原,用活细胞ELISA法筛选50例白癜风血清,选择其中7例抗黑素细胞膜抗体强阳性的患者外周血分离得到CD19+B淋巴细胞,以10%甲醛溶液固定,蛋白酶K溶液通透化处理,进行in-cell PCR.用特异性引物进行in-cell RT-PCR扩增人免疫球蛋白轻链和重链可变区基因,采用Cre/loxP系统在细胞内完成轻重链的连接,然后经巢式PCR扩增获得具有细胞内原始亲本配对特性的单链抗体基因.结果 经in-cell PCR扩增,获得了与预期单链抗体大小一致的单一800bp产物.结论 利用in-cell PCR技术可以扩增白癜风原始亲本配对的自身抗体基因.

关键词: 白癜风, 聚合酶链反应, 自身抗体

Abstract: Objective To obtain in situ pairing of the variable region genes of the immunoglobulin heavy and light chains of B cells from vitiligo patients.Methods Fifty vitiligo patients were screened by a live melanocyte enzyme-linked immunoabsorbent assay.The sera from seven patients were proved to be strongly positive.Then CD19+ B cells were isolated from the peripheral blood lymphocytes of the 7 patients,then fixed in 10% buffered formaldehyde and permeabilised by Proteinase K.The mRNA was amplified within the cells by reverse transcription-polymerase chain reaction (RT-PCR) with specific primers.The immunoglobulin VH and VL DNA assembled within the same cells using the Cre/loxP system.Nested primers were designed to amplify the known major human VH and VL gene familes.Result A unique 800bp band was obtained corresponding in size to single chain Fv fragments.Conclusion The in situ pairing of the variable region genes of the immunoglobulin heavy and light chains of B cells is obtained from vitiligo patients by in-cell PCR.

Key words: Vitiligo, Polymerase chain reaction, Auto antibodies