In-cellPCR技术扩增白癜风自身抗体基因  

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

作　　者：田艳丽[1] 李春英[1] 王刚[1] 高天文[1] 卢涛[1] 李淼[1] 

机构地区：[1]第四军医大学西京医院全军皮肤性病中心,西安710032

出　　处：《中华皮肤科杂志》2006年第1期38-40,共3页Chinese Journal of Dermatology

基　　金：国家自然科学基金资助项目(30300307)

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-cellPCR。用特异性引物进行in-cellRT-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-cellPCR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-cellPCR技术可以扩增白癜风原始亲本配对的自身抗体基因。Objeotive 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 bc 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.

关 键 词：白癜风 聚合酶链反应 自身抗体 

分 类 号：R758.41[医药卫生—皮肤病学与性病学]