外周血B淋巴细胞半套式PCR法扩增人抗体基因

目的用半套式PCR法，以一组人抗体重链和轻链引物直接从人外周血淋巴细胞中扩增人全套抗体基因片段．方法从不同人群外周血淋巴细胞中提取总RNA，经反转录后，以免疫球蛋白信号肽序列引物和家族特异性免疫球蛋白可变区基因引物，进行半套式PCR扩增人全套抗体基因片段．结果采用不同的引物进行重链、轻链Kappa和Lambda链的半套式PCR扩增，均能获得相应大小的PCR产物，其结果扩增率达到100％．结论在建立抗体基因文库时，半套式PCR法能进一步丰富扩增的抗体基因的多样性，可弥补由于转化效率不高而降低抗体库多样性的不足．

Amplification of Immunoglobulin Genes from Human Peripheral Blood Lymphocytes by Half-nested PCR

ObjectiveTo amplife the genes of human immunoglobulin Fd and light chains from human peripheral blood lymphocytes by half-nested PCR.MethodsEmploying half-nested PCR,use Larrick's leader primers to amplify cDNA variable region. The product was used as templates to amplify with primers integrated with Kang's. ResultsHominine immunoglobulin heavy chain and light chain genes were amplified respectively by RT-PCR from hominine lymphocytes using family specific primers and signal sequences of immunoglobulin as half-nested PCR primers. The amplify rate got to 100%. ConclusionHalf-nested PCR enlarges the variety of the product, more or less making up for the low transformation efficiency.