聚合酶链反应扩增人动脉粥样硬化抗体全套可变区基因

Amplification of All Immunoglobulin V Genes of Human Atherosclerosis by Polymerase Chain Reaction

目的利用二次聚合酶链反应(PCR)扩增人动脉粥样硬化抗体全套可变区基因,并将其克隆到T载体进行测序验证。方法收集50例动脉粥样硬化患者外周动脉血标本,密度梯度离心法分离淋巴细胞,TR Izol法提取总RNA,用逆转录-PCR扩增轻链可变区基因(包括VK和Vλ)和重链可变区基因(VH),分别酶切后克隆到T载体上,随机挑取2个克隆进行测序验证。结果总RNA的纯度和完整性都较好,经过2次PCR成功扩增出了抗体全套轻、重链可变区基因。测序结果与已有的人抗体基因的序列高度同源。结论该方法成功扩增出了人动脉粥样硬化抗体全套可变区基因,为下一步噬菌体单链抗体库的构建和筛选奠定了基础。

Objective To amplify all immunoglobulin V genes of human atherosclerosis by PCR and to sequence the products of PCR cloned into T-vector. Methods Peripheral blood samples of 50 patients with atherosclerosis were collected, from which lymphocytes were segregated by density gradient centrifugation and total RNA was extracted by TRIzol reagent. V genes of VL （ including VK and Vλ） and VH were amplified by RT-PCR, and the products were digested by restriction enzyme and then cloned into T-vector. Two clones were picked randomly to sequence. Results Total RNA were extracted purely with integrity, and all V genes of VL and VH were amplified by PCR successfully. The sequences were highly homologous to human immunoglobulin genes. Conclusion All immunoglobulin V genes of human artherosclerosis were amplified successfully, which lays a foundation for the construction and selection of phage library.