应用抑制性消减杂交技术筛选膦甲酸钠免疫调节基因

目的 应用抑制性消减杂交(SSH)技术构建膦甲酸钠(PFA)处理的人T淋巴细胞Jurkat差异表达基因的cDNA消减文库，筛选并克隆PFA免疫调节相关基因，阐明PFA免疫调节的分子生物学机制。方法 以PFA处理Jurkat细胞，同时以生理盐水处理的相同细胞作为对照；24h后制备细胞裂解液，提取mRNA并逆转录为cDNA，经Rsa1酶切后，将实验组cDNA分成两组．分别与两种不同的接头衔接，再与对照组cDNA进行2次消减杂交及2次抑制性多聚酶链反应(PCR)扩增，将产物与T／A载体连接，构建cDNA消减文库，并转染大肠杆菌进行文库扩增，随机挑选克隆PCR扩增后进行测序及同源性分析。结果 成功构建了PFA处理淋巴细胞差异表达基因的cDNA消减文库。文库扩增后得到46个阳性克隆，进行菌落PCR分析，均得到200～1000bp插入片段。挑取含有插入片段的14个克隆进行测序，并通过生物信息学分析获得11种已知基因序列和3个未知基因。结论 应用SSH技术成功构建了PFA处理的淋巴细胞差异表达基因的cDNA消减文库，为进一步阐明PFA的免疫调节机制及深入了解PFA防治病毒性肝炎的药理作用机制提供了依据。

Screening and cloning genes differentially expressed in jurkat cells treated with phosphonoformate by suppression subtractive hybridization technique

Objective To construct a subtractive cDNA library of genes differentially expressed in human lymphoma cell line Jurkat cells treated with phosphonoformate (PFA), and to clone genes associated with its immune regulation. Methods Using suppression subtractive hybridization (SSH) technique, the mRNA was isolated from Jurkat cells treated with phosphonoformate or 0 9 percent sodium chloride, respectively, and then cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. The tester cDNA, which was hybridized with driver cDNA twice and amplified by nested polymerase chain reaction (PCR) twice, was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109. The clones, which were selected randomly, were amplified by PCR, and then they were sequenced and analyzed in GenBank with Blast search. Results The subtractive cDNA library of genes differentially expressed in Jurkat cells treated with PFA was constructed successfully. The amplified library contained 46 positive clones, which contained 200- 1 000 bp of inserts. Fourteen clones were analyzed by sequencing and bioinformatics, which were identified as eleven known genes and three genes with unknown function. Conclusions The subtractive cDNA library of genes differentially expressed in Jurkat cells treated with PFA using SSH technique was constructed successfully, which brought some new clues for the study of the immune regulation mechanism of PFA