IL28B基因多态性检测的质粒标准品构建

人类白介素28b（IL28b）的SNP位点rs12980275的多态性（AA、AG和GG）与聚乙二醇干扰素、利巴韦林联合治疗的效果具有显著相关性。为了确保rs12980275预测丙型肝炎患者抗病毒治疗效果的价值,需要构建标准品作为rs12980275检测的标准对照。提取人类外周血基因组DNA,以IL28B SNP rs12980275为目的基因片段设计引物,进行PCR扩增;纯化目的片段与pGM-T Vector连接并转化到大肠杆菌中;提取重组质粒DNA,并进行PCR、测序鉴定。结果 IL28B SNP rs12980275目的片段制备成功,获得稳定的重组质粒,保证了目的片段的特异性与序列完整性。成功构建了IL28B基因SNP rs12980275突变检测的AA、AG和GG 3种质粒标准品,可作为预测丙型肝炎患者抗病毒治疗效果rs12980275突变检测的阳性质控物。

Standard plasmid construction of gene IL28B polymorphism detection

Genetic polymorphism（ genotypes of AA,AG and GG） of rs12980275 near IL28 B gene was closely associated with peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. To predict the effect of antiviral therapy on patients with hepatitis C,standard plasmids need to be built for a reliable detection system for rs12980275. Human genomic DNA was extracted,and rs12980275 as the target DNA fragment primers was designed for PCR amplification. The purified target fragment of PCR was ligated with a pGMT vector and then transferred into E. coli for replication. The DNA of recombinant plasmid picked out from positive clones was amplified by PCR and then sequenced. The target fragment of DNA fragment near rs12980275 was successfully prepared and the three standard plasmids（ genotypes of AA,AG and GG） for rs12980275 detection were built successfully,which could be used as positive quality controls to predict the effect of antiviral therapy on patients with hepatitis C.