hTRX-PR39融合基因cDNA克隆的构建及鉴定

Construction and identification of recombinant plasmids expressing hTRX-PR39

目的研究人硫氧还蛋白(hTRX)和抗菌肽PR39基因在脑缺血疾病治疗中的作用,构建hTRX-PR39cDNA融合基因。方法设计合成PR39、hTRX正向和反向引物,采用PCR方法,扩增获得两端具有BamHI和Ecor721、Eco721和EcoI酶切位点的PR39 cDNA和hTRXcDNA片段,并分别克隆到pGEM-Teasy中,转化细菌,筛选阳性克隆,酶切鉴定并测序。BamHI和EcoR721双酶切pGEM-T-hTRX和pGEM-T-PR39,将获取的PR39/BamHI,EcoR721片段克隆到pGEM-T-hTRX/BamHI,EcoR721载体中,构建pGEM-T-hTRX-PR39载体。结果经DNA测序证实,修饰过的hTRXcDNA、PR39 cDNA片段的核酸序列和推导的氨基酸同数据库资料一致。重组质粒pGEM-T-hTRX-PR39酶切图谱证实hTRX-PR39融合肽cDNA已克隆到pGEM-T表达载体中。结论成功克隆出具有EcoR721和EcoRI、BamHI和EcoR721酶切位点的hTRXcDNA和PR39 cDNA片段,并构建pGEM-T-hTRX-PR39载体。

Objective To explore the effects of hTRX and PR39 as target genes on cerebral ischemic disease, and to construct a fusion gene of hTRX-PR39 cDNA. Methods The forward and reverse primers of PR39 were designed and synthesized. By means of PCR, the fragment encoding PR39 was gained, including EcoR721 and BamH I restriction enzyme sites, and the new hTRX cDNA including EcoR721 and EcoRI restriction enzyme sites was also gained. Then the synthesized fragments were easily cloned into vector pGEM-T. The positive clone was identified by restriction enzymes, and then the cloned amplified fragments were sequenced by the dideoxy-mediated chain-termination method. The cloned hTRX and PR39 cDNA were compared with the GeneBank sequence by the DNASIS. After pGEM-T-hTRX and pGEM-T-PR39 were both digested by BamH I and EcoRI, the PR39/BamHI, EcoRI was cloned into the recombinant vector pGEM-T-hTRX/BamHI, EcoRl . The recombinant vector pGEM-T- hTRX-PR39 was gained. Results Sequences of modified hTRX and PR39 were consistent with those of the gene bank. After analyzing the ORF of the cloned hTRX-PR39 cDNA by the DNASIS, we found that amino acids encoded by the cloned hTRX-PR39 cDNA were identical to published results. Conclusion The sequence of hTRX including EcoR721 and EcoRI restriction enzyme sites and sequence of PR39 including EcoR721 and BamH I restriction enzyme sites were successfully obtained by PCR. Also, the recombinant vector pGEM-T-hTRX-PR39 was successfully constructed.