Objective To construct the eukaryotic expression vector of heme oxygenase-1 ( HO-1) gene, and transfect the recombinant expression vector to rat peritoneal mesothelial cells ( RPMCs) in vitro. Methods Total RNA was extracted from SD rat spleen, and the full length fragment of HO-1 CDS part was amplified by RT-PCR. The product of PCR was cloned to pMD-18T vector. The pMD-18T-HO-l and pcDNA3. 1 ( - ) vector were digested with restriction endonuclease BamH I and Kpn I , then T4 DNA ligase was used to ligate HO-1 gene fragment and digested pcDNA3. 1 ( - ) vector to construct the eukaryotic expression vector pcDNA3. 1 ( - )-HO-1. The recombinant plasmid pcDNA3. 1 ( - )-HO-1 was transfected into RPMCs. The expression of HO-1 in RPMCs was detected by RT-PCR and Western blotting respectively. Results The full length of rat HO-1 gene was cloned, and the eukaryotic expression vector pcDNA3. 1 ( - ) -HO-1 was successfully constructed. Restriction endonucleases digestion confirmed the target DNA fragment was inserted into the expression vector, and was verified by DNA sequencing. RT-PCR and Western blotting revealed that HO-1 was highly expressed in RPMCs after transfection with pcDNA3-HO-l. Conclusion HO-1 eukaryotic expression vector has been successfully constructed, and HO-1 expresses effectively in RPMCs, which lays a foundation for the further study of the effect of HO-1 in RPMCs injury during peritoneal dialysis.%目的 构建大鼠血红素氧合酶-1(HO-1)真核表达载体,并以该重组表达载体体外转染培养的大鼠腹膜间皮细胞(RPMCs).方法 从SD大鼠脾脏中提取总RNA,并经RT-PCR扩增HO-1基因的CDS区全长片段,将PCR产物克隆至pMD-18T载体,BamH Ⅰ和Kpn Ⅰ双酶切后获取HO-1片段,用T4DNA连接酶将HO-1片段与双酶切后的真核表达载体pcDNA3.1(-)连接,构建大鼠HO-1真核表达载体pcDNA3.1(-)-HO-1.酶切鉴定和测序验证后,利用脂质体介导将重组质粒转染原代培养的RPMCs,通过RT-PCR和Western blotting检测RPMCs中HO-1的表达.结果 成功克隆了大鼠HO-1基因并构建了其真核表达载体pcDNA3.1(-)-HO-1,酶切鉴定证实基因正确地插入表达载体中,并经DNA测序验证.pcDNA3.1(-)-HO-1转染RPMCs后,经RT-PCR和Western blotting检测,HO-1在RPMCs中获得高效表达.结论 HO-1基因真核表达载体的成功构建以及HO-1在RPMCs中的高效表达,为进一步研究HO-1在腹膜透析所致RPMCs损伤中的作用奠定了基础.
展开▼
