摘要
目的构建人caspase-1真核表达载体,观察其在人肝癌细胞HepG2中的表达。方法分离人外周血淋巴细胞,LPS刺激提取细胞总RNA,RT-PCR方法分别扩增caspase-1两片段S_1、S_2,利用重叠延伸PCR方法(SOE)获得caspase-1全长序列,将其克隆入pIRES_2-EGFP载体,进行茵落PCR、限制性酶谱分析、DNA序列测定。通过jetPEI方法转染HepG2肝癌细胞,倒置相差荧光显微镜下观察其表达情况,RT-PCR分析caspase-1的表达水平。结果从外周淋巴细胞中经RT-PCR分别扩增出365bp和883bp片段,纯化后的PCR产物通过SOE扩增得到一1234bp的条带,与预期序列长度一致。SOE产物与pIRES_2-EGFP经EcoR Ⅰ和BamH Ⅰ双酶切后连接,茵落PCR鉴定获得数个阳性克隆,质粒经EcoR Ⅰ和BamH Ⅰ限制性酶谱分析酶解出1234bp条带,DNA序列分析证实重组载体中的DNA序列与GeneBank中人caspase-1序列完全一致。将该载体转染至HepG2细胞中,经RT-PCR证实,与转染pIRES_2-EGFP空载体的对照组细胞相比,caspase-1水平明显升高。结论本研究成功构建人caspase-1表达载体pIRES_2-EGFP-caspase-1,该载体能够在人肝癌细胞中高效表达外源基因。
Objective To establish a human expression vector and observe its expression in HepG2 cells. Methods Total RNA was extracted from peripheral blood monocytes （PBMCs } of human treated with LPS （Sg/ml ） , S1 and S2 of caspase-1 gene were obtained through RT-PCR. The two DNA fragments were spliced through overlapping PCR（ SOE） to form the full length caspase-1 gene. The purified product digested by EcoR Ⅰ and BamH Ⅰ was cloned into pIRES2-EGFP to construct the pIRES2-EGFP-caspase-1 expression vector which was verified by PCR screening, restriction enzyme assay （EcoR Ⅰ and BamH Ⅰ） and DNA sequencing. Then the purified pIRES2-EGFP-caspase-1 plasmid was transfected into HepG2 hepatoma cells through a jetPEI mediated method. The level of expression of caspase-1 was analyzed by RT-PCR 48h after transfection and compared with cells with pIRES2-EGFP. Results A 365bp and a 883bp DNA segments were amplified respectively from total RNA isolated from PBMCs treated with LPS which was compatible to the sequence expected. The purified PCR products then spliced by SOE-PCR which produced a long DNA sequence of 1234bp as expected. SOE product and plRES2-EGFP digested with EcoR Ⅰ and BamH Ⅰ respectively were ligated together by T4 liagase,in the following colony PCR it was showed that we got several positive clones. Assessed by restriction enzyme assay,it indicated that the positive clones gave rise to a 1234bp DNA fragment which was finally proved to be identical to the full length human caspase-1 reported by GeneBank. The recombinant vector were then transfected into HepG2 hepatoma cells by jetPE1,48h after transfection, RT-PCR indicated that the expression level of caspase-1 was significantly increased compared with cells transfected with pIRES2-EGFP. Conclusion We successfully constructed a human caspase-1 expression vector, the recombinant vector could express exogenous gene effectively in human hepatoma cells.
出处
《潍坊医学院学报》
2009年第3期205-207,共3页
Acta Academiae Medicinae Weifang
基金
国家自然科学基金(课题编号:30772497)
山东省卫生高层次人才1020工程专项基金
山东省科技攻关计划(课题编号:2008GG10002007)
