pEGFP-ING4真核表达载体的构建及在人脐静脉内皮细胞中的表达

Construction of pEGFP-ING4 eukaryotic expression vector and its expression in human umbilical vein endothelial cells

目的构建携带生长抑制因子4(ING4)基因的真核表达载体pEGFP-ING4并转染人脐静脉内皮细胞(HUVEC),为进一步研究ING4对胶质瘤血管生成影响提供基础。方法提取人胎盘细胞中总RNA,RT-PCR扩增ING4并克隆至pEGFP-C2载体,酶切电泳鉴定出阳性克隆送测序,利用靶向转染试剂jetPEI-HUVEC转染HUVEC,通过免疫组化检测转染细胞中ING4的表达。结果 成功扩增ING4基因,基因全长747 bp。重组质粒pEGFP-ING4的酶切鉴定及测序结果均证明ING4基因成功克隆到真核表达载体中。酶切片段电泳结果表明:目的条带与ING4的开放读码框大小相符;测序结果和ING4的开放读码框比对相似性为100%。转染重组质粒后的HUVEC中ING4蛋白表达水平增强。结论本实验成功构建携带ING4基因的真核表达载体pEGFP-ING4,并可在HUVEC中获得ING4蛋白的增强表达。

Objective To construct the eukaryotic expression vector pEGFP-ING4 carrying growth inhibitor 4（ING4） and transfect human umbilical vein endothelial cells（HUVEC） with the vector for researching the influence of ING4 on the glioma angiogenesis.Methods Total RNA was extracted from placental cells and the coding sequence of human ING4 gene was amplified by RT-PCR.After its purification,the fragment and eukaryotic expression vector pEGFP-C2 plasmid were ligated.The recombinant plasmid was verified by agarose gel electrophoresis and sequenced.pEGFP-ING4 was transfected into HUVEC by targeted agent jetPEI-HUVEC and its expression was evaluated by immunohistochemistry.Results The length of ING4 amplified by PCR was 747 bp,and the recombinant plasmids pEGFP-ING4 was verified by digestion using restriction endonuclease and sequencing results.The target band was matched up with the open reading frame of ING4 in the size.The similarity of sequence alignment was 100% between the sequencing results and the Open reading frame of ING4.Immunohistochemistry revealed that HUVEC transfected with recombinant plasmids expressed higher level of ING4 protein.Conclusions The recombinant eukaryotic expression vectors pEGFP-ING4 of ING4 gene is successfully constructed and ING4 protein in the transfected cells can be more strongly expressed in HUVEC.