人表皮生长因子基因真核表达载体的构建及其基因转染

Construction of eukaryotic expressing vector of human epidermal growth factor gene and its gene transfection

目的:构建人表皮生长因子基因真核表达载体,并将其导入细胞中表达表皮生长因子重组蛋白。方法:经HindⅢ、BamHI酶切PGEM-TEasy-hEGF,将人表皮生长因子基因定向克隆到真核表达载体pcDNA3.1中。在脂质体的包裹下将pcDNA3.1-hEGF导入角膜组织细胞中,分别从DNA、RNA、蛋白质3个水平上检测人表皮生长因子基因表达情况。结果:经T7/SP6和hEGF内套引物进行PCR扩增及酶切鉴定重组体,分别得到310bp和162bp两条基因片段,将162bp条带经纯化后用AVaⅢ限制性内切酶切鉴定,可分别得到89bp和72bp两条片段;将人表皮生长因子基因序列与Genbank上公布的序列相比,碱基同源性达98.2%,氨基酸完全相同;PCR,RT-PCR在转染后21d之内,在DNA、RNA水平上可检测到人表皮生长因子基因;免疫组织化学检测法在转染后第3天可检测到人表皮生长因子蛋白表达,2周之后,人表皮生长因子蛋白表达结果转为阴性。结论:pcDNA3.1-hEGF重组质粒与LipofectamineTM2000以适当比例混合形成的微囊可携带人表皮生长因子基因进入细胞内并表达功能蛋白。

AIM： To construct the eukaryotie expressing vector of recombinpnt human epidermal growth factor （hEGF）, and PeDNA3.1-hEGF is brought into the cells to express hEGF reco/nbinant protein. METHODS： Restriction enzymes, HindⅢ and Barn HI, were used to remove hEGF gene from PGEM-hEGF plasmid and the plesmid was transferred into eukaryotic expressing vector-peDNA3.1. PeDNA3.1-hEGF were mixed to transfect the corneal cells. The expression of hEGF gene was detected from DNA, RNA and protein levels. RESULTS： PCR ampfication was performed through T 7/SP6 and hEGF primer and recombinant was identified through restriction enzyme, and 310 bp and 162 bp two gene segments were obtained. 162 bp band was identified with AVa III restriction enzyme after being purified, then 89 bp and 72 bp two segments could be obtained; Basic pair homology was 98.2% between hEGF gene and Genbank sequence. Amino acid was identical In the 21 days, DNA and RNA of hEGF could be detected with PCR and RT-PCR methods. Immunohistochemistry was used to detect the protein expressing level, hEGF protein could be detected in 2 weeks. After that time, hEGF could not be detected by this method. CONCLUSION： Mierecapsule composed by different ratios of PeDNA3. 1 hEGT recombinant plesmid and LipofectamineTM2000 can bring hEGF gene into the cells of rabbit cornea and express functional protein.