HER2基因真核表达载体的构建及其抑瘤效应研究01.doc

HER2基因真核表达载体的构建【】HER2基因真核表达载体，研究肿瘤生长的抑制作用。RT-PCR方法从HER-2+SKBR3细胞株中获得HER-2基因胞外段编码区cDNA，构建pRC-CMV-信号肽-hHER2-ECD载体大肠杆菌DH5αELISA法ELISPOT法分别检测体液及细胞免疫情况，然后用EMT-6细胞肿瘤pRC-CMV-信号肽-hHER2-ECD载体DNA测序分析，结果表明该基因的序列与设计的序列完全相同。 pRC-CMV-信号肽-HER2真核表达质粒转染细胞后，目的基因在mRNA 水平的表达与预期结果一致。T细胞反应，并且可明显降低小鼠成瘤率。结论 构建的HER2基因真核表达载体可在细胞中表达且对小鼠实体瘤的生长具有一定的抑制作用。 【】HER2 转染 肿瘤 Construction and antitumor effect of HER2 DNA eukaryotic expression vector He li-qing Zhang cun（The department of biotechnology center，The Fourth Military Medical University， Xi’an 710033，China） 【Abstract】AIM: To construct an eukaryotic expressing vector of HER2 and investigate the inhibitory effect on the growth of tumor by the expression of the plasmid in vivo．．Methods：The cDNA of HER2 extracellular domain was prepared through RT-PCR from HER-2+SKBR cell line then it was cloned into pRC-CMV vector and DH5α,the immunocompetence of the plasmid was study by ELISA and ELISPOT. Incubated mice with EMT-6 and the tumor growth was observed. RESULTS: DNA sequencing and restriction enzyme digestion proved that HER-2 gene was correctly connected. After transfection with plasmid pRC-CMV-signal peptide-HER2,293 cells could produce HER2 mRNA. The plasmid can induce low immune but high T cell response and it can restrain the growth of tumor obviously. CONCLUSION: P1asmid can express in cells,The plasmid Can inhibit the growth of solid tumor． 【Key words】eukaryotic expressing vector ; HER-2; transfection; tumor HER2分子量为185kDa的，大量的疫苗研究表明，HER-2全长并不能诱导有效的免疫反应，而胞外区的近膜端免疫原性较强。故本实验首先截取HER-2胞外区的近膜区构建了rhHER-2-ECD真核表达载体为进行肿瘤疫苗的研究奠定基础。 主要试剂材料。1 细菌菌种 E．coliDH5(本室保存。质粒pRC-CMV为真核表达载体，生化教研室馈赠。HER-2/neu+EMT6细胞株SKBR3细胞株、HEK293细胞株由本室保存。BALB/c小鼠，雌性，由第四军医大学实验动物中心提供。。2 分子生物学试剂 质粒纯化试剂盒与核酸片段纯化试剂盒购自上海博亚公司，寡核苷酸片段由北京澳科公司合成。限制性核酸内切酶BamH I，HindⅢ，XbaI等均购自TaKaRa公司。T4DNA连接酶，TaqDNA聚合酶购自TaKaRa公司。TaqDNA聚合酶（PCRmix），DNA Marker购自天根公司。1640培养基为Gibco公司产品。小牛血清为杭州四季青生物工程材料研究所产品。 HER-2/Fc蛋白RD公司HRP标记的羊抗小鼠IgG和FITC标记的羊抗小鼠IgG购中杉生物工程公司。ELISPOT试剂盒购自MABTECH公司。LipofectamineTM2000脂质体PVDF膜的96孔板购自