pcDNA3．1-hTERT真核表达载体的构建及其抗肿瘤效应

Construction of pcDNA3.1-hTERT eukaryotic recombinant plasmid and its effects of anti-tumor immunization

目的:构建人端粒酶逆转录酶(hTERT)真核表达载体pcDNA3.1-hTERT,并观察其对荷瘤小鼠的抗肿瘤效应。方法:用RT-PCR方法扩增出带有HindⅢ,BamHⅠ酶切位点的hTERT基因片段,与pGEM-TEasy克隆载体连接,筛选出阳性克隆,酶切获得目的片段,与pcDNA3.1载体连接,构建pcDNA3.1-hTERT表达载体。将真核表达载体pcDNA3.1-hTERT注射移植性H22肝癌模型C57BL/6小鼠,并设PBS组及转染空质粒pcDNA3.1组,观察3组的抗肿瘤效应。结果与结论:成功构建pcDNA3.1-hTERT真核表达载体。该载体中的hTERT基因片段全长为951 bp,可编码相对分子量为37 000、由317个氨基酸构成的多肽。该基因片段与GenBank公布的相应基因进行同源性比较,核苷酸序列的同源性为98.73%,编码产物的氨基酸序列的同源性为99.68%。该真核表达载体免疫荷瘤小鼠后,pcDNA3.1-hTERT组肿瘤生长受到抑制,且该组生存期分别长于PBS组及pcDNA3.1组(P<0.05);pcDNA3.1-hTERT组的IL-2、IFN-γ细胞因子水平也均高于PBS组与pcDNA3.1组(P<0.05)。提示该真核表达载体在受试动物体内可有效地诱导特异性抗肿瘤免疫应答。

Aim：To construct pcDNA3. 1-hTERT, an eukaryotic recombinant plasmid of human telomerase reverse transcription （hTERT） , and evaluate its antl-tumor immunization effects in the mice bearing tumor. Methods ：Total RNA was extracted from the tumor tissue and the fragment of hTERT cDNA was amplified by RT-PCR. The amplified fragment was cloned into pGEM-T Easy vector. The target fragment was sub-cloned into eukaryotic expression vector pcDNA3. 1. The C57BL/6 mice model with transplantable H22 liver cancer,were randomly allocated into three groups as PBS, pcDNA3.1, and pcDNA3.1-hTERT. The anti-tumor effects were measured. Results and Conclusion ： The recombinant plasmid pcDNA3.1-hTERT was constructed successfully. The hTERT fragment in pcDNA3.1-hTERT was 951 bp. It encoded the polypeptide with 317 amino acid residues corresponding to calculated molecular masses of 37 000. The homology in nucleotide acid of the hTERT fragment compared with the hTERT gene reported in GenBank was 98.73% , and the homology in amino acids was 99.68%. The result showed that the growth of tumor was slower and the survival time of pcDNA3.1- hTERT group was obviously longer than those of PBS and pcDNA3.1 groups （both P 〈 0.05）. The levels of IL-2 and IFN-γ in the mice of pcDNA3.1-hTERT group were significantly higher than those of PBS and peDNA3.1 groups （ all P 〈 0.05）.The recombinant plasmid pcDNA3.1-hTERT can induce anti-tumor immunization effect in the mice bearing tumor.