携带TRAIL基因慢病毒载体的构建及其体外感染淋巴瘤细胞效率

本研究目的构建携带人肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因的慢病毒载体,并研究其对不同细胞系的淋巴瘤细胞的感染效率。采用分子克隆技术,将针对TRAIL基因mRNA的cDNA片段插入到克隆载体pGM-T,构建pGM-T-TRAIL,然后将测序正确的TRAIL基因克隆至慢病毒表达载体pWPI,构建慢病毒表达载体pWPI-TRAIL。最后利用pWPI/VSV-G/Δ8.2慢病毒包装系统,通过转染293T细胞,制备重组慢病毒plenti-TRAIL,并进行浓缩和滴度测定。将重组慢病毒载体感染不同来源的淋巴瘤细胞系,测定荧光表达评价感染效率,利用RT-PCR和Western blot法评价TRAIL基因的表达情况。结果表明,酶切鉴定及测序结果表明pGM-T-TRAIL和pWPI-TRAIL载体构建成功,滴度测定结果显示浓缩的重组慢病毒plenti-TRAIL浓度可达109IU/ml。感染效率结果显示YTS细胞较DOHH2和Jurkat细胞更易被慢病毒感染(P<0.05)。RT-PCR和Western blot实验表明淋巴瘤细胞后plenti-TRAIL感染后可有效地表达TRAIL基因。结论:成功构建了慢病毒表达载体pWPI-TRAIL,并制备出重组慢病毒plenti-TRAIL。plenti-TRAIL可以有效感染不同细胞来源的淋巴瘤细胞系,同时感染后的淋巴瘤细胞可以有效表达TRAIL基因。

Construction of a lentiviral vector carrying TRAIL gene and its infection efficiency to lymphoma cells in vitro

This study was purposed to construct a lentiviral vector carrying the TNF-related apoptosis-inducing ligand (TRAIL) gene and investigate its infection efficiency to several lymphoma cells lines. A pGM-T-TRAIL vector was constructed by inserting the cDNA segment derived from TRAIL mRNA into the cloning vector pGM-T, which was then inserted into the lentiviral vector pWPI. The recombinant lentiviral vector plenti-TRAIL was produced by transfecting 293T cells with pWPI-TRAIL, packaging plasmid Δ8.2, and envelope plasmid pCMV-VSVG and then harvested from the culture supernatant. Infection efficiency was measured in several lymphoma cell lines by live cell GFP fluorescence, while TRAIL expression was assessed by RT-PCR and Western blot. The results showed that the enzyme cut identification and sequencing demonstrated the successful construction of both pGM-T-TRAIL and pWPI-TRAIL. The results of testing drop showed that the concentration of the restructured lentiviral plenti-TRAIL reached 10(9) IU/ml. Comparison of infection efficiency revealed that YTS cells were more likely to be infected than DOHH2 or Jurkat cells (P < 0.05). Finally, RT-PCR and Western blot showed that lymphoma cells infected with plenti-TRAIL were able to efficiently express the TRAIL mRNA and protein. It is concluded that the lentiviral vector pWPI-TRAIL is successfully constructed and the recombinant lentiviral plenti-TRAIL is manufactured. The plenti-TRAIL vector is able to infect several lymphoma cell lines, and the infected lymphoma cells can effectively express TRAIL genes.