反义HLA-A2和GDNF共表达逆转录病毒载体的构建和鉴定

为构建GDNF和反义HLA -A2共表达的逆转录病毒载体,将人GDNF酶切片段(XhoI/SalI)克隆于逆转录病毒载体pLNCX2 的XhoI位点,再将HLA A2cDNA片段反向克隆于上述重组载体的HindIII/SalI位点,对其作酶切鉴定和测序后转染PA317包装细胞系进行病毒的包装。收获重组病毒感染的NIH3T3并进行病毒滴度的测定,GDNF及HLA- A2表达的RT -PCR检测,进一步感染人胚肺成纤维细胞,观察GDNF分泌情况。结果表明:GDNF和反义HLA -A2两片段的序列和插入方向完全正确,包装后获得的重组病毒的平均滴度为5×105 CFU/ml。RT PCR显示:在小鼠源性的PA317细胞中有人GDNF和HLA A2的表达,ELISA法测得病毒感染人胚肺成纤维细胞上清液中GDNF含量为450pg/ml。通过本实验,我们获得能共表达GDNF和反义HLA -A2的重组逆转录病毒,其转染的人成纤维细胞具有合成GDNF的能力。

CONSTRUCTION AND IDENTIFICATION OF A RETROVIRAL VECTOR CONTAINING hGDNF AND ANTISENSE HLA-A2 cDNA

To construct a recombinant retroviral vector that expresses hGDNF and antisense HLA-A2 cDNA, the GDNF fragment digested with Xho I / Sal I was clockwisely subcloned into Xho I site of a retroviral vector, pLNCX_2, and the HLA-A2 cDNA was counter-clockwisely inserted into the Hind III/ Sal I sites of the above constructed vector. After enzymatic analysis and sequencing of GDNF and HLA-A2 cloned in pLNCX_2 vector, the recombinant vector was transfected into a packaging cell line PA317 for virus production. The recombinant viruses collected from the culture medium were used to infect NIH3T3 cells for titer determination and RT-PCR detection of GDNF and antisense HLA-A2 expression. The viruses were also used to infect human embryonic lung fibroblasts for detection of GDNF secretion. The results showed that both sequence and inserting orientation of GDNF and HLA-A2 fragmemts were correct, and the mean titer of the recombinant viruses in the supernatant of the packaging cell line was 5×10 5 CFU/ml. RT-PCR analysis indicated that GDNF and antisense HLA-A2 fragments were efficiently co-expressed in the murine PA317 cells, and ELISA showed that the concentration of GDNF secreted from the infected human fibroblasts was 450 pg/ml in the supernatant. The present study suggested that the recombinant retrovirus harboring GDNF and antisense HLA-A2 can be produced efficiently and the infected human fibroblasts has a potential to synthesize GDNF.