小鼠IL-23基因的克隆和在逆转录病毒中的表达

从肿瘤组织 (含有活化的淋巴细胞 )提取总RNA ,经逆转录多聚酶链反应 (RT PCR )获得小鼠IL 2 3cDNA。经DNA测序分析 ,证实该片段与GeneBank记载的IL 2 3cDNA序列一致。将该目的基因插入LXSN逆转录病毒载体 ,并转染大肠杆菌DH5α中扩增 ,再感染 ψ2 (ecotropic )和PA317(amphotropic )两种包装细胞 ,经G4 18筛选后获得表达IL 2 3分子的PA317阳性细胞克隆。通过用PCR、RT PCR及Northernblot技术检测目的基因表达 ,结果表明 ,成功地将鼠IL 2 3cDNA插入逆转录病毒载体 ,经转染两种包装细胞产生了高表达IL 2 3的PA317细胞克隆。该克隆细胞产生的逆转录病毒可进一步用于转染肿瘤细胞 ,为制备肿瘤疫苗奠定了基础

Cloning of the Mouse IL-23 Gene and Its Expression in Retrovirus

To construct retroviral vector for mouse IL-23 gene, total DNA was extracted from the tumor tissues containing activated lymphocytes,and the cDNA was obtained by RT-PCR,which was just the same as that was reported in GeneBank.This cDNA was inserted into retroviral vector LXSN,transfected E.coli DH5α,and expanded. Then,two packing cell lines (ecotrophic ψ2 and amphotrophic PA317) were transfected,and the positive cellular clones were screened by G418. The expression of the desired gene was determined by PCR,RT-PCR and Northern blotting analysis. By using the above mentioned methods,the mouse IL-23 cDNA had been inserted into retroviral vector successfully,and the cellular clone PA317 with high level expression of IL-23 was produced through transfections of two packing cell lines with retroviruses.