人白细胞介素-10基因的克隆与逆转录病毒载体的构建

目的克隆、测序人白细胞介素(hIL)-10基因片段,构建逆转录病毒载体(MSCV- hIL-10)。方法采用刀豆蛋白A活化的人外周血单个核细胞培养提取总RNA,设计随机引物并逆转录反应合成hIL-10 cDNA第1链,并以hIL-10特异性引物行PCR扩增,获得hIL-10克隆基因片段,将hIL-10克隆基因片段,经双酶切后定向插入到逆转录病毒载体(MSCV)中,用脂质体法转染PT67包装细胞,以G418筛选阳性克隆。结果聚合酶链反应(PCR)扩增出534 bp特异性片段,测序结果表明同源性与GenBank报道完全一致,hIL-10基因片段插入MSCV载体中经转染包装后得到2×10~7 CFU／ml的分泌hIL-10的病毒悬液,hIL-10的分泌量为1220ng／10~6细胞·d~(-1)。结论成功克隆hIL-10开放阅读框架,成功构建MSCV-hIL-10重组质粒,获得高滴度的分泌hIL- 10的病毒悬液,hIL-10的表达可以应用于移植排斥的基因治疗。

C1oning of human inter1eukin 10 and construction of retrovirus vectors

Objective To clone and sequence human interleukin-10 gene and construct retrovirus vectors.Methods Concanavalin A-activated mononuclear cells from peripheral blood were cultured for total RNA extraction.With the use of random primers,cDNA was synthesized and then PCR was per- fored using the specific human interleukin-10 primers to obtain human IL-10 gene fragment.Human IL- 10 was inserted into MSCV vectors with double digestion.Then the recombinant vectors were transfected into PT67 package cells by lipofectin method and screened by G418 selection medium.Results A 534 bp special gene fragment was amplified with RT-PCR and sequence analysis revealed 100% homology to published data in GeneBank.Human IL-10 gene has been inserted into MSCV vectors and virus solution secreting hIL-10 obtained.Conclusion Human interleukin-10 cDNA open reading frame (ORF) was successfully cloned and MSCV-hIL-10 was constructed.The condensed virus solution with high titration of secretion hIL-10 was acquired.