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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。
Objective To clone and sequence human interleukin (hIL) -10 gene and construct retroviral vector (MSCV-hIL-10). Methods Total RNA was extracted from human peripheral blood mononuclear cells activated by concanavalin A. Total RNA was extracted from hIL-10 cDNA by random primers and reverse transcription reaction. The hIL-10 specific primers were used to amplify hIL -10 cloned gene fragment, the hIL-10 cloned gene fragment was double digested and inserted into the retroviral vector (MSCV), liposome transfected PT67 packaging cells to G418 screening positive clones. Results The 534 bp fragment was amplified by polymerase chain reaction (PCR). The sequencing results showed that the homology was completely consistent with that reported in GenBank. The hIL-10 gene fragment was inserted into MSCV vector and transfected to obtain 2 × 10 ~ 7 CFU / Ml secretion of hIL-10 virus suspension, hIL-10 secretion of 1220ng / 10 ~ 6 cells · d ~ (-1). Conclusion The hIL-10 open reading frame was successfully cloned and the recombinant plasmid of MSCV-hIL-10 was constructed successfully. High titer hIL-10 secreting virus suspension was obtained. The hIL-10 expression could be used in gene therapy for transplant rejection.