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目的 构建小鼠IL 12单链融合基因 (msIL 12 ) ,进行基因治疗初探。方法 用RT PCR法从小鼠腹腔巨噬细胞总RNA分别获取p4 0cDNA和p35cDNA ,2次PCR引入linker后 ,依次克隆入pcDNA3质粒 ,构建成msIL 12真核表达质粒 (pmsIL 12 ) ;用DEAE dextran法将PEG纯化的pmsIL 12转染COS 7细胞 ,用ELISA法检测其培养上清 (转染上清 )IL 12蛋白含量。皮内注射PEG纯化的pmsIL 12 ,用MTT法检测其对正常小鼠脾细胞NK活性的影响 ,观察其对荷H2 2腹水型肝癌细胞瘤小鼠生存期的影响。结果 所得p35cDNA序列与GenBank登录号M86 6 72完全一致 ;所得IL 12p4 0cDNA序列与GenBank登录号AH0 0 4 85 9报道完全相同 ,除第 10 0 0位碱基是G而不是A(但所编码氨基酸相同 ,都是丝氨酸 )外也与GenBank登录号M86 6 71报道相同。成功构建了小鼠IL 12单链融合基因真核表达质粒pmsIL 12 ,其转染COS 7细胞后的转染上清IL 12蛋白含量达 (2 .5 5± 0 .6 0 )ng ml。皮内注射pmsIL 12后 ,使正常小鼠脾细胞NK活性显著增强 (P <0 .0 1) ,使荷H2 2肝癌细胞瘤小鼠的生存期明显延长(P <0 .0 5 )。结论 构建了小鼠IL 12单链融合基因的真核表达质粒 ,可用于基因治疗研究。
Objective To construct murine IL 12 single chain fusion gene (msIL 12) for gene therapy. Methods p40 cDNA and p35 cDNA were respectively obtained from mouse peritoneal macrophages total RNA by RT-PCR. Two PCR reactions were performed to introduce the linker into pcDNA3 plasmid. The recombinant plasmid was constructed into msIL 12 eukaryotic expression plasmid (pmsIL 12) COS-7 cells were transfected with PEG-purified pmsIL 12, and IL 12 protein content of the culture supernatant (transfection supernatant) was detected by ELISA. Intraperitoneal injection of PEG-purified pmsIL 12, the MTT assay of NK cells in normal mouse spleen cells, and observe the impact on the survival of mice bearing H 2 ascites hepatocellular carcinoma. Results The sequence of p35 cDNA was identical to GenBank accession number M86 6 72. The sequence of IL12p40 cDNA obtained was exactly the same as reported in GenBank accession number AH0 0 4 85 9 except that the base at position 10 0 was G but not A (but the encoded amino acid Same, both serine) are also reported as GenBank Accession No. M86 671. The mouse IL 12 single-chain fusion gene eukaryotic expression plasmid pmsIL 12 was successfully constructed. The transfected COS 7 cells transfected with IL 12 protein content (2.55 ± 0.60) ng ml. After intradermal injection of pmsIL12, the NK activity of splenocytes in normal mice was significantly increased (P <0.01), which significantly prolonged the survival of mice bearing H2-HCC (P <0.05). Conclusion The eukaryotic expression plasmid of murine IL 12 single-strand fusion gene was constructed and could be used for gene therapy research.