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目的 探讨用生物学技术获取具有生物学活性的重组人白细胞介素 12 (rhIL 12 )的方法。方法 从经佛波醇酯 (PDBu)刺激的EB病毒转化的人B淋巴母细胞株NC 37中提取mRNA ,经逆转录 聚合酶链反应分别获得hIL 12p40和p35亚单位编码序列的cDNA ,运用重组聚合酶链反应技术 ,将两段基因通过多肽接头 (Gly4Ser) 3 DNA序列连接后进行基因重组 ,构建了重组人单链IL 12融合基因 (rh scIL 12 )。进一步将rhscIL 12融合基因插入pcDNA3 .1(+ )真核表达载体中 ,构建rhscIL 12真核表达载体 [pcDNA3 .1(+ ) rhscIL 12 ],转染COS 7细胞。结果 经G418筛选获COS rhscIL 12融合蛋白稳定表达株。Westernblot显示该融合蛋白相对分子质量为 70× 10 3,可与鼠抗人IL 12p40 p70单克隆抗体特异性结合。生物学活性测定表明 ,rhscIL 12融合蛋白具有促进外周血淋巴细胞增殖、增强NK细胞活性及提高IFN γ产生等功能。结论 用rhscIL 12融合基因构建的表达载体表达的rhscIL 12融合蛋白具有很高的生物学活性 ,为rhscIL 12的基因治疗奠定了基础。
OBJECTIVE: To explore a method for obtaining biologically active recombinant human interleukin-12 (rhIL 12) using biological techniques. Methods mRNA was extracted from EB virus-transformed human B lymphoblastoid cell line NC 37 stimulated by phorbol ester (PDBu), cDNAs encoding hIL 12p40 and p35 subunits were obtained respectively by reverse transcription polymerase chain reaction Polymerase chain reaction (PCR) was used to construct recombinant human single-stranded IL12 fusion gene (rh scIL 12) by gene recombination after the two genes were linked through the Gly4Ser 3 DNA sequence. RhscIL 12 fusion gene was further inserted into pcDNA3. 1 (+) eukaryotic expression vector to construct rhscIL 12 eukaryotic expression vector [pcDNA3. 1 (+) rhscIL 12] and transfected into COS 7 cells. Results The COS rhscIL 12 fusion protein was stably expressed by G418 screening. Western blot showed that the fusion protein had a molecular weight of 70 × 10 3 and could specifically bind to the murine anti-human IL 12p40 p70 monoclonal antibody. The biological activity assay showed that the rhscIL 12 fusion protein could promote the proliferation of peripheral blood lymphocytes, enhance the activity of NK cells and enhance the production of IFNγ. Conclusion The rhscIL 12 fusion protein expressed by rhscIL 12 fusion gene has high biological activity and lays the foundation for the gene therapy of rhscIL 12.