人IL-12在昆虫细胞中的表达及其体外生物活性的检测

目的：建立表达具有生物活性的人IL-12 的杆状病毒/昆虫细胞表达系统。方法：将p40和p35 cDNA一起构建入杆状病毒转移载体pAcUW42，然后与杆状病毒AcUW1.lacZ DNA共同转染昆虫细胞Sf9，使两者产生同源重组；随后利用病毒空斑实验筛选重组的杆状病毒，再由ELISA筛选及鉴定表达IL-12的重组病毒，并进行Western Blotting和体外生物活性的检测。结果：经病毒空斑试验、ELISA和Western Blotting逐步阳性选择的IL-12重组杆状病毒，其感染Sf9细胞的上清与IL-12标准品一样具备刺激正常人外周血T淋巴细胞增生的生物活性。结论：本研究建立了能表达具有体外生物活性的人重组IL-12的杆状病毒/昆虫表达系统。

Expression of recombinant human IL-12 by baculovirus/insect cell system and evaluation of its bioactivity in vitro

Objective: To express recombinant human IL-12 which has the activity of stimulating the proliferation of T cells in vitro by baculovirus/insect cell system. Methods: Insert p35 and p40 cDNA into baculovirus transfer vector pAcUW42, then co-transfect insect Sf9 cells with AcUW1.lacZ baculobvirus DNA for constructing IL-12 expression recombinant baculovirus by homologous recombination. Results: Culture supernatant of Sf9 cells infected by IL-12 recombinant baculovirus which selected by a series of assay of baculovirus plaques, ELISA and Western Blotting, could stimulate the proliferation of T cells from normal human peripheral blood as standard IL-12 did. Conclusion: We have established the IL-12 baculovirus/insect cell expression system whose product has the activity of stimulating the proliferation of T cells in vitro