人IL-12在昆虫细胞中的表达及其体外生物活性的检测

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

目的：建立表达具有生物活性的人 Ｉ Ｌ１２ 的杆状病毒／昆虫细胞表达系统。方法：将ｐ４０ 和ｐ３５ ｃ Ｄ Ｎ Ａ 一起构建入杆状病毒转移载体ｐ Ａｃ Ｕ Ｗ４２，然后与杆状病毒 Ａｃ Ｕ Ｗ １．ｌａｃ Ｚ Ｄ Ｎ Ａ 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 Ｅ Ｌ Ｉ Ｓ Ａ 筛选及鉴定表达 Ｉ Ｌ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、 Ｅ Ｌ Ｉ Ｓ Ａ 和 Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ 逐步阳性选择的 Ｉ Ｌ１２ 重组杆状病毒，其感染 Ｓｆ９ 细胞的上清与 Ｉ Ｌ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 Ｉ Ｌ１２ 的杆状病毒／昆虫表达系统。

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