论文部分内容阅读
目的：建立表达具有生物活性的人 Ｉ Ｌ１２ 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 Ｌ１２ 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 Ｌ１２ 重组杆状病毒，其感染 Ｓｆ９ 细胞的上清与 Ｉ Ｌ１２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 Ｌ１２ 的杆状病毒／昆虫表达系统。
OBJECTIVE: To establish a baculovirus / insect cell expression system that expresses a biologically active human IL-12. Methods: p40 and p35 c D N A were co-constructed into the baculovirus transfer vector p Ac U W42 and then with the baculovirus Ac U W 1. lac Z D N A co-transfected insect cells Sf9, so that the two produced homologous recombination; then the use of viral plaque experiments screening of recombinant baculovirus, and then by E L I S A selection and identification of recombinant expression of I L 12 Virus, and Western Blotting and in vitro bioactivity detection. Results: The virus plaque test, E L I S A and Western Blotting gradually positive selection of I L 12 recombinant baculovirus, the infection of Sf9 cell supernatant and I L 12 standard with the stimulation of normal human peripheral blood T lymphocyte proliferation biological activity. Conclusion: In this study, we established a recombinant baculovirus / insect expression system with human in vitro biological activity.