重组人白介素-22生物学活性检测方法研究  

Study on the Detection Method for Determining the Bioactivity of Recombinant Human Interleukin-22

作　　者：张军要[1] 黄智华 黄予良 许飞[1] 李晓颖[1] 邢瑞青[1] 傅强[1] 

机构地区：[1]四川大学华西基础医学与法医学院生物化学与分子生物学教研室,成都610041 [2]健能隆医药技术上海有限公司

出　　处：《四川大学学报（医学版）》2009年第1期162-165,共4页Journal of Sichuan University(Medical Sciences)

摘　　要：目的建立重组人白介素-22(IL-22)生物学活性的检测方法。方法将pSTAT3-TA-Luc质粒与pcDNA3.1质粒共转染HepG2细胞,经G418筛选出稳定转染的HepG2/STAT3细胞株。用重组人IL-22刺激细胞,定量检测荧光强度确定IL-22的活性。通过正交实验确定最佳检测条件,观察此方法的重复性和特异性,并进行方法对比。结果最佳检测条件为细胞密度4×105/mL,加重组人IL-22刺激4h,加入萤光素酶底物50μL。本方法检测结果为半数有效量(ED50)=16.89ng/mL,变异系数(RSD)=7.09%。中和抗体实验证实其特异性好。与ELISA法比较,该方法稳定性更好,耗时少且价格更便宜。结论选择萤光素酶为报告基因,将报告基因的检测与细胞的转录诱导机制相结合,构建了生物活性检测细胞株,建立了IL-22活性检测方法。Objective To develop a method for determining the bioactivity of recombinant human IL-22. Methods pSTAT3-TA-Luc and pcDNA3. 1 plasmids were co-transfect to HepG2 cells to generate stable HepG2/ STAT3 cell line with G418 screen. After treating cell with IL-22, the luciferase activity was assayed. Orthogonal test was used to optimize the assay condition, and then the reproducibility and specificity of the assay was checked. Results The best condition for this assay are： cell density as 4 × 10^5/mL, stimulating time as 4 hours, luciferase substrate as 50 μL. 50% effective dose （ED50） of IL-22 assayed by this method is 16.89 ng/mL, relative standard deviation （RSD） is 7. 09%. Neutralizing antibody test shows the high specificity. Comparing with ELISA, the method described here has more advantages, including higher stability, easier performance and less cost. Conclusion Luciferase reporter gene assay method is a fast, sensitive, reproducible method for IL-22 hioaetivity determination.

关 键 词：白介素-22 正交实验 报告基因 萤光素酶 

分 类 号：R927[医药卫生—药学]