报告基因法测定人干扰素γ药物的生物学活性

A Reporter Gene Assay for Determination of Biological Activity of InterferonγDrugs

目的建立一种用于人干扰素γ药物生物学活性测定的报告基因法,以提高人干扰素γ药物生物学活性测定的生物安全性、简便性和精密度。方法构建含有人干扰素γ反应性DNA元件和报告基因的转基因细胞,建立使用该细胞测定人干扰素γ药物生物学活性的报告基因法,进行方法学验证,评价其与细胞病变抑制法测定结果的一致性。结果构建了含有干扰素γ活化位点和荧光素酶报告基因的转基因HeLa细胞,建立了人干扰素γ药物生物学活性的测定方法,该方法的剂量反应曲线符合四参数Logistic曲线,具有良好的专属性,3批样品的加标回收率在80%~120%之间,试验内变异系数小于8%,试验间变异系数小于11%,操作人员间变异系数小于15%,细胞代次、细胞接种密度和药物作用时间等试验条件的变化对测定结果无显著影响,该方法与病毒抑制法的测定结果之间有很高的一致性。结论建立的报告基因法不需要使用病毒,操作简便,具有良好的专属性、准确度、精密度、耐用性,可作为病毒抑制法的替代方法,用于人干扰素γ药物生物学活性测定。

OBJECTIVE To establish a reporter gene assay for the determination of biological activity of human interferonγdrugs,in order to improve the biosafety,simplicity and precision of the determination of biological activity.METHODS Transgenic cells containing human interferonγreactive DNA elements and a reporter gene were constructed,a reporter gene assay was established based on the cells to determine the biological activity of human interferonγdrugs,methodological verification was conducted,and its consistency with the results of cytopathic effect inhibition assay was evaluated.RESULTS HeLa transgenic cells containingγinterferon activation site and a luciferase reporter gene were constructed,and a assay for determining the biological activity of human interferonγwas established.The dose-response curve of the assay conformed to the four parameter logistic curve,and the assay had good specificity.The recovery rate of the three batches of samples was between 80%and 120%.The coefficient of variation(CV)within the test was less than 8%,the CV value between the tests was less than 11%,and the CV value between the operators was less than 15%.Changes in test conditions such as cell generation,inoculation density and drug action time had no significant effect on the determination results.There was a high consistency between the determination results of this assay and cytopathic effect inhibition assay.CONCLUSION The established reporter gene assay does not require the use of viruses,is easy to operate,has good specificity,accuracy,precision,and durability.It can be used as an alternative assay to cytopathic effect inhibition assay for the determination of the biological activity of human interferonγdrugs.