鸡IL-4在杆状病毒表达系统中的表达及其生物学活性分析

摘要

鸡(Gallus gallus)白细胞介素4(interleukin-4,IL-4)是家禽2型辅助性T细胞(type 2 T helper,Th2)免疫应答的重要指示分子,对家禽免疫应答、免疫功能分析具有重要作用,由于缺乏有效的检测工具,关于鸡IL-4(ChIL-4)的研究目前较滞后.为制备具有生物学活性的重组ChIL-4,本研究采用分子生物学技术,从实验室前期构建的重组质粒pVAX1-ChIL-4中双酶切获得ChIL-4基因片段,并将其克隆入杆状病毒表达系统的转移载体pFastBac1,构建重组质粒pFast-ChIL-4,通过位点特异性转座,将ChIL-4基因整合到Bacmid穿梭载体中,构建重组质粒pBac-ChIL-4并转染草地贪夜蛾细胞系9(Spodoptera frugiperda,Sf9),进行蛋白表达;采用间接免疫荧光法(indirect immunofluorescence,IFA)、间接酶联免疫吸附测定(enzyme-linked immuno sorbent assay,ELISA)对重组ChIL-4(Bac-ChIL-4)的表达进行分析;采用鸡脾脏淋巴细胞增殖实验对重组蛋白的生物学活性进行初步分析.实验结果表明,重组ChIL-4在杆状病毒表达系统中获得成功表达,转染重组质粒Bac-ChIL-4的Sf9细胞中可见亮绿色荧光;将含有重组蛋白的Sf9细胞上清浓缩后进行间接ELISA检测,结果显示,抗ChIL-4的多抗血清仅识别重组ChIL-4;鸡脾脏淋巴细胞增殖实验结果表明,重组ChIL-4能够刺激鸡脾脏淋巴细胞的增殖,说明该重组蛋白具有较好的生物学活性.ChIL-4重组蛋白的获得为其进一步开发应用以及功能学研究提供了重要的生物材料.%Chicken (Gallus gallus) interleukin-4 (IL-4),which is a key indicator for Th2 type immune response in poultry,is very important in immune response and immune function analysis of chickens (Gallus gallus).Due to the lack of effective assays,the study of chicken IL-4 (ChIL-4) is still lagged behind other cytokines.To prepare recombinant ChIL-4 with high bioactivities,molecular biotechnology was used to construct recombinant vector pBac-ChIL-4,and then pBac-ChIL-4 expressed in Spodopterafurgiperda 9 (Sf9)cells.Firstly,ChIL-4 cDNA fragment was digested from plasmid pVAX1-ChIL-4 constructed previously with restriction enzymes and then subcloned to vector pFastBacl to construct a new plasmid pFast-ChIL-4.With site-specific transposition,ChIL-4 cDNA fragment was transformed to vector bacmid in the baculovirus vector system to form recombinant plasmid pBac-ChIL-4.Secondly,pBac-ChIL-4 was transfected to Sf9 cells to generate recombinant ChIL-4 protein (rChIL-4 or Bac-ChIL-4).Finally,infected Sf9 cells were identified by immunofluorescent assay (IFA) for its expression of ChIL-4,and the supernatants of Sf9 cells infected were harvested and further analyzed by indirect enzyme-linked immuno sorbent assay (ELISA) and lymphocytes proliferation assay.The results of IFA indirect ELISA showed that rChIL-4 successfully expressed in the baculovirus vector system with bright green fluorescence observed in transfected Sf9 cells,polyclonal antibody against ChIL-4 generated previously could only reacted with rChIL-4 (Bac-ChIL-4,His-ChIL-4),but not other irrelative proteins (bovine IL-4,chicken IFN-γ) revealed by ELISA.Splenocytes proliferation assay demonstrated that rChIL-4 had good bioactivity in stimulating and activating T lymphocytes.This study not only facilitates the structural and functional analysis of ChIL-4,but also provides helpful reference value for the expression of other cytokines.