马γ-干扰素单克隆抗体的制备及细胞免疫评价方法的研究

发布时间：2018-07-08 20:14

本文选题：马IFN-γ + 单克隆抗体　；参考：《中国农业科学院》2008年博士论文

【摘要】： γ-干扰素(IFN-γ)是机体内一种具有抗病毒、抗肿瘤和免疫调节功能的重要细胞因子。研究表明,内源性IFN-γ水平的高低在很大程度上可以反映机体的细胞免疫状态,因此对样本中的IFN-γ进行定量分析对免疫机制研究、免疫功能分析、疫苗免疫效果评估、器官移植、过敏反应以及多种胞内病原感染的诊断等方面都具有极其重要的理论意义和应用价值。然而,迄今没有应用于检测马IFN-γ的单克隆抗体,用以建立相应的检测方法。本研究旨在建立检测马IFN-γ(Equine interferon-γ, EIFN-γ)抗原捕获ELISA、细胞内细胞因子染色(ICS)和酶联免疫斑点试验(ELISPOT))等方法,从而为监测马机体免疫状态、研究机体感染或免疫过程中的免疫效应及探讨相关免疫机制等提供技术平台。为此,本研究在克隆表达EIFN-γ之后将其免疫小鼠制备了马IFN-γ单克隆抗体,然后分别对其进行生物素和荧光素(FITC)标记制备了生物素标记的马IFN-γ单克隆抗体和FITC标记的马IFN-γ单克隆抗体,在此基础上成功建立了检测EIFN-γ的相关免疫学方法。首先,从ConA刺激的马外周血单核细胞(PBMC)中采用RT-PCR扩增获得含信号肽的马IFN-γ的编码序列,将其克隆至载体pCR2.1-TOPO中。然后以从重组质粒pCR-EIFN-γ为模版扩增获得编码马IFN-γ成熟蛋白(mature EIFN-γ, mEIFN-γ)的基因,并将其亚克隆至原核表达载体pET-28a(+)。经PCR、酶切及序列测定后所获得的重组子转化至大肠杆菌感受态细胞BL21(DE3),经IPTG诱导,将其产物进行SDS-PAGE分析和Western blot鉴定。利用表达绿色荧光蛋白(GFP)的重组水泡性口炎病毒(VSV*GFP)作为指示病毒对所表达的EIFN-γ进行抗病毒活性测定。结果表明,mEIFN-γ开放阅读框全长为441bp,编码146个氨基酸;其在大肠杆菌表达系统的表达产物以可溶性和包涵体两种形式存在,重组蛋白相对分子量约为18 ku,且具有免疫反应活性。采用Ni+亲和层析对大肠杆菌表达系统所表达的mEIFN-γ在非变性和变性条件下分别进行纯化,均获得了高纯度的重组马IFN-γ,且该重组蛋白具有抗病毒活性,其活性单位为1×103 AU/mL。将原核表达的重组EIFN-γ免疫BALB/c小鼠(Mus musculus),经4次免疫后,取脾细胞与SP2/0骨髓瘤细胞进行融合,以昆虫杆状病毒表达系统获得的重组马IFN-γ作为检测抗原进行间接ELISA检测筛选,最终获得12株能稳定分泌抗体的单细胞克隆株。利用间接免疫荧光(IFA)实验对12株单克隆抗体进行鉴定,结果表明,所获得的12株细胞分泌的抗体均为针对EIFN-γ的特异性抗体。单克隆抗体亚型鉴定结果显示,其中4株为IgG1、2株为IgG2a、3株为IgG2b和其余3株为IgM,而且所有12株单克隆抗体的轻链均为κ链。将此12株单克隆抗体分别与两个截短表达的重组马IFN-γ蛋白(GST-mEIFN-γ(1-84)和GST-mEIFN-γ(80-146))进行特异性ELISA检测,结果表明,其中有7株单克隆抗体识别GST-mEIFN-γ(1-84),而另5株单克隆抗体与GST-mEIFN-γ(80-146)发生反应,说明所获得的12株单克隆抗体至少针对两个或两个以上不同的抗原表位。而且,其中一株单抗(SB10)具有抑制重组EIFN-γ抗病毒活性作用。将两株识别不同表位的抗马IFN-γ单克隆抗体腹水(A5和SB10)纯化后,对其中一株纯化后的单抗(SB10)进行生物素标记,通过对重组EIFN-γ进行ELISA检测来建立马γ-干扰素双抗体夹心ELISA。经过方阵滴定实验确定捕获抗体的最佳浓度为1μg/mL,检测抗体的工作效价为1: 1000。利用建立的方法对不同稀释度的重组马IFN-γ和丝裂原刺激的马外周血单核细胞培养上清进行检测。结果表明,此方法检测敏感度达到1ng/mL,特异性良好。利用荧光素FITC对纯化后的抗马IFN-γ单克隆抗体(SB10)进行标记,制备FITC标记的抗EIFN-γ单克隆抗体,然后采用该抗体对马外周血单核细胞进行胞内EIFN-γ单染色,之后进行流式细胞术检测,并且与商品化的FITC标记的抗牛IFN-γ单克隆抗体(CC302)进行对比试验。结果表明,FITC标记的抗EIFN-γ单抗能够有效地应用于刺激后的马外周血单核细胞的胞内染色检测。分别将识别EIFN-γ不同表位的配对单抗作为捕获抗体和检测抗体,对经刺激的马PBMC所分泌的IFN-γ进行ELISPOT检测,从而建立检测马γ-干扰素ELISPOT方法。经过方阵滴定实验确定捕获抗体的包被浓度为1μg/mL,检测抗体的工作浓度为1: 1000。本研究所初步建立的检测EIFN-γ的抗原捕获ELISA、细胞内细胞因子染色及ELISPOT方法,可为监测马机体免疫状态、研究机体感染或免疫过程中的免疫效应及探讨相关免疫机制等提供技术平台。
[Abstract]:Interferon gamma (IFN- gamma) is an important cytokine with antiviral, anti-tumor and immunomodulatory functions in the body. The study shows that the level of endogenous IFN- gamma can reflect the cellular immune state of the body to a large extent. Therefore, the quantitative analysis of IFN- gamma in the sample is used to study the immune mechanism, the immune function analysis, and the vaccine. Immunological evaluation, organ transplantation, anaphylaxis and diagnosis of multiple intracellular pathogens are of great theoretical and application value. However, no monoclonal antibodies used to detect IFN- gamma have been used so far to establish a corresponding detection method. The aim of this study is to establish the detection of horse IFN- gamma (Equine interferon- gamma, EIFN- gamma) antigen capture ELISA, intracellular cytokine staining (ICS) and enzyme linked immunosorbent assay (ELISPOT), which provide a technical platform for monitoring the immune state of the body, studying the immune effect in the body infection or immune process and exploring the related immune mechanism.
In this study, the monoclonal antibody of horse IFN- gamma was prepared after cloning and expression of EIFN- gamma. Then, biotin and fluorescein (FITC) were used to produce biotin labeled McAbs of Ma IFN- gamma and McAb labeled by FITC. On this basis, the relationship between EIFN- gamma and EIFN- gamma was successfully established. Immunological methods.
First, the encoding sequence of horse IFN- gamma containing signal peptides was amplified by RT-PCR amplification from ConA stimulated peripheral blood mononuclear cells (PBMC), and was cloned into the carrier pCR2.1-TOPO. Then, the gene encoding Ma IFN- gamma mature protein (mature EIFN- gamma, mEIFN-) was amplified from the recombinant plasmid pCR-EIFN- gamma, and was subcloned to the original. Nuclear expression vector pET-28a (+). The recombinant protein obtained by PCR, enzyme digestion and sequencing was converted to BL21 (DE3) of Escherichia coli cells, and was induced by IPTG, and its products were identified by SDS-PAGE and Western blot. Recombinant vesicular stomatitis virus (VSV*GFP) expressed by the expression of green fluorescent protein (GFP) was used as a indicator of the expression of the virus. The antiviral activity of EIFN- gamma was measured. The results showed that the total length of the mEIFN- gamma open reading frame was 441bp and encoded 146 amino acids, and the expression product of the Escherichia coli expression system existed in two forms of soluble and inclusion bodies. The relative molecular weight of the recombinant protein was about 18 Ku, and it had the activity of immunoreaction. Ni+ affinity chromatography was used for the Escherichia coli. MEIFN- gamma expressed in the expression system was purified under the conditions of non denaturation and denaturation respectively, and high purity recombinant horse IFN- gamma was obtained. The recombinant protein had antiviral activity and its active unit was 1 x 103 AU/mL..
The recombinant EIFN- gamma immunized BALB/c mice (Mus musculus) were immunized with the prokaryotic expression. After 4 times immunization, the spleen cells were fused with the SP2/0 myeloma cells, and the recombinant horse IFN- gamma obtained by the insect baculovirus expression system was used as the detection antigen for indirect ELISA detection. Finally, 12 single cell clones capable of stabilizing the antibody were obtained. 12 monoclonal antibodies were identified by indirect immunofluorescence (IFA) test. The results showed that the antibodies secreted by 12 cells were specific antibodies against EIFN- gamma. The results of monoclonal antibody subtype identification showed that 4 of them were IgG1,2 and 3 were IgG2b and the other 3 strains were IgM, and all 12 monoclonal antibodies were light. The chain was kappa. The 12 monoclonal antibodies and two truncated recombinant horse IFN- gamma protein (GST-mEIFN- gamma (1-84) and GST-mEIFN- gamma (80-146)) were detected by specific ELISA. The results showed that 7 monoclonal antibodies identified GST-mEIFN- gamma (1-84), and the other 5 monoclonal antibodies responded to GST-mEIFN- gamma (80-146). 12 monoclonal antibodies were obtained for at least two or more than two different epitopes. Moreover, one of the monoclonal antibodies (SB10) could inhibit the antiviral activity of recombinant EIFN- gamma.
After purification of two different epitopes of anti horse IFN- gamma monoclonal antibody ascites (A5 and SB10), one of the purified monoclonal antibodies (SB10) was labeled with biotin. By ELISA detection of recombinant EIFN- gamma, the optimum concentration of the captured antibody was determined to be 1 u g/mL by a square matrix titration experiment. The work titer of detection antibody was 1: 1000.. The method was used to detect the supernatant of horse peripheral blood mononuclear cell culture of recombinant horse IFN- gamma and mitogen stimulated by different dilution degrees. The results showed that the sensitivity of this method was 1ng/mL and the specificity was good.
The purified anti horse IFN- gamma monoclonal antibody (SB10) was labeled with fluorescein FITC, and the anti EIFN- gamma monoclonal antibody labeled with FITC was prepared. Then the antibody was used to carry out intracellular EIFN- gamma staining of the horse peripheral blood mononuclear cells, then the flow cytometry was performed, and the anti bovine IFN- gamma monoclonal antibody (C) labeled with commercialized FITC (C) was used. C302) comparative test. The results showed that FITC labeled anti EIFN- gamma monoclonal antibody could be effectively applied to detect intracellular staining of peripheral blood mononuclear cells after stimulation.
The matched McAbs of different epitopes of EIFN- gamma were used to capture and detect antibodies, respectively, to detect the ELISPOT of IFN- gamma secreted by the stimulated horse PBMC, and to establish the detection method of IFN- gamma interferon ELISPOT. The concentration of the captured antibody was determined by the square matrix titration experiment and the concentration of the antibody was 1 mu g/mL, and the working concentration of the antibody was 1: 1000..
The antigen capture ELISA of EIFN- gamma, intracellular cytokine staining and ELISPOT method initially established in this study can provide a technical platform for monitoring the immune status of the horse body, studying the immune effect in the body infection or immune process and exploring the related immune mechanism.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【引证文献】