鲤细胞因子多克隆抗体的制备及检测

Preparation and detection of cytokines polyclonal antibodies of Cyprinus carpio

摘要:

为从蛋白质水平研究细胞因子在鲤体内免疫应答过程中的合成变化，本研究采用PCR技术克隆TNF-α、IL-1β、IL-6、IL-12、IL-10和TGF-β基因含有部分抗原决定簇的片段，引入双酶切位点BamH Ⅰ和Hind Ⅲ后连接至pET-32a/21a，构建相应的表达载体，制备多克隆抗体。采用ELISA检测抗体效价，并以此作为实验工具，检测经嗜水气单胞菌感染后鲤血清中炎性细胞因子的合成变化。结果显示，基因TNF-α、IL-1β、IL-6、IL-12、IL-10和TGF-β融合蛋白分子量分别约为31.8、31.7、35.3、32.5、18.0和33.6 ku；抗体效价达到2.4×10⁶；在病原菌感染后的不同阶段，促炎细胞因子TNF-α、IL-1β、IL-6、IL-12和抗炎细胞因子IL-10、TGF-β呈现出不同的合成变化。研究表明，制备的抗体具有较高的效价、亲和力和特异性，可用于鲤细胞因子的定量研究，该抗体的获得为鲤免疫应答与细胞因子合成的系统研究奠定了基础。同时，获取的鲤细胞因子TNF-α、IL-1β、IL-6、IL-12、IL-10和TGF-β的抗体亦可用于其他鱼类细胞因子蛋白质水平的定量研究。

关键词:鲤;细胞因子;原核表达;抗体效价;病原菌;ELISA

Abstract:

Inflammatory cytokines are often used as one of the indicators to detect host immune responses. The lack of cytokines antibodies for fish has hindered analysis of cytokines protein expression by ELISA. In this paper, the fragments of TNF-α, IL-1β, IL-6, IL-12, IL-10 and TGF-β genes of Cyprinus carpio, containing partial antigenic determinants were cloned by PCR. The target sections of cytokines were cloned into plasmid pET-32a/21a vectors which had restriction sites for BamH Ⅰ and Hind Ⅲ. The recombinant plasmids were transformed into E. coli BL21 (DE3). The fusion proteins were highly expressed in E. coli BL21 (DE3) after being induced with IPTG. The purified fusion proteins were used as antigen to immunize New Zealand Rabbits by ear margin veins injection combined with subcutaneous injection to obtain rabbit anti carp polyclonal antibodies of cytokines. Enzyme-Linked Immuno Sorbent Assay (ELISA) was used to evaluate the antibody titers, and the antibodies were used as a test tool to detect the expression of inflammatory cytokines in carp serum after infection with Aeromonas hydrophila. The results showed that the sizes of fusion proteins were about 31.8, 31.7, 35.3, 32.5, 18.0 and 33.6 ku, respectively. The titers of the antiserum were about 2.4×10⁶. The pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-12 and anti-inflammatory cytokines IL-10 and TGF-β showed different expression patterns at different stages after A. hydrophila infection. The results indicated that the prepared antibodies performed high titer, affinity and specificity and could be applied to study the expression of cytokines in C. carpio. The availability of these polyclonal antibodies laid the foundation for the systematic study of immune response and cytokines expression in C. carpio. Meanwhile, these polyclonal antibodies could also be used to exploratively study the cytokines protein expression of TNF-α, IL-1β, IL-6, IL-12, IL-10 and TGF-β in other fishes.

Key words:Cyprinus carpio;cytokines;prokaryotic expression;antibody titer;pathogenic bacteria;ELISA