淇河鲫IL-8原核表达系统的构建及多克隆抗体的制备与鉴定  

Construction of prokaryotic expression system for IL-8 of Carassius auratus var. Qihe and preparation of polyclonal antibody

作　　者：王俊丽[1,2] 陈金顶[1] 卢荣华[3,4] 雒燕婷[2] 聂国兴[3,4] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]河南师范大学生命科学学院,河南新乡453007 [3]河南师范大学水产学院,河南新乡453007 [4]河南省水产动物养殖工程技术研究中心,河南新乡453007

出　　处：《中国水产科学》2017年第5期970-976,共7页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(31372545);河南省高校科技创新团队支持计划项目(14IRTSTHN013);河南省科技计划基金项目(142300410159);河南省教育厅科学技术重点研究项目(14A180007);河南省自然科学基金项目(162300410165)

摘　　要：为揭示淇河鲫(Carassius auratus var.Qihe)白细胞介素-8(interleukin-8,IL-8)的功能,本研究构建了原核表达系统,并制备了兔抗鲫IL-8多克隆抗体。首先采用RT-PCR法扩增淇河鲫IL-8基因编码序列中不含信号肽的基因片段,克隆到pET-32a(+)载体后转化入Rosetta菌株构建原核表达系统。经IPTG诱导表达出相对分子质量约27.8 kD的目标融合蛋白。将纯化的融合蛋白免疫家兔制备多克隆抗体,效价达1︰10~5。经免疫亲和层析纯化的抗体能特异性识别重组和天然的淇河鲫IL-8蛋白。比较不同组织的免疫组化和荧光定量PCR结果,IL-8蛋白量和mRNA的表达量在不同组织间变化趋势一致,在肌肉、脾和头肾均检测到较高的表达量,肠道的表达量较低。本研究为进一步探讨淇河鲫IL-8的生物学功能奠定了基础。Interleukin-8 （IL-8）, a chemokine that participates in the early inflammatory process, plays a key role in mediating resistance to infection in animals. However, there has been no information about IL-8 at the protein level for characterizing the regulatory role of this molecule during the immune response in fish. In this work, we constructed a prokaryotic expression system and prepared a polyclonal antibody against IL-8 of Qihe crucian carp （Carassius auratus var. Qihe）. The coding region without a signal peptide sequence was first amplified and cloned into pET-32a（＋）, a prokaryotic expression plasmid. The recombinant plasmid was then transformed into Es- cherichia coli Rosetta. A soluble fusion protein was expressed after induction by isopropyl 13-D-1-thiogalac- topyranoside. The purified protein was detected as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a predicted molecular mass of 27.8 kD, suggesting that the prokaryotic expression system for IL-8 of Qihe crucian carp was successfully constructed. The purified recombinant protein was used as an immu- nogen to raise polyclonal antibodies in New Zealand rabbits. The serum antibody titer reached 1 ： 105, as detected by indirect enzyme-linked immunosorbent assay. The antibody was purified by affinity chromatography and had a high binding activity and specificity for the recombinant protein antigen, as measured by Western Blot. Immuno- histochemistry results showed that the polyclonal antibody can also specifically bind to endogenous IL-8 in cru- cian carp tissues. Comparing the results of immunohistochemistry with that of fluorogenic quantitative polymerase chain reaction, the expression of IL-8 was consistent between the protein and mRNA levels, and its expression ranked among tissues as follows： muscle 〉 spleen 〉 head kidney 〉 intestine. This study provides a material basis for further research on the role of IL-8 in the immune response of Carassius auratus var. Qihe.

关 键 词：淇河鲫 白细胞介素-8 原核表达系统 多克隆抗体 组织表达 免疫组化 

分 类 号：S917[农业科学—水产科学]