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

Abstract

PDF HTML阅读 XML下载 导出引用 引用提醒 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 DOI: 作者: 作者单位: 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 作者简介: 王俊丽(1971-),女,副教授,研究方向为动物营养与免疫.E-mail:wangjunli1971@126.com 通讯作者: 中图分类号: S917 基金项目: 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）. Construction of prokaryotic expression system for IL-8 of Carassius auratus var. Qihe and preparation of polyclonal antibody Author: Affiliation: 1. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;2. College of Life Sciences, Henan Normal University, Xinxiang 453007, China;3. College of Fisheries, Henan Normal University, Xinxiang 453007, China;4. Engineering Technology Research Center of Henan Province for Aquatic Animal Cultivation, Xinxiang 453007, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为揭示淇河鲫（）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1：105。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。 Abstract: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 ). 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 Escherichia coli Rosetta. A soluble fusion protein was expressed after induction by isopropyl β-D-1-thiogalactopyranoside. 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 crucian carp was successfully constructed. The purified recombinant protein was used as an immunogen 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. Immunohistochemistry results showed that the polyclonal antibody can also specifically bind to endogenous IL-8 in crucian 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 . 参考文献 相似文献 引证文献