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

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。

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

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 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 Qihe 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 Carassius auratus var.Qihe.