【作者】 肖凡书；

【导师】 肖调义； 聂品；

【作者基本信息】 湖南农业大学 ， 水产养殖， 2006， 硕士

【摘要】 白细胞介素10(IL-10)是一种作用广泛的抗炎细胞因子，主要功能是限制和最终终止炎症反应。本研究采用RACE(rapid amplification of cDNA ends)-PCR方法首次扩增出鲢IL-10的cDNA，其全长为1248bp，包含5′非编码区156bp，3′非编码区552bp，开放阅读框540bp。鲢IL-10的开放阅读框编码179个氨基酸，其中包含构成两对二硫键的四个保守半胱氨酸。以15种动物及病毒的IL-10氨基酸序列构建系统发育树，并进行了相应的探讨。RT(reverse transcriptase)-PCR结果显示鲢IL-10 mRNA主要在脾脏、鳃、头肾和肌肉中表达。 根据鲢IL-10的cDNA序列设计含有酶切位点的表达引物，将扩增的鲢IL-10完整开放阅读框和原核表达载体pET-32a-c(+)双酶切之后，连接、构建重组表达质粒，经测序无误后，将重组质粒转入大肠杆菌Rosetta-gami(DE3)中，用IPTG诱导表达。经SDS-PAGE电泳后显示重组融合蛋白在分子量约39kD左右处有明显表达条带，与预期分子量大小一致，且主要以不可溶的包涵体形式存在。变性条件下利用His·Bind树脂成功纯化融合蛋白，将其免疫日本大耳兔，获得兔多克隆抗鲢IL-10抗体，Western blot检测到兔多克隆抗鲢IL-10抗体可有效地与重组蛋白结合。这为进一步研究IL-10的功能奠定了基础。更多还原

【Abstract】 Interleukin-10 (IL-10) is an important anti-inflammation cytokine, functioning mainly in inflammation. In this study, IL-10 cDNA was cloned from silver carp (Hypophthalmichthys molitrix) using RACE (rapid amplification of cDNA ends)-PCR. The full IL-10 cDNA consisted of 1248 bp, including a 156 bp 5’ untranslated region (UTR), a 552 bp 3’-UTR, and a 540 bp open reading frame (OPR) encoding a 179 amino acid peptide with four conservative cysteine (Cys) forming two pairs of disulphide bridges. As revealed by the reverse transcriptase PCR (RT-PCR), the IL-10 mRNA was mainly expressed in the spleen, gill, head kidney and muscle of the silver carp.The expression primers coding for the open reading frames of IL-10 were designed. The DNA products and vector pET-32a were digested by the two same enzymes, and then ligated to construct a prokaryotic expression vector pET-32a-IL-10 with a T7 promoter. The recombinant plasmid was examined by sequencing, and then transformed into the prokaryotic cell E. coli Rosetta-gami (DE3) for expression under the induction of IPTG After the ultrasonic disruption, the expression products were analyzed by sodium dodecyl sulfate-polyacrylamide gel elactrophoresis (SDS-PAGE). The IL-10 was expressed efficiently in E. coli Rosetta-gami (DE3) and the expression products were 39 kD fusion protein in the form of inclusion. The expressed protein was purified using HisBind Resin in the denatured condition and the purified protein was injected into rabbit in order to obtain polyclonal antisera. Using Western blot analyses, the recombinant protein was recognized by the rabbit antisera effectively. The results establish the foundation for further study of the function of IL-10.更多还原