鸡白细胞介素18成熟蛋白基因克隆、表达及多克隆抗体制备
摘要
白细胞介素-18(Interleukin-18，IL-18)是一种能诱导IFN-γ产生的、新发现的细胞因子。目前在分子水平上已经证明人、哺乳动物和某些鸟类中都存在IL-18。IL-18具有多种生物学功能，它不仅可以增强Th1型免疫反应，而且在Th2型免疫反应中也发挥一定的作用。人类医学研究证明IL-18在抗微生物感染、抗肿瘤免疫中具有应用潜力。在集约化畜禽养殖业中，包括IL-18在内的细胞因子作为天然的免疫调节剂，目前已被公认为是一种具有吸引力的、可以替代传统疗法的新型治疗剂。鸡IL-18(Chicken interleukin-18，ChIL-18)基因是最近才被发现的，由于它具有与人和哺乳动物相似的生物学功能，因而ChIL-18在比较免疫学研究和禽病治疗中具有重要意义。
本实验根据已发表的ChIL-18 cDNA基因序列设计引物，用反转录-聚合酶链式反应(Reverse-translation polymerase-chain reaction，RT-PCR)技术从有丝分裂原刀豆蛋白A(Concanavalin A，ConA)刺激48小时活化的鸡脾细胞中扩增出编码ChIL-18成熟蛋白的基因，并进行序列测定。结果表明本研究克隆到的ChIL-18成熟蛋白编码基因全长为507bp，含有一个开放阅读框架。与Schneider等报道的序列有一处碱基不同。即本序列在482位为C，而Schneider等的序列该处为T，这一核苷酸序列的变化导致了推导的对应氨基酸残基由苯丙氨酸变为丝氨酸。
在克隆到编码ChIL-18成熟蛋白的基因后，将此基因亚克隆至原核表达载体pPROEX~(TM)HTb中，构建重组质粒并进行确证性序列测定。然后将重组质粒转化大肠杆菌DH5α并用IPTG于37℃诱导培养获得表达，表达形式为包涵体。凝胶薄层灰度扫描显示表达的融合蛋白约占菌体蛋白的30％。SDS-PAGE和Western blot分析显示，表达的鸡IL-18融合蛋白分子量约为23ku。包涵体通过6M盐酸胍裂解后，利用镍离子亲和树脂进行纯化。用所获得的重组ChIL-18融合蛋白及其纯化产物经三次肌肉注射豚鼠，制备豚鼠抗ChIL-18多克隆抗体。琼脂扩散实验表明制备的多克隆抗体与ChIL-18具有良好的反应性。
本实验成功克隆到编码ChIL-18成熟蛋白的基因，原核表达、纯化了ChIL-18融合蛋白，并制备了豚鼠抗ChIL-18多克隆抗体，为下一阶段ChIL-18生物学特性及其应用的研究打下了坚实基础。
Interleukin-18 (IL-18) is a novel discovered cytokine, which is the potent inducer of IFN-Y production. It has been demonstrated that interleukin-18 exist in cells of human, mammal and some kinds of birds. Interleukin-18 plays an important role in host defense against various microorganisms. In addition, interleukin-18 is expected to be a target for tumor immunotherapy. Cytokines including interleukin-18, as natural mediators of the immune response, offer exciting alternatives to conventional therapeutics in control of disease in intensive livestock and poultry industries. The discovery of chicken interleukin-18 gene is very meaningful for both the study on comparative immunology and the therapeutics for poultry diseases.In this study, specific primers for chicken interleukin-18 (ChIL-18) cDNA have been designed and synthesized according to the previously reported nucleic acid sequence of chicken interleukin-18 cDNA. Using total RNA from ConA-stimulated chicken spleen cells, chicken IL-18 cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis indicated that there is one nucleotide different from the published ChIL-18 cDNA sequence in 482 (T-C).To highly express chicken interleukin-18 in E.coli and purify the fusion protein, cDNA fragment encoding the mature ChIL-18 was subcloned into the prokaryotic expression vector pPROEX橦Tb. The recombinant plasmid was transformed into E.coli, and then induced by IPTG at 37 C. The recombinant ChIL-18 (rChIL-18) was expressed efficiently in forms of inclusion body with the yield accounting for 30% total bacteria protein. SDS-PAGE and Western blot analysis showed that the recombinant fusion protein had a molecular weight approximately 23ku. The inclusion body was solubilized by 6M Guanidine hydrochloric acid and purified by ProBond?Resin. The antiserum against rChIL-18 was obtained by injecting the guinea pig with fusion ChIL-18 protein and its purified form.As a result, we successfully cloned the ChIL-18 mature protein gene, expressed and purified the rChIL-18 fusion protein from E.coli and obtained the polyclonal antibody against rChIL-18, laid a solid foundation and prepared experimental material for the future studies on the bioactivity of ChIL-18.Candidate: Pan Weiqi Major: Basic Veterinary Advisor: Vice Prof. Li Guangxing
本实验根据已发表的ChIL-18 cDNA基因序列设计引物，用反转录-聚合酶链式反应(Reverse-translation polymerase-chain reaction，RT-PCR)技术从有丝分裂原刀豆蛋白A(Concanavalin A，ConA)刺激48小时活化的鸡脾细胞中扩增出编码ChIL-18成熟蛋白的基因，并进行序列测定。结果表明本研究克隆到的ChIL-18成熟蛋白编码基因全长为507bp，含有一个开放阅读框架。与Schneider等报道的序列有一处碱基不同。即本序列在482位为C，而Schneider等的序列该处为T，这一核苷酸序列的变化导致了推导的对应氨基酸残基由苯丙氨酸变为丝氨酸。
在克隆到编码ChIL-18成熟蛋白的基因后，将此基因亚克隆至原核表达载体pPROEX~(TM)HTb中，构建重组质粒并进行确证性序列测定。然后将重组质粒转化大肠杆菌DH5α并用IPTG于37℃诱导培养获得表达，表达形式为包涵体。凝胶薄层灰度扫描显示表达的融合蛋白约占菌体蛋白的30％。SDS-PAGE和Western blot分析显示，表达的鸡IL-18融合蛋白分子量约为23ku。包涵体通过6M盐酸胍裂解后，利用镍离子亲和树脂进行纯化。用所获得的重组ChIL-18融合蛋白及其纯化产物经三次肌肉注射豚鼠，制备豚鼠抗ChIL-18多克隆抗体。琼脂扩散实验表明制备的多克隆抗体与ChIL-18具有良好的反应性。
本实验成功克隆到编码ChIL-18成熟蛋白的基因，原核表达、纯化了ChIL-18融合蛋白，并制备了豚鼠抗ChIL-18多克隆抗体，为下一阶段ChIL-18生物学特性及其应用的研究打下了坚实基础。
Interleukin-18 (IL-18) is a novel discovered cytokine, which is the potent inducer of IFN-Y production. It has been demonstrated that interleukin-18 exist in cells of human, mammal and some kinds of birds. Interleukin-18 plays an important role in host defense against various microorganisms. In addition, interleukin-18 is expected to be a target for tumor immunotherapy. Cytokines including interleukin-18, as natural mediators of the immune response, offer exciting alternatives to conventional therapeutics in control of disease in intensive livestock and poultry industries. The discovery of chicken interleukin-18 gene is very meaningful for both the study on comparative immunology and the therapeutics for poultry diseases.In this study, specific primers for chicken interleukin-18 (ChIL-18) cDNA have been designed and synthesized according to the previously reported nucleic acid sequence of chicken interleukin-18 cDNA. Using total RNA from ConA-stimulated chicken spleen cells, chicken IL-18 cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis indicated that there is one nucleotide different from the published ChIL-18 cDNA sequence in 482 (T-C).To highly express chicken interleukin-18 in E.coli and purify the fusion protein, cDNA fragment encoding the mature ChIL-18 was subcloned into the prokaryotic expression vector pPROEX橦Tb. The recombinant plasmid was transformed into E.coli, and then induced by IPTG at 37 C. The recombinant ChIL-18 (rChIL-18) was expressed efficiently in forms of inclusion body with the yield accounting for 30% total bacteria protein. SDS-PAGE and Western blot analysis showed that the recombinant fusion protein had a molecular weight approximately 23ku. The inclusion body was solubilized by 6M Guanidine hydrochloric acid and purified by ProBond?Resin. The antiserum against rChIL-18 was obtained by injecting the guinea pig with fusion ChIL-18 protein and its purified form.As a result, we successfully cloned the ChIL-18 mature protein gene, expressed and purified the rChIL-18 fusion protein from E.coli and obtained the polyclonal antibody against rChIL-18, laid a solid foundation and prepared experimental material for the future studies on the bioactivity of ChIL-18.Candidate: Pan Weiqi Major: Basic Veterinary Advisor: Vice Prof. Li Guangxing
引文
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