【作者】 严琳；

【导师】 陈焕春； 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 由病毒、细菌等病原微生物侵染动物所引起的各种传染性疾病严重制约了各个国家和地区养殖业的健康发展,同时也对人类健康存在着巨大隐患。因此,对动物传染性疾病的防治措施研究具有重大的经济价值和社会意义。疫苗免疫是当前预防和控制传染性疾病的主要措施,但目前使用的常规疫苗并不理想,存在免疫效率低与安全性差的两大难题。因此,如何在安全剂量疫苗接种下提高动物机体的免疫力,成为当今世界疫苗开发研究的热点之一。 细胞因子是由免疫效应细胞和相关细胞产生的,具有多种生物学活性的细胞调节蛋白。它在免疫应答的产生和调节中具有重要作用,可作为新一代免疫佐剂增强疫苗的免疫效果。二十多年来,各国学者致力于细胞因子重组蛋白作为疫苗佐剂研究,取得了许多振奋人心的结果:多种细胞因子重组蛋白包括IL、IFN、TNF、GM-CSF等,均能在不同程度上增加常规疫苗,包括灭活苗、弱毒苗、亚单位疫苗等的免疫效应。近年来,将细胞因子基因作为DNA疫苗佐剂,由于其简便性与有效性,亦受到了人们愈来愈多的关注与重视。 鉴于此,本研究以猪白细胞介素2(pIL-2)与猪白细胞介素6(pIL-6)为研究对象,开展了这两种细胞因子以及他们的融合蛋白(pIL6-IL2)以重组蛋白的形式对常规疫苗PRV灭活疫苗的佐剂效应研究,及以基因形式对PRRSV ORF5核酸疫苗的佐剂效应研究。主要研究内容包括: 1.pIL-2、pIL-6及pIL6-IL2的原核表达及纯化 构建pIL-2、plL-6及pIL6-IL2原核表达载体,均在E.coli BL21(DE3)中获得高效表达。其中rpIL-2包含信号肽序列,rpIL-6不含信号肽序列,rpIL6-IL2由一段亲水性、低电荷效应的Linker连接均不含信号肽的pIL-6与pIL-2分子组成。三种原核表达产物均以包涵体形式产生,因此通过提取包涵体—SKL变性—透析复性的生化提纯步骤来获取有生物学活性的重组蛋白。另将提纯了的原核表达产物rpIL-2、rpIL-6免疫家兔制备多克隆抗体。 2.19IL-2、pIL-6及pIL6-IL2的酵母表达及纯化 构建pIL-2、pIL-6及pIL6-IL2酵母表达载体,在P.pastoris GS115菌株获得分泌型表达。对3种酵母表达产物分别进行了Western blot检测,验证为目的蛋白。通过N端糖基化分析,发现酵母表达产物rpIL-2带有约5kDa的N端糖链,rpIL-6无N端糖基化修饰,rpIL6-IL2则以有糖链、无糖链两种分子状态存在,之间相差约2kDa;采用硫酸铵沉淀—PBS盐析—PEG20000浓缩的纯化办法,除去培养基中的毒性成分。对3种重组酵母菌进行了分泌外源蛋白最佳诱导时间及菌株持续稳定表达情况分析,验证诱导培养第4、5天外源蛋白分泌量最高;只需保持一定的酵母生长浓度,重组酵母菌可持续培养,稳定表达外源蛋白。提纯的酵母表达产物冻干活更多还原

【Abstract】 In pork-producing community, various infectious diseases which are caused by viral or bacterial pathogens, badly restricted the development of natural and regional farming industries, and also seriously affect human health in whole world. Hence, it’s necessary and great meaningful in both economic development and societal stabilization to prevent and control infectious diseases. Since now vaccination is still the most effective and economical means for the aim. But current routine vaccines are shown not good enough to defense pathogen infection because of the shortages on efficiency and safety. Hence, how to improve the efficiency of vaccine under a safe dosage, become one of the hottest topics in vaccine research area.Cytokine, produced by immune cells and other correlative cells, is a kind of pleiotropic protein which plays important roles during induction and modulation various immune responses. It’s cytokine’s bioactivity in immune system that inspires people to use it as adjuvant to improve vaccine efficiency. After beyond 20yr’s research, scholar in whole world had devoted themselves in exploring the adjuvant effects of cytokine on vaccine and got many excited achievements. They found various cytokines, including Interleukins, Interferon, TNF, GM-CSF et al, could enhance more or less the efficiency of different routine vaccine such as inactivated vaccine, attenuated vaccine and subunit vaccine. Recently, research in cytokine genes as DNA vaccine adjuvants, became popular and absorbed more and more attention because of its construct conveniently and function effectively.In this study, porcine interleukin-2 (pIL-2) and porcine interleukin-6 (pIL-6) were used to explore their adjuvant effects on routine vaccine (PRV inactivated vaccine) with the form of recombinant proteins, and on DNA vaccine (PRRSV ORF5 DNA vaccine) with the form of gene. The main research included as below: 1. Expression of pIL-2、 pIL-6 and their fusion protein pIL6-IL2 in E. coli andpurificationConstructed three prokaryotic expression plasmids pET-IL2, pGEX-IL6, 28a-IL6-IL2, and transferred into E. coli BL21 (DE3) expression strain. Three insoluble recombinant proteins were produced. Among them, rpIL-2 was included its signal peptide, rpIL-6 was designed to cut off signal peptide for expression successfully, rpIL6-IL2 was linked by a fragment which consisted of hydrophilic and low charge base pairs. The method contained with inclusion body collection — SKL denaturalization — dialysis re-naturalization was used to purified these three prokaryotic expression products. Also,purified rpIL-2 and rpIL-6 were used to immune rabbit for specific rpIL-2/rpIL-6 antibody generation2. Expression of pIL-2n pIL-6 and their fusion protein pIL6-IL2 in P. pastoris and purificationConstructed three P. pastoris expression plasmids pPIC3.5K-IL2, pPIC9K-IL6, pPIC9K-IL6-IL2, and transferred into P. pastoris GS115 strain. Three recombinant proteins were secreted into the culture medium with expected molecular weights after the recombinant P. p strains were induced by 1% methanol. All three P. pastoris products were analyzed by Western blot, treated with PNGase F and purified through (NH4)2SO4 deposition — PBS dialysis — PEG20000 concentration to eliminate the toxic in cultural medium. The PNGase F treatment results show that P. pastoris product rpIL-2 contained approximately 5kDa N-linked glycosylation, rpIL-6 hadn’t N-linked glycosylation, and a mixture of the N-linked glycosylated (about 2kDa) and nonglycosylated forms was appeared in rpIL6-IL2. Three recombinant P. pastoris strains were analyzed the best induction time and the stabilization for foreign protein expression. The results show 4th or 5th day was the best induction time, and recombinant yeast cells could grow and secret foreign protein stably only if cultural content is proper. Lyopholozed purified P. pastoris products were stable in 4°C for 4 months.3. Identification and comparison of bio-activities of recombinant proteins in two expression systemsThe biological activities of three recombinant cytokine expressed in E. coli and P. pastoris systems were determined by cell proliferation assays using IL2-dependent cell line CTLL-2 and IL6-dependent cell line B9. The results show the bio-activities of P. pastoris products were higher approximately 10-100 times than E. coli products. Then three P. pastoris recombinant cytokines were used in following animal test.4. Pig assay to explore the adjuvant effects of three recombinant cytokines on PRV inactivated vaccine.Pigs were injected 2mL of PRV inactivated vaccine once and inoculated l*105 U candidate cytokine adjuvant three times. 8 weeks after immunization, all pigs were challenged by high dosage PRV (Ea) strain. The results from VN tests and CTL assays showed that among three recombinant cytokines, rpIL-2 could furthest enhance PRV-specific humoral and cellular immune responses. Moreover, pigs of PRV+IL2 group got a significant higher protection than ones of control group after PRV infection. Unfortunately, we did not observe obvious positive effect of rpIL-6 and rpIL6-IL2 administration on PRV IAV vaccination, in both immune responses and protection ability.5. Construction of DNA vaccines containing PRRSV ORF5 gene and different cytokine gene in two expression cassettes.Constructed 4 eukaryotic expression plasmids containing two expression cassettes: pCI-ORF5-neo, pCI-ORF5-IL2, pCI-ORF5-IL6 and pCI-ORF5-IL6-IL2, and then transfected into Hela cell. IL2-dependent cell line CTLL-2 and IL6-dependent cell line B9 were used to identify whether the foreign cytokine had been expressed in this mammalian cell.6. Mouse assay to explore the adjuvant effects of three recombinant cytokine genes on PRRSV ORF5 DNA vaccine.Mice were immunized twice and sampled at 4, 6 and 8 weeks for analyzing antibody levels by ELISA and VN tests. All three interleukin genes showed adjuvant efficiencies on PRRSV ORF5 DNA vaccines by enhancing mice humoral immune responses.Adjuvant efficiencies of porcine cytokine proterins in PRV inactivated vaccine were tested in this study, aimed to obtain an easy-prepared and highly potent cytokine adjuvant to improve the efficiency of PRV inactivated vaccine and other routine vaccines, defense and control infectious disease outbreak, promote pork-producing industry development. Moreover, in order to enhance DNA vaccines efficiency, develop a new potent DNA vaccine adjuvant, adjuvant efficiencies of porcine cytokine genes in PRRSV ORF5 DNA vaccine were explored in this study.更多还原