鸡堆型艾美耳球虫Hsp90增强型核酸疫苗免疫保护研究

发布时间：2018-03-04 00:20

本文选题：堆型艾美耳球虫　切入点：Hsp90　出处：《东北农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：鸡球虫病(Coccidiosis)是由艾美耳科,艾美耳属(Eimeria)的一种或几种鸡艾美耳球虫引起的以肠道感染为主的细胞内原虫病,鸡球虫寄生于各段肠道引起发病,导致腹泻、便血和体重降低,对养禽业造成严重的经济损失。目前对鸡球虫病的防治主要以药物为主,但由于容易出现耐药性及药物残留,使得一些药物难以发挥其有效的作用。目前,安全而有效的基因工程疫苗用于鸡球虫病的防治已成为研究热点。基因工程疫苗候选基因的选择至关重要,Hsp90家族是分子大小质量81~99 Ku的一种进化保守的蛋白质,在分子伴侣功能、细胞循环调控、抗原传递方面发挥着重要作用,已有研究表明其与多种寄生虫生长发育过程关系密切。本研究根据已经发表的堆型艾美耳球虫(E.acervulina)Hsp90基因序列设计一对特异性引物,通过RT-PCR方法从E.acervulina子孢子中成功克隆得到大小为2 433 bp的Hsp90基因序列,其中包含一段大小为2 139 bp的开放性阅读框,编码713个氨基酸残基,蛋白分子质量大小为82.5 Ku,将ORF通过多克隆位点插入p ET-30a(+)空载体中,成功构建了重组表达质粒p ET30a-Hsp90,转化入E.coli Rosetta感受态细胞并经IPTG诱导,通过SDS-PAGE及His单抗Western-blot检测,结果显示一个大小为89 Ku的可溶性融合蛋白得以高效表达。以纯化的Hsp90蛋白免疫新西兰大白兔制备兔抗Hsp90多克隆抗体,间接ELISA和Western-blot检测表明其反应性和特异性良好。通过利用一段抗原性小的柔性片段(G4S)3多肽linker,将Hsp90与Ch IL-18基因连接后构建p VAX-Hsp90及p VAX-IL18-Hsp90重组真核表达质粒,体外瞬时转染BHK21细胞进行IFA检测,结果显示Hsp90及IL-18均可在体外成功表达,可较好的发挥其免疫原性并用于后续动物免疫保护实验。实验鸡分为5组,分别为未免疫未攻虫组(N组)、PBS注射攻虫组(P组)、p VAX1质粒注射组(E组)、p VAX-Hsp90质粒免疫组(H组)、p VAX-IL18-Hsp90质粒免疫组(I组)。实验组分别以100μL/鸡的PBS或100μg/鸡的p VAX1、p VAX-Hsp90或p VAX-IL18-Hsp90剂量于14 d和21 d胸部肌肉注射免疫雏鸡,在二次免疫后每只鸡口服接种1×105个孢子化卵囊。检测不同处理组雏鸡淋巴细胞增殖功能变化、血清抗体Ig G变化、增重情况、十二指肠病变计分、OPG及抗球虫指数等各项指标,用以评价真核表达质粒的免疫保护效果,结果表明两个实验组各项指标均优于对照组,且I组数值均高于H组;H组与I组的淋巴细胞增殖活性在免疫后显著升高;ELISA方法检测血清抗体Ig G显示,H组与I组抗Hsp90血清抗体效价两次免疫持续升高,且在35日龄时达到最高值,而对照组未见升高;H组与I组综合抗球虫指数值分别为166.17和175.32,均可产生有效的抗球虫保护,这表明Hsp90是一种潜在的球虫保护性抗原,且Ch IL-18对其免疫效果起到佐剂样的作用,本研究为Hsp90的深入研究及鸡球虫病的免疫防治提供了理论和实验基础。
[Abstract]:Coccidiosis is a kind of intracellular protozoa that is mainly intestinal infection caused by the Eimeriaae (Eimeria). The parasitism of chicken coccidiosis causes diarrhea, blood stool and weight loss in each section of the intestine. It has caused serious economic losses to the poultry industry. At present, the prevention and control of chicken coccidiosis is mainly controlled by drugs, but because of the easy emergence of drug resistance and drug residues, it is difficult for some drugs to play their effective role. At present, The safe and effective genetic engineering vaccine has become a research hotspot in the control of chicken coccidiosis. The selection of candidate genes for genetic engineering vaccine is very important, and the Hsp90 family is an evolutionarily conserved protein with molecular weight of 81 ~ 99 Ku. It plays an important role in molecular chaperone function, cell cycle regulation and antigen transmission. In this study, a pair of specific primers were designed based on the published sequence of E. acervulinae Hsp90 gene. The Hsp90 gene sequence of 2 433 BP was cloned from E. acervulina sporozoite by RT-PCR method. It contains an open reading frame of 2 139 BP and encodes 713 amino acid residues. The molecular weight of the protein was 82.5 Ku. the ORF was inserted into the pET-30a () empty vector by polyclonal site. The recombinant expression plasmid pET30a-Hsp90 was successfully constructed and transformed into E. coli Rosetta receptive cells and induced by IPTG, and detected by SDS-PAGE and His monoclonal antibody Western-blot. The results showed that a soluble fusion protein of 89Ku was highly expressed. The purified Hsp90 protein was used to immunize New Zealand white rabbits to prepare rabbit anti-#en1# polyclonal antibody. Indirect ELISA and Western-blot analysis showed that its reactivity and specificity were good. The recombinant eukaryotic expression plasmids of p VAX-Hsp90 and p VAX-IL18-Hsp90 were constructed by ligating Hsp90 with Ch IL-18 gene by using a flexible fragment of G4Sf3 with small antigenicity, Linker. Transient transfection of BHK21 cells in vitro was performed by IFA detection. The results showed that both Hsp90 and IL-18 could be expressed successfully in vitro, which could play a good role in immunogenicity and be used in the subsequent animal immune protection experiment. The chickens were divided into 5 groups. The experimental group was treated with 100 渭 L PBS of 100 渭 L / chicken or 100 渭 g / chicken of p VAX1p VAX-Hsp90 or p VAX-IL18-Hsp90 of 100 渭 L / Chicken or 100 渭 g / chicken of pVAX1p VAX-Hsp90 or pVAX1p VAX-Hsp90, respectively. The chicks were immunized with intramuscular injection at 14 and 21 days. After twice immunization, each chicken was inoculated orally with 1 脳 105 sporulated oocysts. The changes of lymphocyte proliferation, serum IgG, weight gain, duodenal lesion score, OPG and anti-coccidiosis index were detected in different treatment groups. The results showed that each index of the two experimental groups was superior to that of the control group. The value of lymphocyte proliferation in group I was significantly higher than that in group H and group I. Elisa assay showed that the titer of anti Hsp90 serum antibody in H group and I group was continuously increased after immunization. And reached the highest value at the age of 35 days, but the combined anti-coccidiosis index values of group H and group I were 166.17 and 175.32, respectively, which indicated that Hsp90 was a potential protective antigen for coccidiosis. Ch IL-18 plays an adjuvant role on the immune effect of Cho IL-18. This study provides a theoretical and experimental basis for the further study of Hsp90 and the immune control of coccidiosis.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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