毒害艾美耳球虫配子体cDNA文库的构建与筛选及其动力蛋白基因的表达

发布时间：2018-03-17 19:14

本文选题：毒害艾美耳球虫　切入点：配子体　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：鸡球虫病是由一种或数种艾美耳球虫寄生于鸡消化道上皮细胞内而引起的一种原虫寄生虫病,严重危害养鸡业。毒害艾美耳球虫(Eimerianecatrix)是鸡球虫病的重要病原之一,主要危害8～18周龄的鸡,引起鸡的急性小肠球虫病。目前,球虫病的防治主要依赖化学药物或鸡球虫病活疫苗。随着抗球虫药物的广泛与大量使用,球虫耐药性以及人们对药物残留肉蛋、污染环境等诸多问题也随之出现。同时活球虫疫苗又存在生产成本高、容易扩散病原、致弱虫株毒力易返强、虫株的抗原变异、疫苗导致饲料报酬降低、疫苗接种的剂量难以控制等弊端。因此,鸡球虫保护性抗原基因的筛选与克隆表达及其免疫保护力的研究一直是鸡球虫病研究的热点。为此,本文以毒害艾美耳球虫配子体为材料,构建毒害艾美耳球虫配子体cDNA文库,并用免疫学方法从文库中筛选抗原基因,对ENH_00080190基因进行了克隆表达和免疫原性分析,为研究毒害艾美耳球虫亚单位疫苗奠定基础。1毒害艾美耳球虫配子体的分离与纯化24日龄无球虫雏鸡,每鸡经嗉囊感染10 000个毒害艾美耳球虫孢子化卵囊。感染148 h后收取第二代裂殖子。经外科手术方法,将第二代裂殖子直接注入鸡盲肠内,使第二代裂殖子同步发育至配子体;手术后30 h剖检鸡,刮取盲肠黏膜,研磨后用0.5 mmol/L透明质酸酶消化,释放配子体;随后经过60目铜筛、260目锦纶筛兜和17 μm PET膜过滤,滤液经离心洗涤后用裂解液裂解红细胞;最后用30%和50%的Percoll,5 000 rpm离心15 min,分离纯化配子体。结果显示,本法获得的配子体纯度高、数量多,这为研究毒害艾美耳球虫配子体阶段的基因组学、蛋白质组学和免疫学等奠定了基础。2毒害艾美耳球虫配子体cDNA文库的构建用Trizol试剂提取毒害艾美耳球虫配子体总RNA,随后采用SMART技术构建了毒害艾美耳球虫配子体cDNA噬菌体表达文库。经鉴定,结果显示,总RNA的OD260/OD280值为1.95,样品的28S和18S条带清晰,原始文库容量为3.42×106 pfu/mL,重组率为90%,扩增后的文库容量为2.6× 1 010 pfu/mL,插入片段长度250～1000 bp。3毒害艾美耳球虫配子体cDNA文库的筛选首先,制备抗毒害艾美耳球虫的鸡康复血清和鼠抗毒害艾美耳球虫配子体多抗血清;两种血清经ELISA检测,显示两种多抗均有很高的效价;随后,用制备的鼠多抗对文库进行筛选,初次筛选共筛出疑似5个阳性克隆,复筛后确定阳性克隆3个;最后,对复筛后获得的阳性克隆进行PCR鉴定,对获得的EST序列进行生物信息学分析。结果显示,3个EST序列的长度分别为734 bp、270 bp和606 bp,分别编码毒害艾美耳球虫特有蛋白基因、毒害艾美耳球虫动力蛋白基因和毒害艾美耳球虫假定蛋白基因。4毒害艾美耳球虫动力蛋白基因表达及免疫原性分析首先,依据ENH_00080190序列合成毒害艾美耳球虫动力蛋白基因,并将其插入到与载体pET-28a(+)连接,构建重组表达质粒,转化大肠杆菌BL21(DE3)感受态细胞。其次,用IPTG诱导表达,对表达产物进行SDS-PAGE电泳分析。最后,以毒害艾美耳球虫感染鸡康复血清为一抗进行Western-blot检测。结果显示,基因全长为270 bp,编码89个氨基酸;表达产物大小约为13 kDa,主要以包涵体形式存在;重组蛋白能被鸡康复血清识别,显示重组蛋白具有免疫原性。
[Abstract]:Chicken coccidiosis is a kind of by one or several species of Eimeria parasites in the epithelial cells in the digestive tract of chickens caused by protozoan parasites, serious harms to the poultry industry. E.necatrix (Eimerianecatrix) is an important pathogen of chicken coccidiosis. The main harm of 8~18 week old chickens, chickens caused by acute intestinal coccidiosis. At present, the prevention and treatment of coccidiosis mainly depends on chemical drugs or chicken coccidiosis vaccine. With anticoccidial drugs widely and largely used, meat and people on drug residues in coccidia resistance, many environmental pollution and other issues also will appear. At the same time there live coccidial vaccine production cost is high, easy to spread the pathogen, attenuated virulence easily return, the antigenic variation of strains, vaccine induced feed reduced vaccination dose defects are difficult to control. Therefore, screening and cloning and expression of chicken coccidiosis protective antigen gene Study on the protective immunity of chicken coccidiosis is a research hotspot. Therefore, this paper takes E.necatrix gametophyte as materials, the construction of E.necatrix gametophyte cDNA library, and immunological methods from the library screening of antigen gene and ENH_00080190 gene were analyzed for primary cloning expression and immune of poison Eimeria subunit vaccine based.1 isolation and purification of E.necatrix gametophytes of 24 day old chicks per chicken coccidia free, the 10000 crop infection of Eimeria necatrix sporulated oocysts. After 148 h of infection for second generation merozoites. After surgery, the second generation merozoites injected directly into the cecum of chicken second, the synchronous development of the gametophyte generation merozoite to 30 h after operation; necropsy chicken, scraping cecal mucosa, after grinding with 0.5 mmol/L hyaluronidase digestion, release of gametophyte; followed by 6 0 mesh copper screen, 260 mesh nylon sieve bag and 17 m PET membrane filtration, the filtrate was centrifuged after washing with lysis of red blood cells; finally, 30% and 50% Percoll, 5000 rpm centrifugation for 15 min, separation and purification of gametophyte. The results showed that the purity of gametophyte obtained by this method is high, the number of. This study E.necatrix gametophyte stage genomics, proteomics and immunology has laid a foundation for.2 E.necatrix gametophyte cDNA library using Trizol reagent to extract E.necatrix gametes total RNA then uses the SMART technology to construct E.necatrix gametophyte cDNA phage expression library. The identification results showed that the total RNA, OD260/OD280 value is 1.95, the samples of 28S and 18S bands were clear, the original library size is 3.42 * 106 pfu/mL, the recombination rate was 90%. The amplified library size is 2.6 * 1010 pfu/mL, the insert length of 250 ~ Screening 1000 bp.3 E.necatrix gametophyte cDNA library first, preparation of anti E.necatrix chicken rehabilitation serum and mouse anti E.necatrix gametophyte antiserum; two serum titer detected by ELISA, two kinds of antibodies are high; then, with the preparation of polyclonal antibody of rat library screening, initial screening were suspected of 5 positive clones, screening identified 3 positive clones; finally, PCR identification of positive clones obtained after rescreening, bioinformatics analysis of the EST sequence. The results showed that 3 EST sequence length were 734 BP, 270 BP and 606 BP, respectively, encoding E.necatrix specific protein gene of Eimeria necatrix dynein gene and the putative protein.4 gene of Eimeria dynein gene expression and immunogenicity analysis first, on the basis of ENH_0 0080190 sequence synthesis of E.necatrix dynein gene, and inserted into the plasmid pET-28a (+) connection, the recombinant expression plasmid was transformed into E.coli BL21 (DE3) competent cells. Secondly, the expression induced by IPTG. The expression products were analyzed by SDS-PAGE electrophoresis analysis. Finally, in order to Eimeria infected chickens rehabilitation serum as the primary antibody was detected by Western-blot. The results showed that the gene was 270 BP, encoding 89 amino acids; the expression product was about 13 kDa, mainly in the form of inclusion body; recombinant protein can be recovered chicken sera, showed that the recombinant protein had immunogenicity.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

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