本文選題：毒害艾美耳球蟲　切入點(diǎn)：配子體　出處：《揚(yáng)州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：雞球蟲病是由一種或數(shù)種艾美耳球蟲寄生于雞消化道上皮細(xì)胞內(nèi)而引起的一種原蟲寄生蟲病,嚴(yán)重危害養(yǎng)雞業(yè)。毒害艾美耳球蟲(Eimerianecatrix)是雞球蟲病的重要病原之一,主要危害8～18周齡的雞,引起雞的急性小腸球蟲病。目前,球蟲病的防治主要依賴化學(xué)藥物或雞球蟲病活疫苗。隨著抗球蟲藥物的廣泛與大量使用,球蟲耐藥性以及人們對(duì)藥物殘留肉蛋、污染環(huán)境等諸多問(wèn)題也隨之出現(xiàn)。同時(shí)活球蟲疫苗又存在生產(chǎn)成本高、容易擴(kuò)散病原、致弱蟲株毒力易返強(qiáng)、蟲株的抗原變異、疫苗導(dǎo)致飼料報(bào)酬降低、疫苗接種的劑量難以控制等弊端。因此,雞球蟲保護(hù)性抗原基因的篩選與克隆表達(dá)及其免疫保護(hù)力的研究一直是雞球蟲病研究的熱點(diǎn)。為此,本文以毒害艾美耳球蟲配子體為材料,構(gòu)建毒害艾美耳球蟲配子體cDNA文庫(kù),并用免疫學(xué)方法從文庫(kù)中篩選抗原基因,對(duì)ENH_00080190基因進(jìn)行了克隆表達(dá)和免疫原性分析,為研究毒害艾美耳球蟲亞單位疫苗奠定基礎(chǔ)。1毒害艾美耳球蟲配子體的分離與純化24日齡無(wú)球蟲雛雞,每雞經(jīng)嗉囊感染10 000個(gè)毒害艾美耳球蟲孢子化卵囊。感染148 h后收取第二代裂殖子。經(jīng)外科手術(shù)方法,將第二代裂殖子直接注入雞盲腸內(nèi),使第二代裂殖子同步發(fā)育至配子體;手術(shù)后30 h剖檢雞,刮取盲腸黏膜,研磨后用0.5 mmol/L透明質(zhì)酸酶消化,釋放配子體;隨后經(jīng)過(guò)60目銅篩、260目錦綸篩兜和17 μm PET膜過(guò)濾,濾液經(jīng)離心洗滌后用裂解液裂解紅細(xì)胞;最后用30%和50%的Percoll,5 000 rpm離心15 min,分離純化配子體。結(jié)果顯示,本法獲得的配子體純度高、數(shù)量多,這為研究毒害艾美耳球蟲配子體階段的基因組學(xué)、蛋白質(zhì)組學(xué)和免疫學(xué)等奠定了基礎(chǔ)。2毒害艾美耳球蟲配子體cDNA文庫(kù)的構(gòu)建用Trizol試劑提取毒害艾美耳球蟲配子體總RNA,隨后采用SMART技術(shù)構(gòu)建了毒害艾美耳球蟲配子體cDNA噬菌體表達(dá)文庫(kù)。經(jīng)鑒定,結(jié)果顯示,總RNA的OD260/OD280值為1.95,樣品的28S和18S條帶清晰,原始文庫(kù)容量為3.42×106 pfu/mL,重組率為90%,擴(kuò)增后的文庫(kù)容量為2.6× 1 010 pfu/mL,插入片段長(zhǎng)度250～1000 bp。3毒害艾美耳球蟲配子體cDNA文庫(kù)的篩選首先,制備抗毒害艾美耳球蟲的雞康復(fù)血清和鼠抗毒害艾美耳球蟲配子體多抗血清;兩種血清經(jīng)ELISA檢測(cè),顯示兩種多抗均有很高的效價(jià);隨后,用制備的鼠多抗對(duì)文庫(kù)進(jìn)行篩選,初次篩選共篩出疑似5個(gè)陽(yáng)性克隆,復(fù)篩后確定陽(yáng)性克隆3個(gè);最后,對(duì)復(fù)篩后獲得的陽(yáng)性克隆進(jìn)行PCR鑒定,對(duì)獲得的EST序列進(jìn)行生物信息學(xué)分析。結(jié)果顯示,3個(gè)EST序列的長(zhǎng)度分別為734 bp、270 bp和606 bp,分別編碼毒害艾美耳球蟲特有蛋白基因、毒害艾美耳球蟲動(dòng)力蛋白基因和毒害艾美耳球蟲假定蛋白基因。4毒害艾美耳球蟲動(dòng)力蛋白基因表達(dá)及免疫原性分析首先,依據(jù)ENH_00080190序列合成毒害艾美耳球蟲動(dòng)力蛋白基因,并將其插入到與載體pET-28a(+)連接,構(gòu)建重組表達(dá)質(zhì)粒,轉(zhuǎn)化大腸桿菌BL21(DE3)感受態(tài)細(xì)胞。其次,用IPTG誘導(dǎo)表達(dá),對(duì)表達(dá)產(chǎn)物進(jìn)行SDS-PAGE電泳分析。最后,以毒害艾美耳球蟲感染雞康復(fù)血清為一抗進(jìn)行Western-blot檢測(cè)。結(jié)果顯示,基因全長(zhǎng)為270 bp,編碼89個(gè)氨基酸;表達(dá)產(chǎn)物大小約為13 kDa,主要以包涵體形式存在;重組蛋白能被雞康復(fù)血清識(shí)別,顯示重組蛋白具有免疫原性。
[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.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.7

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