本文選題：堆型艾美耳球蟲　切入點(diǎn)：Hsp90　出處：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：雞球蟲病(Coccidiosis)是由艾美耳科,艾美耳屬(Eimeria)的一種或幾種雞艾美耳球蟲引起的以腸道感染為主的細(xì)胞內(nèi)原蟲病,雞球蟲寄生于各段腸道引起發(fā)病,導(dǎo)致腹瀉、便血和體重降低,對(duì)養(yǎng)禽業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失。目前對(duì)雞球蟲病的防治主要以藥物為主,但由于容易出現(xiàn)耐藥性及藥物殘留,使得一些藥物難以發(fā)揮其有效的作用。目前,安全而有效的基因工程疫苗用于雞球蟲病的防治已成為研究熱點(diǎn)�；蚬こ桃呙绾蜻x基因的選擇至關(guān)重要,Hsp90家族是分子大小質(zhì)量81~99 Ku的一種進(jìn)化保守的蛋白質(zhì),在分子伴侶功能、細(xì)胞循環(huán)調(diào)控、抗原傳遞方面發(fā)揮著重要作用,已有研究表明其與多種寄生蟲生長(zhǎng)發(fā)育過(guò)程關(guān)系密切。本研究根據(jù)已經(jīng)發(fā)表的堆型艾美耳球蟲(E.acervulina)Hsp90基因序列設(shè)計(jì)一對(duì)特異性引物,通過(guò)RT-PCR方法從E.acervulina子孢子中成功克隆得到大小為2 433 bp的Hsp90基因序列,其中包含一段大小為2 139 bp的開放性閱讀框,編碼713個(gè)氨基酸殘基,蛋白分子質(zhì)量大小為82.5 Ku,將ORF通過(guò)多克隆位點(diǎn)插入p ET-30a(+)空載體中,成功構(gòu)建了重組表達(dá)質(zhì)粒p ET30a-Hsp90,轉(zhuǎn)化入E.coli Rosetta感受態(tài)細(xì)胞并經(jīng)IPTG誘導(dǎo),通過(guò)SDS-PAGE及His單抗Western-blot檢測(cè),結(jié)果顯示一個(gè)大小為89 Ku的可溶性融合蛋白得以高效表達(dá)。以純化的Hsp90蛋白免疫新西蘭大白兔制備兔抗Hsp90多克隆抗體,間接ELISA和Western-blot檢測(cè)表明其反應(yīng)性和特異性良好。通過(guò)利用一段抗原性小的柔性片段(G4S)3多肽linker,將Hsp90與Ch IL-18基因連接后構(gòu)建p VAX-Hsp90及p VAX-IL18-Hsp90重組真核表達(dá)質(zhì)粒,體外瞬時(shí)轉(zhuǎn)染BHK21細(xì)胞進(jìn)行IFA檢測(cè),結(jié)果顯示Hsp90及IL-18均可在體外成功表達(dá),可較好的發(fā)揮其免疫原性并用于后續(xù)動(dòng)物免疫保護(hù)實(shí)驗(yàn)。實(shí)驗(yàn)雞分為5組,分別為未免疫未攻蟲組(N組)、PBS注射攻蟲組(P組)、p VAX1質(zhì)粒注射組(E組)、p VAX-Hsp90質(zhì)粒免疫組(H組)、p VAX-IL18-Hsp90質(zhì)粒免疫組(I組)。實(shí)驗(yàn)組分別以100μL/雞的PBS或100μg/雞的p VAX1、p VAX-Hsp90或p VAX-IL18-Hsp90劑量于14 d和21 d胸部肌肉注射免疫雛雞,在二次免疫后每只雞口服接種1×105個(gè)孢子化卵囊。檢測(cè)不同處理組雛雞淋巴細(xì)胞增殖功能變化、血清抗體Ig G變化、增重情況、十二指腸病變計(jì)分、OPG及抗球蟲指數(shù)等各項(xiàng)指標(biāo),用以評(píng)價(jià)真核表達(dá)質(zhì)粒的免疫保護(hù)效果,結(jié)果表明兩個(gè)實(shí)驗(yàn)組各項(xiàng)指標(biāo)均優(yōu)于對(duì)照組,且I組數(shù)值均高于H組;H組與I組的淋巴細(xì)胞增殖活性在免疫后顯著升高;ELISA方法檢測(cè)血清抗體Ig G顯示,H組與I組抗Hsp90血清抗體效價(jià)兩次免疫持續(xù)升高,且在35日齡時(shí)達(dá)到最高值,而對(duì)照組未見(jiàn)升高;H組與I組綜合抗球蟲指數(shù)值分別為166.17和175.32,均可產(chǎn)生有效的抗球蟲保護(hù),這表明Hsp90是一種潛在的球蟲保護(hù)性抗原,且Ch IL-18對(duì)其免疫效果起到佐劑樣的作用,本研究為Hsp90的深入研究及雞球蟲病的免疫防治提供了理論和實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.31

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本文編號(hào)：1563328