本文選題：豬圓環(huán)病毒2型 + Cap蛋白��； 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：豬圓環(huán)病毒病(PCVDs)由豬圓環(huán)病毒2型(PCV2)引起,是當(dāng)前對(duì)養(yǎng)豬業(yè)危害嚴(yán)重的傳染性疾病。疫苗免疫接種是預(yù)防本病的主要手段,常用的疫苗有全病毒滅活疫苗,免疫效果較好,但存在病毒滴度較低、需要進(jìn)行病毒濃縮、價(jià)格較高的問(wèn)題�？乖味雀�、免疫效果好、使用更安全的PCV2疫苗研發(fā)是一個(gè)重要目的。腺病毒活載體疫苗具有制備相對(duì)簡(jiǎn)單、病毒滴度高、免疫刺激持久、效果確實(shí)等優(yōu)點(diǎn),在PCV2基因工程疫苗研究中受到關(guān)注。目前的重組病毒構(gòu)建策略一般是將PCV2的衣殼蛋白(Cap)基因插入到病毒基因組中進(jìn)行表達(dá)。假結(jié)核耶爾森菌侵襲素(Inv)是一種外膜蛋白,能夠增強(qiáng)宿主細(xì)胞對(duì)抗原的攝取能力,提高疫苗免疫效果,蛋白的C端(InvC)在這一過(guò)程中發(fā)揮重要作用。本研究將PCV2的Cap基因和InvC基因在重組腺病毒共表達(dá),構(gòu)建一種同時(shí)表達(dá)目的抗原和免疫促進(jìn)因子的重組腺病毒,進(jìn)行動(dòng)物免疫試驗(yàn),對(duì)免疫效果進(jìn)行評(píng)價(jià),以期獲得一種新型的PCV2腺病毒疫苗。本研究獲得了以下結(jié)果:1.構(gòu)建了共表達(dá)PCV2 Cap基因和InvC基因的重組腺病毒。將PCV2的Cap基因和InvC基因依次嵌入重組腺病毒穿梭載體,轉(zhuǎn)化入BJ5183感受態(tài)細(xì)胞進(jìn)行同源重組,獲得重組腺病毒骨架質(zhì)粒pAd-Cap-InvC,轉(zhuǎn)染HEK293細(xì)胞,包裝出重組腺病毒rAd-Cap-InvC。經(jīng)PCR、RT-PCR、Western blot、IFA檢測(cè),rAd-Cap-InvC遺傳基因穩(wěn)定分析,證明Cap-InvC融合蛋白能夠在重組病毒中得到表達(dá)。滴度可達(dá)109.32 TCID50/mL。2.重組病毒的動(dòng)物免疫試驗(yàn)顯示,其能有效刺激機(jī)體產(chǎn)生特異性的抗體。以108TCID50/只的重組腺病毒rAd-Cap-InvC和rAd-Cap分別免疫接種PCV2抗體陰性昆明系小鼠,首免后14 d加強(qiáng)免疫一次(108TCID50/只)。首免后14、21、35、42 d分別測(cè)定試驗(yàn)鼠血清中PCV2抗體水平。結(jié)果發(fā)現(xiàn),兩種重組腺病毒免疫小鼠均產(chǎn)生了針對(duì)PCV2 Cap的抗體,其中rAd-Cap-InvC免疫組比rAd-Cap免疫組的抗體滴度要高,在整個(gè)檢測(cè)時(shí)間段均顯著差異(P0.05)。以1010TCID50/只的重組腺病毒rAd-Cap-InvC,109TCID50/只的重組腺病毒rAd-Cap-InvC、rAd-Cap及rAd分別免疫接種試驗(yàn)豬2頭,首免后14 d加強(qiáng)免疫一次(各組病毒劑量同首免)。首免后第14、21、28、36 d測(cè)定試驗(yàn)豬血清中PCV2抗體水平。結(jié)果發(fā)現(xiàn),rAd-Cap-InvC組的抗體滴度在各檢測(cè)時(shí)間點(diǎn)均要高于rAd-Cap組;1010TCID50/只的重組腺病毒rAd-Cap-InvC免疫組抗體滴度也高于109TCID50/只免疫組。動(dòng)物免疫試驗(yàn)表明,重組腺病毒rAd-Cap-InvC和rAd-Cap均能使試驗(yàn)動(dòng)物產(chǎn)生針對(duì)PCV2 Cap蛋白的特異性抗體,并且rAd-Cap-InvC能夠產(chǎn)生更高的抗體滴度。本研究構(gòu)建了共表達(dá)PCV2 Cap蛋白和免疫增強(qiáng)因子InvC重組腺病毒,其能使試驗(yàn)動(dòng)物產(chǎn)生更高水平的特異性的免疫抗體,由此可知InvC對(duì)抗體的產(chǎn)生具有促進(jìn)作用。
[Abstract]:Porcine circovirus disease (PCVDs) is caused by porcine circovirus type 2 (PCV2) and is a serious infectious disease which is harmful to pig industry at present. Vaccination is the main method to prevent this disease. The commonly used vaccine is the whole virus inactivated vaccine, the immune effect is good, but the virus titer is low, the virus concentration is needed, and the price is high. High antigen titer, good immune effect, the use of a more secure PCV 2 vaccine development is an important goal. Adenovirus live vector vaccine has many advantages, such as relatively simple preparation, high titer of virus, long-lasting immune stimulation and definite effect, so it has attracted much attention in the research of PCV2 gene engineering vaccine. Currently, the construction strategy of recombinant virus is to insert PCV2 capsid gene into the virus genome for expression. The involute of Yersinia pseudotuberculosis is an outer membrane protein, which can enhance the ability of the host cells to ingest antigen and improve the immune effect of the vaccine. The C terminal InvC of the protein plays an important role in this process. In this study, the Cap gene and InvC gene of PCV2 were co-expressed in recombinant adenovirus, and a recombinant adenovirus expressing both target antigen and immune promoting factor was constructed. In order to obtain a new type of PCV 2 adenovirus vaccine. The results of this study are as follows: 1: 1. A recombinant adenovirus co-expressing PCV2 Cap gene and InvC gene was constructed. The Cap gene and InvC gene of PCV2 were inserted into the recombinant adenovirus shuttle vector, and then transformed into BJ5183 cells for homologous recombination. The recombinant adenovirus skeleton plasmid pAd-Cap-InvCwas transfected into HEK293 cells, and the recombinant adenovirus rAd-Cap-InvCwas packaged. The stability analysis of rAd-Cap-InvC gene by PCR RT-PCR Western blotsimetry showed that Cap-InvC fusion protein could be expressed in recombinant virus. Titer can reach 109.32 TCID 50 / ml. 2. Animal immunoassay of recombinant virus shows that it can effectively stimulate the body to produce specific antibodies. Recombinant adenovirus rAd-Cap-InvC and rAd-Cap were inoculated into mice with negative PCV2 antibody respectively. The serum levels of PCV2 antibody in mice were measured on day 14, 21 and 3 542 after the first immunization. The results showed that both recombinant adenovirus immunized mice produced antibodies against PCV2 Cap. The titer of antibody in rAd-Cap-InvC immunized group was higher than that in rAd-Cap immunization group, and the difference was significant in the whole detection period. The recombinant adenovirus rAd-Cap-InvCID50 / rAd-Cap-InvCnrAd-Cap and rAd were inoculated with two pigs respectively. Serum PCV2 antibody levels were measured at day 14, 21, 28 and 36 after the first immunization. The results showed that the titer of antibody in rAd-Cap-InvC group was higher than that in rAd-Cap group (1010TCID50 / rAd-Cap group) and 109TCID50 / mouse group. Animal immunoassay showed that both the recombinant adenovirus rAd-Cap-InvC and rAd-Cap could produce specific antibodies against PCV2 Cap protein, and rAd-Cap-InvC could produce higher antibody titers. In this study, co-expression of PCV2 Cap protein and immune enhancer InvC recombinant adenovirus was constructed. The recombinant adenovirus can produce a higher level of specific immune antibody in experimental animals, so InvC can promote the production of antibodies.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S855.3

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相關(guān)期刊論文 前2條

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本文編號(hào)：2016092