本文選題：PED + 畢赤酵母��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：豬流行性腹瀉(PED)是由豬流行性腹瀉病毒(PEDV)引起的一種豬的急性、高度接觸性腸道傳染病,主要特征為腹瀉、嘔吐、脫水和高死亡率。自2010年以來,以PEDV變異株為主引起的PED在我國各省份廣泛流行,對我國養(yǎng)豬業(yè)造成了嚴(yán)重的經(jīng)濟損失。疫苗免疫是目前防控PED的最有效途徑,目前市場主要預(yù)防PED的疫苗為多聯(lián)滅活、或弱毒疫苗,以上疫苗存在保護(hù)率低、安全性低、成本昂貴等缺點,未對PED實現(xiàn)有限地防控和凈化,市場亟待開發(fā)更加安全、經(jīng)濟、高效的新型疫苗(尤其是亞單位疫苗)用于PED的防制。PEDV S1結(jié)構(gòu)域是介導(dǎo)PED保護(hù)性免疫的主要功能區(qū),核心中和表位(COE)是S1的最重要功能區(qū),也是目前用于PED亞單位疫苗研究的最小保護(hù)抗原。因此,本研究通過畢赤酵母表達(dá)系統(tǒng)對PEDV S1的COE進(jìn)行表達(dá)。同時嘗試將COE與人和豬的Ig G1 Fc進(jìn)行融合來提高COE抗原的免疫效果,取得的主要結(jié)果如下:a)利用PCR分別擴增出COE、P1Fc、H1Fc片段,通過重疊PCR技術(shù)拼接COE-P1Fc和COE-H1Fc片段,成功構(gòu)建了酵母表達(dá)載體p PICZαA-COE、p PICZαA-COEP1Fc和p PICZαA-COE-H1Fc;b)利用酵母表達(dá)系統(tǒng)分泌表達(dá)產(chǎn)物經(jīng)Western blotting鑒定,結(jié)果表明成功的表達(dá)了r COE、r COE-P1Fc、r COE-H1Fc 3種蛋白。c)免疫小鼠42d后采集各組血清通過間接ELASA和中和試驗評價機體產(chǎn)生抗體情況。間接ELASA結(jié)果表明3組實驗組都產(chǎn)生了較高水平的抗體。血清中和試驗結(jié)果顯示r COE、r COE-P1Fc、r COE-H1Fc實驗組中和效價依次為1:160、1:640、1:512,r COE-P1Fc、r COE-H1Fc實驗組中和效價顯著高于r COE實驗組和PBS對照組,結(jié)果表明在COE基因基礎(chǔ)上融合佐劑P1Fc、H1Fc,明顯提高了PED亞單位疫苗效果。本研究首次利用畢赤酵母表達(dá)系統(tǒng)表達(dá)S1蛋白核心中和表位融合蛋白,對研究PED亞單位疫苗進(jìn)行首次嘗試,且Ig G1 Fc片段具有佐劑功能,能提高機體免疫力,成為防控PED的新型亞單位疫苗。
[Abstract]:Porcine epidemic diarrhea (PED) is an acute, highly contact intestinal infection caused by porcine epidemic diarrhea virus (PEDVV), characterized by diarrhea, vomiting, dehydration and high mortality. Since 2010, PED, mainly caused by PEDV variant strains, has been widely prevalent in various provinces of China, which has caused serious economic losses to the pig industry in China. Vaccine immunization is the most effective way to prevent and control PED at present. At present, the main vaccine to prevent PED is multiplex inactivation or attenuated virus vaccine. These vaccines have the disadvantages of low protection rate, low safety, high cost and so on. In the absence of limited control and purification of PED, it is urgent for the market to develop more safe, economical and efficient new vaccines (especially subunit vaccines) for PED control. PEDV S1 domain is the main functional region that mediates the protective immunity of PED. Core neutralizing epitope (Coe) is the most important functional region of S1 and the least protective antigen for PED subunit vaccine research. Therefore, the COE of PEDV S1 was expressed by Pichia pastoris expression system. At the same time, we try to improve the immune effect of COE antigen by fusion of COE with human and pig IgG1 FC. The main results are as follows: (1) using PCR to amplify COEN P1FcH1 Fc fragment, COE-P1Fc and COE-H1Fc fragments were spliced by overlapping PCR technique. Yeast expression vectors, p PICZ 偽 A-COEG, p PICZ 偽 A-COEP1Fc and p PICZ 偽 A-COE-H1 Fcb, were successfully constructed. The expressed products were identified by Western blotting using yeast expression system. The results showed that the mice were immunized with rCOEE-P1Fcnr COE-H1Fc successfully for 42 days. After immunization, the sera of each group were collected to evaluate the antibody production by indirect ELASA and neutralization test. Indirect ELASA results showed that the three experimental groups produced a higher level of antibodies. The results of serum neutralization test showed that the neutralization titer of the experimental group was 1: 160, 1: 640,1: 512rCOE-P1Fccr COE-H1Fc, and the neutralization titer of the experimental group was significantly higher than that of the experimental group of r COE and the control group of PBS, and the neutralization titer of the experimental group was significantly higher than that of the experimental group of r COE and the control group of PBS. The results showed that the fusion of the adjuvant P1Fc1 Fc1 Fc on the basis of COE gene significantly improved the effect of PED subunit vaccine. In this study, we used Pichia pastoris expression system to express S1 protein core neutralizing epitope fusion protein for the first time, and we tried to study PED subunit vaccine for the first time, and Ig G1 FC fragment has adjuvant function and can improve body immunity. To become a new subunit vaccine for the prevention and control of PED.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.28

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