本文選題：豬流行性腹瀉 + 豬傳染性胃腸炎　； 參考：《山東農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：仔豬病毒性腹瀉主要是由豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PEDV)、豬傳染性胃腸炎病毒(Transmissible gastroenteritis virus,TGEV)和豬輪狀病毒(Porcine rotavirus,PoRV)單獨或混合感染引起,該病對仔豬的危害較大,病死率高。自2010年冬季以來,山東省部分地區(qū)仔豬發(fā)生以腹瀉為主要癥狀的疾病,給養(yǎng)豬業(yè)造成了嚴(yán)重的經(jīng)濟損失。其中豬傳染性胃腸炎是引起仔豬腹瀉的主要病因之一,該病可造成10日齡以內(nèi)的仔豬100%死亡。TGEV主要感染胃腸道引起局部組織發(fā)生病變,胃腸道黏膜免疫可以有效預(yù)防病毒感染,而口服疫苗可刺激機體產(chǎn)生胃腸道黏膜免疫。本實驗室已經(jīng)成功構(gòu)建好TGEV的乳酸桿菌口服疫苗。為了解山東省仔豬病毒性腹瀉病病原的感染流行情況和TGEV乳酸桿菌口服疫苗的免疫情況,本研究進行了以下幾個方面的工作:1.仔豬病毒性腹瀉病病原流行病學(xué)調(diào)查本研究采用RT-PCR方法對2013年10月~2014年10月從山東濟南、濰坊、濟寧等10個地市豬場采集的226份腹瀉樣品進行檢測。結(jié)果顯示,PEDV、TGEV與PoRV的檢出率分別為34.96%、2.21%和28.76%,并且不同季節(jié)PEDV和PoRV均有感染,表明PEDV和PoRV是引起山東省仔豬腹瀉的主要病因,并且山東省PoRV的檢出率明顯高于全國(劉云波等,2013)。規(guī)�；i場與散養(yǎng)戶相比,PEDV、TGEV、PoRV 3種病原的檢出率明顯降低,而且相對于未免疫腹瀉二聯(lián)苗的母豬而言,免疫后的母豬生產(chǎn)的仔豬發(fā)生腹瀉后,該病原的檢出率有所降低,表明合理的飼養(yǎng)環(huán)境和免疫對該病具有防控作用。2.TGEV SA基因的原核表達及間接ELISA方法的建立設(shè)計針對SA基因的特異性檢測引物,以實驗室保存的質(zhì)粒pUC-S為模板PCR擴增TGEV的SA基因,并連接到pET-30a載體上,構(gòu)建了重組表達質(zhì)粒,導(dǎo)入表達菌E.coli BL21,并進行了表達條件(IPTG濃度、溫度、轉(zhuǎn)速、時間)優(yōu)化。結(jié)果表明,在1m M/mL的IPTG,37℃,200r/min,表達6h時SA蛋白表達量最大。經(jīng)過Ni-Agarose柱純化獲得純度較高的目的蛋白,Western blot檢測表明,表達的重組SA蛋白能與TGEV陽性血清發(fā)生特異性反應(yīng),不與其他常見豬病陽性血清發(fā)生反應(yīng),說明該蛋白具有一定的反應(yīng)原性。以表達的SA蛋白為包被抗原建立間接ELISA方法,采用方陣滴定法確定抗體最佳稀釋度是1:100,抗原的包被濃度為0.75ug/ml。3.豬傳染性胃腸炎乳酸桿菌口服疫苗研究本研究將已經(jīng)構(gòu)建好的含有TGEV的A、D抗原位點和乳酸桿菌復(fù)制子基因Rep.8014的重組真核質(zhì)粒pRc/CMV2-SAD-Rep.8014電轉(zhuǎn)到豬源乳酸桿菌中,口服免疫6~8周齡BALB/c小鼠,并以乳酸桿菌組和商品疫苗組作為對照。分別在免疫前、二免前、二免疫后7d、14d、21d、28d采集小鼠血液收集血清檢測特異性IgG抗體、IL-4和IFN-γ,并在免疫前和免疫后每間隔2周各處死2只小鼠,取脾臟檢測CD4+T和CD8+T細(xì)胞的百分率,同時收集并制備小鼠的腸液樣本檢測特異性SIgA抗體。檢測結(jié)果顯示,口服免疫組中IgG抗體、IFN-γ以及CD4+T和CD8+T細(xì)胞在淋巴細(xì)胞中的百分含量與商品疫苗組差異較小,但均明顯高于乳酸桿菌對照組;IL-4只有在二免后14d含量才有所提高,其余時間變化不明顯;口服免疫組14d后檢測的腸液中的特異性SIgA抗體含量高于商品疫苗組,且差異顯著(P0.05)。結(jié)果表明,該口服疫苗具有一定的免疫效果,且能刺激機體產(chǎn)生較高的黏膜免疫抗體。
[Abstract]:Viral diarrhea in piglets is mainly caused by swine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), swine infectious gastroenteritis virus (Transmissible gastroenteritis virus, TGEV) and pig rotavirus (Porcine rotavirus, PoRV) alone or mixed infection. The disease has great harm to piglets and high mortality. Since 2010, the disease has high mortality. In some areas of Shandong, piglets have suffered from diarrhea as the main symptom of the disease, causing serious economic loss to the pig industry. Among them, swine infectious gastroenteritis is one of the main causes of diarrhea in piglets. The disease can cause 100% death.TGEV of piglets within 10 days of age and the main infection of the gastrointestinal tract and gastrointestinal tract caused by gastrointestinal tract. Mucosal immunity can effectively prevent virus infection, and oral vaccine can stimulate the body's gastrointestinal mucosal immunity. This laboratory has successfully constructed a good oral vaccine of TGEV lactobacilli. In order to understand the epidemic situation of viral diarrhea pathogens in Shandong piglets and the immunization of oral vaccine of TGEV lactobacillus, this study was conducted. The following aspects of the work: 1. the epidemiological investigation of viral diarrhoea in piglets: the RT-PCR method was used to detect 226 diarrhoea samples collected from 10 local pig farms in Ji'nan, Weifang, Jining, Shandong, October 2013 and October. The results showed that the detection rates of PEDV, TGEV and PoRV were 34.96%, 2.21% and 28.76%, respectively. PEDV and PoRV were infected in different seasons, indicating that PEDV and PoRV were the main causes of diarrhea in Shandong piglets, and the detection rate of PoRV in Shandong province was significantly higher than that of the whole country (Liu Yunbo, 2013). Compared with the scattered families, the detection rates of 3 pathogens of PEDV, TGEV and PoRV were significantly lower than those of unimmune diarrhea, and compared with those of unimmune diarrhea. As for the sows, the detection rate of the pathogen was reduced after the diarrhea of the piglets produced by the immune sows. It showed that the rational feeding environment and the immune response to the disease had the prevention and control effect of the prokaryotic expression of.2.TGEV SA gene and the establishment of the indirect ELISA method. The specific detection primers for the SA gene were designed, and the plasmid pUC-S in the laboratory was stored in the laboratory. The SA gene of TGEV was amplified by the template PCR and connected to the pET-30a vector. The recombinant expression plasmid was constructed, and the expression bacteria E.coli BL21 was introduced, and the expression conditions (IPTG concentration, temperature, speed, time) were optimized. The results showed that the expression of the protein was the largest at 1m M/mL IPTG, 37, and 200r/min. The purified protein was purified and purified by column purification. Western blot detection showed that the expression of recombinant SA protein could react with the positive serum of TGEV, and did not react with other common pig disease positive sera, indicating that the protein had a certain reactivity. The expression of SA protein was the indirect ELISA method of the envelope by Kang Yuanjian, and the formula was used to determine the resistance by square matrix titration. The best dilution of the body is 1:100, the concentration of the antigen is the concentration of 0.75ug/ml.3. in the oral vaccine of Lactobacillus in swine infectious gastroenteritis. This study will build a good A containing TGEV, the recombinant of D in situ point and the replication of Lactobacillus replicator Rep.8014, the true nuclear grain pRc/ CMV2-SAD-Rep.8014 is transferred to the Lactobacillus porcine source, and the oral immune 6~8 is taken orally. BALB/c mice of week age were compared with Lactobacillus and commercial vaccine groups. Before immunization, two immunization before and two immunization, 7d, 14d, 21d, and 28d were collected to detect specific IgG antibodies, IL-4 and IFN- gamma, and 2 mice were killed every 2 weeks before and after immunization, and the percentage of the spleen to detect CD4+T and CD8+T cells was obtained. The results showed that the percentage of IgG antibody, IFN- gamma, CD4+T and CD8+T cells in the lymphocytes of the oral immune group were less different than those of the commercial vaccine group, but all were significantly higher than those of the Lactobacillus group, and the 14d content of IL-4 was only raised after two exempts of two. The changes in the remaining time were not obvious, and the specific SIgA antibody content in the intestinal liquid of the oral immunization group was higher than that of the commercial vaccine group, and the difference was significant (P0.05). The results showed that the oral vaccine had a certain immune effect and could stimulate the body to produce a higher immune antibody to the mucous membrane of 14d.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.28

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