仔猪病毒性腹泻病流行病学调查及猪传染性胃肠炎乳酸杆菌口服疫苗研究

发布时间：2018-04-20 02:23

本文选题：猪流行性腹泻 + 猪传染性胃肠炎　；参考：《山东农业大学》2015年硕士论文

【摘要】：仔猪病毒性腹泻主要是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)和猪轮状病毒(Porcine rotavirus,PoRV)单独或混合感染引起,该病对仔猪的危害较大,病死率高。自2010年冬季以来,山东省部分地区仔猪发生以腹泻为主要症状的疾病,给养猪业造成了严重的经济损失。其中猪传染性胃肠炎是引起仔猪腹泻的主要病因之一,该病可造成10日龄以内的仔猪100%死亡。TGEV主要感染胃肠道引起局部组织发生病变,胃肠道黏膜免疫可以有效预防病毒感染,而口服疫苗可刺激机体产生胃肠道黏膜免疫。本实验室已经成功构建好TGEV的乳酸杆菌口服疫苗。为了解山东省仔猪病毒性腹泻病病原的感染流行情况和TGEV乳酸杆菌口服疫苗的免疫情况,本研究进行了以下几个方面的工作:1.仔猪病毒性腹泻病病原流行病学调查本研究采用RT-PCR方法对2013年10月~2014年10月从山东济南、潍坊、济宁等10个地市猪场采集的226份腹泻样品进行检测。结果显示,PEDV、TGEV与PoRV的检出率分别为34.96%、2.21%和28.76%,并且不同季节PEDV和PoRV均有感染,表明PEDV和PoRV是引起山东省仔猪腹泻的主要病因,并且山东省PoRV的检出率明显高于全国(刘云波等,2013)。规模化猪场与散养户相比,PEDV、TGEV、PoRV 3种病原的检出率明显降低,而且相对于未免疫腹泻二联苗的母猪而言,免疫后的母猪生产的仔猪发生腹泻后,该病原的检出率有所降低,表明合理的饲养环境和免疫对该病具有防控作用。2.TGEV SA基因的原核表达及间接ELISA方法的建立设计针对SA基因的特异性检测引物,以实验室保存的质粒pUC-S为模板PCR扩增TGEV的SA基因,并连接到pET-30a载体上,构建了重组表达质粒,导入表达菌E.coli BL21,并进行了表达条件(IPTG浓度、温度、转速、时间)优化。结果表明,在1m M/mL的IPTG,37℃,200r/min,表达6h时SA蛋白表达量最大。经过Ni-Agarose柱纯化获得纯度较高的目的蛋白,Western blot检测表明,表达的重组SA蛋白能与TGEV阳性血清发生特异性反应,不与其他常见猪病阳性血清发生反应,说明该蛋白具有一定的反应原性。以表达的SA蛋白为包被抗原建立间接ELISA方法,采用方阵滴定法确定抗体最佳稀释度是1:100,抗原的包被浓度为0.75ug/ml。3.猪传染性胃肠炎乳酸杆菌口服疫苗研究本研究将已经构建好的含有TGEV的A、D抗原位点和乳酸杆菌复制子基因Rep.8014的重组真核质粒pRc/CMV2-SAD-Rep.8014电转到猪源乳酸杆菌中,口服免疫6~8周龄BALB/c小鼠,并以乳酸杆菌组和商品疫苗组作为对照。分别在免疫前、二免前、二免疫后7d、14d、21d、28d采集小鼠血液收集血清检测特异性IgG抗体、IL-4和IFN-γ,并在免疫前和免疫后每间隔2周各处死2只小鼠,取脾脏检测CD4+T和CD8+T细胞的百分率,同时收集并制备小鼠的肠液样本检测特异性SIgA抗体。检测结果显示,口服免疫组中IgG抗体、IFN-γ以及CD4+T和CD8+T细胞在淋巴细胞中的百分含量与商品疫苗组差异较小,但均明显高于乳酸杆菌对照组;IL-4只有在二免后14d含量才有所提高,其余时间变化不明显;口服免疫组14d后检测的肠液中的特异性SIgA抗体含量高于商品疫苗组,且差异显著(P0.05)。结果表明,该口服疫苗具有一定的免疫效果,且能刺激机体产生较高的黏膜免疫抗体。
[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.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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