【摘要】：口蹄疫(Foot-and-Mouth Disease,FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)引起的一種烈性、急性家畜傳染病,傳播速度快,影響范圍大,給畜禽養(yǎng)殖業(yè)造成重大的經(jīng)濟(jì)損失。隨著規(guī)�；B(yǎng)殖程度的提高,FMD等傳染性疫病對(duì)家畜養(yǎng)殖業(yè)的危害越來(lái)越嚴(yán)重,防治難度不斷加大,傳統(tǒng)防治措施已突顯其不足,對(duì)新的防控技術(shù)、特別是安全有效的新型疫苗的需求日益迫切。VP1是編碼FMDV-VP1蛋白的免疫原性基因,表達(dá)的蛋白帶有誘導(dǎo)FMD免疫反應(yīng)的關(guān)鍵表位。本研究將FMDV VP1基因克隆并進(jìn)行優(yōu)化,構(gòu)建重組表達(dá)質(zhì)粒并檢測(cè)其在大腸桿菌和乳酸桿菌中的表達(dá),最后將重組乳酸桿菌免疫豚鼠檢測(cè)其誘導(dǎo)的黏膜免疫保護(hù)效果。具體研究方法和結(jié)果如下:1.FMDV VP1基因的原核表達(dá)載體的構(gòu)建及重組乳酸桿菌的制備從A型FMDV中克隆出VP1基因后,根據(jù)乳酸桿菌密碼子偏好性對(duì)VP1進(jìn)行優(yōu)化,與大腸桿菌-乳酸桿菌穿梭表達(dá)載體p SIP411進(jìn)行連接,并將連接產(chǎn)物轉(zhuǎn)化到DH5α中篩選出陽(yáng)性克隆菌,重組質(zhì)粒酶切和PCR鑒定成功后再轉(zhuǎn)化到BL21中進(jìn)行誘導(dǎo)表達(dá),表達(dá)產(chǎn)物經(jīng)SDS-PAGE和western blot檢測(cè)可見(jiàn)在24k Da處有明顯的條帶,證明VP1-p SIP411大腸桿菌-乳酸桿菌穿梭重組表達(dá)載體構(gòu)建成功并且在BL21中得到表達(dá)的蛋白具有反應(yīng)原性。然后將重組質(zhì)粒p SIP411-VP1電轉(zhuǎn)化到乳酸桿菌NC8和WCFS1中,通過(guò)質(zhì)粒提取、雙酶切鑒定和PCR鑒定等方法篩選出的陽(yáng)性菌進(jìn)行Spp IP誘導(dǎo)表達(dá),表達(dá)產(chǎn)物經(jīng)western blot檢測(cè)可見(jiàn)明顯的目的條帶,證明VP1在NC8和WCFS1中得到表達(dá)并且具有反應(yīng)原性。2.重組乳酸桿菌免疫豚鼠以及免疫效果的檢測(cè)動(dòng)物免疫試驗(yàn)中將豚鼠分為5組(p SIP411-VP1-NC8,p SIP411-NC8,p SIP411-VP1-WCFS1,p SIP411-WCFS1和陰性對(duì)照組),免疫期間采集豚鼠唾液、糞便和血清樣品進(jìn)行ELISA檢測(cè),用流式細(xì)胞儀檢測(cè)外周血中CD4+、CD8+淋巴細(xì)胞表達(dá)水平,CFSE法測(cè)定脾臟淋巴細(xì)胞增殖情況,細(xì)胞中和試驗(yàn)檢測(cè)中和抗體含量。結(jié)果顯示,兩組重組乳酸桿菌免疫組抗體水平明顯高于其他三組,差異顯著,CD4+、CD8+淋巴細(xì)胞含量明顯增多,脾淋巴細(xì)胞在抗原刺激后增殖明顯,可在血清中檢測(cè)到中和抗體。以上結(jié)果表明,VP1在重組乳酸桿菌NC8和WCFS1中獲得了表達(dá),并且免疫動(dòng)物后增強(qiáng)了動(dòng)物的免疫功能,抵抗了FMDV的感染,為研制預(yù)防FMDV的重組乳酸桿菌疫苗奠定了實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Foot-and-mouth disease (Foot-and-Mouth Disease,FMD) is a kind of acute infectious disease caused by foot-and-mouth disease virus (Foot-and-Mouth Disease Virus,FMDV). With the development of large-scale aquaculture, FMD and other infectious diseases are becoming more and more serious to livestock breeding, and the difficulty of prevention and control is increasing. The traditional prevention and control measures have highlighted its shortcomings, and the new technology of prevention and control has been brought to light. VP1 is the immunogenicity gene encoding FMDV-VP1 protein, and the expressed protein has the key epitope to induce FMD immune response. In this study, FMDV VP1 gene was cloned and optimized to construct recombinant expression plasmid and detect its expression in Escherichia coli and Lactobacillus coli. The specific research methods and results are as follows: construction of prokaryotic expression vector of 1.FMDV VP1 gene and preparation of recombinant Lactobacillus coli VP1 gene were cloned from type A FMDV and VP1 was optimized according to codon preference of Lactobacillus. The recombinant plasmid was ligated with Escherichia coli-Lactobacillus shuttle expression vector p SIP411, and the product was transformed into DH5 偽 to screen positive clones. The recombinant plasmid was digested and identified by PCR and then transformed into BL21 to induce expression. SDS-PAGE and western blot analysis showed that there were obvious bands at 24k Da, which proved that the recombinant expression vector of VP1-p SIP411 Escherichia coli-Lactobacillus shuttle was successfully constructed and expressed in BL21. Then the recombinant plasmid p SIP411-VP1 was electrotransformed into NC8 and WCFS1 of Lactobacillus. The positive bacteria screened by plasmid extraction, double enzyme digestion and PCR identification were induced by Spp IP expression. The expression of VP1 in NC8 and WCFS1 showed obvious target bands by western blot, which showed that VP1 was expressed in NC8 and WCFS1. 2. 2. Guinea pigs immunized with recombinant lactobacillus and their immunological effects the guinea pigs were divided into 5 groups (p SIP411-VP1-NC8,p SIP411-VP1-WCFS1,p SIP411-WCFS1 and negative control group) in the animal immune test. Saliva was collected from guinea pigs during immunization. The expression of CD4 and CD8 in peripheral blood was detected by flow cytometry, the proliferation of splenic lymphocytes by CFSE, and the content of neutralizing antibody by neutralization test. The results showed that the antibody levels of the two groups were significantly higher than those of the other three groups. The levels of CD4 and CD8 lymphocytes were significantly increased and the spleen lymphocytes proliferated after antigen stimulation. Neutralizing antibodies could be detected in serum. The results showed that VP1 was expressed in recombinant Lactobacillus NC8 and WCFS1, and the immune function of the animals was enhanced after immunization, and the infection of FMDV was resisted, which laid an experimental foundation for the development of recombinant Lactobacillus vaccine for the prevention of FMDV.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.4

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本文編號(hào)：2314156