本文選題：豬流行性腹瀉病毒 + 疫苗　； 參考：《河南農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：豬流行性腹瀉病(Porcine epidemic diarrhea,PED)是由豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PEDV)引發(fā)的一種腸道傳染病,自從該病在我國流行以來,給我國養(yǎng)豬業(yè)造成了重大的經(jīng)濟(jì)損失。豬場主要通過日常消毒和接種疫苗來預(yù)防該病,但目前在市場上流行的疫苗并不能到達(dá)理想的免疫效果。研究表明仔豬主要通過初乳獲得母源SIgA(分泌型免疫球蛋白A)產(chǎn)生對PEDV的被動(dòng)免疫保護(hù),血液中循環(huán)的IgG抗體不能對仔豬產(chǎn)生有效的保護(hù),非經(jīng)腸道黏膜免疫不能獲得SIgA,因此通過腸道黏膜免疫是使仔豬獲得免疫保護(hù)的重要途徑,研制安全有效可以通過腸道黏膜免疫的新型疫苗尤為重要。S蛋白是由S基因編碼的PEDV一個(gè)表面結(jié)構(gòu)蛋白,也是PEDV的主要結(jié)構(gòu)蛋白,它含有介導(dǎo)病毒侵入宿主細(xì)胞的受體結(jié)合域,還含有介導(dǎo)機(jī)體產(chǎn)生病毒中和抗體的抗原表位。有可以誘導(dǎo)機(jī)體產(chǎn)生中和抗體和提供黏膜免疫保護(hù)的作用。COE抗原表位是S基因中的一個(gè)重要抗原表位,是由chang等人發(fā)現(xiàn)并命名的,這個(gè)抗原表位位于S基因的499-638aa區(qū)域。自從COE抗原表位被發(fā)現(xiàn)后世界各國學(xué)者均嘗試應(yīng)用COE抗原表位基因研究制備PEDV的基因工程疫苗。本實(shí)驗(yàn)是研究PEDV主要抗原決定簇COE的原核表達(dá)與純化,為研制新型的豬流行性腹瀉病毒疫苗奠定基礎(chǔ)。本實(shí)驗(yàn)首先從河南本地的疑似得豬流行性腹瀉病的病豬小腸分離出PEDV,然后根據(jù)GenBank發(fā)表的PEDV-COE序列設(shè)計(jì)出一對引物,用PCR的方法擴(kuò)增出長度為420bp的COE目的片段。將目的片段連接在載體PMD19-T上,然后轉(zhuǎn)化到JM109大腸桿菌中,涂板過夜生長,挑菌接種到液體LB中培養(yǎng)擴(kuò)增、提質(zhì)粒,鑒定正確后送公司測序,將測序結(jié)果與GenBank公布的最相近的4個(gè)PEDV-COE序列做比較,發(fā)現(xiàn)河南本地PEDV發(fā)生了一定的突變,與河南周邊地區(qū)的毒株具有一定的差異。將COE基因片段連接至PET-32a載體上構(gòu)建原核表達(dá)載體PET-32a-COE,鑒定正確后將表達(dá)載體轉(zhuǎn)化到BL21(DE3)pLysS大腸桿菌中。為了探索最佳的誘導(dǎo)條件,我們對IPTG誘導(dǎo)的濃度和時(shí)間進(jìn)行了優(yōu)化,結(jié)果顯示0.6mmol/L的IPTG誘導(dǎo)4h能夠得到最大量的目的蛋白。我們將誘導(dǎo)表達(dá)目的蛋白的大腸桿菌經(jīng)超聲破碎、經(jīng)鎳株蛋白純化試劑盒純化,透析復(fù)性,最后得到了HIS-COE目的蛋白。HIS-COE目的蛋白的成功表達(dá)與純化為制備能夠預(yù)防在本地區(qū)流行的PEDV的新型疫苗提供了參考,對PEDV基因工程疫苗的研究與制備也具有重要意義。
[Abstract]:Porcine epidemic diarrhea (PED) is a kind of intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). Pig farm mainly through daily disinfection and vaccination to prevent the disease, but the current popular vaccine in the market can not reach the ideal immune effect. The results showed that maternal Siga (secretory immunoglobulin A) produced passive immune protection against PEDV in piglets, and circulating IgG antibody in blood could not protect piglets effectively. Siga can not be obtained without intestinal mucosal immunization, so it is an important way for piglets to get immunity protection through intestinal mucosal immunization. It is particularly important to develop a new vaccine that can be immunized safely and effectively through intestinal mucosa. S protein is a surface structural protein encoded by S gene and the main structural protein of PEDV. It contains a receptor binding domain that mediates the invasion of the virus into host cells. It also contains antigenic epitopes that mediate the production of viral neutralizing antibodies. Coe epitope is an important antigen epitope in S gene which was discovered and named by chang et al. It is located in the 499-638aa region of S gene. Since the discovery of Coe epitopes, researchers all over the world have tried to prepare PEDV genetic engineering vaccine by using Coe epitope gene research. This study was aimed to study the prokaryotic expression and purification of PEDV major antigen determinant Coe, which laid a foundation for the development of a novel porcine epidemic diarrhea virus vaccine. A pair of primers were designed according to the PEDV-COE sequence published in GenBank to amplify the Coe fragment with length of 420bp. The target fragment was ligated into the vector PMD19-T, then transformed into E. coli JM109, and then grew overnight on the coated plate, inoculated into liquid LB, cultured and amplified, extracted the plasmid, and identified correctly and sent to the company for sequencing. The sequencing results were compared with the four most similar PEDV-COE sequences published by GenBank. It was found that the local PEDV mutation in Henan Province was different from that in the surrounding regions of Henan Province. Coe gene fragment was ligated into PET-32a vector to construct the prokaryotic expression vector PET-32a-COE, and the expression vector was transformed into BL21 (DE3) pLysS Escherichia coli. In order to explore the optimal induction conditions, we optimized the concentration and time of IPTG induction. The results showed that 0.6 mmol / L IPTG induction for 4 h could obtain the maximum amount of target protein. We will induce the expression of the target protein of Escherichia coli by ultrasonic fragmentation, purification of nickel strain protein purification kit, dialysis renaturation, Finally, the successful expression and purification of HIS-COE target protein 路HIS-COE target protein provided a reference for the preparation of new PEDV vaccine, which could prevent PEDV from becoming prevalent in our region. It is also of great significance for the research and preparation of PEDV gene engineering vaccine.
【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.651

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本文編號(hào)：2071864