本文選題：傳染性法氏囊病 + 乙肝病毒核心抗原��； 參考：《南京農(nóng)業(yè)大學(xué)》2010年碩士論文

【摘要】：傳染性法氏囊病(Infectious Bursal Disease, IBD)是由傳染性法氏囊病病毒(Infectious Bursal Disease Virus, IBDV)引起的以侵害雛雞淋巴組織,特別是中樞免疫器官—法氏囊為主要特征的傳染病。該病不僅引起患病動(dòng)物死亡,而且還導(dǎo)致機(jī)體免疫抑制,使機(jī)體的免疫防御能力降低和疫苗免疫接種失敗,對(duì)易感雞群實(shí)施疫苗接種是預(yù)防該病最有效的方法,但由于各地流行的IBDV毒株的毒力與抗原性的差異,給疫苗毒株的選擇造成較大的困難,每年由于IBD免疫失敗或免疫抑制造成的經(jīng)濟(jì)損失巨大。為了抵抗超強(qiáng)毒的攻擊以及母源抗體的干擾,目前廣泛應(yīng)用中等毒力的IBD活疫苗,給IBD的防控帶來(lái)極大的隱患,滅活疫苗存在著抗原制備的困難。鑒于此,進(jìn)一步分析當(dāng)前IBDV的分子流行病學(xué)、研制新型的基因工程疫苗,仍是當(dāng)前禽病防控工作中亟待解決的重要課題。研究?jī)?nèi)容包括兩個(gè)部分： 試驗(yàn)1：IBDV抗原表位與HBcAg嵌合體基因的構(gòu)建及其表達(dá)產(chǎn)物分析 為了提高模擬IBDV多表位基因5epis的免疫效力,運(yùn)用重疊延伸PCR技術(shù),在乙型肝炎病毒核心抗原(Hepatitis B core antigen, HBcAg)基因的231位～232位核苷酸(77位～78位氨基酸殘基)之間插入BamH I和EcoR I酶切位點(diǎn),構(gòu)建基于HBcAg修飾基因(Modified HBc, mHBc)的抗原表位嵌合體基因分子載體pET-mHBc。將模擬IBDV多表位基因5epis定向插入到pET-mHBc的mHBc基因中,獲得嵌合體基因mHBc-5epis表達(dá)質(zhì)粒pET-mHBc-5epis,在大腸桿菌中表達(dá)。用SDS-PAGE分析,重組嵌合體蛋白的分子量約為29ku；在Western-blotting試驗(yàn)中,重組嵌合體蛋白與IBDV抗體反應(yīng)形成1條特異性蛋白帶。將重組菌超聲破碎后,用電鏡觀察,可見重組嵌合體蛋白呈病毒樣顆粒(VLP),直徑約100nm～120nm；用IBDV抗體夾心ELISA檢測(cè),抗原效價(jià)達(dá)到1：200。本試驗(yàn)成功構(gòu)建了5epis與HBcAg嵌合體基因,在大腸桿菌中高效表達(dá)具有IBDV抗原反應(yīng)性的重組病毒樣顆粒嵌合體蛋白,為表位型IBD基因工程疫苗的研究提供了一條新途徑。 試驗(yàn)2：重組mHBc-5epis嵌合體蛋白的免疫功能分析 將重組菌pET-mHBc-5epis誘導(dǎo)表達(dá),用SDS-PAGE膠回收方法純化重組mHBc-5epis嵌合體蛋白,免疫1月齡SPF雞,雙抗體夾心ELISA法檢測(cè)免疫雞血清中的IFN-y、 IL-2、IL-4、IL-6動(dòng)態(tài)變化,在免疫后的第2天IL-2、IL-4表達(dá)量都有明顯上升;用間接ELISA去檢測(cè)免疫雞血清中IBDV抗體,在免疫后的第14天IBDV抗體效價(jià)達(dá)到1：100。試驗(yàn)表明,嵌合體基因HBcAg分子載體能夠增強(qiáng)雞產(chǎn)生特異性的體液免疫及細(xì)胞免疫應(yīng)答。本試驗(yàn)首次證明了HBcAg在雞體內(nèi)能增強(qiáng)抗原表位的免疫保護(hù)效力,為新型IBD顆�；啾砦灰呙绲难芯康於嘶A(chǔ)。
[Abstract]:Infectious bursal disease (Bursal Disease, IBD) is an infectious disease caused by infectious bursal disease virus (IBDV) Infectious Bursal Disease Virus, IBDV), which is characterized by invasion of chicken lymphoid tissue, especially central immune organ-bursa of Fabricius. The disease not only causes the death of diseased animals, but also leads to immune suppression, which reduces the immune defense ability of the body and fails to vaccinate the susceptible chickens. The most effective way to prevent the disease is to vaccinate susceptible chickens. However, because of the difference of virulence and antigenicity of IBDV strains in different places, it is difficult to select vaccine strains. Every year, the economic loss caused by the failure of IBD immunization or immunosuppression is enormous. In order to resist the attack of super virulence and the interference of maternal antibody, the medium virulence IBD live vaccine is widely used, which brings great hidden trouble to the prevention and control of IBD, and the inactivated vaccine has the difficulty of antigen preparation. In view of this, further analysis of molecular epidemiology of IBDV and development of new genetic engineering vaccine are still an important task to be solved in poultry disease prevention and control. The study consists of two parts: Construction of 1:IBDV epitope and HBcAg chimera gene and analysis of its expression products In order to improve the immune potency of 5epis, a mimic IBDV polyepitope gene, the overlapping extension PCR technique was used. To construct the chimeric gene vector pET-mHBcbased on modified HBcAg modified gene, the restriction sites of BamH I and EcoR I were inserted between the amino acid residues at the 77th and 78th position of nucleotide at the 231st and 232nd nucleotides of hepatitis B virus core antigen (B core antigen, HBcAg), and the molecular vector pET-mHBcwas constructed based on the modified HBcAg gene (modified HBc, mHBcc). The chimeric gene mHBc-5epis expression plasmid pET-mHBc-5epis was obtained by inserting the mimic IBDV polyepitope gene 5epis into the mHBc gene of pET-mHBc and expressed in Escherichia coli. By SDS-PAGE analysis, the molecular weight of recombinant chimeric protein was about 29ku. in Western-blotting test, the recombinant chimeric protein reacted with IBDV antibody to form a specific protein band. The recombinant chimeric protein was observed by electron microscope after ultrasonic fragmentation. The recombinant chimeric protein was shown to be a virus like particle with a diameter of about 100 nm and 120 nm in diameter. The titer of the recombinant chimeric protein was 1: 200 detected by IBDV antibody sandwich ELISA. In this study, 5epis and HBcAg chimeric genes were successfully constructed and highly expressed in Escherichia coli with IBDV antigen-responsive recombinant virus-like granular chimeric proteins, which provided a new approach for the study of epitope IBD gene engineering vaccine. Test 2: analysis of immune function of Recombinant mHBc-5epis Chimeric protein The recombinant mHBc-5epis chimeric protein was purified by SDS-PAGE gel recovery method. The recombinant mHBc-5epis chimeric protein was immunized with 1-month-old SPF chicken. The dynamic changes of IFN-y, IL-2, IL-4 and IL-6 in serum of immunized chicken were detected by double antibody sandwich ELISA method. On the second day after immunization, the expression of IL-4 increased obviously, and the titer of IBDV antibody reached 1: 100 on the 14th day after immunization by indirect ELISA to detect the IBDV antibody in the serum of immunized chicken. The results showed that the chimeric gene HBcAg molecular vector could enhance the specific humoral and cellular immune responses of chickens. It was proved for the first time that HBcAg could enhance the immuno-protective effect of antigen epitopes in chicken and lay a foundation for the study of novel IBD granulated multiepitope vaccine.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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