本文選題：傳染性法氏囊病病毒 + 反向遺傳　； 參考：《浙江大學(xué)》2006年博士論文

【摘要】：傳染性法氏囊病(IBD)是由傳染性法氏囊病病毒(IBDV)引起的急性接觸性傳染病,是危害世界養(yǎng)禽業(yè)的主要傳染病之一。IBDV主要侵害3-8周齡雛雞的中樞免疫器官法氏囊,破壞帶有sIgM的B淋巴細(xì)胞,引起脾臟、胸腺及外周血液淋巴細(xì)胞的凋亡,導(dǎo)致以淋巴細(xì)胞衰竭壞死為主要特征的免疫缺陷性和免疫抑制性疾病。 通過易感動(dòng)物雛雞,構(gòu)建了一株傳染性法氏囊病病毒基因組節(jié)段雜合病毒。應(yīng)用PCR定點(diǎn)突變方法,在IBDV野毒株ZJ2000株A節(jié)段2366-2371nt引入沉默突變NaeI后,克隆入真核表達(dá)載體pCI,獲得pCI-AKZJ2000,分別制備pCI-AKZJ2000、以及含有細(xì)胞致弱疫苗株HZ2株A和B節(jié)段全長(zhǎng)的真核表達(dá)載體pCI-AKHZ2和pCI-mBHZ2的超純質(zhì)粒。不同組合的上述質(zhì)粒(pCI-AKZJ2000/pCI-mBHZ2和pCI-AKHZ2/pCI-mBHZ2),在脂質(zhì)體介導(dǎo)下,肌肉途徑分別注射雛雞。通過血清抗體、病毒抗原和病毒粒子檢測(cè)、雞胚傳代試驗(yàn)、以及分子遺傳標(biāo)記鑒定,表明從雞體內(nèi)拯救出了雜合病毒rAKZJ/HZ和重組病毒Xihu2002株。本試驗(yàn)為IBDV的反向遺傳系統(tǒng)提供了一個(gè)新策略,尤其是對(duì)于不能適應(yīng)細(xì)胞的IBDV強(qiáng)毒株的拯救。結(jié)果還提示IBDV基因組B節(jié)段序列對(duì)病毒毒力具有重要作用,為研究VP1蛋白在病毒復(fù)制和致病中的作用提供了思路。 制備了兔抗IBDV VP5多克隆抗體。利用RT-PCR技術(shù)從IBDV地方分離株TL2004株感染雞胚尿囊液中擴(kuò)增到VP5基因,進(jìn)而構(gòu)建了T7啟動(dòng)子控制下的N端GST-Tag融合表達(dá)質(zhì)粒pGEX-VP5。序列測(cè)定表明VP5基因全長(zhǎng)438bp,編碼一個(gè)由145個(gè)氨基酸組成的VP5蛋白。將pGEX-VP5轉(zhuǎn)化大腸桿菌BL21,在IPTG的誘導(dǎo)下高效表達(dá)了GST-VP5融合蛋白(44kD)。通過包涵體純化的方法,獲得的較高純度的融合蛋白,免疫新西蘭兔,Western blot和ELISA分析表明,制備的融合蛋白抗血清效價(jià)在1:12800以上,并具有良好的免疫反應(yīng)性,為進(jìn)一步研究VP5在IBDV復(fù)制與致病中的作用,以及IBDV VP5基因缺失病毒打下了良好的基礎(chǔ)。 在上述基礎(chǔ)上,進(jìn)而探索了VP5基因序列及編碼蛋白對(duì)IBDV復(fù)制的影響。通過PCR定點(diǎn)突變方法,將IBDV HZ2株A節(jié)段ORF A2(VP5)起始密碼子
[Abstract]:Infectious bursal disease (IBD) is an acute contact infectious disease caused by infectious bursal disease virus (IBDV). The destruction of B lymphocytes with sIgM caused apoptosis of lymphocytes in spleen thymus and peripheral blood and resulted in immunodeficient and immunosuppressive diseases characterized by lymphocyte failure and necrosis. A novel infectious bursal disease virus (IBDV) genomic segment heterozygous virus was constructed from susceptible chickens. The silencing mutation NaeI was introduced into the A segment 2366-2371nt of IBDV wild strain ZJ2000 by PCR site-directed mutation. The pCI-AKZJ2000 and the eukaryotic expression vectors pCI-AKZJ2000 and pCI-mBHZ2 were cloned into the eukaryotic expression vector pCI-AKZJ2000 and the eukaryotic expression vectors pCI-AKZZ2 and pCI-mBHZ2 containing the full-length segments of A and B segments of the attenuated vaccine strain HZ2 were prepared, respectively. Different combinations of these plasmids (pCI-AKZJ2000 / pCI-mBHZ2 and pCI-AKHZ2 / pCI-mBHZ2) were injected into chicks by liposome mediated muscle pathway. The results of serum antibody, virus antigen and virus particle detection, chicken embryo passage test and molecular genetic marker identification showed that the hybrid virus rAKZJ / HZ and recombinant virus Xihu2002 strain were rescued from chicken. This study provides a new strategy for the reverse genetic system of IBDV, especially for the rescue of IBDV virulent strains that can not adapt to cells. The results also suggest that the B segment sequence of IBDV genome plays an important role in virulence of the virus and provides a way to study the role of VP1 protein in viral replication and pathogenicity. Rabbit anti IBDV VP5 polyclonal antibody was prepared. The VP5 gene was amplified by RT-PCR from chicken embryo allantoic fluid infected with TL2004 strain of IBDV, and then the N-terminal GST-Tag fusion expression plasmid pGEX-VP5 was constructed under the control of T7 promoter. Sequence analysis showed that the VP5 gene was 438 BP in length, encoding a 145 amino acid VP5 protein. PGEX-VP5 was transformed into Escherichia coli BL21, and GST-VP5 fusion protein (44kD) was highly expressed under the induction of IPTG. The high purity fusion protein was obtained by the method of inclusion body purification. Western blot and Elisa analysis showed that the antiserum titer of the fusion protein was over 1: 12800 and had good immunoreactivity. The results provide a good basis for the further study of the role of VP5 in the replication and pathogenesis of IBDV and the deletion of VP5 gene of IBDV. On the above basis, the effects of VP5 gene sequence and encoded protein on IBDV replication were explored. The ORF A2 (VP5) initiation codon of A segment of IBDV HZ2 strain was determined by PCR site-directed mutation.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R373

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 張寧;傳染性法氏囊病病毒非結(jié)構(gòu)蛋白的功能研究[D];新疆農(nóng)業(yè)大學(xué);2007年

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本文編號(hào)：2101490