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標題: 大腸桿菌表現雞傳染性華氏囊病類病毒顆粒產量及保護效力之提升
Enhancement of the production and protection efficiency of IBDV virus like particle expressed in E. coli
作者: 廖君儀
Liao, Jung-Yi
關鍵字: 雞傳染性華氏囊病;Infectious Bursal Disease Virus;類病毒顆粒;大腸桿菌;Virus like particle;E. coli
出版社: 微生物暨公共衛生學研究所
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雞傳染性華氏囊病病毒(Infectious Bursal Disease Virus; IBDV)顆粒由VP2和VP3組成,分別位於顆粒之外層及內層。在病毒顆粒形成過程中,首先IBDV之polyprotein VP243藉由其中的VP4切割產生VP2前驅物(pVP2)及VP3。一部分的pVP2經宿主細胞酵素切割後與pVP2形成病毒顆粒前驅物。其中之pVP2經VP4多次切割後,再藉由VP2 cis-cleavage活性切割產生VP2,而形成成熟之病毒顆粒。然而VP4在病毒顆粒之形成過程中的影響仍然有待進一步研究。因此本試驗目的為(1)探討VP4在pVP2及VP3存在的情形下,對類病毒顆粒(Virus like particle; VLP)的形成是否扮演重要的角色;(2)經由對VLP形成之了解,設計不同之重組質體以檢測大腸桿菌系統生產IBDV VLP及subviral particle (SVP)之情形,並測試此重組蛋白對雞隻感染強毒株IBDV (very virulent IBDV; vvIBDV)之保護力。本試驗共構築五種重組質體:(1) pTri VP2-452,表現經由宿主細胞酵素作用後之pVP2 (表現VP2前端之452個胺基酸;VP2-452)、(2) pET VP243,表現polyprotein VP243、(3) pET 512/VP3/452,共同表現全長之pVP2 (VP2-512)、VP3及VP2-452、(4) pET 487/VP3/452,共同表現經VP4完整切割之pVP2 (VP2-487)、VP3及VP2-452及(5) pET VP243/452共同表現polyprotein VP243及VP2-452。將重組質體以大腸桿菌進行蛋白質表現後,收取可溶性蛋白質,經由蔗糖密度梯度超高速離心並以酵素連結免疫吸附試驗(enzyme linked immunosorbent assay; ELISA)分析重組蛋白之組成,各質體皆有T=1 SVP之形成,但僅pET VP243及pET VP243/452具較多VLP表現量,進一步以穿透式電子顯微鏡(transmission electron microscope; TEM)可觀察到大小約70 nm之VLP,然而這些VLP並未在不包含VP4之重組質體表現之蛋白中發現。將不同之重組蛋白進行保護力之動物實驗,第一次動物實驗結果顯示在2×102 EID50 vvIBDV的攻毒劑量下,pET 512/VP3/452及pET 487/VP3/452之重組蛋白僅提供雞隻部分保護力,其餘之重組蛋白則提供80 %以上之保護力。第二次動物實驗結果顯示pET VP243/452之重組蛋白確實能提供雞隻對vvIBDV感染80 %以上的保護力;而市售佐劑ISA 71誘發的抗體力價顯著高於佛氏佐劑,但是於注射部位造成嚴重的副作用。因此本研究認為VP4在pVP2的成熟及VLP的形成扮演重要的角色。而pET VP243及pET VP243/452不僅能產生VLP,並能提供雞隻對抗vvIBDV感染之良好保護力。

The viral particle of infectious bursal disease virus (IBDV) is composed of VP2 and VP3 on the outside and inside, respectively. During the formation of viral particle, a polyprotein VP243 was first produced and self-cleaved into precursor VP2 (pVP2) and VP3 by VP4. The researchers had shown that pVP2 C-terminal was further cut by VP4 and partially by host cell enzyme, and finally by VP2-itself to form mature VP2 and finally produce viral particle. However, the role of VP4 in the final steps of viral partical formation is still not clear. The objectives of this study are (1) to investigate the requirement of VP4 in the formation of virus like particle (VLP) after that pVP2 and VP3 have been already produced; (2) to construct various recombinant plasmids to investigate the production of VLP and subviral particle (SVP) in E. coli and evaluate the protection efficacy of theses recombinant proteins against very virulent IBDV (vvIBDV) challenge. Five plasmids were constructed, including (1) pTri VP2-452 (encoding for the first 452 amino acids of VP2, VP2-452, the pVP2 after cutting by host cell enzyme); (2) pET VP243 (encoding for VP243); (3) pET 512/VP3/452 (encoding for the full length of pVP2 (VP2-512), VP3 and VP2-452); (4) pET 487/VP3/452 (encoding for the pVP2 after the last cut by VP4 (VP2-487), VP3 and VP2-452); (5) pET VP243/452 (encoding for VP243 and VP2-452). The plasmids were transformed into E. coli and the expressed virus like particles were separated by sucrose gradient centrifugation and analyzed by ELISA and electron microscopy. The pET VP243 and pET VP243/452, but not the plasmids without VP4 gene, had higher 70-nm-VLP production efficiency. After challenge with 2×102 EID50 vvIBDV, the proteins expressed by pTri VP2, pET VP243 and pET VP243/452 could provide more than 80 % protection of chickens against vvIBDV. However, proteins expressed by pET 512/VP3/452 and pET 487/VP3/452 could only provide protection less than 60%. When ISA 71was used as adjuvant, the anti-IBDV antibody titer was significantly higher than that when Freund’s adjuvant was used. The severe inflammations were observed in the injection sites of proteins with ISA 71. The results indicated that VP4 play an important role in the final steps of VP2 maturation and the formation of VLP. Moreover, pET VP243 and pET VP243/452 can not only produce VLP in E. coli but also provide efficacious protection of chicks against vvIBDV.
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