Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36242
標題: H7亞型流感病毒重組HA似病毒顆粒疫苗的免疫效果分析
Immunogenicity and Efficacy of Baculovirus Expressing Recombinant H7 Subtypic Influenza HA Virus-like Particles
作者: 張若麟
Chang, Ro-Lin
關鍵字: influenza virus
流感病毒
virus-like particles
hemagglutinin
vaccine.
似病毒顆粒
血球凝集素
疫苗
出版社: 生物科技學研究所
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摘要: 自從高致病性流感病毒疫情在禽鳥之間爆發以來,對家禽養殖產業與大眾健康造成了重大威脅。為了避免流感病毒的感染,接種疫苗是最主要的預防措施。在許多預防病毒傳染性疾病所使用的疫苗類型中,似病毒顆粒具備安全與效果佳的優點,因其外在結構類似真實病毒顆粒且不含病毒基因體,因此被視為是可取代傳統流感疫苗。本研究為了探討流感似病毒顆粒形成的必需蛋白組成與其在雞隻中所引發的保護效果,遂將H7N1病毒的hemagglutinin (HA) 基因單獨構築、或與來自H6N1病毒之matrix 1 (M1) 基因或neuraminidase (NA) 基因共同構築表達。將所構築之3株重組桿狀病毒分別感染Hi-5細胞,證實所帶的流感病毒基因 HA、HA/M1或HA/NA/M1皆可表現,以20%~60%蔗糖梯度離心分析,發現單獨表達HA和共表現HA/M1和HA/NA/M1的樣品在密度1.10~1.16 g/cm3的區間都有很強的紅血球凝集反應,而在兩組共表現的梯度密度區間樣品則可同時偵測到M1或M1/NA,進一步以Sepharose CL-4B層析管柱純化,證實在上述的區間中含有大於2,000 kD的蛋白複體。上述結果顯示單獨HA就可以驅動budding的發生而形成只含有HA的似病毒顆粒。穿透式電子顯微鏡的觀察證實由HA或HA/M1或HA/NA/M1所組成的3種流感似病毒顆粒外型與真實流感病毒顆粒相似,皆具多型性,大小為80~150 nm。HA似病毒顆粒的發現為首次報導,此結果顯示HA蛋白為利用桿狀病毒表現系統生產流感似病毒顆粒所需的最小結構單元。雞隻免疫試驗的結果顯示含有2 μg HA抗原的VLPs於第一次注射後的二週內即可以令所有雞隻血清中的HI抗體力價成功達到16倍以上,第二次注射後,所有血清的HI力價明顯升高,並且持續至少三個月皆大於16倍。將VLP中的HA抗原含量降低至0.5或0.2 μg,單次注射最多僅有80% 雞隻血清呈現HI>16,而在第二次注射後的兩週內,HI>16的雞數達到100%。綜合以上的結果顯示,利用桿狀病毒表現系統生產流感似病毒顆粒具有開發成為安全且有效的流感疫苗的潛力,更重要的是,本研究中所報導的單單HA-VLPs流感疫苗就可提供一個簡單且快速的生產。
Outbreaks of highly pathogenic influenza viruses in avian species have resulted in great impacts on poultry industry and especially on public health. Influenza virus-like particles (VLPs) have been suggested to be a promising vaccine approach because of their safety and effectiveness in vaccination. For improving the production of VLP-based influenza vaccines, Hemagglutinin (HA) gene alone and in combination with matrix 1 (M1) or M1/ neuraminidase (NA) genes were constructed in baculovirus vectors for the expression of pertinent membrane proteins of these viruses. HA, HA/M1 or HA/NA/M1 proteins expressed and clarified by sucrose gradient ultracentrifugation produced strong hemagglutination responses in the fractions with sucrose densities between 1.10 and 1.16 g/cm3. Further purification by Sepharose CL-4B column demonstrated that these fractions contained proteins with an expected size larger than 2000 kDa. These results suggest that VLPs were formed and released into culture medium when HA was either individually expressed or co-expressed with M1 or with NA/M1. Transmission electron microscopy analysis showed that these VLPs with a diameter of 80-150 nm were pleiomorphic and similar to the authentic influenza virions. VLPs consisting of HA alone observed herein for the first time suggests that HA protein was the minimal protein requirement for the production of influenza VLPs using baculovirus expression system. With sera collected from 2 μg VLP (based on the HA contents)-vaccinated chickens two weeks after the prime injection, the hemagglutination inhibition titers induced by these three kinds of VLPs were found to be higher than 16. The titers were promoted after the boost injection and persisted for at least three months higher than 16. These results indicate that using insect cells to harvest the recombinant influenza VLPs may be a promising approach for the development of a safe and effective vaccine against the influenza virus infection. Most of all, VLPs consisting of HA only provides a simple and fast technical platform for annual influenza virus vaccine manufacturing.
URI: http://hdl.handle.net/11455/36242
其他識別: U0005-1511201016371900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1511201016371900
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