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Development of Massive Production Techniques for Newcastle Disease Virus (NDV) Hn Protein and Screening of Adjuvant Required for Ndv HN Protein as a Subunit Vaccine
我們試驗室過去幾年間，接受農業生物技術國家型計畫經費資助，進行以禽痘毒(FPV)為載體攜帶新城病毒(NDV) HN 及/或華氏囊病毒(IBDV) VP2 基因重組禽痘毒(rFPV)的構築。rFPV 經純化和鑑定後，進一步以雞隻進行安全性及效力試驗。在幾種不同組合的rFPV 中，其中含有NDV HN 基因的重組禽痘毒 (rFPV/HN)感染細胞後，表現的HN 蛋白質混合佐劑免疫雞隻具有很好的保護效果。因此，本計畫擬進行產學合作以rFPV/HN 病毒為載體感染細胞後，將HN 蛋白質製成油質次單位疫苗。本計畫擬分兩年進行。第一年將以迴旋培養瓶培養細胞並感染rFPV/HN，開發以細胞培養量產HN 蛋白質技術，接著以不同佐劑混合HN 蛋白質製成多種油質疫苗或Al-Gel 疫苗並免疫雞隻及攻毒試驗，依結果尋找最適當佐劑及製劑配方，之後，利用尋找到的最佳配方疫苗，依國家檢驗標準及田間應用需要，分別以2、1、1/2 劑量疫苗分別免疫4 週齡及2~3 日齡雞隻，經攻擊試驗及HI 抗體檢測試驗評估HN 次單位疫苗之免疫效力。最後，將所得資料整理成冊，撰寫田間試驗計畫申請書，向政府主管機關提出田間試驗申請。第二年計畫擬完成疫苗製劑安定性試驗。同時依田間試驗計畫申請書進行田間試驗，之後將結果整理、分析並彙集成冊，準備技術轉移廠商，並協助申請疫苗製造許可。
In previous years, we constructed the recombinant fowlpox virus (rFPV) which contained Newcastle diseasevirus (NDV) HN gene and / or infectious bursal disease (IBDV) VP2 genes. The rFPV was identified andpurified, then was further used for the evaluation of safety and efficacy tests when used as vaccines. Amongseveral rFPV recombinants, rFPV/HN virus which contained NDV HN gene infected cells and the expressedHN protein was formulated with incomplete Freund＇s adjuvant to form a HN subunit vaccine. Aftervaccination, SPF birds were fully protected from the virulent NDV challenge. Therefore, this researchproposal is to cooperate with vaccine company to develop a HN protein oil-adjuvanted subunit vaccine. Theresearch project will be carried out for two years. In the first year, rFPV/HN virus is used to infect DF1 cellswhich are growing in roller bottles to scale-up the massive production of NDV HN proteins.rFPV/HN-infected cell lysates containing HN proteins will be formulated with various adjuvants orAl-Gel-adjuvanted subunit vaccines. The vaccines will be used to immunize birds, followed by challenge.The most effective protection from NDV challenge will be screened and selected for further test. Based onthe National Rules for the Inspection of Biological Products and on the deamans in clinical application, thevaccines with 2, 1, and 1/2 doses will be used to vaccinate 4-week-or 2-3-day-old birds, followed bychallenge and HI test to evaluate the immune response against HN protein and the protection from NDVchallenge. Finally, all obtained data will be collected, analysed, and prepared for the filed trial. In the secondyear, stability test of vaccine developed will be complete. Also, field trials will be carrier out according to theproposal finished as expected in the first year. Then, all data will be collected, assessed, and prepared for theapplication for technique transfer and the permission for vaccine production.
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