201018480 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種鴨類肝炎疫苗及其匍 久丹策備方法，特別係 種以次單位疫苗技術為基礎，並結合鴨類肝 两每上PreS套膜恭 白所製備的鴨類肝炎疫苗及其製備方法。 【先前技術】 鴨子是一般的經濟水禽動物，但鴨隻在飼養過程中若遭逢 〇疾病或Λ災而導致死亡，將會使得養鴨戶蒙受龐大的經濟損 失，而鴨類與人類一樣都具有罹患肝炎的機率，其中鴨類的Β 型肝炎病毒（Duck •沿B virus，以下稱作DHB V )則是在 1980年即被研究人員所分離檢測出（Mas〇nWSeiw，198〇)， DHBV在鴨群中具有高度傳染能力，可以藉由血液體液、糞 便或是垂直感染進行傳播，輕則持續在鴨群間進行慢性感染， 重則可以導致鴨子肝臟發生病變，進而引發肝炎、肝硬化等症 狀。 © 一般來說，生物體大多以施打疫苗的方式預防疫病或傳染 病的發生’疫苗依其中所含的抗原種類又可分為減毒疫苗或是 死毒疫苗’然而減毒疫苗卻存在可能發生毒力回復或病毒活性 過低而導致疫苗效力不足的現象，此外，上述之疫苗亦具有製 備困難、成本高、品質不穩定及保存不易等問題。 目前使用於鴨類的疫苗係將利用雞蛋馴化的活毒疫苗接種 於種鴨群或是一日齡或十日齡的幼鴨腳蹼，使鴨類產生抗體以 達防疫的目的’然而，此疫苗卻屬於前述的減毒疫苗，故仍然 5 201018480 具有經剔化的病毒轉變為具有毒力的病原體，進而形成疾病防 治上的漏洞。 近年來，農產試驗研發人員則將分子生物學及疫苗學技術 加以整合，期望能藉此獲得經濟、安全、高有效穩定性及使用 方便之疫苗，其中「次單位疫苗（subunit vaccine )」技術即為 其中較新的技術之一，該次單位疫苗係將病毒粒（viri〇n )中無 法激發免疫反應或是對生物體有害的組成去除，將其中有效的 ❹疫苗成分保留來製備疫苗，這類的疫苗並非為一個完整的病原 體，該次單元疫苗係經由注射抗原蛋白以激發體液免疫反應， 因此不會引發細胞免疫。 然而’該「次單位疫苗」技術並未應用在製作鴨類肝炎疫 苗’因此在鴨類養殖上’養鴨戶因未能有破實有效的疫苗提供 鴨隻避免疾病的發生，故仍必須承擔鴨隻因感染疾病而大量集 體死亡之風險’因此在鴨類肝炎疫苗的製備上，仍需參酌現有 技術的研發，配合地進行研究開發以提供具該「次單位疫苗」 U優點的鴨類肝炎疫苗。 【發明内容】 本發明的主要目的亦即為了解決上述既有技術所存在之問 題’進而提供一種以次單位疫苗技術為基礎所製備之鴨類肝炎 疫苗及其製備方法。 本發明的鴨類肝炎疫苗製備方法係將鴨類B型肝炎病毒中 PreS套膜蛋白之DNA序列選殖至一載體以形成一重組質體， 再將該重組質體轉殖至一微生物菌株中，經由培養環境調控和 6 201018480 蛋白質誘導後，即可產生大量該PreS套膜蛋白以製備成為疫 苗。 本發明的鴨類肝炎疫苗至少包含鴨類B型肝炎病毒的PreS套膜 蛋白及一添加劑，該添加劑為一賦形劑或是一佐劑，藉由將該鴨類 肝炎病毒疫苗以注射或是與飼料混合之方式，使鴨隻能經由該 鴨類肝炎病毒疫苗以產生免疫反應。 該PreS套膜蛋白係在鴨類B型肝炎病毒表面所形成的表面 抗原（Ducksurface antigen，DHBsAg)之一，以提 供病毒做為外層顆粒的保護，而其存在則與病毒的内噬作用 (endocytosis )及產生病毒進行複製時所需的聚合酶酵素有 關，故能藉由該PreS套膜蛋白進行疫苗製備，進而引發鴨類的 免疫作用以達到鴨隻抵禦鴨類B型肝炎傳染或發作之可能。 一般來說，本發明之鴨類B型肝炎疫苗具有次單位疫苗所擁 有的抗原不會在生物體内增殖、不具傳染性、無副作用和研製 簡便等優點。 【實施方式】 本發明之鴨類肝炎疫苗製備方法，包括下列步驟： (1) 製備一 DNA，將該DNA嵌合至一載體形成一重 組質體； (2) 將該重組質體轉殖至一微生物細胞株；201018480 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a duck hepatitis vaccine and a method for preparing the same, which is based on a sub-unit vaccine technology and combined with duck liver The duck hepatitis vaccine prepared by the PreS cover film and its preparation method. [Prior Art] Ducks are common economic waterfowl animals, but if ducks die in the process of feeding, they will cause huge economic losses to duck farmers, and ducks have the same human beings. The chance of suffering from hepatitis, in which the duck hepatitis virus (Duck • along B virus, hereinafter referred to as DHB V ) was detected by the researchers in 1980 (Mas〇nWSeiw, 198〇), DHBV The ducks are highly contagious and can be transmitted by blood body fluids, feces or vertical infections. They can continue to be chronically infected between ducks, and the liver can cause liver disease and liver cirrhosis. © In general, most organisms use vaccines to prevent the occurrence of epidemics or infectious diseases. 'Vaccines can be classified into attenuated vaccines or deadly vaccines depending on the type of antigens contained therein. However, there is a possibility of attenuated vaccines. The virulence recovery or the low activity of the virus results in insufficient efficacy of the vaccine. In addition, the vaccine described above has problems such as difficulty in preparation, high cost, unstable quality, and difficulty in storage. The vaccine currently used in ducks will be vaccinated with a domesticated live vaccination of eggs in breeding ducks or young ducks of one day old or ten days old, so that ducks can produce antibodies for epidemic prevention. However, this vaccine However, it belongs to the aforementioned attenuated vaccine, so it is still 5 201018480 to have a sterilized virus turned into a virulent pathogen, thereby forming a loophole in disease prevention and control. In recent years, agricultural product research and development personnel have integrated molecular biology and vaccination technology, and hope to obtain a vaccine that is economical, safe, highly effective, stable and easy to use, among which "subunit vaccine" technology One of the newer technologies, the sub-unit vaccine removes the virion (viri〇n) that cannot stimulate the immune response or is harmful to the organism, and retains the effective sputum vaccine component to prepare the vaccine. This type of vaccine is not a complete pathogen. This unit of vaccine is based on the injection of antigenic proteins to elicit a humoral immune response and therefore does not trigger cellular immunity. However, the 'secondary unit vaccine' technology has not been applied to the production of duck hepatitis vaccine. Therefore, in duck farming, the duck farmers have to provide the ducks to avoid the disease because they have not provided a solid and effective vaccine. The risk of a large number of collective deaths of ducks due to infections. Therefore, in the preparation of duck hepatitis vaccines, it is still necessary to take into account the research and development of existing technologies, and cooperate with research and development to provide duck hepatitis with the advantages of this "sub-unit vaccine" U. vaccine. SUMMARY OF THE INVENTION The main object of the present invention is to solve the problems of the above-mentioned prior art and to provide a duck hepatitis vaccine prepared based on the subunit vaccine technology and a preparation method thereof. The preparation method of the duck hepatitis vaccine of the present invention is to clone the DNA sequence of the PreS envelope protein in the duck hepatitis B virus into a vector to form a recombinant plastid, and then transfer the recombinant plastid to a microorganism strain. After induction by culture environment and 6 201018480 protein induction, a large amount of the PreS envelope protein can be produced to prepare a vaccine. The duck hepatitis vaccine of the present invention comprises at least a PreS envelope protein of duck hepatitis B virus and an additive which is an excipient or an adjuvant by injecting the duck hepatitis virus vaccine or The method of mixing with the feed allows the duck to pass the duck hepatitis virus vaccine only to generate an immune response. The PreS envelope protein is one of the surface antigens (DHBsAg) formed on the surface of duck hepatitis B virus to provide protection for the outer layer of the virus, and its presence is associated with endocytosis of the virus (endocytosis) ) and the production of the polymerase enzyme required for the virus to replicate, so the vaccine can be prepared by the PreS envelope protein, thereby triggering the immune function of the duck to prevent the duck from resisting the infection or attack of the duck hepatitis B. . In general, the duck hepatitis B vaccine of the present invention has the advantages that the antigen possessed by the subunit vaccine does not proliferate in the living body, is non-infectious, has no side effects, and is simple to develop. [Embodiment] The method for preparing a duck hepatitis vaccine of the present invention comprises the following steps: (1) preparing a DNA, the DNA is chimeric to a vector to form a recombinant plastid; (2) transferring the recombinant plastid to a microbial cell line;
(3) 利用一培養基大量培養該微生物細胞株，以產生一 PreS 套膜蛋白； (4) 萃取及純化PreS套膜蛋白；及 201018480 (5)將該Pres套膜蛋白與一添加物進行均勻混合。 以下即以實施例進行具體之說明。 實施例 1. /VeSDNA 製傷 (1) pVAC-EGFp_卿$質體製備：利用聚合酶鍵反應（pcR) 自罹患B型肝炎的鴨隻血液中增幅基因，並將該基因轉殖至 pVAC載體中’形成一 ρνΑΟγΜ質體，再將該質 ❹體與一常用之PEGFP_C3載體進行重組接合，形成一同時帶有 綠色螢光蛋白基因和pre基因的pVAC-EGFP-preS質體。 (2) pGEM-T Easy-prei1質體製備：利用聚合酶鏈反應自罹患 B型肝炎的鴨隻血液中增幅（ampiifjcatj〇n )〆沾基因，並將該基因轉 殖至市售pGEM-T Easy載體套件’即形成帶有户〜基因的質體 pVAC-EGFP-preS。 2. 建構鴨類B型肝炎病毒重組質體及大腸桿菌表現系 統 q ( 1 )表現質體： (1-1 ) pQE-trisystem-EGFP-preS 重組質體：將質體 pVAC-EGFP-z^eS 以限制酵素（restriction enzyme )尺p«I 及 iVcoI 切割，並且選殖至質體pQE_triSyStem ( QIAGEN )以建構成可 產生EGFP-PreS融合蛋白質的表現質體pQE-trisystem-EGFP-’該EGFP-PreS融合蛋白質之分子量為51.4KDa，其蛋白 質序列末端具有8個連續的組氨酸標籤（His tag )(其蛋白質 序列表如附件一所示）； 8 201018480 (1-2 ) pQE-trisystem-preS重組質體：將帶有鴨類B 型肝炎病毒之基因的pGEM-T Easy-preS質體以限制酵素 £c〇RI和尺切割，並選殖到pQE_trisystem質體以建構成可 產生PreS套膜蛋白之表現質體pQE_trisystem_pre，該preS套 膜蛋白之分子量為25.4KDa ’其蛋白質序列末端具有8個連續 的組氨酸標籤（Histag)(其蛋白質序列表如附件二所示）。 (2 )將前述（1 -1 )和（1 _2 )之表現質體分別以電穿孔 ❹ （elecroporation)方式殖入反應潛能細胞（competent cell)以 得到EGFP-PreS和PreS兩種轉殖細胞株，在本實施例中，該反 應潛能細胞係選用大腸桿菌XL-blue-Ι細胞株》 (3)將該轉殖細胞株以含有i〇〇gg/mi胺节青黴素 (ampicillin)的LB培養基進行選殖後，進行放大培養。 3.鴨類B型肝炎病毒疫苗製備 將大量培養後的轉殖細胞株進行蛋白質萃取及純化，培養 轉殖細胞株之培養基係選用每毫升添加100微克（pg/ml )胺节 II青黴素的LB培養基進行培養，培養溫度為37。(：，當轉殖細胞 株之濃度以分光光度計測試之讀值達到0.5時，即可將轉殖細 胞株收集進行蛋白質萃取及純化。 蛋白質之萃取係可利用化學性或物理性方式將的轉殖細胞 株之細胞壁進行破碎並去除DNA的殘留後，即可獲得蛋白質， 在本實施例中，係採用超音波細胞破碎機將轉殖細胞破碎。 蛋白質純化係可利用一般之蛋白質純化方式或快速的蛋白 質純化套組進行重組蛋白質之純化，本實施例係將萃取出之重 201018480 組蛋白質以純化备細忆τ Δ ΤΜ . , lml column 吨化苌組 KTA prime P1US 和 HisTrap 將蛋白質純化出。 將純化出之蛋白質與一添加劑進行均勻混合其中該添 加劑係可選用佐劑（adjuvant)，例如阜芽類（）佐劑、 完全佐劑或不完全佐劑，或具有生物相容性之賦形劑，藉由將 該鴨類肝炎病毒疫苗以注射或是與飼料混合之方式，使鴨隻能 經由該鴨類肝炎病毒疫苗以產生免疫反應，本實施例中，係使 ❹用弗氏完全佐劑（Freund’s adjuvant)與蛋白質均勻混合，在本 一實施例中，係將0.5微克（叩）的pres套膜蛋白加入！微 升（μΐ)的弗氏完全佐劑中β劑量為1〇〇μι/隻。 4.誘導藥劑、培養時間對蛋白質表現量及鹽類濃度促進蛋 白質沈澱的影響 (1)誘導藥劑對於蛋白質表現量的影響：在微生物培養的 過程中，可藉由誘導藥劑的添加來促進蛋白質的生產，在本實 施例中係使用異丙基·β_0_硫代半乳糖普（is〇pr〇pyl_beta_ © D-thi〇galactopyranoside，以下稱作ΙρΐΌ)為誘導藥劑來誘導 蛋白質表現，其係在前述每毫升添加1〇〇微克（μ§/ιη1)胺苄青 徽素的LB培養基中添加IPTG，其濃度對於EGFp_pres和pres 轉殖細胞株誘導EGFP-PreS套膜蛋白或PreS套膜蛋白產量的影 響係利用抗組氨酸（Anti-His )為抗體藉由西方點墨法檢測蛋 白質的表現量及專一性（結果如表一所示）。 201018480 一、IPTG 對於誘導蛋白質表現的影響 重組蛋白質 EGFP-PreS套膜蛋白(3) using a medium to culture the microbial cell strain in large quantities to produce a PreS envelope protein; (4) extracting and purifying the PreS envelope protein; and 201018480 (5) uniformly mixing the Pres envelope protein with an additive . The following is a detailed description of the embodiments. Example 1. /VeSDNA Injury (1) pVAC-EGFp_qing$ plastid preparation: Polymerase bond reaction (pcR) was used to augment genes from the blood of ducks suffering from hepatitis B, and the gene was transferred to pVAC. A ρνΑΟγ plastid is formed in the vector, and the plastid is recombined with a commonly used PEGFP_C3 vector to form a pVAC-EGFP-preS plastid carrying both the green fluorescent protein gene and the pre gene. (2) Preparation of pGEM-T Easy-prei1 plastid: Polymerase chain reaction was used to amplify (ampiifjcatj〇n) sputum genes from duck blood of patients with hepatitis B, and the gene was transferred to commercially available pGEM-T The Easy Vector Kit's form the plastid pVAC-EGFP-preS with a home to gene. 2. Construction of recombinant plastids and E. coli expression system of duck hepatitis B virus q ( 1 ) Expression of plastids: (1-1 ) pQE-trisystem-EGFP-preS Recombinant plastid: plastid pVAC-EGFP-z^ eS is cleaved with restriction enzymes p«I and iVcoI and cloned into plastid pQE_triSyStem (QIAGEN) to construct a plastid pQE-trisystem-EGFP-'EGFP- that produces EGFP-PreS fusion protein The PreS fusion protein has a molecular weight of 51.4KDa and has eight consecutive histidine tags (the protein sequence listing is shown in Annex I); 8 201018480 (1-2) pQE-trisystem-preS Recombinant plastids: pGEM-T Easy-preS plastids with the gene for duck hepatitis B virus were cut with restriction enzymes £c〇RI and stalks, and cloned into pQE_trisystem plastids to construct a PreS envelope. The protein expresses the plastid pQE_trisystem_pre, the preS envelope protein has a molecular weight of 25.4KDa and has eight consecutive histidine tags (Histag) at the end of its protein sequence (the protein sequence is shown in Annex II). (2) The plastids of the above (1 -1 ) and (1 _2 ) were separately eucroporated into a reactive cell to obtain EGFP-PreS and PreS transgenic cell lines. In the present embodiment, the reaction potential cell line is selected from Escherichia coli XL-blue-Ι cell line. (3) The transgenic cell line is subjected to LB medium containing i〇〇gg/miamine penicillin (ampicillin). After colonization, scale up the culture. 3. Preparation of duck hepatitis B virus vaccine A large number of cultured transgenic cell lines were subjected to protein extraction and purification, and the medium for culturing the transgenic cell line was selected to add 100 μg (pg/ml) of LB of penicillin II per ml. The medium was cultured at a culture temperature of 37. (: When the concentration of the transgenic cell line is 0.5 in the spectrophotometer, the transfected cell line can be collected for protein extraction and purification. The protein extraction can be chemically or physically After the cell wall of the transgenic cell strain is disrupted and the DNA residue is removed, the protein can be obtained. In this embodiment, the transfected cells are disrupted by an ultrasonic cell disruptor. The protein purification system can utilize general protein purification methods or The rapid protein purification kit is used to purify the recombinant protein. In this example, the protein of the 201018480 group is extracted to purify the protein, and the protein is purified by the KL prime P1US and HisTrap. The purified protein is uniformly mixed with an additive, wherein the additive may be an adjuvant such as an axillary bud adjuvant, a complete adjuvant or an incomplete adjuvant, or a biocompatible shape. By allowing the duck hepatitis virus vaccine to be injected or mixed with the feed, the duck can only pass the duck hepatitis The vaccine is used to generate an immune response. In this embodiment, the sputum is uniformly mixed with the protein using Freund's adjuvant. In this embodiment, 0.5 μg of the pres envelope protein is added! The β dose of Freund's complete adjuvant in microliters (μΐ) is 1〇〇μι/. 4. The effect of inducing agent, culture time on protein expression and salt concentration on protein precipitation (1) Inducing agent for protein expression Effect of the amount: During the cultivation of the microorganism, the production of the protein can be promoted by the addition of the inducing agent, and in the present embodiment, isopropyl·β_0_thiogalactose (is〇pr〇pyl_beta_ © D) is used. -thi〇galactopyranoside, hereinafter referred to as ΙρΐΌ), is an inducing agent for inducing protein expression by adding IPTG to LB medium supplemented with 1 μg of microgram (μ§/ιη1) of ampicillin per ml, at a concentration of The effect of EGFp_pres and pres transgenic cell lines on the production of EGFP-PreS envelope protein or PreS envelope protein was detected by Western blotting using anti-His as antibody. Volume and specificity (results are shown in Table 1.) 201018480 I. Effect of IPTG on induced protein expression Recombinant protein EGFP-PreS envelope protein
PreS套膜蛋白 IPTG 濃度(mM ) 0.5 1.0 2.0 0.5 1.0 2.0 西方點墨法 (Western Blot) (2)培養時間對於蛋白質表現量的影響：在前述每毫升添 加1〇〇微克Ug/ml)胺苄青黴素的LB培養基中添加 IPTG，在3 7 C下對轉殖細胞株進行培養，不同培養時間對誘導 蛋白質表現的影響係利用抗組氨酸（Anti_His)為抗體藉由西 方點墨法檢測蛋白質的表現量及專一性（結果如表二所示）。 導蛋白質表現的影孿。 j組蛋白零一^ EGFP-PreS套膜番·白 IPTG 濃度（mM) 培養時間（小時）PreS envelope protein IPTG concentration (mM) 0.5 1.0 2.0 0.5 1.0 2.0 Western Blot (2) Effect of culture time on protein expression: Add 1 μg of Ug/ml per ml of ampicillin IPTG was added to the LB medium of penicillin, and the transgenic cell line was cultured at 3 7 C. The effect of different culture time on the expression of induced protein was detected by Western blotting using anti-histidine (Anti_His) as an antibody. Performance and specificity (results shown in Table 2). The effect of protein expression. j histone zero one ^ EGFP-PreS cover film · white IPTG concentration (mM) culture time (hours)
PreS套膜蛋白 2 3 2 3 西方點墨法 (Western Blot )PreS envelope protein 2 3 2 3 Western Blot
(3)鹽類濃度對於促進蛋白質沈澱的影響：萃取蛋白質 時’可利用鹽析法使蛋白質聚#沈丨殿，在此—實施例中係選用 硫酸銨（（NH4)2so4)進行鹽析，在前述每毫升添加1〇〇微克 Ug/ml)胺节青黴素的LB培養基中添加111^1打(}與不同濃 度硫酸銨，硫酸銨濃度對蛋白質沈澱的影響係利用抗組氨酸 201018480 (Anti-His )為抗體藉由西方點墨法檢測蛋白質的表現量及專 一性（結果如表三所示）。 表三、硫酸銨濃度對於誘導蛋白質沈澱的影響。 重組蛋白質 EGFP-PreS套膜蛋白 IPTG 濃度（mM) 1.0 硫酸銨濃度（％ ) 0 10 20 30 40 50 60 70 80 西方點墨法 (Western Blot 重組蛋白質(3) The effect of salt concentration on promoting protein precipitation: When extracting protein, the protein can be aggregated by salting out method. In this example, ammonium sulfate ((NH4)2so4) is used for salting out. Add 1 1 μg of Ug/ml of penicillin to the LB medium and add 111 ^ 1 dozen (} with different concentrations of ammonium sulfate. The effect of ammonium sulfate concentration on protein precipitation is based on anti-histidine 201018480 (Anti-His For the antibody, the expression and specificity of the protein were detected by the Western blotting method (the results are shown in Table 3). Table 3. Effect of ammonium sulfate concentration on the induction of protein precipitation. Recombinant protein EGFP-PreS envelope protein IPTG concentration ( mM) 1.0 Ammonium Sulfate Concentration (%) 0 10 20 30 40 50 60 70 80 Western Blot Recombinant Protein
PreS套膜蛋白 IPTG 濃度（mM) 1.0 硫酸銨濃度（％ ) 0 10 20 30 40 50 60 70PreS envelope protein IPTG concentration (mM) 1.0 ammonium sulfate concentration (%) 0 10 20 30 40 50 60 70
西方點墨法 (Western Blot 5.免疫試驗 (1 )試驗用鴨隻篩選：利用下列引子（primer )以聚合酶 鏈反應（PCR )檢測鴨隻血液是否具有DHB V，以確認鴨隻是 否感染DHBV: ΟΗΒν-/^5-778〜805 (EcoRV) 5，- GATATCAGTCACATCAAGTTCCTGATGGG -3, ϋΗΒΥ-ρΑΆ2165 〜2145 (五 coRI) 12 201018480 5，- GGAATCGCTACACGTGGCAAAGGTACAG -3, (2)以未感染DHBV鴨隻為檢測樣本進行如表四之分組 及處理。 表四、利用本發明鴨類肝炎病毒疫苗進行免疫試驗樣本分組。 組別 樣本數 接種鴨類肝炎病毒疫苗 攻毒 控制組 陽性控制組 11 施打DHBV血清 陰性控制組 11 每 . 實驗組 三日齡小鴨 6 1OOul ( 0.5pg/pl ) 施打DHBV血清 十曰齡小鴨 7 1 OOul ( 0.5μ§/μ1 ) 施打DHBV血清 其中，以三曰齡小鴨和十曰齡小鴨（簡稱為實驗組）於第 一天及第八天進行鴨類肝炎病毒疫苗注射，並在第35天進行攻 毒（viral-challenge )，攻毒後第三天及第十天進行採血，以dot blotting檢測其中抗體（IgG )，並以PCR檢測DHBV之PreS ❹基因的存在，其結果如表五所示，由表五中可看出，實驗組的 鴨隻在施打本發明之鴨類肝炎病毒疫苗後，確實可以避免 DHBV的感染。 本發明之鴨類肝炎病毒疫苗係選殖病毒上的抗原決定位做 為主要的疫苗原組成*並不具有病毒的毒性*因此不會有毒力 .回復的顧慮，且該抗原並無複製的能力，所以不會在生物體内 增殖，也不會有傳染的危險性，此外，本發明之鴨類肝炎病毒 疫苗製備方式十分簡單，容易大量製備生產供飼鴨戶使用。 13 201018480 表五、利用本發明鴨類肝炎病毒疫苗進行免疫試驗結果。 組別 樣本數 接種 鴨類肝炎 病毒疫苗 攻毒（施 打 DHBV 血清） 攻毒後 產生IgG 抗體 感染 DHBV 控制組 陽性控 制組 11 0 11 8 3 陰性控 制組 11 0 0 11 0 實驗組 三日齡 小鴨 6 6 6 6 0 十日齡 小鴨 7 7 7 6 0Western blotting (Western Blot 5. Immunoassay (1) Test duck screening: Using the following primers to detect duck blood with DHB V by polymerase chain reaction (PCR) to confirm whether the duck is infected with DHBV : ΟΗΒν-/^5-778~805 (EcoRV) 5,- GATATCAGTCACATCAAGTTCCTGATGGG -3, ϋΗΒΥ-ρΑΆ2165 ～2145 (five coRI) 12 201018480 5,- GGAATCGCTACACGTGGCAAAGGTACAG -3, (2) Uninfected DHBV duck as test sample The grouping and treatment are as shown in Table 4. Table 4. Grouping of immunoassay samples using the duck hepatitis virus vaccine of the present invention. Group number of samples inoculated with duck hepatitis virus vaccine challenge control group positive control group 11 DHBV serum negative control Group 11 per. Experimental group three-day-old duckling 6 1OOul (0.5pg/pl) DHBV serum ten-year-old duckling 7 1 OOul (0.5μ§/μ1) Apply DHBV serum to three-year-old ducklings and ten sisters The ducklings (referred to as the experimental group) were injected with duck hepatitis virus on the first day and the eighth day, and were challenged on the 35th day (viral-challenge) on the third and tenth day after the challenge. Blood was collected, and the antibody (IgG) was detected by dot blotting, and the presence of the PreS ❹ gene of DHBV was detected by PCR. The results are shown in Table 5. As can be seen from Table 5, the ducks of the experimental group were in the invention. After the duck hepatitis virus vaccine, DHBV infection can be avoided. The antigenic epitope of the duck hepatitis virus vaccine of the present invention is the main vaccinogen composition* and does not have the toxicity of the virus* The virulence. Responsive concerns, and the antigen has no ability to replicate, so it does not proliferate in the organism, and there is no risk of infection. In addition, the preparation method of the duck hepatitis virus vaccine of the present invention is very simple and easy. A large number of preparations for the production of ducks for domestic use. 13 201018480 Table V, the results of the immunoassay using the duck hepatitis virus vaccine of the present invention. The number of group samples was vaccinated with duck hepatitis virus vaccine (using DHBV serum) to produce IgG after challenge Antibody-infected DHBV control group positive control group 11 0 11 8 3 negative control group 11 0 0 11 0 experimental group three-day-old duckling 6 6 6 6 0 10 day old duckling 7 7 7 6 0
14 201018480 【圖式簡單說明】 無。 【附件說明】 附件一為本發明製備方法所使用的EGFP-PreS蛋白質之氨 基酸序列。 附件二為本發明製備方法所使用的PreS套膜蛋白之氨基酸 序列。 【主要元件符號說明】 無。 ❿ 1514 201018480 [Simple description of the schema] None. [Description of Attachment] Annex 1 is the amino acid sequence of the EGFP-PreS protein used in the preparation method of the present invention. Annex 2 is the amino acid sequence of the PreS envelope protein used in the preparation method of the present invention. [Main component symbol description] None. ❿ 15