TWI496595B - Manufacture method of fish rna interference reagent - Google Patents
Manufacture method of fish rna interference reagent Download PDFInfo
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本發明係一種製備魚類核醣核酸試劑之方法，尤其係關於一種防治魚類感染性疾病之魚類核醣核酸試劑之製備方法。The invention relates to a method for preparing a fish ribonucleic acid reagent, in particular to a method for preparing a fish ribonucleic acid reagent for controlling fish infectious diseases.
近年來，水產養殖已成為國際化的重要產業。由於需求不斷地提升以及經濟效應考量，養殖戶多以密集的方式進行養殖。然而，擁擠的生長環境會直接影響魚隻健康，造成傳染病盛行與氾濫的結果，影響水產養殖業甚鉅。其中，抗生素雖可控制細菌性疾病之疫情，卻無法防制病毒性疾病，使病毒性疾病躍升為水產魚類大規模死亡的主要原因。這其中，又以神經壞死病毒對石斑魚造成的疫情最為嚴重。In recent years, aquaculture has become an important industry in internationalization. Due to the increasing demand and economic effects, farmers are often farming in an intensive manner. However, the crowded growth environment will directly affect the health of fish, resulting in the prevalence and spread of infectious diseases, affecting the aquaculture industry. Among them, although antibiotics can control the epidemic of bacterial diseases, they can not prevent viral diseases, making viral diseases the main cause of large-scale death of aquatic fish. Among them, the epidemic caused by the necrosis virus to the grouper is the most serious.
目前市面上並未有商品化的魚類病毒試劑，但有學者提出許多不同的疫苗方式進行防治，包括死毒疫苗、活毒疫苗、減毒疫苗、次單位疫苗，係將病毒或病毒表面之分子引入魚體中，誘發魚體免疫系統辨識抗原而產生體液性及細胞性免疫記憶。進而，未來受病毒感染時，免疫系統能有效地辨識抗原並攻擊病毒， 避免魚體病情惡化，達到防治的效果。There are no commercially available fish virus reagents on the market, but some scholars have proposed many different vaccine methods for prevention and treatment, including dead vaccines, live vaccines, attenuated vaccines, sub-unit vaccines, and molecules on the surface of viruses or viruses. Introduced into the fish body, the fish immune system is induced to recognize the antigen and produce humoral and cellular immune memory. Furthermore, in the future, when infected by a virus, the immune system can effectively identify antigens and attack viruses. Avoid the deterioration of the fish body condition and achieve the effect of prevention and treatment.
要將這些疫苗送入魚體內，其方式有三種，即注射法、浸泡法、口服法。其中，注射法是將藥物直接注射至魚隻的腹腔或肌肉中；浸泡法是將魚集中在含有高濃度藥液的小容器中，強迫進行短期藥浴；口服法則是將魚類飼料、藥物及無毒的黏合劑混合拌勻，製成藥餌進行投餵。There are three ways to deliver these vaccines into fish, namely, injection, soaking, and oral methods. Among them, the injection method is to directly inject the medicine into the abdominal cavity or muscle of the fish; the immersion method is to concentrate the fish in a small container containing a high concentration of the liquid medicine, forcing a short-term medicated bath; the oral method is to feed the fish, the medicine and the medicine. The non-toxic adhesive is mixed and mixed to make a bait for feeding.
口服法有施用方便、成本低廉、對魚隻的傷害及緊迫壓力較小等優點，適用於敏感脆弱的稚魚。然而，魚隻前腸的消化功能會破壞大部分疫苗中的蛋白質抗原。因此，如何保護試劑成分，使抵抗消化道之酸性環境及各種酵素破壞，抵達主司吸收的尾腸，乃口服方式最重要的課題。The oral method has the advantages of convenient application, low cost, harm to fish and less pressing pressure, and is suitable for sensitive and fragile juveniles. However, the digestive function of the fish's foregut destroys most of the protein antigens in the vaccine. Therefore, how to protect the reagent components, to resist the acidic environment of the digestive tract and the destruction of various enzymes, to reach the tail intestine absorbed by the main division, is the most important subject of oral administration.
在所有學者提出的口服疫苗中，被研究得較清楚的係一種用以對抗魚類神經壞死病毒的疫苗，其方法如下。首先，製作神經壞死病毒抗原的重組蛋白質DNA序列，***質體DNA中，再轉殖到大腸桿菌內，使大腸桿菌大量表現該重組蛋白質。接著，以大腸桿菌餵食豐年蝦，再以豐年蝦餵食魚類。如此，該大腸桿菌所表現的神經壞死病毒重組蛋白即可藉由食物鏈關係進入魚體，且受豐年蝦外殼及大腸桿菌細胞壁保護，得順利抵達主司吸收的尾腸(請參閱後述參考文獻1)。進而，誘發魚體產生體液性免疫記憶及細胞性免疫記憶，使魚體獲得對抗神經壞死病毒的能力。Among the oral vaccines proposed by all scholars, one that has been studied more clearly is a vaccine against fish necrosis virus, and the method is as follows. First, a recombinant protein DNA sequence of a necrosis virus antigen is produced, inserted into a plastid DNA, and then transferred into Escherichia coli to cause Escherichia coli to express the recombinant protein in a large amount. Next, feed the brine shrimp with E. coli and feed the fish with brine shrimp. In this way, the recombinant protein of the necrosis virus expressed by the Escherichia coli can enter the fish body through the food chain relationship, and is protected by the brine shrimp shell and the E. coli cell wall, and can smoothly reach the tail sausage absorbed by the main body (refer to Reference 1 below). ). Furthermore, the body fluid is induced to produce humoral immune memory and cellular immune memory, so that the fish body has the ability to resist neuronecrosis virus.
然而，魚類免疫系統須至孵化後20-30天才會成熟，始有可能進行主動免疫，且免疫後還需要3-4週才能誘發足夠抗體對抗病毒感染(請參閱後述參考文獻2)。因此，疫苗方法僅適用於 體長一吋(平均魚齡40天)以上之魚苗，魚卵孵化後至體長一吋的魚苗都無法使用這樣的方法進行保護。However, the fish immune system must not mature until 20-30 days after hatching, and it is possible to carry out active immunization, and it takes 3-4 weeks after immunization to induce sufficient antibodies against viral infection (see Reference 2 below). Therefore, the vaccine method is only applicable to Fish fry with a body length of more than 40 days (average fish age 40 days) can not be protected by such methods after hatching.
但現實情況是，魚苗往往在剛孵化不久即受病毒感染(通常是經由母體垂直感染或環境共養之方式感染)，且在魚卵孵化後十幾天到吋苗期間是死亡率最高之時，約80-90%，養殖業者根本來不及等到魚體免疫系統發育成熟即已損失慘重。由此可知，亟待發展一種新的魚類感染性疾病防治方法，以大幅提高魚類的存活率，特別是極需要解決體長小於或等於一吋之稚魚(抑或是魚齡小於等於40天之稚魚)等魚類發育早期，因感染而大量死亡的問題。However, the reality is that fry are often infected by virus (usually through parental vertical infection or environmental co-culture) just after hatching, and the highest mortality rate occurs during the ten days after the hatching of the eggs. About 80-90%, the breeders have no time to wait until the fish's immune system matures and has suffered heavy losses. It can be seen that there is a need to develop a new method for the prevention and treatment of fish infectious diseases, in order to greatly improve the survival rate of fish, especially the need to solve the juvenile fish with a body length less than or equal to one squat (or a juvenile fish with a fish age less than or equal to 40 days) In the early stages of fish development, the problem of massive death due to infection.
前述參考文獻1：魚用生物包埋型口服疫苗製備與有效性測試，2004，(林青丘、楊惠郎/國立成功大學生物科技研究所)。The aforementioned reference 1: preparation and effectiveness test of bio-embedded oral vaccine for fish, 2004, (Lin Qingqiu, Yang Huilang/National University of Biotechnology, National Institute of Biotechnology).
前述參考文獻2：石斑魚神經性壞死症病毒重組單源抗體的製備及表現，2009，(張瑞昕、齊肖琪/台灣大學動物學研究所)。Reference 2 above: Preparation and performance of recombinant single-source antibody to grouper neuronecrosis virus, 2009, (Zhang Ruizhen, Qi Xiaoqi/Institute of Zoology, National Taiwan University).
本案發明人鑑於習知魚類感染性疾病口服疫苗的各項缺點，乃亟思加以改良創新，並經多年潛心研究後，終於成功研發完成本件魚類核醣核酸試劑。In view of the shortcomings of the conventional oral vaccine for fish infectious diseases, the inventors of the present invention have improved and innovated, and after years of painstaking research, finally successfully developed the fish ribonucleic acid reagent.
有鑑於儘早防治感染性疾病之需求，本發明之目的在於提供一種適用於魚類發育各個時期之感染性疾病試劑之製備方法。亦即，提供一種亦可適用於魚類發育早期之感染性疾病試劑之製備方法。In view of the need to prevent infectious diseases as early as possible, it is an object of the present invention to provide a method for preparing an infectious disease agent suitable for various stages of fish development. That is, a method for preparing an infectious disease agent which is also applicable to early fish development is provided.
本發明之次一目的，在於提供一種可順利通過魚類消化道中酸性環境及各種酵素破壞的試劑。A second object of the present invention is to provide an agent which can smoothly pass through an acidic environment and various enzymes in the digestive tract of fish.
本發明之另一目的，在於提供一種可對抗病毒之試劑。Another object of the present invention is to provide an agent which is resistant to viruses.
本發明之再一目的，在於提供一種可對抗魚類神經壞死病毒之試劑。Still another object of the present invention is to provide an agent which is resistant to fish necrosis virus.
於一較佳實施例中，本發明提供一種魚類核醣核酸試劑，包括：一核酸分子，用以抑制一魚類病原體之一蛋白質表現；以及一脂質體(liposome)，用以包覆該核酸分子。In a preferred embodiment, the invention provides a fish ribonucleic acid reagent comprising: a nucleic acid molecule for inhibiting protein expression of a fish pathogen; and a liposome for coating the nucleic acid molecule.
於一較佳實施例中，其中該魚類病原體係一魚類致病病毒。In a preferred embodiment, the fish pathogen system is a fish-borne virus.
於一較佳實施例中，其中該蛋白質係該致病病毒之一殼蛋白，且該魚類核醣核酸試劑可抑制該魚類致病病毒在一魚體內或一魚類細胞中複製。In a preferred embodiment, the protein is a shell protein of the pathogenic virus, and the fish ribonucleic acid agent inhibits replication of the fish pathogenic virus in a fish or a fish cell.
於一較佳實施例中，可使體長不大於一吋或魚齡不大於40天之受感染之一稚魚的存活率提升。In a preferred embodiment, the survival rate of an infected one of the juveniles having a body length of no more than one ticks or a fish age of not more than 40 days can be increased.
於一較佳實施例中，係施用於一石斑魚。In a preferred embodiment, it is applied to a grouper.
於一較佳實施例中，係直接以口服方式投遞、混合飼料之口服方式投遞、以及浸泡方式投遞等方式中之至少一種。In a preferred embodiment, it is at least one of oral delivery, oral delivery of mixed feed, and immersion delivery.
於一較佳實施例中，其中該魚類病原體係一神經壞死病毒。In a preferred embodiment, the fish pathogen system is a necrosis virus.
於一較佳實施例中，其中該核酸分子係一短鏈核醣核酸分子。In a preferred embodiment, wherein the nucleic acid molecule is a short stranded ribonucleic acid molecule.
於一較佳實施例中，其中該短鏈核醣核酸分子係SEQ ID NO：1序列、SEQ ID NO：2序列、SEQ ID NO：3序列、 以及SEQ ID NO：4序列之至少一者。In a preferred embodiment, wherein the short-stranded ribonucleic acid molecule is SEQ ID NO: 1 sequence, SEQ ID NO: 2 sequence, SEQ ID NO: 3 sequence, And at least one of the sequences of SEQ ID NO: 4.
於一較佳實施例中，其中該核酸分子係一去氧核醣核酸分子，且該去氧核醣核酸分子可透過一聚合酶(polymerase)而表現一短鏈核醣核酸分子。In a preferred embodiment, the nucleic acid molecule is a deoxyribonucleic acid molecule, and the deoxyribonucleic acid molecule can express a short-chain ribonucleic acid molecule through a polymerase.
於一較佳實施例中，其中該去氧核醣核酸分子包含SEQ ID NO：5序列、SEQ ID NO：6序列、SEQ ID NO：7序列、以及SEQ ID NO：8序列之至少一者。In a preferred embodiment, wherein the deoxyribonucleic acid molecule comprises at least one of the sequence of SEQ ID NO: 5, the sequence of SEQ ID NO: 6, the sequence of SEQ ID NO: 7, and the sequence of SEQ ID NO: 8.
於一較佳實施例中，其中該脂質體之組成份包括幾丁質。In a preferred embodiment, wherein the liposome component comprises chitin.
於另一較佳實施例中，本發明提供一種魚類核醣核酸試劑，包括：一核酸分子，序列包含SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、以及SEQ ID NO：8之至少一者；其中，該核酸分子可抑制該神經壞死病毒在一魚體內或一魚類細胞中複製。In another preferred embodiment, the present invention provides a fish ribonucleic acid reagent comprising: a nucleic acid molecule, the sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: At least one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; wherein the nucleic acid molecule inhibits replication of the necrosis virus in a fish or a fish cell .
於再一較佳實施例中，本發明提供一種魚類核醣核酸試劑，包括：一核酸分子，用以抑制一神經壞死病毒之一蛋白質表現；以及一載體，用以包覆或包埋該核酸分子。In still another preferred embodiment, the present invention provides a fish ribonucleic acid reagent comprising: a nucleic acid molecule for inhibiting protein expression of a neuronal necrosis virus; and a vector for coating or embedding the nucleic acid molecule .
於再一較佳實施例中，其中該載體係可為一脂質體(liposome)、一膠囊、一晶球微膠囊、一明膠(Gelatin)膠球、一海藻酸鈉(Sodium Alginate)膠球中之任一種載體。In still another preferred embodiment, the carrier can be a liposome, a capsule, a crystal microcapsule, a gelatin gel, and a sodium alginate (Sodium Alginate) gel. Any one of the carriers.
於再一較佳實施例中，其中該魚類核醣核酸試劑可使體長不大於一吋或魚齡不大於40天之受感染之一稚魚的存活率提升。In still another preferred embodiment, wherein the fish ribonucleic acid reagent can increase the survival rate of an infected one of the juveniles having a body length of no more than one ticks or a fish age of not more than 40 days.
於再一較佳實施例中，其中該核酸分子係一短鏈核醣核酸分子。In still another preferred embodiment, wherein the nucleic acid molecule is a short stranded ribonucleic acid molecule.
於再一較佳實施例中，其中該短鏈核醣核酸分子係SEQ ID NO：1序列、SEQ ID NO：2序列、SEQ ID NO：3序列、以及SEQ ID NO：4序列之至少一者。In still another preferred embodiment, the short-stranded ribonucleic acid molecule is at least one of the sequence of SEQ ID NO: 1, the sequence of SEQ ID NO: 2, the sequence of SEQ ID NO: 3, and the sequence of SEQ ID NO: 4.
於再一較佳實施例中，其中該核酸分子係一去氧核醣核酸分子，且該去氧核醣核酸分子可透過一聚合酶(polymerase)而表現一短鏈核醣核酸分子。In still another preferred embodiment, wherein the nucleic acid molecule is a deoxyribonucleic acid molecule, and the deoxyribonucleic acid molecule can express a short-stranded ribonucleic acid molecule through a polymerase.
於再一較佳實施例中，其中該去氧核醣核酸分子包含SEQ ID NO：5序列、SEQ ID NO：6序列、SEQ ID NO：7序列、以及SEQ ID NO：8序列之至少一者。In still another preferred embodiment, wherein the deoxyribonucleic acid molecule comprises at least one of the sequence of SEQ ID NO: 5, the sequence of SEQ ID NO: 6, the sequence of SEQ ID NO: 7, and the sequence of SEQ ID NO: 8.
圖1：神經壞死病毒殼蛋白之訊息核醣核酸示意圖暨核醣核酸干擾序列。Figure 1: Schematic diagram of the neuronal necrosis virus capsid protein ribonucleic acid and ribonucleic acid interference sequence.
圖2A：核醣核酸干擾序列質體對病毒殼蛋白表現量影響的cDNA電泳圖。Figure 2A: cDNA electropherogram of the effect of ribonucleic acid sequence plastids on viral capsid expression.
圖2B：圖2A之定量分析圖。Figure 2B: Quantitative analysis of Figure 2A.
圖3：pSi730核醣核酸干擾序列質體對受病毒感染之魚類細胞響之顯微照片。Figure 3: Photomicrograph of the pSi730 ribonucleic acid sequence plastids against virus-infected fish cells.
圖4：pSi730核醣核酸干擾序列質體對受病毒感染之魚類細胞內病毒數量影響之長條圖。Figure 4: Bar graph of the effect of the pSi730 ribonucleic acid sequence on the number of viruses in virus-infected fish cells.
圖5：被餵食魚類核醣核酸試劑之斑馬魚的螢光顯微照片。Figure 5: Fluorescent micrograph of zebrafish fed a fish ribonucleic acid reagent.
圖6：被餵食魚類核醣核酸試劑之斑馬魚的即時聚合酶鏈鎖反應結果。Figure 6: Results of an instant polymerase chain reaction of zebrafish fed a fish ribonucleic acid reagent.
圖7：魚類核醣核酸試劑對受病毒感染之石斑魚存活影響之分析圖。Figure 7: Analysis of the effect of fish ribonucleic acid reagent on the survival of virus-infected grouper.
鑑於習知技術的限制及缺陷，亟待發展一種適用於魚類發育早期之感染性疾病試劑。本案發明人基於斑馬魚的核醣核酸干擾作用(RNAi)系統幾乎在魚苗孵化後就建立完全之事實，積極探究養殖魚類是否有類似的情形，並設法開發適用於魚類發育早期之口服感染性疾病試劑，終於研發完成本案一種防治魚類感染性疾病之魚類核醣核酸試劑。In view of the limitations and drawbacks of the prior art, it is urgent to develop an agent for infectious diseases suitable for early fish development. The inventor based on the zebrafish RNA interference (RNAi) system establishes the complete fact almost after the hatching of the fry, actively explores whether the farmed fish has a similar situation, and tries to develop an oral infectious disease reagent suitable for early fish development. Finally, the research and development of a fish ribonucleic acid reagent for the prevention and treatment of fish infectious diseases.
以下係提供本發明實施例之詳細說明書、本發明之技術及特點，係以防治石斑魚受神經壞死病毒感染為例。然本實施例並非用以限定本發明，任何熟悉此技術者，在不脫離本發明之精神和範圍內，當可應用於其他養殖魚類及其他病原性疾病，或作各種更動與潤飾。The following is a detailed description of the embodiments of the present invention, the technology and features of the present invention, and the control of grouper being infected with a necrosis virus as an example. The present examples are not intended to limit the invention, and any one skilled in the art can be applied to other farmed fish and other pathogenic diseases, or to various modifications and refinements without departing from the spirit and scope of the present invention.
核醣核酸干擾作用簡介Introduction to RNA interference
核醣核酸干擾作用(RNA interference，簡稱RNAi)始於長度為21~23個核甘酸的一短鏈核醣核酸分子(RNA)，被稱為小干擾核醣核酸分子(small interfering RNA，siRNA)。當該短鏈核醣核酸分子與細胞轉錄(Transcription)出的訊息核醣核酸分子(Messenger RNA,mRNA)互補性結合時，將活化沉默結合體(RNA-induced silencing complex)，導致該訊息核醣核酸分子降解，而無法進一步轉譯(Translation)出蛋白質。RNA interference (RNAi) begins with a short-stranded ribonucleic acid molecule (RNA) of 21 to 23 nucleotides in length and is called small interfering RNA (siRNA). When the short-stranded ribonucleic acid molecule binds complementarily to the Transcription-derived message RNA molecule (Messenger RNA, mRNA), the RNA-induced silencing complex is activated, resulting in degradation of the message ribonucleic acid molecule. And can't further translate the protein.
因此，設計特定序列的短鏈核醣核酸分子，使與一目標基因之訊息核醣核酸分子序列互補，即可能藉由核醣核酸干擾作用而專一性地抑制該基因表現。Thus, a particular sequence of short-stranded ribonucleic acid molecules is designed to be complementary to a message ribonucleic acid molecule sequence of a target gene, i.e., the expression of the gene may be specifically inhibited by ribonucleic acid interference.
目前人們在設計短鏈核醣核酸分子序列上仍有些許進步空間，有時會發生預測出的序列無法抑制基因表現，或即便能抑制基因表現，其抑制效果也不一定好之情形(例如僅讓蛋白質表現量由1降低到0.9)。因 此，除了借重序列的互補性進行預測外，還需倚賴實驗進行篩選驗證，才能在巨大的基因分子上挑出適合設計短鏈核醣核酸分子的位置。At present, there is still some room for improvement in designing the sequence of short-stranded ribonucleic acid molecules. Sometimes, the predicted sequence cannot inhibit the expression of the gene, or even if the expression of the gene is inhibited, the inhibition effect is not necessarily good (for example, only Protein expression decreased from 1 to 0.9). because In addition to predicting the complementarity of the heavy sequences, it is also necessary to rely on experiments for screening and verification in order to select suitable locations for designing short-stranded ribonucleic acid molecules on huge gene molecules.
實驗一：報導質體及核醣核酸干擾(RNAi)序列質體的選殖Experiment 1: Reporting the plastid and ribonucleic acid interference (RNAi) sequence plastid colonization
病毒進入魚體後，會利用宿主細胞內的酵素及原料大量複製病毒核酸及殼蛋白，經組裝後再由宿主細胞釋出大量的新病毒，使魚體內的病毒數量越來越多，病況也越來越嚴重。其中，病毒表面的殼蛋白是病毒複製並從宿主細胞釋出時不可或缺的元件。因此，可針對病毒的殼蛋白(或其他必要蛋白)設計短鏈核醣核酸分子序列，並將該短鏈核醣核酸分子送入魚體細胞內，以阻斷病毒之殼蛋白(或其他必要蛋白)基因表現，進而抑制病毒在宿主細胞內複製，以防止魚類病毒疾病之惡化或發生。After the virus enters the fish body, it will use the enzymes and raw materials in the host cell to replicate the viral nucleic acid and the shell protein in large quantities. After assembly, a large amount of new virus will be released from the host cell, and the number of viruses in the fish body will increase. getting more serious. Among them, the shell protein on the surface of the virus is an indispensable element when the virus replicates and is released from the host cell. Therefore, a short-chain ribonucleic acid molecule sequence can be designed against the viral capsid protein (or other essential protein), and the short-stranded ribonucleic acid molecule can be introduced into the fish body cell to block the viral shell protein (or other essential protein). Gene expression, which in turn inhibits replication of the virus in the host cell, prevents the deterioration or occurrence of fish virus disease.
首先，於美國國家生技資訊中心(NCBI)資料庫中搜尋魚類神經壞死病毒(Nervous Necrosis Virus,NNV)殼蛋白基因的互補去氧核醣核酸(complementary DNA，cDNA)序列，查得一NCBI序列編號為NC_008041之序列。其中，該魚類神經壞死病毒為Nodaviridae科，betanodavirus屬，RGNNV基因型。將該魚類神經壞死病毒的殼蛋白基因序列輸入軟體，經該軟體分析取得所有有潛力進行核醣核酸干擾作用的位置，再挑選出其中四個可能最有潛力進行核醣核酸干擾作用的位置，其序列分別為：First, search for the complementary DNA sequence of the Nervous Necrosis Virus (NNV) capsid gene in the National Center for Biotechnology Information (NCBI) database, and find an NCBI sequence number. Is the sequence of NC_008041. Among them, the fish necrosis virus is a Nodaviridae family, a betanodavirus genus, and an RGNNV genotype. The fish protein necrosis virus capsid gene sequence is input into the software, and the software analyzes all the positions that have potential for RNA interference, and then selects four of the most potential sites for ribonucleic acid interference. They are:
(1)5’-CAATCGTCGGCGTAGCAAT-3’(1) 5'-CAATCGTCGGCGTAGCAAT-3’
(2)5’-CACTGATGTGGTCAACGTG-3’(2) 5’-CACTGATGTGGTCAACGTG-3’
(3)5’-TCCATCCTCCTAGGATCCA-3’(3) 5’-TCCATCCTCCTAGGATCCA-3’
(4)5’-AACTAACCGGGTCATCCGG-3’(4) 5’-AACTAACCGGGTCATCCGG-3’
請參閱圖一，係神經壞死病毒殼蛋白之訊息核醣核酸示意圖暨核醣核酸干擾序列。將該四個可能最有潛力進行核醣核酸干擾作用的位置序列轉換為短鏈核醣核酸分子序列(即SEQ ID NO：1、SEQ ID NO： 2、SEQ ID NO：3、SEQ ID NO：4)。抑或是將該些短鏈核醣核酸分子序列再進一步轉換為雙股之去氧核醣核酸分子序列(即SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8，依序命名為siRNA 93、siRNA 585、siRNA 730、siRNA 1024)。Please refer to Figure 1. The message of the neuronal necrosis virus shell protein ribonucleic acid and the ribonucleic acid interference sequence. Converting the four possible positions that are most likely to undergo ribonucleic acid interference to a short-stranded ribonucleic acid molecule sequence (ie, SEQ ID NO: 1, SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4). Or is the sequence of the short-stranded ribonucleic acid molecules further converted into a double-stranded deoxyribonucleic acid molecule sequence (ie, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, Named siRNA 93, siRNA 585, siRNA 730, siRNA 1024).
將siRNA 93、siRNA 585、siRNA 730、siRNA 1024分別選殖到pIASIR這個核醣核酸干擾表現質體(產品名稱為pAAV-IRES-Neo Expression Vector，購自美國Cell Biolabs，產品編號為VPK-416，亦可選用其他核醣核酸干擾表現質體)的魚類啟動子序列下游，分別將這些新的「核醣核酸干擾序列質體」命名為pSi93、pSi585、pSi730、pSi1024。則魚類聚合酶(Polymerase，例如位於魚體內之魚類聚合酶、位於游離之魚類細胞中的魚類聚合酶、或於試管中外加之魚類聚合酶)在辨識到該魚類啟動子序列後，即能進一步依據下游的siRNA 93、siRNA 585、siRNA 730、或siRNA 1024序列轉錄出短鏈核醣核酸分子。siRNA 93, siRNA 585, siRNA 730, and siRNA 1024 were separately selected into pIASIR, a ribonucleic acid-expressing plastid (product name: pAAV-IRES-Neo Expression Vector, purchased from Cell Biolabs, USA, product number VPK-416, also These new "ribonuclear interference sequence plastids" can be named as pSi93, pSi585, pSi730, pSi1024, respectively, downstream of the fish promoter sequence using other ribonucleic acids to interfere with the expression of plastids. The fisher polymerase (Polymerase, such as fish polymerase in fish, fish polymerase in free fish cells, or fish polymerase added to the test tube) can further rely on the fish promoter sequence. The downstream siRNA 93, siRNA 585, siRNA 730, or siRNA 1024 sequences transcribe short-stranded ribonucleic acid molecules.
其中，pIASIR中也含有遠紅螢光蛋白(hc-red)基因及抗新黴素(neomycin)基因，可作為後續篩選之用。選殖後，再經酵素切割及DNA定序以確認該些序列是否已正確***質體中。Among them, pIASIR also contains far red fluorescent protein (hc-red) gene and anti-neomycin gene, which can be used for subsequent screening. After colonization, enzyme cleavage and DNA sequencing are performed to confirm whether the sequences have been correctly inserted into the plastid.
實驗二：核醣核酸干擾序列對病毒殼蛋白表現之影響Experiment 2: The effect of ribonucleic acid interference sequence on the performance of viral capsid protein
為了評估各個序列是否能抑制病毒殼蛋白之表現，再進一步以健康的魚類細胞株SSN-1(購自英國European Collection of Animal Cell Cultures，簡稱ECACC，產品編號為96082808)進行測試。首先，將魚類神經壞死病毒殼蛋白基因選殖到質體(可購自德國QIAGEN，產品名稱為pQE-30 Xa Vector)中，經定序確認後命名為pIRNNV質體。利用轉染套組(購自美國Roche，產品名稱為FuGENE)，將pIRNNV質體轉染(transfect)到健康的SSN-1細胞株中，使SSN-1細胞表現魚類神經壞死病毒之殼蛋白。To assess whether each sequence inhibited the expression of the viral capsid protein, the healthy fish cell line SSN-1 (purchased from the European Collection of Animal Cell Cultures, ECACC, product number 96082808) was further tested. First, the fish necrosis virus capsid gene was cloned into a plastid (available from QIAGEN, Germany, under the product name pQE-30 Xa Vector) and identified as pIRNNV plastid after sequence confirmation. The pIRNNV plastid was transfected into a healthy SSN-1 cell line using a transfection kit (purchased from Roche, USA, under the product name FuGENE), and the SSN-1 cells were expressed as a capsid protein of fish necrosis virus.
分別將pSi93、pSi585、pSi730、pSi1024轉染到上述可表現魚類神經壞死病毒殼蛋白之健康SSN-1細胞中，觀察各組別的干擾結果。請參閱圖2A，係核醣核酸干擾序列質體對病毒殼蛋白表現量影響的cDNA電泳圖。圖2B之橫軸方向由左至右依序為對照組、負控制組、pSi93組、pSi585組、pSi730組、pSi1024組；由上而下依序為神經壞死病毒殼蛋白之表現量、β肌動蛋白(β-actin)之表現量。pSi93, pSi585, pSi730, and pSi1024 were transfected into the above healthy SSN-1 cells expressing the fish necrosis virus shell protein, and the interference results of each group were observed. Please refer to Figure 2A, which is a cDNA electropherogram of the effect of ribonucleic acid interference sequence plastids on viral capsid expression. The horizontal axis direction of Fig. 2B is from left to right in order of control group, negative control group, pSi93 group, pSi585 group, pSi730 group, and pSi1024 group; from top to bottom, the expression of neuron necrosis virus shell protein, β muscle The amount of actin (β-actin).
其中，對照組細胞僅轉染pIRNNV質體，並未轉染核醣核酸干擾序列質體，因此，可觀察到其大量表現pIRNNV質體中帶有的病毒殼蛋白基因。負控制組為完全未經處理的細胞，可觀察到完全沒有表現病毒殼蛋白基因，顯示所使用的SSN-1細胞株並未受神經壞死病毒感染，也不曾被轉殖外來的病毒殼蛋白基因。pSi93組、pSi585組、pSi730組及pSi1024組為同時轉染pIRNNV質體及核醣核酸干擾序列質體的細胞(但各組的核醣核酸干擾序列不同)，可觀察到病毒殼蛋白基因的表現量確實受到核醣核酸干擾序列抑制。請參閱圖2B，其為圖2A之定量分析圖。圖2B中，pSi93組、pSi585組、pSi730組及pSi1024組之病毒殼蛋白表現量分別為65.1%、69.3%、56.2%、60.4%，顯示pSi730組抑制病毒殼蛋白表現的效果最好。Among them, the control cells were only transfected with pIRNNV plastids and were not transfected with ribonucleic acid to interfere with the plastids. Therefore, a large number of virion protein genes in pIRNNV plastids were observed. The negative control group was completely untreated, and no virus capsid gene was observed at all, indicating that the SSN-1 cell strain used was not infected with necrosis virus, nor was it transferred to a foreign viral capsid protein gene. . The pSi93 group, pSi585 group, pSi730 group and pSi1024 group were cells transfected with pIRNNV plastids and ribonucleic acid splicing plastids simultaneously (but the ribonucleic acid interference sequences of each group were different), and the expression of the viral capsid gene was observed. Inhibited by ribonucleic acid interference sequences. Please refer to FIG. 2B, which is a quantitative analysis diagram of FIG. 2A. In Fig. 2B, the viral shell protein expressions of the pSi93 group, the pSi585 group, the pSi730 group and the pSi1024 group were 65.1%, 69.3%, 56.2%, and 60.4%, respectively, indicating that the pSi730 group had the best effect in inhibiting viral shell protein expression.
實驗三：核醣核酸序列保護魚類神經細胞之情形Experiment 3: The situation of ribonucleic acid sequence protecting fish nerve cells
將pSi730轉染入受病毒感染的魚類細胞株SSN-1中，以觀察抑制病毒殼蛋白之表現是否確實能抑制魚類神經細胞中的病毒數量，並保護魚類神經細胞不受病毒所害。pSi730 was transfected into the virus-infected fish cell line SSN-1 to observe whether inhibition of viral capsid protein actually inhibited the number of viruses in fish nerve cells and protected fish nerve cells from viruses.
首先，利用轉染套組(購自美國Roche，產品名稱為FuGENE)將pSi730轉染到健康的魚類細胞株SSN-1中，再以抗新黴素篩選出含有pSi730質體之細胞。接著，在L-15培養基(購自美國Gibco，產品名稱Leibovitz L-15 medium)中加入與細胞數量相等的魚類神經病毒(MOI=1)，使帶有pSi730質體的SSN-1細胞與魚類神經病毒共同培養5天，觀察細胞中的病毒數量 及細胞之存活情形(5天內不更換培養基，以避免細胞流失)。First, pSi730 was transfected into healthy fish cell line SSN-1 using a transfection kit (purchased from Roche, USA, under the product name FuGENE), and cells containing pSi730 plastids were screened with anti-neomycin. Next, a fish neuron (MOI=1) equal to the number of cells was added to L-15 medium (purchased from Gibco, USA, under the product name Leibovitz L-15 medium) to make SSN-1 cells with pSi730 plastids and fish The neurovirus was co-cultured for 5 days and the number of viruses in the cells was observed. And the survival of the cells (do not change the medium within 5 days to avoid cell loss).
請參閱圖3，其為pSi730核醣核酸干擾序列質體對受病毒感染之魚類細胞響之顯微照片。圖3之橫軸方向由左至右依序為負控制組、對照組、實驗組；縱軸方向由上而下依序為培養36小時、3天、5天。其中，負控制組為完全未處理的健康SSN-1細胞，其細胞數量隨著時間而增加，且細胞貼附情形良好，幾乎未出現細胞剝落、變圓等現象；對照組為經病毒共培養及轉染溶劑處理之細胞，可觀察到細胞逐漸出現剝落、變圓等細胞病變效應(cytopathic effect，CPE，即細胞感染病毒後產生剝落、變圓、萎縮等現象)；實驗組為經病毒共培養及pSi730質體處理之細胞，相較於對照組，可觀察到pSi730明顯改善細胞剝落、變圓之情形。本實驗顯示，阻斷病毒殼蛋白表現確實能降低魚類神經細胞受病毒感染所發生的病變。Please refer to Figure 3, which is a photomicrograph of the pSi730 ribonuclease sequence plastid on the virus-infected fish cells. The horizontal axis direction of Fig. 3 is from the left to the right in the negative control group, the control group, and the experimental group; the vertical axis direction is from the top to the bottom for 36 hours, 3 days, and 5 days. Among them, the negative control group was completely untreated healthy SSN-1 cells, the number of cells increased with time, and the cell attachment was good, almost no cell flaking, rounding, etc.; the control group was co-cultured by virus And the cells treated with the solvent treatment can observe the cytopathic effect (CPE, which is the phenomenon of flaking, rounding and atrophy after the cells are infected with the virus), and the experimental group is a virus. Compared with the control group, cells cultured with pSi730 plastids showed that pSi730 significantly improved cell flaking and rounding. This experiment shows that blocking the expression of viral capsid protein can indeed reduce the pathological changes of fish nerve cells infected by viruses.
請參閱圖4，其為pSi730核醣核酸干擾序列質體對受病毒感染之魚類細胞內病毒數量影響之長條圖。橫軸為感染後的天數，由左而右依序為對照組1、對照組2、實驗組，縱軸則為病毒數量。其中，對照組1僅經病毒共培養5天，完全未經其他處理，數據顯示第5天之總病毒數量達1010 ；對照組2經轉染溶劑處理後，再經病毒共培養5天，數據顯示第5天之總病毒數量達109 ，與對照組1之間無顯著差異；實驗組經pSi730質體轉染後，再經病毒共培養5天，數據顯示第5天之總病毒數量達103 ，與對照組1及對照組2之間均有顯著差異，病毒數量為原來的10-7 倍。Please refer to Figure 4, which is a bar graph of the effect of the pSi730 ribonucleic acid sequence on the number of viruses in virus-infected fish cells. The horizontal axis is the number of days after infection, from left to right, in the control group 1, the control group 2, the experimental group, and the vertical axis is the number of viruses. Among them, the control group 1 was only cultured for 5 days by virus, and was completely free of other treatments. The data showed that the total number of viruses on the fifth day reached 10 10 ; the control group 2 was treated with the transfection solvent, and then co-cultured for 5 days. The data showed that the total number of viruses on day 5 reached 10 9 , which was not significantly different from that of control group 1 ; the experimental group was transfected with pSi730 plastids and then co-cultured for 5 days with virus, the data showed the total number of viruses on the 5th day. Up to 10 3 , there was a significant difference between the control group 1 and the control group 2, and the number of viruses was 10 -7 times.
由圖3及圖4的結果可知，核醣核酸干擾試劑阻斷病毒殼蛋白表現(請參見圖2)，使得病毒複製所需的殼蛋白原料不足，確實會進一步反應在受感染細胞的病毒數量上，即會抑制病毒在魚類細胞中複製，使病毒數量顯著減少(請參見圖4)，且會進一步改善受感染細胞之細胞病變及死亡情形(請參見圖3)。這些結果顯示，核醣核酸干擾試劑有機會成為疫苗以外的另一項利器，用以防制魚類病毒性疾病。From the results of Fig. 3 and Fig. 4, it is known that the ribonucleic acid interference reagent blocks the expression of the viral capsid protein (see Fig. 2), so that the shell protein raw material required for virus replication is insufficient, and the number of viruses in the infected cells is actually further reacted. That will inhibit the replication of the virus in fish cells, significantly reducing the number of viruses (see Figure 4), and will further improve the cytopathic and death of infected cells (see Figure 3). These results show that ribonucleic acid interference agents have the opportunity to be another tool besides vaccines to prevent fish viral diseases.
應能理解的是，病毒在魚隻體內的細胞中也會採行一樣的複製方式，因此，使用同樣的核醣核酸干擾方式應會有類似的結果，而能抑制病毒在魚體內複製。因此，以下將進一步確認這樣的方法及系統是否確實能保護魚隻不受病毒所害。It should be understood that the virus will adopt the same method of replication in the cells of the fish. Therefore, the same ribonucleic acid interference method should have similar results, and can inhibit the replication of the virus in the fish. Therefore, the following will further confirm whether such methods and systems can indeed protect fish from viruses.
實驗四：製備魚類核醣核酸試劑Experiment 4: Preparation of fish ribonucleic acid reagent
本發明魚類核醣核酸試劑之主要成分為核酸分子(例如序列如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、以及SEQ ID NO：4之至少一者，抑或是序列包括SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、以及SEQ ID NO：8之至少一者)，可利用口服方式投遞、混合飼料之口服方式投遞、以及浸泡方式投遞之至少任一種。較佳者，利用一載體包覆或包埋核酸分子，例如利用一膠囊、一晶球微膠囊、一明膠(Gelatin)膠球、一海藻酸鈉(Sodium Alginate)膠球或其他載體包覆或包埋核酸分子，以保護核酸分子不被外界環境中的物質破壞。The main component of the fish ribonucleic acid reagent of the present invention is a nucleic acid molecule (for example, at least one of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or the sequence includes SEQ ID NO: 5, at least one of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8) may be administered by oral delivery, oral delivery of mixed feed, and immersion delivery. . Preferably, the nucleic acid molecule is coated or embedded by a carrier, for example, by using a capsule, a crystal microcapsule, a gelatin gel, a sodium alginate (Sodium Alginate) rubber ball or other carrier or The nucleic acid molecule is embedded to protect the nucleic acid molecule from being destroyed by substances in the external environment.
較佳者，利用一脂質體(liposome，係一空心微球)包覆核酸分子，以保護該核酸分子通過魚類消化道，進而被尾腸吸收。例如脂質體包覆之該核酸分子為pSi730質體，則此種魚類核醣核酸試劑可透過脂質體之保護而順利抵達魚隻的尾腸，且其內的pSi730質體可抑制魚類神經壞死病毒的殼蛋白表現，進而抑制該魚類神經壞死病毒在魚體內或魚類細胞中複製，達到較佳的防治魚類神經壞死疾病之效果。Preferably, the nucleic acid molecule is coated with a liposome (a hollow microsphere) to protect the nucleic acid molecule from the digestive tract of the fish and then absorbed by the tail. For example, if the nucleic acid molecule coated with the liposome is pSi730 plastid, the fish ribonucleic acid reagent can smoothly reach the tail of the fish through the protection of the liposome, and the pSi730 plastid therein can inhibit the fish necrosis virus. The expression of the capsid protein, thereby inhibiting the replication of the fish neuronecrosis virus in fish or fish cells, achieves a better effect of preventing and treating fish nerve necrosis.
該脂質體之組成份包括例如卵磷脂、膽固醇，亦可替換為其他脂質類。較佳者，該脂質體之組成份更包括幾丁質，使該脂質體更能保 護該核酸分子通過魚類消化道。本實施例之魚類核醣核酸試劑之製備方法如下，但應能理解的是，並不以此為限：The components of the liposome include, for example, lecithin, cholesterol, and may be replaced with other lipids. Preferably, the liposome component further comprises chitin, which makes the liposome more Protect the nucleic acid molecule through the digestive tract of fish. The preparation method of the fish ribonucleic acid reagent of the present embodiment is as follows, but it should be understood that it is not limited thereto:
將1.5μg卵磷脂、1.5μg膽固醇、及1mL氯仿(chloroform)加入圓底瓶中，混合均勻。於70℃水浴槽中均勻搖晃，使氯仿揮發。再加入1mL含有20μg pSi730質體之二次水溶液，搖晃均勻使脂質溶解。最後，再加入1ug幾丁質粉末，混合均勻，即製備出核醣核酸干擾試劑。應能理解的是，所包覆之核酸分子可以如上述般為去氧核醣核酸(DNA)分子，抑或是核醣核酸(RNA)分子，只要送入魚體或是魚類細胞後，能透過核醣核酸干擾作用抑制病毒之蛋白質(例如殼蛋白)表現即可。1.5 μg of lecithin, 1.5 μg of cholesterol, and 1 mL of chloroform were added to a round bottom bottle and mixed well. Shake evenly in a 70 ° C water bath to volatilize chloroform. Further, 1 mL of a secondary aqueous solution containing 20 μg of pSi730 plastid was added, and the lipid was dissolved by shaking uniformly. Finally, 1 ug of chitin powder was added and mixed uniformly to prepare a ribonucleic acid interference reagent. It should be understood that the coated nucleic acid molecule can be a DNA molecule as described above, or a ribonucleic acid (RNA) molecule, which can pass through the ribonucleic acid as long as it is fed into a fish or fish cell. The interference inhibits the expression of a protein of the virus (for example, a capsid protein).
實驗五：以魚類核醣核酸試劑餵食魚類後之吸收情形Experiment 5: Absorption after feeding fish with fish ribonucleic acid reagent
利用斑馬魚評估魚類核醣核酸試劑是否可以順利通過魚隻的腸胃，進而被魚隻吸收。Use the zebrafish to assess whether the fish ribonucleic acid reagent can pass through the gastrointestinal tract of the fish and be absorbed by the fish.
斑馬魚可向國家衛生研究院台灣斑馬魚核心設施(衛清分支)購買。將魚類核醣核酸試劑(脂質體中包覆的是pSi730質體)溶液與飼料混合(魚類核醣核酸試劑溶液與飼料的體積比為5：2)，直接餵食魚齡為4天的斑馬魚。16小時後利用螢光顯微鏡以及即時聚合酶鏈鎖反應(Real time PCR)觀察魚隻是否表現遠紅螢光蛋白(hc-red)，以確認魚類核醣核酸試劑是否已進入魚體、魚隻細胞內的聚合酶是否能辨識pSi730上的啟動子而表現下游基因。Zebrafish can be purchased from the National Zebra's Taiwan Zebrafish Core Facility (Weiqing Branch). A solution of fish ribonucleic acid reagent (pSi730 plastid in liposome) was mixed with the feed (volume ratio of fish ribonucleic acid reagent solution to feed was 5:2), and zebrafish with a fish age of 4 days was directly fed. After 16 hours, fluorescence microscopy and real-time polymerase chain reaction (Real time PCR) were used to observe whether the fish showed far-red fluorescent protein (hc-red) to confirm whether the fish ribonucleic acid reagent had entered the fish or fish cells. Whether the internal polymerase recognizes the promoter on pSi730 and expresses the downstream gene.
請參閱圖5，係被餵食魚類核醣核酸試劑之斑馬魚的螢光顯微照片。圖5由左至右依序為明視野及暗視野下的結果，明視野係用以觀察魚隻整體形態，暗視野則用以觀察螢光量及螢光表現位置；由上而下依序為控制組及實驗組。控制組之斑馬魚被餵食一般飼料，可觀察到並未表現遠紅螢光蛋白；實驗組之斑馬魚則被餵食混合魚類核醣核酸試劑(包覆 pSi730質體)的飼料，可觀察到確實有表現遠紅螢光蛋白，顯示本發明之魚類核醣核酸試劑能順利通過魚隻的腸胃，進而被魚隻吸收，且魚隻細胞內的聚合酶確實能辨識pSi730上的啟動子而表現下游基因。Referring to Figure 5, a fluorescent micrograph of a zebrafish fed a fish ribonucleic acid reagent. Figure 5 shows the results from the left to the right in the bright field and the dark field. The bright field is used to observe the overall shape of the fish, and the dark field is used to observe the amount of fluorescence and the position of the fluorescent light. From top to bottom, Control group and experimental group. The zebrafish in the control group were fed a general diet, and no far red fluorescent protein was observed. The zebrafish in the experimental group were fed with mixed fish ribonucleic acid reagent (coated). In the feed of pSi730 plastid, it was observed that the far red fluorescent protein was indeed expressed, indicating that the fish ribonucleic acid reagent of the present invention can smoothly pass through the gastrointestinal tract of the fish, and is absorbed by the fish, and the polymerase in the fish cell is indeed The promoter on pSi730 can be recognized to represent downstream genes.
請參閱圖6，係被餵食魚類核醣核酸試劑之斑馬魚的即時聚合酶鏈鎖反應結果，橫軸為餵食後經歷的時間長度，由左而右依序為控制組及實驗組，縱軸則為遠紅螢光蛋白mRNA之相對定量結果。圖6同樣顯示本發明之魚類核醣核酸試劑能順利通過魚隻的腸胃，且魚隻細胞內的聚合酶能辨識pSi730上的啟動子而表現下游基因。Please refer to Figure 6. The results of real-time polymerase chain reaction of zebrafish fed with fish ribonucleic acid reagent, the horizontal axis is the length of time after feeding, from left to right, the control group and the experimental group, and the vertical axis. It is the relative quantitative result of far red fluorescent protein mRNA. Figure 6 also shows that the fish ribonucleic acid reagent of the present invention can smoothly pass through the gastrointestinal tract of the fish, and the polymerase in the fish cell can recognize the promoter on pSi730 to express the downstream gene.
實驗六：魚類核醣核酸試劑保護石斑魚之情形Experiment 6: The situation of fish ribonucleic acid reagent protecting grouper
石斑魚，泛指鱸形目(Perciformes)鮨科(Serranidae)石斑魚屬(Epinephelus )裡的各種魚類。石斑魚為暖水性魚類，廣佈全世界熱帶及亞熱帶海域，由於其肉質鮮美，目前已成為全世界重要的高經濟海水養殖魚種。然而，目前人工養殖之石斑魚普遍受神經壞死病毒所苦，影響養殖業甚鉅。因此，下列將以防治點帶石斑魚(Epinephelus coioides )受神經壞死病毒感染為例，測試本發明魚類核醣核酸試劑之效果及系統之可行性。Grouper, broadly referred to as various fish in the genus Serenaidae ( Epinephelus ). Grouper is a warm-water fish that is widely distributed in tropical and subtropical waters around the world. Due to its delicious meat, it has become an important high-economic marine culture species in the world. However, the currently farmed grouper is generally suffering from neuronecrosis virus, which affects the breeding industry. Therefore, the following will test the effect of the fish ribonucleic acid reagent of the present invention and the feasibility of the system by taking the infection of the grouper grouper ( Epinephelus coioides ) with neuronecrosis virus as an example.
以下係評估核醣核酸試劑是否可以保護受神經壞死病毒感染之石斑幼魚，以減少幼魚之死亡率。石斑魚可向海洋大學水生動物實驗中心購買。將3mL神經壞死病毒液(TCID50 為106 )加入700mL水體中，再放入魚齡20天之石斑幼魚，浸泡2小時。接下來，將石斑魚移到乾淨的水體中養殖，並將魚類核醣核酸試劑(包覆pSi730質體)溶液與飼料混合，直接餵食該些石斑幼魚，觀察魚隻存活情形。The following is an evaluation of whether a ribonucleic acid reagent can protect a group of juveniles infected with a necrotic virus to reduce the mortality of juvenile fish. Groupers can be purchased from the Ocean University Aquatic Animal Experimental Center. 3 mL of necrotic virus solution (TCID 50 of 10 6 ) was added to 700 mL of water, and then the grouper of juvenile fish, which was 20 days old, was immersed for 2 hours. Next, the grouper was moved to a clean water body, and the fish ribonucleic acid reagent (coated with pSi730 plastid) solution was mixed with the feed, and the grouper was directly fed to observe the survival of the fish.
請參閱圖7，係魚類核醣核酸試劑對受病毒感染之石斑魚存活影響之分析圖。其中，負控制組的石斑幼魚未浸泡病毒液，飼料中也未做任何處理，可觀察到數量由剛開始的15隻經正常死亡率而剩9隻；對照 組的石斑幼魚經浸泡神經壞死病毒液，飼料中混合二次水，可觀察到數量由剛開始的15隻經大量死亡而剩1隻；實驗組1的石斑幼魚經浸泡神經壞死病毒液，飼料中混合0.5倍劑量之魚類核醣核酸試劑(含有pSi730質體。魚類核醣核酸試劑溶液經對倍稀釋後與飼料混合，該魚類核醣核酸試劑溶液之稀釋液與飼料的體積比為5：2)，可觀察到數量由剛開始的15隻到最後剩9隻，與未感染病毒之負控制組間無顯著差異；實驗組2的石斑幼魚經浸泡神經壞死病毒液，飼料中混合1倍劑量之魚類核醣核酸試劑(含有pSi730質體。魚類核醣核酸試劑溶液與飼料的體積比為5：2)，可觀察到數量由剛開始的15隻到最後剩11隻，與未感染病毒之負控制組間無顯著差異。圖7之結果顯示本發明魚類核醣核酸試劑確實有保護石斑魚的效果，且證實本發明魚類核醣核酸試劑利用脂質體包覆核苷酸而防治魚類感染性疾病之系統確實可行，可施用於體長不大於一吋或魚齡不大於40天之稚魚。Please refer to Figure 7 for an analysis of the effects of fish ribonucleic acid reagent on the survival of virus-infected grouper. Among them, the gray spotted juveniles in the negative control group were not soaked in the virus solution, and no treatment was done in the feed. It was observed that the number of the first 15 normal mortality rates was 9; The group of juveniles of the grouper was soaked with neuronecrosis virus solution, and the feed was mixed with secondary water. It was observed that the number of the first 15 died after a large number of deaths; the group 1 of the experimental group 1 was soaked with necrosis virus solution. The feed is mixed with a 0.5-fold dose of fish ribonucleic acid reagent (containing pSi730 plastid. The fish ribonucleic acid reagent solution is mixed with the feed after double dilution, and the volume ratio of the dilution of the fish ribonucleic acid reagent solution to the feed is 5:2. ), the number can be observed from the first 15 to the last 9 left, and there is no significant difference between the negative control group and the uninfected virus control group; the experimental group 2 of the grouper juvenile fish is soaked with neuronecrosis virus solution, the feed is mixed 1 times Dosage of fish ribonucleic acid reagent (containing pSi730 plastid. The volume ratio of fish ribonucleic acid reagent solution to feed is 5:2), the number can be observed from the first 15 to the last 11 remaining, and the negative of the uninfected virus There were no significant differences between the control groups. The results of Fig. 7 show that the fish ribonucleic acid reagent of the present invention does have the effect of protecting groupers, and it is confirmed that the fish ribonucleic acid reagent of the present invention utilizes liposome-coated nucleotides to prevent and treat fish infectious diseases, and is practically applicable to body length. Not more than one or a juvenile fish with a fish age of no more than 40 days.
把發明利用核醣核酸干擾作用及脂質體包覆運輸系統，成功建立一種適用於稚魚的魚類核醣核酸試劑。在考量不傷害稚魚或對稚魚造成緊迫壓力時，可直接經由口服方式投遞，而不用擔心被消化系統中的酸性環境或酵素破壞。本發明魚類核醣核酸試劑有別於習知技術一魚類疫苗無法防治稚魚受病原體感染之藥物系統缺陷，為一種可成功施用於稚魚的藥物系統，且確實可使受感染稚魚之存活率大幅度提升。The invention successfully established a fish ribonucleic acid reagent suitable for juvenile fish by using ribonucleic acid interference and liposome-coated transport system. When considering not to harm juvenile fish or put pressure on juvenile fish, it can be delivered directly by oral administration without fear of being damaged by the acidic environment or enzymes in the digestive system. The fish ribonucleic acid reagent of the present invention is different from the defect of the pharmaceutical system in which the fish vaccine cannot prevent juveniles from being infected by pathogens, and is a drug system which can be successfully applied to juvenile fish, and can indeed greatly improve the survival rate of infected juveniles. .
應能理解的是，本發明魚類核醣核酸試劑應用的範圍並不以口服方式投遞於稚魚為限。投遞方式可以其它方式實現，例如注射法或浸泡法；被投遞之魚隻亦可以為體長大於一吋或魚齡大於40天之魚隻；被投遞之魚種不限於石斑魚，其他種類的魚隻例如黃臘鯵(yellow-waxpompano)、海鱺(cobia)、歐洲鰻(European eel)、赤鰭笛鯛(fire-spot snapper)、中國鯰魚(Chinese catfish)等亦可。 應能理解的是，上述之投遞方式、魚隻大小及魚種等均為本發明魚類核醣核酸試劑應用範圍之示例，但並不以此為限。It should be understood that the scope of application of the fish ribonucleic acid reagent of the present invention is not limited to oral administration to juveniles. The delivery method can be implemented in other ways, such as injection or soaking; the delivered fish can also be fish with a body length greater than one or a fish age greater than 40 days; the delivered species are not limited to groupers, other species of fish For example, yellow-waxpompano, cobia, European eel, fire-spot snapper, Chinese catfish, etc. It should be understood that the above delivery methods, fish size and fish species are examples of the application range of the fish ribonucleic acid reagent of the present invention, but are not limited thereto.
以上所述僅為本發明之較佳實施例，並非用以限定本發明之申請專利範圍，因此凡其它未脫離本發明所揭示之精神下所完成之等效改變或修飾，均應包含於本案之申請專利範圍內。The above are only the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Therefore, any equivalent changes or modifications made without departing from the spirit of the present invention should be included in the present invention. Within the scope of the patent application.
<110> 國立臺灣海洋大學<110> National Taiwan Ocean University
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| Title |
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| 蔡翰甽，在斑馬魚中利用小片段核醣核酸干擾疫苗抑制神經壞死病毒，國立臺灣大學漁業科學研究所碩士論文，2006年 摘要、第20頁第2段、第24頁第1段、第25頁第1-2段、第28頁第1段、第29頁第3段、第30頁第1段、表1 林青丘，魚用生物包埋型口服疫苗製備與有效性測試，2004年，國立成功大學生物科技研究所碩士論文。 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9705274B2 (en) | 2015-02-13 | 2017-07-11 | Foxconn Interconnect Technology Limited | Electrical connector and method of making the same |
Also Published As
| Publication number | Publication date |
|---|---|
| TW201446285A (en) | 2014-12-16 |
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