WO2017079936A1 - 表现异源基因的系统及其用途 - Google Patents

表现异源基因的系统及其用途 Download PDF

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WO2017079936A1
WO2017079936A1 PCT/CN2015/094412 CN2015094412W WO2017079936A1 WO 2017079936 A1 WO2017079936 A1 WO 2017079936A1 CN 2015094412 W CN2015094412 W CN 2015094412W WO 2017079936 A1 WO2017079936 A1 WO 2017079936A1
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gene
promoter
heterologous gene
heterologous
expressing
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French (fr)
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许家恺
陈历历
许德根
元淑冰
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昕颖生医技术股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to a system for expressing a heterologous gene and uses thereof, and more particularly to a system and method for expressing a target gene in a eukaryote using a Listeria and a expression vector.
  • viral proteins as antigens is one of the common pathogen control methods, for example, the use of microorganisms to deliver pathogenic proteins into mammalian cells, thereby inducing cellular immune responses.
  • E. coli as a protein delivery system for the treatment of human diseases.
  • the possibility of using other bacterial species to transport pathogenic proteins into the human body has been explored, for example, the increase in mononuclear cells.
  • Listeria monocytogenes Listeria is a facultative anaerobic Gram-positive bacterium with strong environmental adaptability, can grow at 3-45 °C, and can survive in a high salt environment.
  • the present invention provides a system for expressing a heterologous gene comprising: a Listeria bacterium; and a recombinant expression vector, wherein the recombinant expression vector comprises: (a) a bacterial promoter; (b) an eukaryotic a biological promoter; and (c) a heterologous gene.
  • the eukaryotic promoter is a water producer promoter.
  • the water producer promoter is a pCMV promoter.
  • the bacterial promoter is a phly promoter.
  • the Listeria bacterium is an inactive bacterium.
  • heterologous gene comprises a reporter gene
  • the reporter gene comprises, but is not limited to, an enhanced green fluorescent protein gene (GFP) or a pigment gene.
  • GFP enhanced green fluorescent protein gene
  • heterologous gene comprises a growth gene
  • heterologous gene comprises an anti-stress gene.
  • heterologous gene comprises a pathogenic gene
  • the pathogenic gene is an aquatic animal pathogenic gene.
  • the aquatic animal pathogenic gene is a chitinvirus protein gene.
  • the chitinvirus protein gene includes but is not limited to: shrimp white spot virus (White spot syndrome virus) protein gene, Penaeus monodon baculovirus protein gene, Taura syndrome virus protein gene, Baculoviral midgut necrosis virus protein gene, shrimp Baculovirus penaei protein gene, infectious hypodermal and haematopoietic necrosis virus protein gene, yellowhead virus protein gene, Hepatopancreaic parvovirus protein gene, Or the Reo-like virus protein gene.
  • shrimp white spot virus White spot syndrome virus
  • Penaeus monodon baculovirus protein gene Penaeus monodon baculovirus protein gene
  • Taura syndrome virus protein gene Baculoviral midgut necrosis virus protein gene
  • shrimp Baculovirus penaei protein gene infectious hypodermal and haematopoietic necrosis virus protein gene
  • yellowhead virus protein gene yellowhead virus protein gene
  • Hepatopancreaic parvovirus protein gene Or the Reo-like virus protein gene.
  • the aquatic animal pathogenic gene is a fish viral protein gene.
  • the fish viral protein gene includes, but is not limited to, an Oncorhynchus masou virus protein gene, a grouper iridovirus protein gene, and a red sea bream iridovirus protein.
  • Gene spring viraemia of carp virus protein gene, epidemic haematopoietic necrosis virus protein gene, channel catfish virus protein gene, infectious hematopoietic Infectious haematopoietic necrosis virus protein gene, Lymphocystis virus protein gene, Nervous Necrosis virus protein gene, Viral Haemorrhagic Septicaemia virus protein gene, White sturgeon iridovirus protein gene, Grass carp hemorrhagic virus protein gene, infectious scorpion anemia virus (Infectious salmon anae) Mia virus) a protein gene, a koi herpesvirus protein gene or an infectious pancreatic necrosis virus protein gene.
  • the present invention further provides a biological preparation comprising a system for expressing a heterologous gene, wherein the system for expressing a heterologous gene comprises a Listeria bacterium; and a recombinant expression vector, wherein the weight
  • the panel expression vector comprises (a) a bacterial promoter; (b) a eukaryotic promoter; and (c) a heterologous gene.
  • the eukaryotic promoter is a water producer promoter.
  • the water producer promoter is a pCMV promoter.
  • the bacterial promoter is a phly promoter.
  • the Listeria bacterium is an inactive bacterium.
  • heterologous gene comprises a reporter gene
  • the reporter gene comprises, but is not limited to, an enhanced green fluorescent protein gene (GFP), a pigment gene.
  • GFP enhanced green fluorescent protein gene
  • heterologous gene comprises a growth gene
  • heterologous gene comprises an anti-stress gene.
  • heterologous gene comprises a pathogenic gene
  • the pathogenic gene is an aquatic animal pathogenic gene.
  • the aquatic animal pathogenic gene is a chitinvirus protein gene.
  • the chitinvirus protein gene includes, but is not limited to, a white spot syndrome virus protein gene, a Penaeus monodon baculovirus protein gene, and a Taura syndrome.
  • Virus gene protein gene of Baculoviral midgut necrosis virus, protein gene of Baculovirus penaei, infectious hypodermal and haematopoietic necrosis virus , yellowhead virus protein gene, Hepatopancreaic parvovirus protein gene, or Reo-like virus protein gene.
  • the aquatic animal pathogenic gene is a fish viral protein gene.
  • the fish virus protein gene includes but is not limited to: sakura virus (Oncorhynchus masou virus) protein gene, grouperiridovirus protein gene, Red sea bream iridovirus protein gene, spring viraemia of carp virus protein gene, epidemic Epizootic haematopoietic necrosis virus protein gene, Channel catfish virus protein gene, Infectious haematopoietic necrosis virus protein gene, Lymphocystis virus Protein gene, Nervous Necrosis virus protein gene, Viral Haemorrhagic Septicaemia virus protein gene, White sturgeon iridovirus protein gene, Grass carp hemorrhagic virus Virus) protein gene, infectious protein of infectious salmon anaemia virus, protein gene of koi herpesvirus Pancreatic necrosis virus (Infectious pancreatic necrosis virus) protein gene.
  • sakura virus Oncorhynchus masou virus
  • grouperiridovirus protein gene Red sea bre
  • the biological preparation includes, but is not limited to, a pharmaceutical, a cosmetic, a food or a farm animal product.
  • the biological agent is an aquatic animal feed ingredient, a feed additive or an animal drug.
  • the present invention further provides a method for preparing a biological preparation comprising the first step of using a carrier for expression in a Listeria bacterium, wherein the vector comprises a bacterial promoter; a eukaryotic promoter; The source gene sequence; and step two, the Listeria bacteria are not active.
  • the eukaryotic promoter is a water producer promoter.
  • the water producer promoter is a pCMV promoter.
  • the bacterial promoter is a phly promoter.
  • a method for expressing a target gene comprising the first step of using a carrier for a Listeria Characterizing a target gene, wherein the vector comprises (a) a first promoter; (b) a second promoter; and (c) a heterologous gene; and step two, through the Listeria
  • the bacteria are transfected with a eukaryote.
  • the eukaryotic organism expresses the heterologous gene through the second promoter.
  • the second promoter is a pCMV promoter.
  • the Listeria bacterium exhibits the heterologous gene through the first promoter.
  • said first promoter is a phly promoter.
  • the eukaryotic organism is an aquatic animal.
  • a method for controlling a pathogenic infection comprising administering an effective amount of a biological preparation to a eukaryotic organism to induce a cellular reaction of the eukaryote, wherein the biological preparation comprises a Listeria bacterium; a promoter; a second promoter; and a heterologous gene.
  • the heterologous gene comprises, but is not limited to, a reporter gene, an anti-stress gene or a pathogenic gene.
  • the eukaryotic organism expresses the heterologous gene through the second promoter.
  • the second promoter is a pCMV promoter.
  • the Listeria bacterium exhibits the heterologous gene through the first promoter.
  • said first promoter is a phly promoter.
  • the eukaryotic organism is an aquatic animal.
  • the Listeria is administered to an aquatic animal by oral or injection.
  • a method for promoting cell growth comprising administering an effective amount of a biological preparation to a eukaryotic organism to induce a cellular reaction of the eukaryotic organism, wherein the biological preparation comprises a Listeria bacterium; a promoter; a second promoter; and a heterologous growth gene.
  • the eukaryotic organism expresses the heterologous gene through the second promoter.
  • the second promoter is a pCMV promoter.
  • the Listeria bacterium exhibits the heterologous gene through the first promoter.
  • said first promoter is a phly promoter.
  • the eukaryotic organism is an aquatic animal.
  • the Listeria is administered to an aquatic animal by oral or injection.
  • Embodiment 1 is a map showing a carrier of Embodiment 1 of the present invention.
  • Example 2 is a result of analyzing the relative expression of WSSV vp28 and vp53a genes in different groups of Example 1 of the present invention
  • Figure 3 is a Western blot analysis of a sample of shrimp intestines fed with 9, 5, and 20 days of powdered feed according to Example 1 of the present invention
  • Dscam Down syndrome cell adhesion molecule
  • Figure 5 is a cumulative mortality result of the WSSV virus infection experiment of Example 1 of the present invention.
  • Example 6 is an experiment showing the heterologous gene expression of white shrimp in Example 2 of the present invention.
  • Fig. 7 is a graph showing the results of gene expression analysis of the Listeria to grouper GF-1 cells transfected with the GFP gene expression vector in Example 3 of the present invention.
  • WSSV protein genes vp53a and vp28 were used as the viral protein target genes.
  • WSSV protein genes vp53a and vp28 could be expressed as WSSV virus protein VP53A and VP28,
  • VP53A is a protein that binds WSSV to host cell receptors and is a key protein for virus invasion.
  • VP28 is the main protein on WSSV envelope, which plays an important role in triggering shrimp immunity.
  • the test is divided into a control group (Control, feeding a general commercial feed), a virus protein genome (abbreviated as: plasmid, fed with a performance carrier without a viral protein gene), and a vp28 genome alone ( Abbreviation: VP28), with vp53a genome alone (abbreviation: VP53A), with vp28 and vp53a genome (abbreviation: VP28+VP53A), etc., constructing the expression vector of the above group and transforming into Listeria, making The mushroom powder feeds the shrimp and infects WSSV to test the efficacy of the powder feed against WSSV.
  • the WSSV gene vp28 or vp53a was separately selected into a special expression vector of Listeria, and the map of the expression vector is shown in Fig. 1.
  • the expression vector is characterized by a dual promoter. When the Listeria infection enters the host cell, the phly promoter is activated to display the colonization gene, and the phagosome is escaped from the host. After the cytoplasm, the Listeria will release the viral protein and the expression vector into the cytoplasm, and the expression vector enters the host cell nucleus, and the host cell uses the pCMV promoter to express the cloning gene to produce the target protein.
  • the constructed performance vector was transferred into Listeria, and Listeria was cultured with Brain heart infusion broth (BHI broth). After overnight culture, the medium was replaced with PBS and placed at 65 ° C for 1 hour. In order to cause the death of Listeria, the dried Listeria is mixed into the dry shrimp feed, and then a small amount of tantalum powder and salad oil are put on, and then dried, and stored at a low temperature for use.
  • BHI broth Brain heart infusion broth
  • the white shrimps were randomly divided into five groups and fed the test feed, which included the general commercial feed (Control group), the bacterial powder feed containing only the expression carrier (Plasmid group), and the vp28 genome alone (abbreviation: VP28 group).
  • the phage powder feed containing the vp53a genome (abbreviation: VP53A group) and the vp28 and vp53a genomes (abbreviation: VP28+VP53A group) was fed for 20 days.
  • the amount of gene expression of the expression vector in the shrimp cells was confirmed by real-time quantitative PCR.
  • Real-time quantitative PCR analysis of WSSV vp28 and vp53a genes was performed on the intestinal cells of shrimps fed with 9, 15 and 20 days of mushroom powder feed, as shown in Figure 2.
  • the gene expression of vp28 or vp53a was increased in VP28 group, VP53A group and VP28+VP53A group.
  • the gene expression of vp53a was even higher than that of Control and plasmid group, indicating that the feed of Listeria powder could be reconstituted.
  • the vector is sent to shrimp cells.
  • the intestinal samples of shrimps fed with 9, 15 and 20 days of powder feed were analyzed by Western transfer method, as shown in Fig. 3.
  • the results showed that the protein expression of the vp28 and vp53a genes was measured in the intestinal samples of the shrimp fed for 20 days.
  • the intestines of the shrimps were tested for the intestinal samples of shrimps fed with 9, 15 and 20 days of powdered feed. The results are shown in Figure 4.
  • the performance of the shrimp-specific immune protein "Down syndrome cell adhesion molecule (Dscam)" was used as an indicator of the change of the immune index.
  • the analysis showed that the amount of Dscam in the shrimp body fed the VP53A powder feed significantly increased, and it was found that the powder feed did have the effect of improving the immunity of the shrimp.
  • the white shrimps were randomly grouped and fed for 15 days, then the sick shrimps infected with WSSV were fed. Simulate natural infections. The experiment was carried out twice: the first test of each group of 1000 white shrimps, the second test of each group of 3000 white shrimps, the test results shown in Figure 5, feeding the powder feed with WSSV viral protein gene It can effectively reduce the mortality of WSSV infected by white shrimp, which can increase the survival rate by 40%. Among them, the survival rate of the group containing VP28+VP53A powder feed is higher (about 3-4% higher).
  • the green fluorescent protein (GFP) gene is inserted as a heterologous gene into the expression vector of the present invention and sent to Listeria.
  • the Listeria monocytogenes carrying the expression vector of the present invention are then fed to the host white shrimp.
  • White shrimps were fed with normal feed and Listeria monosaccharide (twice daily feeding).
  • the white and shrimp tissues of the experimental group and the control group were taken out on the 3rd, 9th and 15th days, and observed by inverted fluorescence microscope.
  • the test results are shown in Fig. 6; it was found that fluorescence appeared after the ninth day, and green fluorescence was observed throughout the entire intestine on the 15th day, and no fluorescence signal was observed in the control group.
  • the test results confirmed that the present invention can indeed deliver the expression vector into the host and smoothly express the heterologous gene.
  • the permanent cell line GF-1 of Epinephelus coioides was used as the host cell, and the green fluorescent protein (GFP) gene was used as a reporter gene. Inserting the GFP gene into the expression vector of the present invention, and breeding into the Listeria, and then transfecting the Listeria with GF-1 cells to confirm that the Listeria can indeed deliver the expression vector to the grouper In the cell, the grouper cell can also express the gene on the expression vector.
  • the analysis was carried out by reverse transcription-PCR (RT-PCR), and the results of the experiment are shown in FIG. The results showed that the zebrafish cells had GFP mRNA in the samples after 5 minutes of transfection, and the expression of GFP mRNA increased with time.
  • This experiment uses 2 ⁇ spots with grouper as experimental animals, which are divided into general commercial feeds (Control group), only The powder feed containing the performance carrier (Plasmid group), with the Major capsid protein genome (abbreviation: MCP group), the virus was infected after 5 days of feeding, and the mortality of the Control group was about 60% after 10 days of drug administration. The mortality of the MCP group was about 25% after 10 days of drug administration, indicating that the powdered feed with the viral protein gene did have an effect of inhibiting viral infection.
  • publications such as CN104178511A and EP1774004B1 mention an example of an embodiment in which the expression of a specific gene sequence of a specific eukaryotic cell is accelerated to accelerate the growth of eukaryotic cells, and is also applicable to a system for expressing a heterologous gene of the present invention.
  • the expression vector can express the colonization gene and can enable the eukaryotic host to produce the target protein or nucleic acid sequence, thereby achieving the effects of pathogen prevention, detection, and acceleration of the host growth, and the principle can also be applied to aquatic animals.
  • the content of the present invention has the following advantages:
  • the present invention can be mixed with feed and administered in comparison with the conventional injection method of the conventional vaccine, which greatly reduces the manpower operation requirement and has low production cost. Helps industrial applications.
  • the present invention allows simultaneous delivery of protein antigens and DNA vectors, and protection by the cells to prevent decomposition by enzymes in the external environment.
  • the present invention can be administered by using dead bacteria without affecting the immune effect, and the dead bacteria powder does not cause pollution to the environment, and is beneficial to a virus control strategy as an aquatic animal.
  • the present invention is applied to crustaceans to effectively transport the invention to the body via its open circulation system to substantially block viral invasion.
  • the invention is applicable to harsh environments, such as high salinity seawater, the effect of which is unaffected by environmental conditions.

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Abstract

提供了一种表达异源基因的系统，其包含李斯特菌属细菌以及重组表达载体；还提供了包含所述系统的表达目的基因的方法、一种生物制剂及制备生物制剂的方法、一种防治病原感染的方法，以及一种促进细胞生长的方法。

Description

表现异源基因的系统及其用途 技术领域
本发明是关于一种表现异源基因的系统及其用途，特别是指应用李斯特菌及表现载体于真核生物中表现目标基因的系统及方法。
背景技术
利用病毒蛋白质做为抗原为常见的病原防治方法之一，例如利用微生物将病原蛋白质送入哺乳类动物细胞，进而诱发细胞的免疫反应。先前曾有研究利用大肠杆菌作为人类疾病治疗的蛋白质运送系统，但有鉴于大肠杆菌对环境的污染特性，近年则开始探讨利用其他细菌品种运送病原体蛋白质进入人体的可能性，例如：单核球增多性李斯特菌(Listeria monocytogenes)。李斯特菌为兼性厌氧革兰氏阳性细菌，其对环境适应力强，可生长于3-45℃，亦可于高盐环境中生存。
先前有研究显示，经基因改良的李斯特菌可运送蛋白质进入哺乳类动物细胞，提升哺乳类动物的免疫效果(美国专利号：US6143511A、US7794728B2，以及Dietrich G等人发表于Nat Biotechnol.1998Feb；16(2):181-5.之研究)。显示李斯特菌对于哺乳类动物确实可作为一蛋白质运送平台，但迄今仍未有研究指出是否可将李斯特菌应用于其他真核生物，例如水产动物。
此外，农业养殖栽培产业为重要的经济产业，贸易量大且持续蓬勃发展，但多年来常遭受天灾、病原的威胁，使得生长不易、收成不佳，造成业者的严重损失，例如水产养殖业，尽管水产养殖业的疫病监测及通报系统已逐步建立，但当疫病爆发时却往往无有效的控制方法，使得经济损失难以估计。目前针对真核生物病原性疾病，例如水产动物病毒染的预防及控制方法大多局限于实验 室规模，像是DNA疫苗或基因减弱(gene knock-down)等，因生产成本过高，不利实际产业应用，其防治病原感染的效益不高。故如何建立一种安全、低成本且可有效防治水产动物等真核生物病原感染的系统及方法成为极重要的课题。有鉴于此，本申请发明人在经过长时间努力后，完成本申请内容，并提出本申请，期能为产业带来新的气象。
发明内容
本发明提供一种表现异源基因的系统，其包含：一李斯特菌属细菌；以及一重组表现载体，其中所述重组表现载体包含：(a)一细菌启动子；(b)一真核生物启动子；以及(c)一异源基因。
根据本发明，其中所述真核生物启动子为一水产生物启动子。
根据本发明，其中所述水产生物启动子为pCMV启动子。
根据本发明，其中所述细菌启动子为phly启动子。
根据本发明，其中所述李斯特菌属细菌为一非活性细菌。
根据本发明，其中所述异源基因包含一报导基因。
根据本发明，其中所述报导基因包含但不限于：增强型绿色荧光蛋白基因(GFP)或色素基因(pigment gene)。
根据本发明，其中所述异源基因包含一生长基因。
根据本发明，其中所述异源基因包含一抗逆境基因。
根据本发明，其中所述异源基因包含一病源基因。
根据本发明，其中所述病源基因为一水产动物病原基因。
根据本发明，其中所述水产动物病原基因为一甲壳类病毒蛋白基因。
根据本发明，其中所述甲壳类病毒蛋白基因包含但不限于：虾白点症病毒 (White spot syndrome virus)蛋白质基因、草虾杆状病毒(Penaeus monodon baculovirus)蛋白质基因、桃拉病毒(Taura syndrome virus)蛋白质基因、中肠腺坏死杆状病毒(Baculoviral midgut necrosis virus)蛋白质基因、对虾杆状病毒(Baculovirus penaei)蛋白质基因、传染性皮下及造血组织坏死病毒(infectious hypodermal and haematopoietic necrosis virus)蛋白质基因、黄头病毒(yellowhead virus)蛋白质基因、肝胰腺细小病毒(Hepatopancreaic parvovirus)蛋白质基因，或肠呼吸道病毒(Reo-like virus)蛋白质基因。
根据本发明，其中所述水产动物病原基因为一鱼类病毒蛋白基因。
根据本发明，其中所述鱼类病毒蛋白基因包含但不限于：樱鳟病毒(Oncorhynchus masou virus)蛋白质基因、石斑鱼虹彩病毒(Grouperiridovirus)蛋白质基因、嘉魶虹彩病毒(Red sea bream iridovirus)蛋白质基因、鲤鱼春季毒血症病毒(spring viraemia of carp virus)蛋白质基因、流行性造血组织坏死症病毒(Epizootic haematopoietic necrosis virus)蛋白质基因、河鲶病毒症病毒(Channel catfish virus)蛋白质基因、传染性造血组织坏死症病毒(Infectious haematopoietic necrosis virus)蛋白质基因、淋巴囊肿症病毒(Lymphocystis virus)蛋白质基因、神经坏死病毒(Nervous Necrosis virus)蛋白质基因、病毒性出血性败血症病毒(Viral Haemorrhagic Septicaemia virus)蛋白质基因、白鲟虹彩病毒(White sturgeon iridovirus)蛋白质基因、草鱼出血性病毒(Grass carp hemorrhagic virus)蛋白质基因、传染性鲑类贫血病毒(Infectious salmon anaemia virus)蛋白质基因、锦鲤疱疹病毒(koi herpesvirus)蛋白质基因或传染性胰坏死症病毒(Infectious pancreatic necrosis virus)蛋白质基因。
本发明另提供一种生物制剂，其包含一表现异源基因的系统，其中所述表现异源基因的系统包含一李斯特菌属细菌；以及一重组表现载体，其中所述重 组表现载体包含(a)一细菌启动子；(b)一真核生物启动子；以及(c)一异源基因。
根据本发明，其中所述真核生物启动子为一水产生物启动子。
根据本发明，其中所述水产生物启动子为pCMV启动子。
根据本发明，其中所述细菌启动子为phly启动子。
根据本发明，其中所述李斯特菌属细菌为一非活性细菌。
根据本发明，其中所述异源基因包含一报导基因。
根据本发明，其中所述报导基因包含但不限于：增强型绿色荧光蛋白基因(GFP)、色素基因(pigment gene)。
根据本发明，其中所述异源基因包含一生长基因。
根据本发明，其中所述异源基因包含一抗逆境基因。
根据本发明，其中所述异源基因包含一病源基因。
根据本发明，其中所述病源基因为一水产动物病原基因。
根据本发明，其中所述水产动物病原基因为一甲壳类病毒蛋白基因。
根据本发明，其中所述甲壳类病毒蛋白基因包含但不限于：虾白点症病毒(White spot syndrome virus)蛋白质基因、草虾杆状病毒(Penaeus monodon baculovirus)蛋白质基因、桃拉病毒(Taura syndrome virus)蛋白质基因、中肠腺坏死杆状病毒(Baculoviral midgut necrosis virus)蛋白质基因、对虾杆状病毒(Baculovirus penaei)蛋白质基因、传染性皮下及造血组织坏死病毒(infectious hypodermal and haematopoietic necrosis virus)蛋白质基因、黄头病毒(yellowhead virus)蛋白质基因、肝胰腺细小病毒(Hepatopancreaic parvovirus)蛋白质基因，或肠呼吸道病毒(Reo-like virus)蛋白质基因。
根据本发明，其中所述水产动物病原基因为一鱼类病毒蛋白基因。
根据本发明，其中所述鱼类病毒蛋白基因包含但不限于：樱鳟病毒 (Oncorhynchus masou virus)蛋白质基因、石斑鱼虹彩病毒(Grouperiridovirus)蛋白质基因、嘉魶虹彩病毒(Red sea bream iridovirus)蛋白质基因、鲤鱼春季毒血症病毒(spring viraemia of carp virus)蛋白质基因、流行性造血组织坏死症病毒(Epizootic haematopoietic necrosis virus)蛋白质基因、河鲶病毒症病毒(Channel catfish virus)蛋白质基因、传染性造血组织坏死症病毒(Infectious haematopoietic necrosis virus)蛋白质基因、淋巴囊肿症病毒(Lymphocystis virus)蛋白质基因、神经坏死病毒(Nervous Necrosis virus)蛋白质基因、病毒性出血性败血症病毒(Viral Haemorrhagic Septicaemia virus)蛋白质基因、白鲟虹彩病毒(White sturgeon iridovirus)蛋白质基因、草鱼出血性病毒(Grass carp hemorrhagic virus)蛋白质基因、传染性鲑类贫血病毒(Infectious salmon anaemia virus)蛋白质基因、锦鲤疱疹病毒(koi herpesvirus)蛋白质基因或传染性胰坏死症病毒(Infectious pancreatic necrosis virus)蛋白质基因。
根据本发明，其中所述生物制剂包含但不限于：医药品、妆品、食品或农畜用品。
根据本发明，其中所述生物制剂为水产动物饲料成分、饲料添加物或动物用药。
本发明另提供一种制备生物制剂的方法，其包含步骤一，使用一载体于一李斯特菌属细菌中表现，其中所述载体包含一细菌启动子；一真核生物启动子；以及一异源基因序列；以及步骤二，使所述李斯特菌属细菌不具活性。
根据本发明，其中所述真核生物启动子为一水产生物启动子。
根据本发明，其中所述水产生物启动子为pCMV启动子。
根据本发明，其中所述细菌启动子为phly启动子。
一种表现目标基因的方法，其包含步骤一，使用一载体于一李斯特菌属细 菌中表现目标基因，其中所述载体包含(a)一第一启动子；(b)一第二启动子；以及(c)一异源基因；以及步骤二，透过所述李斯特菌属细菌转染一真核生物。
根据本发明，其中所述真核生物是透过所述第二启动子表现所述异源基因。
根据本发明，所述第二启动子为pCMV启动子。
根据本发明，其中所述李斯特菌属细菌是透过所述第一启动子表现所述异源基因。
根据本发明，其中所述第一启动子为phly启动子。
根据本发明，其中所述真核生物是水产动物。
根据本发明，其中进一步包含一步骤：进一步使所述李斯特菌属细菌不具活性。
一种防治病原感染的方法，其包含对一真核生物投予有效量的一生物制剂，以诱发所述真核生物的细胞反应，其中所述生物制剂包含一李斯特菌属细菌；一第一启动子；一第二启动子；以及一异源基因。
根据本发明，其中所述异源基因包含但不限于报导基因、抗逆境基因或病源基因。
根据本发明，其中所述真核生物是透过所述第二启动子表现所述异源基因。
根据本发明，所述第二启动子为pCMV启动子。
根据本发明，其中所述李斯特菌属细菌是透过所述第一启动子表现所述异源基因。
根据本发明，其中所述第一启动子为phly启动子。
根据本发明，其中所述真核生物为水产动物。
根据本发明，其中进一步包含一步骤：进一步使所述李斯特菌属细菌不具活性。
根据本发明，其中利用口服或注射方式将所述李斯特菌投予水产动物。
一种促进细胞生长的方法，其包含对一真核生物投予有效量的一生物制剂，以诱发所述真核生物的细胞反应，其中所述生物制剂包含一李斯特菌属细菌；一第一启动子；一第二启动子；以及一异源生长基因。
根据本发明，其中所述真核生物是透过所述第二启动子表现所述异源基因。
根据本发明，所述第二启动子为pCMV启动子。
根据本发明，其中所述李斯特菌属细菌是透过所述第一启动子表现所述异源基因。
根据本发明，其中所述第一启动子为phly启动子。
根据本发明，其中所述真核生物为水产动物。
根据本发明，其中进一步包含一步骤：进一步使所述李斯特菌属细菌不具活性。
根据本发明，其中利用口服或注射方式将所述李斯特菌投予水产动物。
附图说明
图1是本发明实施例1表现载体的图谱；
图2是本发明实施例1不同组别的WSSV vp28与vp53a基因相对表现量分析结果；
图3是本发明实施例1喂食9、15与20天菌粉饲料的虾体肠道样本的西方转印法(Western blot)分析结果；
图4是本发明实施例1喂食9、15与20天菌粉饲料的虾体肠道的唐氏综合症细胞黏附分子(Down syndrome cell adhesion molecule，Dscam)相对基因表现量分析结果；
图5是本发明实施例1WSSV病毒感染实验的累积死亡率结果；
图6是本发明实施例2白虾表现异源基因表现实验；
图7是本发明实施例3转染带有GFP基因表现载体的李斯特菌至石斑鱼GF-1细胞的基因表现量分析结果。
具体实施方式
本发明是以下述实施例予以示范阐明，但本发明不受下述实施例限制。
实施例1虾白点症病毒(White spot syndrome virus,WSSV)蛋白质质表现及感染试验
试验设计：本试验以2～3公克的白虾(Litopenaeus vannamei)为实验动物，并以WSSV蛋白质基因vp53a与vp28作为病毒蛋白质标的基因，WSSV蛋白质基因vp53a与vp28分别可表现为WSSV病毒蛋白质VP53A与VP28，VP53A为WSSV与宿主细胞受器结合的蛋白质，为病毒入侵的关键蛋白质，而VP28为WSSV封套上的主要蛋白质，对引发虾体免疫作用具极重要的功能。本试验共分为控制组(Control，喂食一般市售饲料)、未带有病毒蛋白质基因组(简称：plasmid，喂食不带有病毒蛋白质基因的表现载体的菌粉饲料)、单独带有vp28基因组(简称：VP28)、单独带有vp53a基因组(简称：VP53A)、同时带有vp28与vp53a基因组(简称：VP28+VP53A)等五组，建构上述组别的表现载体并转型进入李斯特菌后，制作菌粉饲料喂食虾只，并感染WSSV，以测试菌粉饲料对抗WSSV的效力。
实施例1.1表现载体的建构
将WSSV基因vp28或vp53a分别选殖进入李斯特菌专门表现载体中，该表现载体的图谱如图1所示。该表现载体的特点在于具双启动子，当李斯特菌感染进入宿主细胞，即启动phly启动子开始表现选殖基因，且在逃离吞噬小体进入宿主 细胞质后，此李斯特菌会将病毒蛋白质及表现载体释放于细胞质，该表现载体则进入宿主细胞核中，由宿主细胞利用pCMV启动子表现选殖基因，产生标的蛋白质。
实施例1.2制备含有李斯特菌死菌饲料
将建构完成的表现载体转殖进入李斯特菌，以脑心浸出物培养液(Brain heart infusion broth,BHI broth)培养李斯特菌，隔夜培养后以PBS置换培养基，置于65℃作用1小时以使李斯特菌死亡，将干燥的李斯特菌混入干性虾饲料吸附后，再扮入少量鳗粉及色拉油后吹干，置于低温中保存备用。
实施例1.3病毒蛋白质质基因表现分析
将白虾随机分为五组，并喂食试验用的饲料，包含一般市售饲料(Control组)、仅含表现载体的菌粉饲料(Plasmid组)、单独带有vp28基因组(简称：VP28组)、单独带有vp53a基因组(简称：VP53A组)、同时带有vp28与vp53a基因组(简称：VP28+VP53A组)的菌粉饲料，共喂食20天。
利用实时定量PCR确认表现载体于虾细胞中的基因表现量。收取喂食9、15与20天菌粉饲料的虾体肠道细胞进行WSSV vp28与vp53a基因实时定量PCR分析，如图2所示。vp28或vp53a的基因表现量于VP28组、VP53A组与VP28+VP53A组中皆呈现上升趋势，其中vp53a的基因表现量甚至远高于Control及plasmid组，显示喂食李斯特菌粉饲料确实可将重组载体送入虾细胞中。
实施例1.4病毒蛋白质表现分析
为确认表现载体是否可于虾细胞中表现出WSSV病毒蛋白质，收取喂食9、15与20天菌粉饲料的虾体肠道样本进行西方转印法分析，如图3所示。结果显示在喂食20天的虾体肠道样本中可测得vp28与vp53a基因的蛋白质表现。
实施例1.5虾体免疫反应分析
针对喂食9、15与20天菌粉饲料的虾体肠道样本进行虾只体内免疫指数变化指标分析，结果如图4所示。本试验以虾类专一性免疫蛋白质「唐氏综合症细胞黏附分子(Down syndrome cell adhesion molecule,Dscam)」表现量作为免疫指数变化指标。分析结果显示喂食VP53A菌粉饲料的组别，其虾体Dscam表现量显着上升，进而可知菌粉饲料确实具备提高虾体免疫的功效。
实施例1.6生物污染防治
为了了解菌粉饲料是否会造成生物体感染或环境污染，针对100只喂食菌粉饲料的白虾进行为期四周的观察，监控虾只累积死亡率及李斯特菌于环境中的生长数(表1)。结果显示不管是在活着的虾体或死亡的虾体表面、水体中、缸子里，都没有检测到重组型李斯特菌的生长。死亡率方面一般饲料为10％，菌粉饲料平均为10.75％，两者无显着差异。外界环境中并没有检测到重组型李斯特菌，显示此质体并不会藉由自然转型进入野生型的菌株中，导致突变株的出现，造成环境中的污染。
表1养殖环境监控结果
Figure PCTCN2015094412-appb-000001
实施例1.7感染试验
先将白虾随机分组，并喂食饲料达15天后，再喂食感染WSSV的病虾尸体以 模拟自然感染。本实验共进行两次：第一次试验每组各1000只白虾，第二次试验每组各3000只白虾，试验结果如图5所示，喂食带有WSSV病毒蛋白基因的菌粉饲料可有效降低白虾感染WSSV只死亡率，约可增加40％的存活率，其中又以食用含VP28+VP53A菌粉饲料的组别存活率较高(高约3～4％)。
实施例2白虾异源基因表现实验
将绿色荧光蛋白(Green fluorescent protein,GFP)基因作为异源基因，插入本发明的表现载体中，并送入李斯特菌。再将带有本发明表现载体之李斯特菌喂食宿主白虾。白虾分别喂食一般饲料和李斯特菌饲料(每日喂食两次组)，连续喂食30天数后，第3、9及15天取出实验组和对照组之白虾肠组织，使用倒立荧光显微镜观察，试验结果如图6所示；发现在第9天后开始有荧光的出现，第15天可观察到绿色荧光遍布全肠道，在对照组中并没有观察到荧光讯号。试验结果确认本发明内容确实可将该表现载体送入宿主中，并顺利表现异源基因。
实施例3石斑鱼报导基因表现试验
本试验以石斑鱼(Epinephelus coioides)永久细胞株GF-1为宿主细胞，以绿色荧光蛋白(Green fluorescent protein,GFP)基因为报导基因。将GFP基因插入本发明的表现载体中，并选殖进入该李斯特菌，再将此李斯特菌转染GF-1细胞，以确认该李斯特菌确实可将该表现载体送入石斑鱼细胞中，且该石斑鱼细胞亦可表现该表现载体上的基因。以反转录PCR(reverse transcription-PCR,RT-PCR)进行分析，试验结果如图7所示。结果显示转染5分钟后的样本中，石斑鱼细胞已生GFP mRNA，随着时间增加，GFP mRNA表现量亦随的增加。
实施例4石斑鱼虹彩病毒(Grouper iridovirus)感染试验
本试验以2吋的点带石斑为实验动物，共分为一般市售饲料(Control组)、仅 含表现载体的菌粉饲料(Plasmid组)、带有Major capsid protein基因组(简称：MCP组)，先喂食饲料5天后再感染病毒，Control组于供毒后10天的死亡率约可达60％，而MCP组于供毒后10天的死亡率则约25％，显示带有病毒蛋白基因的菌粉饲料确实具有抑制病毒感染的效果。
有关类似细菌表现载体的应用广泛，过去已有相当成熟的研究，像是公开号CN101133160A、US8093025B2等文献实施例中提到细菌载体的多种应用，包含作为疫苗给药、提供用于免疫、基因治疗的肽和/或核酸序列或其他生物学活性化合物、疾病检测或作为驱虫剂等用途。此外，公开号CN104178511A、EP1774004B1等文献提到透过提高特定真核细胞之特定基因序列表现量，达到加速真核细胞生长的实施例子，亦可适用于本发明表现异源基因的系统。
上述实施例可知，表现载体可表现选殖基因，且能使真核细胞宿主产生目标蛋白质或核酸序列，进而达到病原防治、检测以及加速宿主生长等效果，其原理也可以应用于水产动物上。
本发明内容与其他常用技术相互比较时，更具有下列的优点：
1.本发明的操作与投予容易，在一实施例中，相较于一般传统疫苗的注射投药方式，本发明可与饲料混合再进行投予，大幅减低人力操作需求，且生产成本低廉，有助于产业应用。
2.在一实施例中，本发明可同时进行蛋白质抗原与DNA载体运送，并透过菌体给予保护，避免于被外在环境中的酵素所分解。
3.在一实施例中，本发明可利用死菌进行投予而不影响其免疫效果，死菌菌粉对环境不会造成污染，有利于作为水产动物的病毒防治策略。
4.在一实施例中，本发明应用于甲壳类动物，可有效藉其开放式循环系统将本发明运送到全身，全面遏阻病毒入侵。
5.在一实施例中，本发明可适用于严苛环境，例如高盐度的海水，其效果不受环境条件的影响。
上述详细说明是针对本发明的一可行实施例的具体说明，但该实施例并非用以限制本发明的专利范围，凡未脱离本发明技术要点的等效实施或变更，均属于本发明保护的范围。

Claims (46)

  1. 一种表现异源基因的系统，其特征在于，包含：
    一李斯特菌属细菌；以及
    一重组表现载体，其中所述重组表现载体包含：
    (a)一细菌启动子；
    (b)一真核生物启动子；以及
    (c)一异源基因。
  2. 如权利要求1所述的表现异源基因的系统，其中所述真核生物启动子为一水产生物启动子。
  3. 如权利要求2所述的表现异源基因的系统，其中所述水产生物启动子为pCMV启动子。
  4. 如权利要求1所述的表现异源基因的系统，其中所述细菌启动子为phly启动子。
  5. 如权利要求1-4任一项所述的表现异源基因的系统，其中所述李斯特菌属细菌为一非活性细菌。
  6. 如权利要求5所述的表现异源基因的系统，其中所述异源基因包含一报导基因。
  7. 如权利要求6所述的表现异源基因的系统，其中所述报导基因包括：增强型绿色荧光蛋白基因、或色素基因。
  8. 如权利要求5所述的表现异源基因的系统，其中所述异源基因包含一生长基因。
  9. 如权利要求5所述的表现异源基因的系统，其中所述异源基因包含一抗逆境基因。
  10. 如权利要求5所述的表现异源基因的系统，其中所述异源基因包含一病源基因。
  11. 如权利要求10所述的表现异源基因的系统，其中所述病源基因为一水产动物病原基因。
  12. 如权利要求11所述的表现异源基因的系统，其中所述水产动物病原基因为一甲壳类病毒蛋白基因。
  13. 如权利要求12所述的表现异源基因的系统，其中所述甲壳类病毒蛋白基因为：虾白点症病毒蛋白质基因、草虾杆状病毒蛋白质基因、桃拉病毒蛋白质基因、传染性皮下及造血组织坏死病毒蛋白质基因、黄头病毒蛋白质基因、肝胰腺细小病毒蛋白质基因，或肠呼吸道病毒蛋白质基因。
  14. 如权利要求11所述的表现异源基因的系统，其中所述水产动物病原基因为一鱼类病毒蛋白基因。
  15. 如权利要求14所述的表现异源基因的系统，其中所述鱼类病毒蛋白基因为：樱鳟病毒蛋白质基因、石斑鱼虹彩病毒蛋白质基因、嘉魶虹彩病毒蛋白质基因、鲤鱼春季毒血症病毒蛋白质基因、流行性造血组织坏死症病毒蛋白质基因、河鲶病毒症病毒蛋白质基因、传染性造血组织坏死症病毒蛋白质基因、淋巴囊肿症病毒蛋白质基因、神经坏死病毒蛋白质基因、病毒性出血性败血症病毒蛋白质基因、白鲟虹彩病毒蛋白质基因、草鱼出血性病毒蛋白质基因、传染性鲑类贫血病毒蛋白质基因、锦鲤疱疹病毒蛋白质基因或传染性胰坏死症病毒蛋白质基因。
  16. 一种生物制剂，其特征在于，包含如权利要求6-15任一项所述的表现异源基因的系统。
  17. 如权利要求16所述的生物制剂，其为医药品、妆品、食品或农畜用品。
  18. 如权利要求16所述的生物制剂，其为水产动物饲料成分、饲料添加物或动物用药。
  19. 一种制备生物制剂的方法，其特征在于，包含：
    步骤一，使用一载体于一李斯特菌属细菌中表现，其中所述载体包含一细菌启动子；一真核生物启动子；以及一异源基因序列；以及
    步骤二，使所述李斯特菌属细菌不具活性。
  20. 如权利要求19所述的方法，其中所述真核生物启动子为一水产生物启动子。
  21. 如权利要求20所述的方法，其中所述水产生物启动子为pCMV启动子。
  22. 如权利要求19所述的方法，其中所述细菌启动子为phly启动子。
  23. 一种表现目标基因的方法，其特征在于，包含：
    步骤一，使用一载体于一李斯特菌属细菌中表现目标基因，其中所述载体包含(a)一第一启动子；(b)一第二启动子；以及(c)一异源基因；以及
    步骤二，透过所述李斯特菌属细菌转染一真核生物。
  24. 如权利要求23所述的方法，其中所述真核生物是透过所述第二启动子表现所述异源基因。
  25. 如权利要求24所述的方法，所述第二启动子为pCMV启动子。
  26. 如权利要求23所述的方法，其中所述李斯特菌属细菌是透过所述第一启动子表现所述异源基因。
  27. 如权利要求26所述的方法，其中所述第一启动子为phly启动子。
  28. 如权利要求23所述的方法，其中所述真核生物是水产动物。
  29. 如权利要求23-28任一项所述的方法，进一步包含一步骤：进一步使所述李斯特菌属细菌不具活性。
  30. 一种防治病原感染的方法，其特征在于，其包含对一真核生物投予有效量的一生物制剂，以诱发所述真核生物的细胞反应，其中所述生物制剂包含：
    一李斯特菌属细菌；
    一第一启动子；
    一第二启动子；以及
    一的异源基因。
  31. 如权利要求30所述的方法，其中所述异源基因包括：报导基因、抗逆境基因或病源基因。
  32. 如权利要求30所述的方法，其中所述真核生物是透过所述第二启动子表现所述异源基因。
  33. 如权利要求32所述的方法，所述第二启动子为pCMV启动子。
  34. 如权利要求30所述的方法，其中所述李斯特菌属细菌是透过所述第一启动子表现所述异源基因。
  35. 如权利要求34所述的方法，其中所述第一启动子为phly启动子。
  36. 如权利要求30所述的方法，其中所述真核生物为水产动物。
  37. 如权利要求30-36任一项所述的方法，进一步包含一步骤：进一步使所述李斯特菌属细菌不具活性。
  38. 如权利要求37所述的方法，其中利用口服或注射方式将所述李斯特菌投予水产动物。
  39. 一种促进细胞生长的方法，其特征在于，其包含对一真核生物投予有效量的一生物制剂，以诱发所述真核生物的细胞反应，其中所述生物制剂包含：
    一李斯特菌属细菌；
    一第一启动子；
    一第二启动子；以及
    一异源生长基因。
  40. 如权利要求39所述的方法，其中所述真核生物是透过所述第二启动子表现所述异源基因。
  41. 如权利要求40所述的方法，所述第二启动子为pCMV启动子。
  42. 如权利要求39所述的方法，其中所述李斯特菌属细菌是透过所述第一启动子表现所述异源基因。
  43. 如权利要求42所述的方法，其中所述第一启动子为phly启动子。
  44. 如权利要求39所述的方法，其中所述真核生物为水产动物。
  45. 如权利要求39-44任一项所述的方法，其中进一步包含一步骤：进一步使所述李斯特菌属细菌不具活性。
  46. 如权利要求45所述的方法，其中利用口服或注射方式将所述李斯特菌投予水产动物。
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