TWI658140B - A system for expressing a heterologous gene and use thereof - Google Patents

Abstract

The present invention provides a performance system comprising a Listeria bacterium and a recombinant expression vector; the present invention also provides a method for expressing a target gene comprising the above expression system, a biological preparation and a method for preparing the biological preparation, and a method for controlling pathogenic infection Method; and a method of promoting cell growth.

Description

System for expressing heterologous genes and uses thereof

The present invention relates to a system for expressing a heterologous gene and its use, and in particular to a system and method for expressing a target gene in a eukaryote using a Listeria and a expression vector.

The use of 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. Previous studies have used E. coli as a protein delivery system for the treatment of human diseases. However, in view of the environmental pollution characteristics of E. coli, in recent years, 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.

Previous studies have shown that genetically modified Listeria can transport proteins into mammalian cells and enhance the immune effects of mammals (US Patent Nos.: US6143511A, US7794728B2, and Dietrich G et al., Nat Biotechnol. 1998 Feb; 16 ( 2): Research of 181-5.). It is shown that Listeria can be used as a protein delivery platform for mammals, but so far no research has been conducted to indicate whether Listeria can be applied to other eukaryotes, such as aquatic animals.

In addition, the agricultural aquaculture industry is an important economic industry. The volume of trade is large and continues to flourish. However, it has been threatened by natural disasters and pathogens for many years, making it difficult to grow and poorly harvested, causing serious losses to the operators, such as aquaculture. Although the aquaculture surveillance and notification system has been gradually established, there are often no effective control methods when the disease erupts, making economic losses difficult to estimate. At present, the prevention and control methods for pathogenic diseases of eukaryotes, such as aquatic animal virus infection, are mostly limited to laboratory scale, such as DNA vaccine or gene knock-down, which is unfavorable to the actual industry due to high production costs. Application, its effectiveness in the prevention and treatment of pathogenic infections is not high. Therefore, how to establish a safe and low-cost system and method for effectively preventing and treating eukaryotic pathogen infections such as aquatic animals has become an extremely important issue. In view of this, the inventor of this case completed the content of the case after a long period of hard work, and proposed the application of this case, which will bring new weather to the industry.

The present invention provides a performance system comprising: a Listeria bacterium; and a recombinant expression vector, wherein the recombinant expression vector comprises: (a) a bacterial promoter; (b) a eukaryotic promoter; c) A heterologous gene.

According to the present creation, the eukaryotic promoter is a water-producing promoter.

According to the present creation, the water producer promoter is the pCMV promoter.

According to the present creation, wherein the bacterial promoter is a phly promoter.

According to the present invention, the Listeria bacterium is an inactive bacterium.

According to the present creation, wherein the heterologous gene comprises a reporter gene.

According to the present creation, the reporter gene includes, but is not limited to, an enhanced green fluorescent protein gene (GFP) or a pigment gene.

According to the present invention, the heterologous gene comprises a growth gene.

According to the present invention, the heterologous gene comprises an anti-stress gene.

According to the present invention, the heterologous gene comprises a pathogenic gene.

According to the present creation, the pathogenic gene is an aquatic animal pathogenic gene.

According to the present invention, the aquatic animal pathogenic gene is a chitinvirus protein gene.

According to the present invention, 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. Protein gene, protein gene of Baculoviral midgut necrosis virus, protein gene of Baculovirus penaei, infectious hypodermal and haematopoietic necrosis virus protein gene, A yellowhead virus protein gene, a Hepatopancreaic parvovirus protein gene, or a Reo-like virus protein gene.

According to the present creation, the aquatic animal pathogenic gene is a fish virus protein gene.

According to the present invention, the fish virus protein gene includes but is not limited to: Oncorhynchus masou virus protein gene, Grouper iridovirus protein gene, Red sea bream iridovirus protein gene, 鲤Spring viraemia of carp virus protein gene, episomic haematopoietic necrosis virus protein gene, channel catfish virus protein gene, infectious hematopoietic tissue necrosis Infectious haematopoietic necrosis virus protein gene, Lymphocystis virus protein gene, Nervous Necrosis virus protein gene, Viral Haemorrhagic Septicaemia virus protein gene, white peony White sturgeon iridovirus protein gene, grass carp hemorrhagic virus protein gene, infectious anaemia virus (Infectious salmon anaemia virus) protein gene, brocade A koi herpesvirus protein gene or an infectious pancreatic necrosis virus protein gene.

The present invention further provides a biological preparation comprising a performance system, wherein the expression system comprises a Listeria bacterium; and a recombinant expression vector, wherein the recombinant expression vector comprises (a) a bacterial promoter; (b) a eukaryotic promoter; and (c) a heterologous gene.

According to the present creation, the eukaryotic promoter is a water-producing promoter.

According to the present creation, the water producer promoter is the pCMV promoter.

According to the present creation, wherein the bacterial promoter is a phly promoter.

According to the present invention, the Listeria bacterium is an inactive bacterium.

According to the present creation, wherein the heterologous gene comprises a reporter gene.

According to the present creation, the reporter gene includes, but is not limited to, an enhanced green fluorescent protein gene (GFP) and a pigment gene.

According to the present invention, the heterologous gene comprises a growth gene.

According to the present invention, the heterologous gene comprises an anti-stress gene.

According to the present invention, the heterologous gene comprises a pathogenic gene.

According to the present creation, the pathogenic gene is an aquatic animal pathogenic gene.

According to the present invention, the aquatic animal pathogenic gene is a chitinvirus protein gene.

According to the present invention, 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. Protein gene, protein gene of Baculoviral midgut necrosis virus, protein gene of Baculovirus penaei, infectious hypodermal and haematopoietic necrosis virus protein gene, A yellowhead virus protein gene, a Hepatopancreaic parvovirus protein gene, or a Reo-like virus protein gene.

According to the present creation, the aquatic animal pathogenic gene is a fish virus protein gene.

According to the present invention, the fish virus protein gene includes but is not limited to: Oncorhynchus masou virus protein gene, Grouper iridovirus protein gene, Red sea bream iridovirus protein gene, 鲤Spring viraemia of carp virus protein gene, episomic haematopoietic necrosis virus protein gene, channel catfish virus protein gene, infectious hematopoietic tissue necrosis Infectious haematopoietic necrosis virus protein gene, Lymphocystis virus protein gene, Nervous Necrosis virus protein gene, Viral Haemorrhagic Septicaemia virus protein gene, white peony White sturgeon iridovirus protein gene, grass carp hemorrhagic virus protein gene, infectious anaemia virus (Infectious salmon anaemia virus) protein gene, brocade A koi herpesvirus protein gene or an infectious pancreatic necrosis virus protein gene.

According to the present invention, the biological preparation includes, but is not limited to, a pharmaceutical, a cosmetic, a food or a farm animal product.

According to the present invention, the biological preparation is an aquatic animal feed ingredient, a feed additive or an animal medicine.

The present invention further provides a method for preparing a biological preparation comprising the first step of using a vector for expression in a Listeria bacterium, wherein the vector comprises a bacterial promoter; a eukaryotic promoter; and a heterologous gene a sequence; and step two, making the Listeria bacterium inactive.

According to the present creation, the eukaryotic promoter is a water-producing promoter.

According to the present creation, the water producer promoter is the pCMV promoter.

According to the present creation, wherein the bacterial promoter is a phly promoter.

A method for expressing a target gene, comprising the first step of using a vector to express a target gene in a Listeria bacterium, wherein the vector comprises (a) a first promoter; (b) a second promoter; (c) a heterologous gene; and step two, transfecting a eukaryotic organism through the Listeria bacterium.

According to the present invention, the eukaryotic organism expresses the heterologous gene through the second promoter.

According to the present creation, the second promoter is the pCMV promoter.

According to the present invention, the Listeria bacterium exhibits the heterologous gene through the first promoter.

According to the present creation, wherein the first promoter is a phly promoter.

According to the present creation, the eukaryote is an aquatic animal.

According to the present invention, there is further included a step of further rendering the Listeria bacterium inactive.

A method for controlling pathogenic infection, 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 first promoter a second promoter; and a heterologous gene.

According to the present invention, the heterologous gene includes, but is not limited to, a reporter gene, an anti-stress gene or a pathogenic gene.

According to the present invention, the eukaryotic organism expresses the heterologous gene through the second promoter.

According to the present creation, the second promoter is the pCMV promoter.

According to the present invention, the Listeria bacterium exhibits the heterologous gene through the first promoter.

According to the present creation, wherein the first promoter is a phly promoter.

According to the present creation, the eukaryote is an aquatic animal.

According to the present invention, there is further included a step of further rendering the Listeria bacterium inactive.

According to the present creation, 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 eukaryote, wherein the biological preparation comprises a Listeria bacterium; a first promoter a second promoter; and a heterologous growth gene.

According to the present invention, the eukaryotic organism expresses the heterologous gene through the second promoter.

According to the present creation, the second promoter is the pCMV promoter.

According to the present invention, the Listeria bacterium exhibits the heterologous gene through the first promoter.

According to the present creation, wherein the first promoter is a phly promoter.

According to the present creation, the eukaryote is an aquatic animal.

According to the present invention, there is further included a step of further rendering the Listeria bacterium inactive.

According to the present creation, the Listeria is administered to an aquatic animal by oral or injection.

This creation is exemplified by the following examples, but the present creation is not limited by the following examples.

Example 1 Protein Spot Expression and Infection Test of White Spot Syndrome Virus (WSSV)

Experimental Design: In this experiment, 2 to 3 grams of white shrimp (Litopenaeus vannamei) as experimental animals, and to WSSV protein gene is a viral protein vp28 vp53a the target gene, and vp28 vp53a WSSV protein gene can be expressed respectively as proteins of WSSV and VP53A 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 a single genomic vp53a (abbreviation: VP53A), simultaneously with vp28 and vp53a genome (abbreviation: after VP28 + VP53A) and other five groups, the above-described construction of the expression vector and transformation into the groups of Listeria prepared The mushroom powder feeds the shrimp and infects WSSV to test the efficacy of the powder feed against WSSV.

Example 1.1 Construction of Expression Carrier

The vp28 WSSV genes are cloned into the vp53a or Listeria specialized expression vectors, the expression vector of pattern 1 shown in FIG. 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 cloning gene, and after escaping the phagosome into the host cytoplasm, the Listeria will be a viral protein. And the expression vector is released into the cytoplasm, and the expression vector enters the host cell nucleus, and the host cell utilizes the pCMV promoter to express the cloning gene to produce the target protein.

Example 1.2 Preparation of a diet containing Listeria monocytogenes

The constructed expression vector was transferred into Listeria, and the 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. Let Listeria die, mix the dried Listeria into the dry shrimp feed, then put a small amount of lotus root starch and salad oil, then blow dry, and store at low temperature for later use.

Example 1.3 Analysis of viral protein gene expression

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). alone with vp53a genome (abbreviation: VP53A group), simultaneously with vp28 and vp53a genome (abbreviation: VP28 + VP53A group) sinensis feed were fed for 20 days.

The amount of gene expression of the expression vector in shrimp cells was confirmed by real-time quantitative PCR. 9,15 charged with 20 days feeding intestinal cells sinensis feed shrimp WSSV vp28 gene and vp53a instant quantitative PCR analysis, as shown in FIG. vp53a vp28 gene expression or in an amount of VP28 group, VP53A group and VP28 + VP53A groups are on the rise, in which gene expression is even much higher than the amount vp53a plasmid and Control group, the display can actually fed Listeria bacteria powder feed recombinant The vector is sent to shrimp cells.

Example 1.4 Analysis of viral protein expression

In order to confirm whether the expression vector can express WSSV virus protein in shrimp cells, the intestinal transfer samples of shrimps fed with 9, 15 and 20 days of powder feed were analyzed by Western transfer method, as shown in Fig. 3, after feeding for 20 days. shrimp intestinal samples can be measured in body protein expression and vp53a vp28 gene.

Example 1.5 Analysis of shrimp immune response

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. In this experiment, the performance of the shrimp-specific immune protein "Down syndrome cell adhesion molecule (Dscam)" was used as an indicator of changes in the immune index. The analysis showed that the amount of Dscam in the shrimp body fed the VP53A powder feed significantly increased, and it is known that the powder feed does have the effect of improving the immunity of the shrimp.

Example 1.6 Biological Pollution Prevention

In order to understand whether the powder feed will cause biological infection or environmental pollution, the white shrimps fed with 100 powder feeds were observed for four weeks to monitor the cumulative mortality of shrimp and the growth of Listeria in the environment (Table 1). ). The results showed that no growth of recombinant Listeria was detected in the surface of the living shrimp or dead shrimp, in the water, or in the tank. The average feed rate was 10% for the mortality rate and 10.75% for the powder feed. There was no significant difference between the two. Recombinant Listeria was not detected in the external environment, indicating that the plastid did not naturally enter into the wild-type strain, resulting in the emergence of mutant strains, causing environmental pollution.

Table 1 Monitoring results of culture environment

Example 1.7 Infection test

The white shrimps were randomly grouped and fed for 15 days, and then the sick shrimps infected with WSSV were fed to 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).

Example 2 Experiment of heterologous gene expression of white shrimp

The green fluorescent protein (GFP) gene was used as a heterologous gene, inserted into the expression vector of this creation, and sent to Listeria. The Listeria monocytogenes carrying the creative expression vector are then fed to the host white shrimp. White shrimps were fed with normal feed and Listeria monosaccharide (two groups per day). After 30 days of continuous feeding, the white and shrimp tissues of the experimental group and the control group were taken out on the 3rd, 9th and 15th days, using inverted fluorescence microscope. Observations, the results of the test are shown in Fig. 6; it was found that the presence of fluorescence began after the ninth day, and green fluorescence was observed throughout the entire intestine on the 15th day, and no fluorescent 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.

Example 3 Grouper Reporting Gene Performance Test

In this experiment, 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. The GFP gene was inserted into the expression vector of the present invention, and cloned into the Listeria, and the Listeria was transfected into GF-1 cells to confirm that the Listeria could indeed deliver the expression vector into the grouper cells. And 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.

Example 4 Grouper iridovirus infection test

This experiment uses 2 吋 spotted grouper as experimental animal, which is divided into general commercial feed (Control group), bacterial powder feed containing only performance carrier (Plasmid group), and Major capsid protein genome (abbreviation: MCP group) ), the virus was infected for 5 days before feeding, and the mortality of the Control group was about 60% after 10 days of drug administration, while the mortality of the MCP group was about 25% after 10 days of drug administration, showing a virus. The powder feed of the protein gene does have an effect of inhibiting viral infection.

There are a wide range of applications for similar bacterial expression vectors, and there have been quite mature studies in the past, such as the publication of bacterial carriers such as CN101133160A, US8093025B2, and the like, including various applications of bacterial carriers, including administration as a vaccine, providing for immunization, gene therapy. Use of peptides and/or nucleic acid sequences or other biologically active compounds, disease detection or as an insect repellent. In addition, publications such as CN104178511A and EP1774004B1 mention an example of an embodiment in which the growth of a specific gene sequence of a specific eukaryotic cell is accelerated to accelerate the growth of eukaryotic cells, and it is also applicable to the present performance system.

The above examples show that 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.

When the content of this creation is compared with other conventional techniques, the following advantages are obtained: 1. The operation and administration of the present creation are easy. In an embodiment, the creation can be combined with the injection method of the conventional vaccine. Mixing and then investing, greatly reducing the manpower operation requirements, and low production costs, and contribute to industrial applications. 2. In one embodiment, 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. 3. In one embodiment, 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. 4. In one embodiment, the present application is applied to crustaceans, which can effectively transport the creation to the whole body through its open circulation system to completely prevent virus invasion. 5. In one embodiment, the creation is applicable to harsh environments, such as high salinity seawater, the effect of which is not affected by environmental conditions.

The detailed description above is a detailed description of a possible embodiment of the present invention, but the embodiment is not intended to limit the scope of the patents, and equivalent implementations or modifications that are not departing from the spirit of the present invention should be included in The scope of the request in this case.

In summary, this case is not only innovative in terms of method, but also enhances the above-mentioned multiple functions compared with the conventional technology. It has fully complied with the statutory invention patent requirements of novelty and progress, and applied for it according to law. You are requested to approve the patent application for this invention. The case, in order to invent, to the sense of virtue.

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Figure 1 is a map of a representation vector.

FIG 2 is a quantitative analysis of different groups of WSSV vp28 gene vp53a relative performance results.

Figure 3 is a Western blot analysis of a sample of shrimp intestines fed with 9, 15 and 20 days of powder feed.

Figure 4 shows the results of relative gene expression analysis of Down syndrome cell adhesion molecule (Dscam) in the intestines of shrimps fed with 9, 15 and 20 days of powder feed.

Figure 5 is the cumulative mortality result of the WSSV virus infection experiment.

Figure 6 is a graph showing the performance of heterologous genes in white shrimp.

Figure 7 is a graph showing the results of gene expression analysis of Listeria to Grouper GF-1 cells transfected with a GFP gene expression vector.

Claims (5)

The invention relates to a biological preparation for use as a feed component of a marine animal aquatic animal, wherein the biological preparation comprises a performance system, wherein the performance system comprises a Listeria bacterium, the Listeria bacterium comprising at least one recombinant expression vector Wherein the at least one recombinant expression vector comprises: (a) a pCMV promoter; and (b) a heterologous gene. The use of a biological preparation as a feed additive, wherein the biological preparation comprises a performance system, the performance system comprising a Listeria bacterium, the Listeria bacterium comprising at least one recombinant expression vector, wherein the at least one A recombinant expression vector comprises: (a) a pCMV promoter; and (b) a heterologous gene. The use of a biological preparation as an animal medicine, wherein the biological preparation comprises a performance system, the performance system comprising a Listeria bacterium, the Listeria bacterium comprising at least one recombinant expression vector, wherein the at least one The recombinant expression vector comprises: (a) a pCMV promoter; and (b) a heterologous gene. A feeding method comprising administering to a eukaryote an effective amount of a biological preparation, wherein the biological preparation comprises a Listeria bacterium, wherein the Listeria bacterium comprises: a pCMV promoter; A heterologous gene; wherein the feeding method is oral. The method of claim 4, wherein the eukaryotic organism is an aquatic animal.