KR20070077307A - Cell surface expression vector for wssv antigen and microorganism transformed by the same - Google Patents

Abstract

A cell surface expression vector for white spot syndrome virus(WSSV) antigen and a bacteria transformed by the same are provided to be used as vaccines and feed additives for treating and preventing diseases induced by the WSSV, thereby preventing outbreak of mass death of Crustacea caused by the virus and contributing to profit increase of fishery and aquaculture. The cell surface expression vector for WSSV antigen comprises at least one poly-gamma-glutamic acid synthetic complex gene selected from the group consisting of pgsA, pgsB and pgsC and a gene coding the WSSV antigen. The bacteria is transformed by the vector, wherein the bacteria is selected from the group consisting of Escherichia coli, Salmonella typhi, Salmonella typhimurium, Vibrio cholera, Mycobacterium bovis, shigella, bacillus, lactobacillus, Staphylococcus, Corynebacteria, Listeria monocytogenes, and Streptococcus.

Description

Cell Surface Expression Vector for WSSV Antigen and Microorganism Transformed by the Same}

Figure 1 shows a genetic map of the surface expression vectors pBT: pgsA-VP26 (A) and pBT: pgsA-VP28 (B) of the antigens of shrimp white spot virus VP26 and VP28.

Figure 2 shows the results of Western blot expression of VP26 (A) and VP28 (B) in the pBT: pgsA-VP26 and pBT: pgsA-VP28 transformants (lane 1: untransformed Lactobacillus casei lane 2: pBT: pgsA-VP26 / Lactobacillus casey; lane 3: pBT: pgsA-VP28 / Lactobacillus casey).

Figure 3 shows the mortality of the experimental and control shrimps infected with muscle white spot virus (A) and immersion (B) over time.

Figure 4 shows the infection of the virus by PCR with a lethal shrimp after immersion infection ([1] ~ [6] is the number of the killed shrimp; lane 1: VP26; lane 2: VP28).

Field of invention

The present invention relates to a surface expression vector of a shrimp white spot virus antigen and a microorganism transformed thereby, and more particularly, to a surface expression vector comprising a polygamma glutamic acid synthesis complex gene and a gene encoding a shrimp white spot virus antigen. To vaccines and feed additives for the treatment or prevention of shrimp white spot viral diseases comprising the bacteria transformed by the vector and the bacterial white spot virus antigen surface-expressing antigen or the crude antigen extracted from the bacteria as an active ingredient. It is about.

Background of the Invention

Shrimp White Spot Syndrome Virus (WSSV) is one of the most fatal shrimp diseases known to date. Shrimp white spot virus is infected with various tissues of mesodermal and ectoderm, including lymphoid organs, hematopoietic tissue, gills, intestines, stomach, central nervous system, subcutaneous tissue and tactile gland, causing serious damage to each infected tissue causing massive mortality. do. Shrimp white spot virus is known to be vertically infected (an infection that passes directly from the mother to offspring) and to horizontal infection (a common infection that spreads to a large number of unspecified individuals) in infected populations or crustaceans, such as crabs and genus around the farm.

Since it was first reported in shrimp farms in Fuan Province, China in August 1992, it has been found in Japan, Taiwan and China in 1993. It is now spreading almost throughout Southeast Asia, from Japan and China to the Philippines, Thailand, Indonesia and India. In Korea, the damage area has spread since it was first discovered in shrimp farms in Chungnam and Jeonbuk in 1993, and in 1996, the mortality rate soared to 75%. Since it was first discovered in East Asia, it has been reported to rapidly spread to shrimp farms in Southeast Asia, the United States and Latin America.

The genome of the shrimp white spot virus consists of a circular double-strand DNA of about 292,697 bp in length, approximately 275 nm long and 120 nm wide. Virions are rod-shaped, tightly enclosed in three layers of envolop, and have tail-like attachments at their ends. Shrimp white spot virus has five major proteins ranging in size from 15 to 28 kDa: the envelope contains VP28 and VP19, and the virion contains VP26, VP24 and VP15. It is known to play an important role in the infection of shrimp white spot virus (WSSV), which is located on the surface of virus particles.

Histopathological examination, electron microscopy, immunoassay, fluorescence antibody (FAT), ELISA, gene probe, polymerase chain reaction (PCR) are used to diagnose viral infections. Shrimp white spot virus (WSSV) has the entire genome decoded (van Hulten MC. Et al ., Virol . , 2001) It is possible to quickly and easily identify infection by PCR.

The incidence of shrimp white spot syndrome occurs mainly at water temperatures above 20 ° C and occurs throughout the year from postlarvae to maturity. It is known to be vertically infected from stocks carrying WSSV and to horizontal infections in crustaceans such as crabs and genus around infected populations or farms. When infected, shrimp can spread white spots along the inside of the shell, spreading all over the body, and the shell and muscles become loose and easily separated. As the onset progresses, white spots can be identified with the naked eye, hemolymph is opaque, and hepatic pancreas in the middle of the onset shows necrosis. In addition, the cell nucleus and lymphoid organs show symptoms that are particularly enlarged than normal, and almost 100% of them die after 3 to 10 days after infection. Therefore, the rapid growth of shrimp white spot virus is causing a huge damage to the shrimp farming industry, and the need for vaccines for the prevention and treatment of shrimp white spot virus infection is increasing, but there is no clear measures. In Korea, a method of improving beta immunity by adding beta glucan preparation to feed, disinfecting ponds by disinfecting shrimp before stocking with chlorine disinfectant, and combining aquaculture with fish and crayfish have not been found. .

In the case of shrimp white spot virus (WSSV), the host cell is not yet accurately identified, and it is difficult to pass passage, so it is practically impossible to use live or attenuated or inactivated virus as an antigen. Shrimp's immune system is different from that of vertebrates, making it difficult to develop vaccines. Obtaining literature or information on the shrimp's immune system is one of the challenges. Invertebrates such as shrimps are known to have no specific immune function using antibodies, but infection control effects have been reported by promoting the propagation activity of shrimp blood cells using inactivated cells of Vibrio peneus. 3-204820. In addition, the challenge test of VP28, the envelope protein of WSSV, reported increased survival of shrimp, suggesting the possibility that a specific immune response could be induced in shrimp (Jeroen Witteveldt et. al ., J. Vriol ., 2004). However, no effective medicines have been developed for such viral infections, so there is no effective control measures.

The technique of attaching and expressing a desired protein on the cell surface of a microorganism is called cell surface display technology. This cell surface expression technology uses surface proteins of microorganisms such as bacteria and yeast as surface anchoring motifs to express foreign proteins on the surface.The scope of application is determined by which proteins are expressed on the cell surface. Therefore, the potential for industrial application using cell surface expression technology is considerable.

Surface expression matrices are the most important for successful cell surface expression techniques. The key to this technology is how to select and develop a matrix capable of expressing foreign proteins on the cell surface.

The surface-expressing matrix known and used so far is largely four kinds such as outer membrane protein, lipid protein (lipoprotein), secretory protein (secretory protein), surface organ proteins such as flagella protein.

For Gram negative bacteria, LamB, PhoE (Charbit et al . , J. Immunol ., 139: 1658, 1987; Agterberg et al . , Vaccine , 8:85, 1990), proteins present in the extracellular membrane, such as OmpA, were mainly used, and the lipoprotein TraT (Felici et. al . , J. Mol . Biol ., 222: 301, 1991), peptidoglycan associated lipoprotein (PAL) (Fuchs et al . , Bio / Technology , 9: 1369, 1991) and Lpp (Francisco et al . , Proc . Natl . Acad . Sci . USA , 489: 2713, 1992) and fimbria proteins (Hedegaard et al ., Such as FimA or FimH adhesin of type 1 fimbriae). al . , Gene , 85: 115, 1989), and pili proteins such as PapA pilu subunit were used as cell surface expression mothers to try to express foreign proteins. In addition, ice nucleation protein (Jung et al . , Nat . Biotechnol ., 16: 576, 1998; Jung et al . , Enzyme Microb . Technol ., 22: 348, 1998; Lee et al . , N at . Biotechnol. 18: 645, 2000), pullulanase of Klebsiela oxytoca (Kornacker et al . , Mol . Microl ., 4: 1101, 1990), IgA protease from Neiseria (Klauser et al . , EMBO J. , 9: 1991, 1990), E. coli adhesin AIDA-1, shigella VirG protein, Lpp and OmpA fusion proteins have been reported to be used as the surface expression matrix.

Gram positive bacteria have been reported to effectively express malaria antigens using Staphylococcus aureus- derived protein A and FnBPB proteins as surface-expressing mothers. report on the surface expression using coat protein and Streptococcus M6 protein from pyogenes (Medaglini, D et al ., Proc . Natl . Acad. Sci . USA ., 92: 6868, 1995), Bacillus It has been reported that the surface proteins of Gram-positive bacteria such as anthracis S-layer protein EA1 and Bacillus subtilis CotB were used as mothers.

The present inventors utilize a synthetic complex gene (pgsBCA) of polygamma glutamic acid derived from Bacillus sp. Strain as a new surface expression parent, to effectively express a foreign vector on the surface of a microorganism and to the surface of the microorganism transformed with the vector. A method for expressing a large amount of foreign protein has been developed (Reg. No. 10-0469800). Many studies have been attempted to stably express antigens or antigenic determinants of pathogens in bacteria that can be mass-produced through genetic engineering using the surface-expressing mothers introduced above.

In particular, it has been reported that oral administration of foreign immunogens on the surface of non-pathogenic bacteria can induce a more persistent and stronger immune response than vaccines using conventionally attenuated pathogenic bacteria or viruses. This induction of immune response is because the surface structures of bacteria act as an adjuvant to increase the antigenicity (antigenicity) of the surface expressed foreign protein, and is known to be due to the immune response in the body to the living organisms. The development of recombinant live vaccines for non-pathogenic bacteria using such surface expression systems is noteworthy.

Many studies have been conducted to prevent and treat the infection of shrimp white spot virus. Antibody development method using antiserum or monoclonal antibody (Korea Patent Registration No. 10-0489617: Water-soluble antibody protein for the prevention and treatment of white spot virus infection and eggs, and preparation thereof; Korean Patent Publication No. 2003-0069506: Egg yolk antibody to shrimp white spot virus) has been reported, but it is not practical due to the cost and technical problems to obtain the antibody. In addition, viable, attenuated, or inactivated viruses should be used to induce viral antibodies. If the virus itself is used as an antigen, since the virus contains several proteins, it is more likely to use a pathogenic antigen than a single antigen. There is a problem that the antibody titer against is low.

Therefore, the present inventors have made intensive efforts to develop a vaccine for treating and preventing shrimp white spot viral diseases. As a result, the recombinant vector for microbial surface expression comprising a polygammaglutamic acid synthesis complex gene and a gene encoding shrimp white spot virus antigen A microorganism transformed with (vaccine manufacturing vector) was produced, and it was confirmed that the microbial strain can effectively treat and prevent shrimp white spot viral diseases, thereby completing the present invention.

An object of the present invention is to provide a surface expression vector comprising a polygamma glutamic acid synthesis complex gene and a gene encoding a shrimp white spot virus antigen.

Another object of the present invention is to provide a bacterium transformed by the vector.

Still another object of the present invention is a vaccine and feed additive for treating or preventing shrimp white spot viral diseases containing the shrimp white spot virus antigen surface-expressing bacteria or shrimp white spot virus antigen crudely extracted from the bacteria as an active ingredient. To provide.

In order to achieve the above object, the present invention is for the surface expression of shrimp white spot virus antigen containing any one or more polygamma glutamic acid synthesis complex gene selected from the group consisting of pgsA, pgsB and pgsC and genes encoding shrimp white spot virus antigen Provide a vector.

In the present invention, the antigen may be characterized as VP19 or VP28 which is an envelope antigen of shrimp white spot virus, and at least one selected from the group consisting of VP15, VP24 and VP26 which are virion antigens of shrimp white spot virus. It can be characterized. In addition, the vector may be characterized by having a SlpA7 promoter. In addition, the vector may be characterized in that the plasmid pBT: pgsA-VP26 having the gene map shown in Figure 1A, plasmid pBT: pgsA-VP28 having the gene map shown in Figure 1B may be characterized. .

The present invention also provides a bacterium transformed with the vector. In the present invention, the bacteria are Escherichia coli, Salmonella typhi, Salmonella typhimurium, Vibrio cholera, Mycobacterium bovis, Shigella, Bacillus, Lactobacillus, Stephylococcus, Corynebacteria, Listeria monocytogenes and strap It may be characterized in that it is any one selected from the group consisting of tococus, preferably characterized in that the lactic acid bacteria.

The present invention also provides a method for producing shrimp white spot virus antigens, wherein the transformed bacteria are cultured to express shrimp white spot virus antigens on the surface of the bacteria.

The present invention is also for the treatment or prevention of shrimp white spot viral diseases containing the shrimp white spot virus antigen obtained by the bacterial culture as an active ingredient or the surface of the shrimp white spot virus antigen crudely extracted from the bacteria. Provide a vaccine. In the present invention, the vaccine may be characterized by ingestion orally or by food, may be characterized by subcutaneous or intraperitoneal injection, and may be characterized by nasal administration.

The present invention also provides a feed additive containing as a active ingredient a bacterium surface-expressed shrimp white spot virus antigen obtained by the bacterial culture or shrimp white spot virus antigen crudely extracted from the bacteria.

Hereinafter, the present invention will be described in detail.

First, in order to express the antigen of the shrimp white spot virus on the cell surface of the microorganism, at least one polygamma glutamic acid synthesis complex gene selected from the group consisting of pgsA, pgsB and pgsC and a gene encoding the shrimp white spot virus antigen Recombinant vectors for microbial surface expression were prepared. In one embodiment of the present invention, as the antigen, the vectors pBT: pgsA-VP26 and pBT: pgsA-VP28 capable of surface-expressing VP26, an antigenic protein of shrimp white spot virus virion, and VP28, an antigenic protein of an envelope. Was produced. Lactobacillus casei, a lactic acid bacterium, was transformed with the microbial surface expression vectors pBT: pgsA-VP26 and pBT: pgsA-VP28 to express the antigen on the surface of the lactic acid bacteria, and then the transformant was mixed and supplied to the feed of shrimp. Shrimp white spot virus was infected (muscle infection and / or soaking infection) to determine the mortality of the shrimp over time.

As a result, it could be observed that the mortality of the shrimp was delayed or decreased in the group treated with the microorganisms expressing the antigen of the shrimp white spot virus compared to the control group (FIG. 3). Through this, it was confirmed that the microorganism expressing the antigen of the shrimp white spot virus according to the present invention has a therapeutic and preventive effect against the infection of shrimp white spot virus.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In the examples below, VP26 and VP28 were used as the shrimp white spot virus antigens, but the present invention is not limited thereto. Any of the shrimp white spot virus antigens VP15, VP19, VP24, VP26 and VP28 can be used.

In addition, in the following examples, the microorganisms used as host cells for transformation are E. coli, Salmonella typhi, Salmonella typhimurium, Vibrio cholera, Mycobacterium bovis, Any one of Shigella, Bacillus, Lactobacillus, Lactococcus, Staphylococcus, Listeria monocytogenes and Stramatococcus can be used, and is an edible microorganism (lactic acid bacteria).

Example  1: Vector for surface expression pBT : pgsA - VP26  And pBT : pgsA - VP28 Made of

Antigens of shrimp white spot virus virions using gram-negative microorganisms and gram-positive microorganisms as phosts using pgsA among genes of extracellular membrane proteins (pgsA, pgsB and pgsC) involved in polygammaglutamic acid synthesis derived from Bacillus sp. The vectors pBT: pgsA-VP26 and pBT: pgsA-VP28, which can surface express the protein VP26 and the envelope protein VP28, were constructed.

Prior to the performance of the present invention, the present inventors used the virus isolated from shrimp killed in Gochang, Korea in 2004 (the isolation process of the virus is the same as described in Example 3 below), and used SEQ ID NOs: 2 and 3 and Each PCR was performed using primers of SEQ ID NOs: 4 and 5, thereby obtaining VP 26 and VP 28 gene fragments, respectively. The resulting VP 26 and VP 28 gene fragments were inserted and stored in pGEM T vector (Promega).

1-1: pBT : pgsA - VP26 Made of

The surface expression vector pBT: pgsA including the pgsA gene among the genes of the SlpA7 promoter (SEQ ID NO: 1) and the extracellular membrane protein involved in polygammaglutamic acid synthesis, which are always high-expression promoters on pAT, the Gram-negative and Gram-positive universal vectors, To increase the surface expression of the vector, the HCE promoter portion of pHCE2LB: pgsA-HPVL1 (E. coli transformed with KCTC 10349BP: pHCE2LB: pgsA-HPVL1) was replaced with the SlpA7 promoter and restriction enzymes Bam HI and by treatment with Kpn I were prepared by removing the HPVL1 gene.

The SlpA7 promoter consists of a total of 214 bp of contiguous sequence of 90 bp sites (SEQ ID NO: 1) based on the consensus sequence of 124 bp derived from the HCE promoter and the slpA gene encoding the S-layer protein known from lactic acid bacteria. )

SEQ ID NO: 5'-tgatctctcc ttcacagatt cccaatctct tgttaaataa cgaaaaagca tcaatcaaaa cggcggcatg tctttctata ttccagcaat gttttatagg ggacatattg atgaagatgg gtattagtat ttttcggtca ttttaacttg ctattttttg aagaggtcag t ttattat gta

In the present invention, the pAT vector is widely known as a Gram-positive microorganism universal vector (Trieu-Cuot, et. al ., Gene , 29:99, 1991; Zhou and Johnson, Biotech . Lett. , 29: 121, 1993), the present invention is also used as a back-bone vector for producing a transformation vector in the production of transformed microorganisms using gram-negative microorganisms (US 2002/309560, US 2002 / 217613, US 2002/197153, Jung CM, et al ., J. Bacteriology , 181: 2816, 1999).

To introduce a polynucleotide encoding VP26 antigen of shrimp white spot virus into the prepared surface expression vector pBT: pgsA, the VP26 gene of the pGEM T vector containing about 615 bp of VP26 gene was used as a template, and SEQ ID NOs: 2 and 3 PCR was performed using the primers of to obtain a VP26 gene having the size as described above.

SEQ ID NO: 5'- cgc ggatcc gaa ttt ggt aac ctt aca-3 '

SEQ ID NO: 5'-gga aagctt tta tta ctt ctt ctt gat tt-3 '

(The underlined parts in SEQ ID NOS: 2 and 3 represent the cleavage sites of Bam HI and Kpn I, respectively)

The C-terminal region of the gene pgsA of the extracellular membrane protein treated with the PCR product VP26 gene with the restriction enzymes Bam HI and Kpn I and involved in polygammaglutamic acid synthesis of the surface expression vector pBT: pgsA prepared in Example 1-1. By coordinating the translation codons, the vector pBT: pgsA-VP26 capable of surface-expressing VP26, an antigen of shrimp white spot virus, was produced using gram-negative microorganisms and gram-positive microorganisms as hosts (FIG. 1A).

1-2: pBT : pgsA - VP28 Made of

In order to introduce a polynucleotide encoding VP28 antigen of shrimp white spot virus into the surface expression vector pBT: pgsA of Example 1-1, the VP28 gene of the pGEM T vector containing about 615 bp of VP28 gene was used as a template. PCR was performed using primers SEQ ID NOs: 4 and 5 to obtain a VP28 gene having a size as described above.

SEQ ID NO: 5'- cgc ggatcc gat ctt tct ttc act ctt-3 '

SEQ ID NO: 5'-gga aagctt tta tta ctc ggt ctc agt gcc-3 '

(The underlined portions in SEQ ID NOs: 4 and 5 represent the cleavage sites of Bam HI and Kpn I, respectively)

The C-terminal region of the gene pgsA of the extracellular membrane protein treated with the PCR product VP28 gene with the restriction enzymes Bam HI and Kpn I and involved in polygammaglutamic acid synthesis of the surface expression vector pBT: pgsA prepared in Example 1-1. By coordinating the translation codons, the vector pBT: pgsA-VP28 capable of surface expression of VP28, an antigen of shrimp white spot virus, was produced using gram-negative and gram-positive microorganisms as hosts (FIG. 1B).

Example  2: antigen of shrimp white spot virus VP26  And VP28 Cell surface expression

Lactobacillus casei strains were transformed with the surface expression vectors pBT: pgsA-VP26 and pBT: pgsA-VP28 of Examples 1-1 and 1-2, and the transformants were then subjected to MRS medium (Lactobacillus MRS, Becton Dickinson and Company Sparks). , USA) was used to induce surface expression at 37 ℃.

In the present invention, the host cell for transformation may alternatively use Gram-negative bacteria E. coli, Salmonella typhi, Salmonella typhimurium, Vibrio cholera, Mycobacterium bovis, Shigella, and the like. Bacillus, Lactobacillus, Lactococcus, Staphylococcus, Listeria monocytogenes, Stramtococcus, etc. can also be used.

The cultured transformant was adjusted to the same cell concentration, and a certain amount of the transformant was extracted to denature the protein to prepare a sample, and then analyzed by SDS-PAGE. Bio-Rad). The PVDF membranes to which proteins were transferred were blocked by blocking for 1 hour in blocking buffer solution (50 mM Tris-HCl, 5% skim milk, pH 8.0), and then the blocking solution was added to the blocking solution with a polyclonal primary antibody derived from rabbit against PgsA. Was diluted 1000-fold in and reacted for 4 hours. After completion of the reaction, the membrane was washed with a buffer solution, washed again by reacting the avidin-biotin reagent for 1 hour, and then subjected to color reaction by adding a substrate (H ₂ O ₂ ) and a coloring reagent (DAB). By induction, the expression of the fusion proteins (PgsA-VP26 and PgsA-VP28) in the transformants was confirmed (FIG. 2).

As a result, as shown in FIG. 2, the expression of the fusion proteins PgsA-VP26 (FIG. 2A) and PgsA-VP28 (FIG. 2B) was confirmed in the transformant. Since PgsA with linker is about 43.6 kDa and VP26 and VP28 are about 22.2 kDa and about 19 kDa, respectively, the fusion proteins PgsA-VP26 and PgsA-VP28 expressed in the transformants correspond to about 65.8 kDa and about 62.6 kDa, respectively. do.

Example  3: Virus Protective Effect of Microorganisms Transformed with Shrimp White Spot Virus Antigen

In the transformants expressing the VP26 and VP28 fusion proteins of Example 2, the immune responses of the transformants to the respective fusion proteins and their inhibitory effect on shrimp white spot virus infection were investigated.

3-1: Determination of Infection Concentration of Shrimp White Spot Virus

In order to investigate the inhibitory effect of shrimp white spot virus, the dose of virus was first determined to perform the challenge test of shrimp white spot virus.

The shrimp used for the immunoassay was penaeus chinensis, and the shrimp white spot virus was isolated from shrimp killed by the virus in Gochang, Korea in 2004.

Hochanged shrimps were homogenized and diluted in phosphate buffer (pH 7.4), and the diluted virus-containing solution was filtered through a 0.45 μm filter, followed by gradient centrifugation using 30% sucrose. Gradient centrifugation was performed to concentrate the virus solution. The concentrated virus was rediluted in phosphate buffer (pH 7.4) to prepare it as a virus for infection. The prepared virus was checked again for the presence of virus by performing PCR using specific primers (SEQ ID NOs: 2 and 3 and SEQ ID NOs: 4 and 5) for shrimp white spot virus.

In order to determine the muscle infection treatment concentration of shrimp white spot virus, the virus solution was dispensed in 5 μl, 10 μl, 20 μl and 30 μl, and each virus aliquot was dispensed with 6 to 8 g penaeus using a 30 gauge syringe. Injections were made between third and fourth ablation of chinensis shrimp (15 mice / group). The injection of phosphate buffer was used as a control. As a result of investigating mortality of shrimp for 2 weeks after injection and cumulative mortality for 7 days after injection, it was confirmed that the cumulative mortality was 50-70% when the virus injection amount was 20 μl. 20 μl of virus solution was determined at the treatment concentration for the infection.

To determine the immersion treatment concentration of shrimp white spot virus, the rediluted virus solution was prepared by diluting 1: 100, 1: 1000, 1: 3000, 1: 5000, and 1: 10000, with each virus dilution. After filling the tank, 6-8 g penaeus chinensis shrimps were immersed in the tank for 7 hours (15 / group). After 7 hours of immersion, the water in the tank was exchanged and the mortality of shrimp was examined for 2 weeks and the cumulative mortality for 14 days after immersion. It was confirmed that it is 70%. Virus solutions with a dilution ratio of 1: 1000 were determined at the treatment concentration for immersion infection.

3-2: Antimicrobial Effect of Antimicrobial Surfaces on Shrimp White Spot Virus

Shrimp white spot virus VP26 and VP28 Antimicrobial inhibitory effect of shrimp white spot virus was investigated using shrimp ingested with Lactobacillus.

Two groups, each consisting of 20 shrimps, were prepared as experimental and control groups. Shrimp feed containing 0.5% of Lactobacillus surface-expressing VP26 and VP28 antigens was supplied to the prepared experimental shrimp for about 10 days, and then commercialized shrimp feed was supplied thereafter. Twenty shrimps received commercialized shrimp feed were used as controls. Ten days after the start of the experiment, the shrimp group of each group set as the experimental group was infected with the shrimp white spot virus of the concentration determined in Example 3-1, and the mortality of the shrimps was observed according to the seal (FIG. 3A).

As a result, as shown in Figure 3A, the shrimp of the control group were all killed between 3-5 days after infection, whereas the shrimp of the experimental group fed Lactobacillus surface-expressed antigen survives 4-8 days after infection Could confirm. This indirectly proves that the infection of the virus was protected by lactic acid bacteria expressing the antigen of the shrimp white spot virus.

3-3: Shrimp white spot virus of an antigen-expressed microorganism Immersion Infection  Defense effect

Shrimp white spot virus was tested using Lactobacillus surface-expressing shrimps with VP26 and VP28 antigens.

Two groups, each consisting of 20 shrimps, were prepared as experimental and control groups. Shrimp feed containing 0.05-5%, preferably 0.1-1%, and more preferably 0.5% of Lactobacillus surface-expressing VP26 and VP28 antigens was supplied to the prepared experimental groups for about 10 days, and then commercialized thereafter. Prepared shrimp feed. Twenty shrimps received commercialized shrimp feed were used as controls. Ten days after the start of the experiment, the shrimp of each group set as the experimental group were immersed in a tank containing a shrimp white spot virus solution having the dilution ratio determined in Example 3-1 for 7 hours, and water was changed. The lethality of the shrimp was observed (FIG. 3B).

As a result, as shown in Figure 3B, the shrimp of the control group showed a mortality of about 70% at 13 days after infection, whereas the shrimp of the experimental group showed a mortality of about 25% at 19 days after infection It was. This indirectly demonstrates that the infection of the virus (infection under conditions similar to natural infection) was protected by lactic acid bacteria expressing the antigen of the shrimp white spot virus.

Based on the results of Examples 3-2 and 3-3, the microorganism surface-expressed antigen of the present invention has the effect of treating and preventing the death of shrimp caused by infection with shrimp white spot virus, the viral disease It can be confirmed that it can be usefully used as a vaccine, feed additives, etc.

3-4: PCR On by Immersion Infection  Confirm

In the immersion infection experiment of Example 3-3, PCR was performed to confirm that lethality of shrimp in the control group and the experimental group was caused by infection of shrimp white spot virus and not by factors other than immersion infection.

First, the killed shrimp of Example 3-3 were homogenized and diluted in phosphate buffer (pH 7.4), and the diluted virus-containing solution was filtered through a 0.45 μm filter, followed by sucrose gradient centrifugation at 30%. Concentrated. The concentrated virus was re-diluted in phosphate buffer (pH 7.4) to be used as a template, and PCR was carried out using primers of SEQ ID NOs: 2 and 3 and SEQ ID NOs: 4 and 5, respectively, so that VP26 and VP28 genes in killed shrimp The presence of was confirmed (FIG. 4).

As a result, as shown in Figure 4, it was confirmed that the VP26 and VP28 genes of about 615bp in the killed shrimp. This demonstrates that the shrimp lethality of Example 3-3 is due to submerged infection of shrimp white spot virus, not other environmental tolerants.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention relates to a surface expression vector comprising a polygammaglutamic acid synthesis complex gene and a gene encoding a shrimp white spot virus antigen, a bacterium transformed by the vector and a bacterium or a bacterium surface expressing the shrimp white spot virus antigen. It is effective to provide a vaccine and feed additive for treating or preventing shrimp white spot viral diseases containing crude extracted shrimp white spot virus antigen as an active ingredient.

Bacteria surface-expressing shrimp white spot virus antigens and shrimp white spot virus antigens extracted from the bacteria can be used as vaccines, feed additives, etc. to treat and prevent diseases caused by shrimp white spot virus. Therefore, it is possible not only to prevent the mass death of shellfish caused by the virus, but also to increase the profits of aquaculture and fishing.

<110> Bioleaders Corp. <120> Cell Surface Expression Vector for WSSV Antigen and Microorganism          Transformed by the Same <130> P06-B003 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 214 <212> DNA <213> Artificial Sequence <220> <223> promoter <400> 1 tgatctctcc ttcacagatt cccaatctct tgttaaataa cgaaaaagca tcaatcaaaa 60 cggcggcatg tctttctata ttccagcaat gttttatagg ggacatattg atgaagatgg 120 gtattagtat ttttcggtca ttttaacttg ctattttttg aagaggtcag taaatatgaa 180 tcgtggtaag tattttcaca cttctggaaa aagg 214 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cgcggatccg aatttggtaa ccttaca 27 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggaaagcttt tattacttct tcttgattt 29 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cgcggatccg atctttcttt cactctt 27 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggaaagcttt tattactcgg tctcagtgcc 30

Claims (15)

A shrimp white spot virus antigen surface expression vector containing any one or more polygammaglutamic acid synthesis complex genes selected from the group consisting of pgsA, pgsB and pgsC and genes encoding shrimp white spot virus antigens. The vector of claim 1, wherein the antigen is VP19 or VP28, which is an envelope antigen of shrimp white spot virus. The vector of claim 1, wherein the antigen is at least one selected from the group consisting of VP15, VP24, and VP26 which are virion antigens of shrimp white spot virus. The vector of claim 1, wherein the vector contains a SlpA7 promoter. The vector according to claim 1, which is a plasmid pBT: pgsA-VP26 having a gene map shown in FIG. 1A. The vector according to claim 1, which is a plasmid pBT: pgsA-VP28 having a gene map shown in FIG. 1B. A bacterium transformed with the vector of any one of claims 1 to 6. 8. The bacterium according to claim 7, wherein the bacterium is Escherichia coli, Salmonella typhi, Salmonella typhimurium, Vibrio cholera, Mycobacterium bovis, Shigella, Bacillus, Lactobacillus, Stephylococcus, Coryne bacteria, Listeria monocytogenes and Bacteria, characterized in that any one selected from the group consisting of Straptococcus. The bacterium of claim 8, wherein the bacterium is a lactic acid bacterium. The method of producing a shrimp white spot virus antigen, comprising culturing the transformed bacteria of claim 7 to express the shrimp white spot virus antigen on the bacterial surface. The shrimp white spot virus antigen obtained by the culture of the transformed bacteria of claim 7 or the shrimp white spot viral antigen containing the shrimp white spot virus antigen crudely extracted from the bacteria as an active ingredient for the treatment or prevention of viral diseases vaccine. The vaccine according to claim 11, which is ingested orally or edible. The vaccine of claim 11, for subcutaneous or intraperitoneal injection. The vaccine of claim 11, wherein the vaccine is for nasal administration. A feed additive comprising a bacterial surface-expressed shrimp white spot virus antigen obtained by the culture of the transformed bacteria of claim 7 or a shrimp white spot virus antigen crudely extracted from the bacteria as an active ingredient.

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