KR-20260067515-A - Recombinant antigen protein for preventing shrimp Acute Hepatopancreatic Necrosis Disease and vaccine composition comprising the same

Abstract

The present application relates to a recombinant antigen protein for preventing acute hepatopancreatic necrosis disease in shrimp and a vaccine composition containing the same. According to one aspect, the recombinant antigen protein can maintain stability under seawater treatment conditions and not only does not affect the growth of shrimp but can also significantly reduce the mortality rate of shrimp, so it can be usefully used as an immune enhancer or feed additive in shrimp farming.

Inventors

• 박희주
• 김태희
• 김가혜
• 임광묵
• 전서현
• 김재훈

Assignees

• 디허스코리아 주식회사
• 녹십자수의약품 주식회사

Dates

Publication Date

20260513

Application Date

20241105

Claims (13)

1. Vibrio parahemolitis PirA (Photorhabdus insect-related toxin A) or PirB (Photorhabdus insect-related toxin B), toxin proteins of ( Vibrio parahaemolyticus ); Maltose-binding protein (MBP); and Recombinant antigen protein containing a histidine tag.
2. A recombinant antigen protein according to claim 1, wherein the histidine-tag is connected to the N-terminus of PirA or PirB.
3. The recombinant antigen protein of claim 1, wherein the MBP is connected to the N-terminus of PirA or PirB.
4. A recombinant antigen protein according to claim 1, wherein the histidine-tag is connected to the N-terminus of the MBP.
5. A recombinant antigen protein according to claim 1, wherein the histidine-tag is 3 to 10 polyhistidine-tags.
6. Vibrio parahemolitis Polynucleotide sequence encoding PirA or PirB, the toxin protein of ( Vibrio parahaemolyticus ); A polynucleotide sequence encoding a maltose-binding protein (MBP); and A recombinant expression vector comprising a polynucleotide sequence encoding a histidine-tag.
7. Host cell transformed with a recombinant expression vector according to claim 6.
8. A host cell according to claim 7, wherein the host cell is one selected from the group consisting of Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi, and yeast.
9. A vaccine composition for preventing acute hepatopancreatic necrosis disease (AHPND) in shrimp comprising a recombinant PirA protein according to claim 1, a recombinant PirB protein according to claim 1, or a combination thereof.
10. A vaccine composition according to claim 9, wherein the composition comprises 0.5 μg to 30 μg of the recombinant PirA protein based on 1 g by weight.
11. A vaccine composition according to claim 9, wherein the composition comprises 2 μg to 120 μg of the recombinant PirB protein based on 1 g by weight.
12. A vaccine composition according to claim 9, wherein the weight ratio of the recombinant PirA protein and the recombinant PirB protein is 1:1 to 20.
13. A shrimp feed additive comprising the vaccine composition of any one of claims 9 to 12.

Description

Recombinant antigen protein for preventing shrimp Acute Hepatopancreatic Necrosis Disease and vaccine composition comprising the same The present application relates to a recombinant antigen protein for the prevention of acute hepatopancreatic necrosis disease in shrimp and a vaccine composition containing the same. Acute hepatopancreatic necrosis disease (AHPND) is a disease that has recently surged in shrimp farming. Caused by Vibrio parahaemolyticus bacteria, it produces insect toxins that cause up to 100% mortality within as little as six hours or one week. These insect toxins are produced through the expression of specific genes present in a particular plasmid (Photorhabdus insect-related toxins, Pir toxin) within the bacteria. Because the bacteria are motile and can easily move from place to place, this disease spreads much faster than viral shrimp diseases that have previously attracted attention, such as White Spot Virus (WSSV), Tauravirus (TSV), and Infectious Myonecrosis Virus (IMNV). AHPND originated in China in 2009 and rapidly spread to various Asian countries, including Thailand, Malaysia, and Vietnam, within a year. Outside of Asia, it emerged in Mexico and spread to other Central American countries, causing significant damage to the majority of the shrimp market. In Korea, it first occurred in 2015–2016 and caused significant damage, and research is currently underway for the prevention and management of this disease. The excessive use of antibiotics to manage AHPND has led to antibiotic resistance, making it urgent to find an alternative. Although it has been known that shrimp, which are invertebrates, lack an adaptive immune response, research results have been reported since the late 1980s indicating that vibriosis in cultured shrimp was prevented by vaccination (Itami T. Takahashi Y. Nakamura Y (1989) Efficacy of vaccination against vibriosis in cultured kuruma prawns Penaeus japonicus. Journal of Aquatic Animal Health, 1: 234-242.;). Against this technological backdrop, there is a growing need for the development of immune boosters or feed additives capable of effectively preventing AHPND, but the current situation remains inadequate. Figure 1 is a figure showing the vector map of a recombinant expression vector pHis6-MBP_PirA containing a polynucleotide encoding a 6XHis-MBP-PirA recombinant protein according to one aspect, and a recombinant expression vector pHis6-MBP_PirB containing a polynucleotide encoding a 6XHis-MBP-PirB recombinant protein according to one aspect. Figure 2 is a figure showing the vector map of a recombinant expression vector pET28a(+)_PirA containing a polynucleotide encoding a 6XHis-PirA recombinant protein according to one aspect and a recombinant expression vector pET28a(+)_PirB containing a polynucleotide encoding a 6XHis-PirB recombinant protein according to one aspect. Figure 3 shows the results of SDS-PAGE analysis performed on E. coli transformed with recombinant expression vectors (pHis6-MBP_PirA, pHis6-MBP_PirB, pET28a(+)_PirA, pET28a(+)_PirB) according to one pattern, cultured under high temperature conditions after IPTG induction, lysed, and separated into whole cell lysate (Total fraction), soluble fraction, and insoluble fraction. Figure 4 shows the results of SDS-PAGE analysis performed on E. coli transformed with recombinant expression vectors (pHis6-MBP_PirA, pHis6-MBP_PirB, pET28a(+)_PirA, pET28a(+)_PirB) according to one aspect, cultured under low temperature conditions after IPTG induction, lysed, and separated into whole cell lysate (Total fraction), soluble fraction, and insoluble fraction. Figure 5 shows the results of purifying the recombinant antigen protein produced from E. coli transformed with recombinant expression vectors (pHis6-MBP_PirA, pHis6-MBP_PirB, pET28a(+)_PirA, pET28a(+)_PirB) according to one aspect, performing SDS-PAGE analysis to confirm the recombinant protein, and measuring the recovery rate by protein quantification. Figure 6 shows the results of evaluating long-term storage stability, seawater salinity condition stability, and high temperature stability for four types of recombinant PirA/PirB proteins (6XHis-MBP-PirA, 6XHis-MBP-PirB, 6XHis-PirA, 6XHis-PirB). Figure 7 shows the results of evaluating the survival rate after feeding whiteleg shrimp with recombinant 6XHis-MBP-PirA protein and recombinant 6XHis-MBP-PirB protein at low or high concentrations, or after feeding them with recombinant 6XHis-PirA protein and recombinant 6XHis-PirB protein at low or high concentrations, and then challenging them with Vibrio parahaemolyticus strains that produce PirA and PirB toxins causing AHPND. Figure 8 shows the results of evaluating the survival rate after 96 hours when whiteleg shrimp were challenged with Vibrio parahaemolyticus strains that produce PirA and PirB toxins causing AHPND, after orally administering recombinant 6XHis-MBP-PirA protein and recombinant 6XHis-MBP-PirB protein at different concentrations (0.5X, 1.0X,