Preparation of novel O antigen and specific serum of vibrio parahaemolyticus

Abstract

The invention provides preparation of a novel O antigen and specific serum of vibrio parahaemolyticus. The classification name of the vibrio parahaemolyticus is vibrio parahaemolyticus O17: K4, and the preservation number of the vibrio parahaemolyticus O17: K4 is CGMCC (China General Microbiological Culture Collection Center) No.31706. The invention further discloses a preparation method of the vibrio The novel thallus antigen O17 vibrio parahaemolyticus is determined by adopting an agglutination reaction and a whole genome sequencing method of existing diagnostic serum (11 kinds of O) of the vibrio parahaemolyticus. The novel O17 specific serum is obtained through preparation of a novel thallus antigen O17, animal immunization and adsorption separation. The novel O17 specific serum prepared by the invention is high in sensitivity and strong in specificity, can be conveniently and quickly used for detecting a brand new novel thallus antigen O17 vibrio parahaemolyticus which is prevalent in recent years, and provides an important detection technology for pathogen confirmation, monitoring and prevention and control of infectious diarrhea and food-borne diseases in laboratories.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and relates to a novel bacterial antigen and a method for preparing a specific serum thereof, and in particular to a novel O antigen of Vibrio parahaemolyticus and the preparation of a specific serum thereof, wherein the bacterial body is Vibrio parahaemolyticus. Background Art

[0002] Vibrio parahaemolyticus (VP) is the main pathogen of foodborne diseases. Humans are mainly infected through seawater exposure and eating raw or semi-raw seafood. It usually causes acute gastroenteritis, and occasionally wound infection and sepsis. In severe cases, it can lead to death. Data from my country's Foodborne Disease Outbreak Monitoring Network show that in coastal areas, foodborne diseases caused by Vibrio parahaemolyticus have surpassed Salmonella and Staphylococcus aureus, becoming the most important biological factor in foodborne disease outbreaks.

[0003] Serotyping of Vibrio parahaemolyticus is of great significance in epidemiological research and outbreak monitoring. At present, most studies on Vibrio parahaemolyticus worldwide use serotype as a special term for international communication to distinguish different biological phenotypes of Vibrio parahaemolyticus. Vibrio parahaemolyticus can be serotyped based on the heat-stable bacterial lipopolysaccharide O antigen and the heat-labile capsular polysaccharide antigen. There are 16 types of O antigen and more than 70 types of K antigen, which are mainly identified by slide agglutination reaction. With the advancement of molecular biology technology, whole genome sequencing technology has been gradually applied to molecular typing research of Vibrio parahaemolyticus in recent years. The resolution of strains has reached the base level. Based on whole genome sequencing, the complete bacterial genome sequence can be obtained, the O / K antigen gene cluster of Vibrio parahaemolyticus can be analyzed, and its serotype can be predicted. Before 1996, Vibrio parahaemolyticus infection did not show a clear dominant serotype. It was not until February 1996 that the new O3:K6 serotype strain appeared in Kolkata, India, which caused a group outbreak of food poisoning. This new strain then quickly spread across continents among people in many coastal countries and regions around the world, causing multiple large-scale outbreaks and becoming the most important type of Vibrio parahaemolyticus. According to recent reports, a new serotype of Vibrio parahaemolyticus O10:K4 was first detected in Guangxi, China in 2020. This strain has the characteristics of a pandemic strain and has caused multiple local food poisoning outbreaks and sporadic incidents. Subsequently, Beijing, Zhejiang and other places reported that O10:K4 Vibrio parahaemolyticus became the most important serotype in the local area. This research team selected 30 strains of O10:K4 Vibrio parahaemolyticus from different years between 2020 and 2023 for whole genome sequencing and analyzed their O / K antigen gene clusters. It was found that the O antigen determinant cluster contains 17 genes with a total length of 16030bp, while the known O10 antigen determinant cluster contains 17 genes with a total length of 14977bp. The consistency rate of the antigenic determinant sequences of the two is only 64%; and the new O antigen determinant is also different from the sequences of the other 15 O antigen determinants except O10. Therefore, it is believed that the O antigen determinant cluster is a new type and can be named O17. The serotyping reagents for O10:K4 Vibrio parahaemolyticus reported in the existing literature all use internationally recognized commercial reagents-Denka Seiken, Tokyo, Japan. The diagnostic serum kit contains 11 types of Vibrio parahaemolyticus O sera commonly used in clinical practice (O1-O11). We speculate that the O10 serum in the existing commercial reagents cross-agglutinates with the new O17 bacterial antigen of Vibrio parahaemolyticus, resulting in inappropriate results. The present invention relates to a novel bacterial antigen O17 of Vibrio parahaemolyticus and a method for preparing specific serum thereof, which can effectively diagnose and monitor the prevalence and outbreak of the serotype of Vibrio parahaemolyticus and is of great significance to the prevention, control and diagnosis and treatment of related diseases. Summary of the invention

[0004] The object of the present invention is to provide a novel O antigen of Vibrio parahaemolyticus, wherein the bacterium is Vibrio parahaemolyticus, the bacterium antigen is named O17, the classification name of the Vibrio parahaemolyticus is Vibrio parahaemolyticus O17:K4, the deposit number is CGMCC No.31706, and the DNA sequence of the novel Vibrio parahaemolyticus bacterium antigen O17 is shown in SEQ ID NO:1.

[0005] Determination of the novel bacterial antigen O17 strain provided by the present invention Vibrio parahaemolyticus was identified by matrix-assisted laser desorption ionization-time of flight (MALDI TOF) mass spectrometry or biochemical reaction. The serotype was determined by using the diagnostic serum of Vibrio parahaemolyticus (Denka Seiken, Tokyo, Japan) (containing 11 O and 69 K). The slide agglutination method was used to identify the strain as the O10K4 strain of Vibrio parahaemolyticus. The whole genome sequencing of O10:K4 Vibrio parahaemolyticus was performed, and its O / K antigen gene cluster was analyzed. It was found that the O antigen determinant cluster contained 17 genes with a total length of 16030 bp (see SEQ ID NO: 1), while the known O10 antigen determinant cluster contained 17 genes with a total length of 14977 bp. The sequence identity of the two antigen determinant clusters was only 64%; and the O antigen determinant cluster was also different from the sequences of the other 15 known O antigen determinant clusters except O10 (see Figure 1 ). It is therefore believed that the O antigen determinant cluster is a new bacterial antigen type of Vibrio parahaemolyticus and can be named O17. The Vibrio parahaemolyticus strain was deposited on October 31, 2024 at the General Microbiology Center of the China Culture Collection Administration (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with a deposit number of CGMCC No. 31706. The classification name recommended by the collection center is Vibrio parahaemolyticus O17K4.

[0006] Another object of the present invention is to provide a method for preparing the novel bacterial antigen O17 serum of Vibrio parahaemolyticus, which is achieved by the following steps: ① Antigen preparation The Vibrio parahaemolyticus strain with bacterial antigen O17 was suspended in 3% NaCl + 5% glycerol solution and inactivated under high pressure; washed twice with PBS and centrifuged to obtain the precipitate; the bacteria were resuspended in 0.9% saline solution and emulsified with Freund's adjuvant at a volume ratio of 1:1 to obtain the immune antigen; ②Collect immune serum Immune antigen (10 10CFU / ml), injected into 9-12 week-old New Zealand white rabbits, immunized in 6 sites (4 in the back and 2 in the groin), 200ul in each site. After the first immunization, booster immunization was performed every 2 weeks, and then weekly (same immunization dose each time). After 5 immunizations, whole blood was collected, placed at 37℃ for 2h, 4℃ overnight, centrifuged, and the supernatant was the immune serum; ③ Preparation of specific immune serum The immune serum is subjected to agglutination reaction with the novel bacterial antigen O17 Vibrio parahaemolyticus and other known O serotype Vibrio parahaemolyticus. If there is a cross reaction, the immune serum is adsorbed with known O serotype Vibrio parahaemolyticus that has been sterilized by high pressure (the strain is suspended in a 3% NaCl + 5% glycerol solution, inactivated by high pressure, and washed twice with PBS) at 4°C overnight, and the supernatant is the specific immune serum of the novel bacterial antigen O17 strain; ④Performance verification and application of specific immune serum The novel O17 serum has strong specificity, and it agglutinates significantly with the novel bacterial antigen O17 Vibrio parahaemolyticus, but does not agglutinate with the other 9 known O bacterial antigens Vibrio parahaemolyticus, and does not agglutinate with other intestinal pathogens Vibrio cholerae (non-O 1 O 139 group), Salmonella typhimurium, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri and Vibrio fluvialis, and did not agglutinate with intestinal colonizing bacteria Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae.

[0007] The new O17 serum has good sensitivity. When diluted to 64 times with normal saline, the serum still has obvious agglutination with the O17 strain.

[0008] The novel bacterial antigen O17 serum of Vibrio parahaemolyticus prepared by the present invention has strong specificity, can be used for the identification of the serotype strain, and is of great significance for the diagnosis and monitoring of Vibrio parahaemolyticus in foodborne diseases and infectious diarrhea.

[0009] The novel O17 serum provided by the present invention was used to perform a slide agglutination test on Vibrio parahaemolyticus from clinical acute diarrhea patients, and a total of 253 novel bacterial antigen O17 strains were identified. 100 strains were randomly selected for whole genome sequencing and O antigen determinant cluster analysis, and the sequencing results showed the same O antigen determinant cluster, which was consistent with the serum agglutination results.

[0010] Advantages and effects of the present invention: The invention has the advantages of providing a novel bacterial antigen (O17) of Vibrio parahaemolyticus and a method for preparing its specific serum. The novel O17 serum has strong specificity and can be conveniently and quickly used to detect a new and novel O serotype Vibrio parahaemolyticus that has become the most popular in recent years. It provides an important detection technology for the diagnosis, monitoring and prevention of pathogens of foodborne diseases and infectious diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the result of similarity comparison between the novel O17 antigenic determinant sequence and 16 known O antigenic determinant sequences.

[0012] Figure 2 It is the agglutination reaction between the immune serum of the novel bacterial antigen O17 Vibrio parahaemolyticus and the O17 and O10 strains. The left side of Figure A indicates that the novel bacterial antigen O17 Vibrio parahaemolyticus does not agglutinate with saline, which is a negative control, and the right side indicates that the novel bacterial antigen O17 Vibrio parahaemolyticus and the O17 immune serum have obvious agglutination phenomenon; the left side of Figure B indicates that the novel bacterial antigen O10 Vibrio parahaemolyticus does not agglutinate with saline, which is a negative control, and the right side indicates that the novel bacterial antigen O10 Vibrio parahaemolyticus and the O17 immune serum have cross-agglutination phenomenon.

[0013] Figure 3 It is the agglutination reaction between the new O17 specific immune serum and 10 known O serotype strains. Figure A shows the agglutination phenomenon between the O17 specific immune serum and different O antigen strains; Figure B shows the agglutination phenomenon between the O17 specific immune serum and different O antigen strains under a microscope.

[0014] Figure 4 It is the application of the new O17-specific immune serum. The slide agglutination test was performed with the prepared new O17-specific serum, and 253 strains of the new bacterial antigen O17 (O17K4) were identified among a total of 527 strains of Vibrio parahaemolyticus from 2019 to 2023. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only used to illustrate the present invention and are not restrictive, and the protection scope of the present invention cannot be limited by the following examples.

[0016] Example 1, determination of a novel bacterial antigen (O17) strain of Vibrio parahaemolyticus: Fecal specimens from patients with acute diarrhea were inoculated in alkaline peptone water, cultured at 37°C for 6 hours, and then transferred to TCBS medium. After incubation at 37°C for 18 hours, a single colony was selected and identified as Vibrio parahaemolyticus using a Bruker matrix-assisted laser desorption ionization-time of flight (MALDITOF) mass spectrometer. The Vibrio parahaemolyticus diagnostic serum (containing 11 O and 69 K) from Japan Seiken was used, and normal saline was used as a negative control to determine the serotype of Vibrio parahaemolyticus by slide agglutination method. 30 strains of Vibrio parahaemolyticus of serotype O10K4 were selected for whole genome sequencing, and the strains' O antigen determinants were analyzed, and the sequences were all identical (see sequence 1). The O antigen determinant of this strain is only 64% similar to the known O10 antigen determinant, and is different from the other 15 known O antigen determinant sequences except O10, so it can be named O17. The similarity comparison results of the novel O17 antigen determinant sequence with the 16 known O antigen determinant sequences are shown in Figure 1 It can be seen that O17 is a new type of bacterial antigen of Vibrio parahaemolyticus.

[0017] Example 2 Detailed process of serum preparation of a new type of bacterial antigen O17 of Vibrio parahaemolyticus: Antigen preparation: The novel bacterial antigen O17 Vibrio parahaemolyticus was inoculated into 3% NaCl BHI medium, cultured at 37°C overnight, and then collected by centrifugation at 4500×g for 30 min. The cells were washed twice with PBS, resuspended in 3% NaCl + 5% glycerol solution and inactivated under high pressure, washed twice with PBS, and centrifuged at 4500×g for 30 min. The collected cells were resuspended in physiological saline solution for immunization of rabbits.

[0018] Immunization of rabbits: Select three healthy New Zealand white rabbits aged 9-12 weeks and allow them to acclimate to the environment for 1-2 weeks. Then, resuspend the bacterial antigen (10 10 CFU / ml) was emulsified with Freund's adjuvant at a volume ratio of 1:1, and each rabbit was immunized at 6 sites (4 in the back and 2 in the groin), with 200ul in each site. After the first immunization, booster immunization was performed at intervals of 2 weeks, and then weekly immunization was performed with the same immunization dose as the first immunization. After 5 immunizations, whole blood was collected by cardiac blood sampling.

[0019] Collect immune serum: After the collected whole blood is placed at 37°C for 2 hours, it is transferred to 4°C overnight. The next day, it is centrifuged at 12000×g for 10 minutes to collect the supernatant, which is the immune serum.

[0020] Preparation of specific immune serum: The collected immune serum was subjected to slide agglutination reaction with the novel bacterial antigen O17 strain. The specific operation steps of slide agglutination are as follows: Use a marker pen to divide the slide into sections and lines, and add 1 drop (about 5 μL) of the prepared novel O17 immune serum to each section. In one section, physiological saline was added instead of the novel O17 serum as a control to exclude self-agglutination. Then, 5 μL of bacterial suspension was added to each section, mixed thoroughly, and after 2 minutes of sufficient reaction, observed whether there was agglutination. The collected immune serum and the novel bacterial antigen O17 strain had a very obvious agglutination phenomenon ( Figure 2 A). Considering that the novel bacterial antigen O17 strain agglutinated with the Japanese Seiken Vibrio parahaemolyticus diagnostic serum O10 (previously reported as the O10K4 strain), this study conducted a slide agglutination test with the prepared O17 immune serum and bacterial antigen O10 Vibrio parahaemolyticus. The results showed that the O17 immune serum and bacterial antigen O10 strain had a cross-agglutination reaction ( Figure 2 B). Select bacterial antigen O10 Vibrio parahaemolyticus, suspend it in 3% NaCl + 5% glycerol solution, inactivate it under high pressure, wash it twice with PBS, and centrifuge to obtain the precipitate to obtain O10 bacterial antigen. Use freshly prepared O10 bacterial antigen to adsorb O17 immune serum overnight at 4°C, and centrifuge it at 12000×g for 10 minutes to obtain the supernatant, which is the specific immune serum (O17 serum) of the new bacterial antigen O17 Vibrio parahaemolyticus.

[0021] Example 3 Performance of the novel O17-specific serum: The prepared Vibrio parahaemolyticus O17-specific serum and the novel bacterial antigen O17 strain can quickly detect the novel O17 serotype strain through agglutination; no agglutination occurs with 9 known O serotypes of Vibrio parahaemolyticus ( Figure 3 , Table 1); and other enteric pathogens Vibrio cholerae (non-O 1 O 139 The O17 serum did not agglutinate with the new bacterial antigen O17 strain (Table 3). The new O17 serum had good specificity and sensitivity.

[0022] Table 1 Agglutination of novel O17-specific sera with different O serotypes of Vibrio parahaemolyticus

[0023] Table 2 Agglutination of novel O17-specific sera with other intestinal pathogens

[0024] Table 3 The dilution ratio of novel O17-specific serum and the agglutination of bacterial antigen O17 Vibrio parahaemolyticus

[0025] Example 4 Application of novel O17 specific serum The inventors have been conducting long-term research on the variation of Vibrio parahaemolyticus from patients with acute infectious diarrhea and established a library of Vibrio parahaemolyticus strains. The slide agglutination experiment was performed using the prepared new O17-specific serum. Among a total of 527 strains of Vibrio parahaemolyticus from 2019 to 2023, 253 strains of Vibrio parahaemolyticus with a new bacterial antigen O17 (O17K4) were identified ( Figure 4 ). Whole genome sequencing was performed on 100 of the strains, and the O antigen determinant cluster was analyzed. The results showed that all of them were novel O17 antigen determinants, that is, they were all novel bacterial antigen O17 Vibrio parahaemolyticus. If the novel O17-specific serum provided by the present invention is not used, it can only be identified by whole genome sequencing of bacteria. The whole genome sequencing method is costly and requires a detection cycle of 3-4 weeks, which is not suitable for routine clinical laboratory use. Therefore, the novel O17-specific serum can quickly and easily identify the novel bacterial antigen O17 Vibrio parahaemolyticus.

Claims

1. A novel O antigen of Vibrio parahaemolyticus, characterized in that: The bacterial antigen of Vibrio parahaemolyticus is named O17, the classification name of Vibrio parahaemolyticus is Vibrio parahaemolyticus O17:K4, the preservation number is CGMCC No.31706, and the DNA sequence of the bacterial antigen O17 of Vibrio parahaemolyticus is shown in SEQ ID NO:

1.

2. The method for preparing a novel O antigen O17 serum of Vibrio parahaemolyticus according to claim 1 is achieved by the following steps: ① Antigen preparation The Vibrio parahaemolyticus strain with bacterial antigen O17 was suspended in 3% NaCl + 5% glycerol solution and inactivated under high pressure; washed twice with PBS and centrifuged to obtain the precipitate; the bacteria were resuspended in 0.9% saline solution and emulsified with Freund's adjuvant at a volume ratio of 1:1 to obtain the immune antigen; ②Collect immune serum Immune antigen 10 10 CFU / ml, injected into New Zealand white rabbits of 9-12 weeks old, immunized 6 parts of each rabbit, 200ul of each part, boosted immunization 2 weeks after the first immunization, and then immunized weekly, each immunization dose was the same, after 5 immunizations, whole blood was collected, placed at 37℃ for 2h, 4℃ overnight, centrifuged, and the supernatant was the immune serum; ③ Preparation of specific immune serum The immune serum is subjected to agglutination reaction with the bacterial antigen O17 Vibrio parahaemolyticus and other known O serotype Vibrio parahaemolyticus. If there is a cross reaction, the immune serum is adsorbed with known O serotype Vibrio parahaemolyticus sterilized by high pressure at 4°C overnight, and the supernatant is the specific immune serum of the bacterial antigen O17 strain.

3. The preparation method according to claim 2, characterized in that: The high-pressure sterilization treatment is as follows: the strain is suspended in a 3% NaCl + 5% glycerol solution, inactivated under high pressure, and washed twice with PBS.

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