CN116486901A - Method for screening aeromonas hydrophila protective antigen based on reverse vaccinology technology - Google Patents
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Abstract
The invention discloses a method for screening aeromonas hydrophila protective antigen based on reverse vaccinology technology, which comprises the following steps of S1: acquiring a whole genome nucleotide coding sequence of pathogenic aeromonas hydrophila; s2: predicting the adhesiveness and antigenicity of outer membrane proteins by using online software, screening out proteins exceeding a specified threshold, and then carrying out inter-species homology comparison between proteins, and screening out outer membrane proteins with good antigenicity, adhesiveness and high inter-species conservation as candidate antigens; s3: and (3) performing outer membrane protein conservation verification, namely detecting the distribution condition of main outer membrane protein coding genes in aeromonas hydrophila of different genotypes by adopting PCR and sequencing, and detecting the sequence similarity of the main outer membrane protein coding genes. Screening and evaluating potential aeromonas hydrophila outer membrane proteins as candidate antigens by bioinformatics using genomic data; and detecting the distribution condition of the main outer membrane protein coding genes in aeromonas hydrophila of different genotypes by adopting PCR and sequencing, and detecting the sequence conservation of the main outer membrane protein coding genes.
Description
Technical Field
The invention relates to the field of medicine, in particular to a method for screening aeromonas hydrophila protective antigen based on reverse vaccinology technology.
Background
Currently, research on aeromonas hydrophila vaccines mainly comprises inactivated vaccines, attenuated vaccines, recombinant subunit vaccines and the like. The preparation of the aeromonas hydrophila inactivated vaccine comprises the steps of firstly extracting pathogenic aeromonas hydrophila strains from diseased fish, selecting optimal conditions for culturing and inactivating after rejuvenation, and preparing the vaccine after sterile inspection and safety inspection are qualified. Although the inactivated vaccine has an immune protection effect on specific strains, the inactivated vaccine cannot achieve a better immune cross protection effect on strains with different serotypes. And the whole bacterial cells are used as antigens, so that the components are complex, and the body is easy to generate antibodies containing irrelevant antigens, thereby causing adverse reactions.
The attenuated vaccine of aeromonas hydrophila is prepared by changing culture conditions or passaging in a heterogeneous animal body to weaken the toxicity of a pathogen or removing a certain fragment of a virulence related gene in the genome of the pathogen by utilizing a genetic engineering technology to make the fragment become a defective pathogen. The disadvantage is that pathogenic bacteria may spread in the water and virulence reversion occurs.
The development of traditional vaccines has problems such as safety problems or lack of proper culture conditions for some pathogens, which has led to the shift of the focus of research on vaccines towards subunit vaccines. Subunit vaccines are vaccines made by extracting certain components of pathogenic microorganisms, such as outer membrane proteins of bacteria, lipopolysaccharides, toxoids, exotoxins, etc. The outer membrane protein of aeromonas hydrophila has good immunogenicity and cross-protection, so that the outer membrane protein becomes one of the candidate immune antigen components with the most potential of the recombinant subunit vaccine. The traditional method for screening subunit vaccine candidate antigens mostly relies on empirical screening, and needs to select possible proteins according to previous experience based on a large amount of researches on pathogens, and determine proper antigens through separation, purification, identification and testing. It is time consuming and often only one or two antigens can be studied, with low efficiency. At present, vaccine antigens are screened based on immune proteomics, namely two-dimensional gel electrophoresis, immunoblotting and mass spectrometry identification technologies are utilized, time and effort are wasted, the methods are rarely used for detecting immunogenic proteins on a large scale, and a method for screening aeromonas hydrophila protective antigens based on reverse vaccinology technology is provided.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and provides a method for screening aeromonas hydrophila protective antigen based on reverse vaccinology technology so as to solve the problems of the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions: the method for screening the aeromonas hydrophila protective antigen based on the reverse vaccinology technology comprises the following specific steps:
s1: acquiring a whole genome nucleotide coding sequence of pathogenic aeromonas hydrophila, screening and analyzing genome coding information of a target aeromonas hydrophila J-1 strain, and finding out genes which can be coded into potential antigens in pathogens through a bioinformatics analysis algorithm; the signal peptide prediction, the transmembrane helix structure prediction and the subcellular localization of the protein are carried out by utilizing online software to screen out outer membrane proteins;
s2: predicting the adhesiveness and antigenicity of outer membrane proteins by using online software, screening out proteins exceeding a specified threshold, and then carrying out inter-species homology comparison between proteins, and screening out outer membrane proteins with good antigenicity, adhesiveness and high inter-species conservation as candidate antigens;
s3: and (3) performing outer membrane protein conservation verification, namely detecting the distribution condition of main outer membrane protein coding genes in aeromonas hydrophila of different genotypes by adopting PCR and sequencing, and detecting the sequence similarity of the main outer membrane protein coding genes.
As a preferred embodiment of the present invention, the signal peptide of the protein in S1 predicts: signal peptide predictions were performed on the collected nucleotide-encoded protein sequences using SignalP 5.0 (http:// www.cbs.dtu.dk/services/SignalP) online websites, only proteins with signal peptides remaining were recorded.
As a preferred technical scheme of the invention, the transmembrane helix structure in S1 is predicted by: protein transmembrane helix structures screened in signal peptides of proteins were predicted using TMHMM 2.0 (http:// www.cbs.dtu.dk/services/TMHMM /) and HMMTOP (http:// www.enzim.hu/HMMTOP /); the protein with the number less than or equal to 2 of the transmembrane helix structure is recorded and reserved, and the transmembrane helix of the protein is predicted by using two pieces of software, so that the result is more accurate, and the smaller the number of transmembrane helices in the protein is, the smaller the expression and preparation difficulty of the recombinant protein is.
As a preferred embodiment of the present invention, the subcellular localization in S1: subcellular localization of proteins screened in transmembrane helices was predicted using PSORTb (https:// www.psort.org /), CELLO (http:// CELLO. Life. Nctu. Edu. Tw /) and Gneg-mPLOC (http:// www.csbio.sjtu.edu.cn/bionf/Gneg-multi /), and the record was kept of outer membrane proteins.
As a preferable technical scheme of the invention, the adhesion and antigenicity of the outer membrane proteins are predicted by using online software in the step S2, wherein the antigenicity is evaluated by adopting VaxiJen software to evaluate the potential antigenicity of the outer membrane proteins screened by subcellular localization, the cut-off value is set to be 0.4, and the proteins with the reserved antigenicity more than or equal to 0.4 are recorded.
As a preferable technical scheme of the invention, the adhesion and antigenicity of the outer membrane protein are predicted by using online software in the S2, wherein the adhesion is evaluated by using Vaxign online software, and the adhesion of the outer membrane protein is predicted according to the standard that the adhesion index is more than 0.5.
As a preferable technical scheme of the invention, the S2 protein is subjected to intraspecies homology comparison: the final screened proteins were subjected to intraspecies BLAST sequence alignment.
As a preferred technical scheme of the invention, the specific steps of the S3 for carrying out the outer membrane protein conservation verification are as follows:
s31: designing corresponding primers according to the screened outer membrane protein sequences, and carrying out PCR amplification and sequencing by taking genome DNA of aeromonas hydrophila strains with different genotypes as templates;
s32: and (3) analyzing the sequencing result, and keeping the similarity value of the protein sequence coverage rate exceeding 80%, and judging the conservation of the protein on the epidemic aeromonas hydrophila strains and the aeromonas bacteria in the database according to the value, wherein the higher the value is, the better the conservation of the outer membrane protein is.
As a preferred embodiment of the present invention, PCR amplification of the CDS region of the candidate protein in S31: and designing a specific primer according to the CDS region sequence of the candidate protein, taking the genome DNA of the aeromonas hydrophila of different gene subtypes as templates, adjusting a PCR reaction system and a reaction program to obtain clear electrophoresis strips, detecting the PCR products by 1.5% agarose gel electrophoresis, and then sequencing.
As a preferable technical scheme of the invention, the analysis sequencing result in the S32 is that BLAST protein sequence comparison is adopted, DNAStar software is utilized to analyze the full-length sequence of the sequencing strain and find out ORFs, 39 candidate protein sequences of the reference strain are compared with BLAST protein sequences of the same genus bacteria in the sequencing strain and NR database, and consistency data with protein sequence coverage rate more than 80% are reserved and recorded.
Identification of pathogenic aeromonas hydrophila:
16S rDNA gene-specific primers: 16S rDNA F:5'-AGAGTTTGATCATGGCTCAG-3';16S rDNA R:5'-GGTTACCTTGTTACGACTT-3'.
The conserved region of the 16S rDNA gene was PCR amplified using the laboratory-collected bacterial genomic DNA of 10 suspected Aeromonas hydrophila strains as a template, followed by sequencing. Finally, full-length sequences were assembled using DNAStar software (http:// www.dnastar.com) and the like, and sequence alignment was performed by BLAST. 16S rDNA is a gene encoding 16S rRNA from prokaryotes, approximately 1500bp in length, composed of 10 conserved regions and 9 variable regions (V1-V9) reflecting the differences between species.
Enterobacter intergenic repeat consensus (ERIC) -PCR typing:
using universal primers, ERIC F:5'-ATGTAAGCTCCTGGGGGATTCAC-3'; ERIC R:5'-AAGTAAGTACTGGGGGGTGAGCG-3' ERIC-PCR was performed using 9 aeromonas hydrophila genomic DNAs as templates. And adjusting the PCR reaction system and the reaction program to obtain clear electrophoresis bands. And detecting the amplified ERIC-PCR product by 1.5% agarose gel electrophoresis, and photographing to obtain the aeromonas hydrophila ERIC fingerprint. Analyzing aeromonas hydrophila ERIC fingerprint by using quality One v4.6.0 software, calculating and recording the molecular weight of each strip according to a Marker, and generating an electrophoresis schematic diagram, wherein the analysis parameters are as follows: sensitivity 8.0,Width2.5,Min Dens 0.0,Filter4.0,Shoulder1.0,Size 5.0. The amplified bands were recorded as "1" when present and "0" when not present. The genetic similarity matrix and the distance matrix are obtained by NTSYS-pc2.1 software, and the fingerprint is subjected to cluster analysis by adopting a non-weighted pairing average method (UPGMA). The similarity is equal to or greater than 0.8, and the similarity is different genotypes.
The beneficial effects of the invention are as follows: the invention screens 39 candidate proteins with higher homology in aeromonas based on reverse vaccinology, verifies the conservation of the candidate proteins among different subtype aeromonas hydrophila and main pathogenic bacteria of aeromonas hydrophila through PCR and sequencing, and provides materials for preparing broad-spectrum subunit vaccines capable of providing cross protection for various serotypes of aeromonas hydrophila. The invention starts from the whole genome sequence of aeromonas hydrophila J-1 strain, is quite different from the traditional vaccine development thought, does not need to culture pathogens in vitro, and avoids the diffusion of the pathogens; all outer membrane proteins of the pathogen are analyzed and predicted, so that the screening range is increased, and candidate antigens with partial neglect can be screened; the vaccine target of the pathogen can be directly predicted, the screening time of the optimal antigen is greatly shortened, the subsequent targeted experiment is convenient, and the time, the labor and the cost are saved.
Drawings
Fig. 1 is: predicting subcellular localization of candidate proteins;
note that: a: prediction of PSORTb; b: prediction results of CELLO; c: predictive outcome of Gneg-mPLOC; d: cross prediction results of three software.
Fig. 2 is: ERIC-PCR fingerprint and UPGMA cluster map of 10 strains.
Fig. 3 is: the heat map shows the identity and distribution of the 39 outer membrane protein sequences of 24 aeromonas and 6 sequenced strains in the NCBI database;
note that: the bottom is the name of the strain, and the left side is the name of the sequence protein; the threshold for protein presence among 30 strains is expressed as uniformity; yellow indicates a consistency of about 20%, red indicates about 60%, and black indicates about 100%; blank indicates the absence of outer membrane protein sequences or less than 80% protein sequence coverage.
FIG. 4 is a schematic diagram of bioinformatics analysis of the genome of Aeromonas hydrophila J-1 strain of the present invention.
Note that: downloading the whole genome nucleotide coding sequence of the aeromonas hydrophila J-1 strain from NCBI database, wherein the total number of the nucleotide coding sequences is 4268 (accession number: CP 006883.1); protein with signal peptide was predicted using SignalP 5.0; predicting transmembrane helix structure using TMHMM 2.0 and HMMTOP; subcellular localization of proteins was predicted using PSORTb, CELLO and Gneg-mPLOC; the potential antigenicity of the protein was assessed using VaxiJen, with a cut-off value set to 0.4; protein adhesion was assessed by Vaxign with an adhesion index greater than 0.5; BLAST sequence alignment to assess candidate protein homology for aeromonas bacteria.
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the attached drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making clear and defining the scope of the present invention.
Examples: as shown in fig. 1-4, the present invention provides a technical solution: the method for screening the aeromonas hydrophila protective antigen based on the reverse vaccinology technology comprises the following specific steps:
s1: and (3) data collection: the full genome nucleotide coding sequence of the Aeromonas hydrophila J-1 strain in FASTA file format was downloaded from NCBI database, and 4268 sequences (accession number: CP 006883.1) were downloaded in total.
S2: signal peptide prediction: the 4268 protein sequences of aeromonas hydrophila J-1 strain were analyzed using SignalP 5.0 on-line software, with gram negative prokaryotes as default settings, to predict the presence of signal peptides. Results: the aeromonas hydrophila J-1 strain genome encodes a total of 4268 proteins, 678 proteins having a signal peptide in the n-terminal region. Among them, the number of proteins having a common secretory pathway (Sec) signal peptide, a double arginine translocation (TAT) signal peptide and a lipoprotein signal peptide was 479, 26 and 173, respectively.
S3: transmembrane helix structure prediction: the transmembrane helix structure of the protein encoded by the Vibrio hydrophila J-1 strain was predicted using TMHMM 2.0 and HMMTOP online software. The results showed that 466 proteins did not have a transmembrane protein alpha-helix and 158 proteins had an alpha-helix, indicating that the structure was predominantly beta-barrel. In a prokaryotic expression system, multiple transmembrane helices affect recombinant expression of the protein, thus eliminating 54 proteins with multiple transmembrane helices, leaving 624 proteins for further analysis.
S4: prediction of subcellular localization: subcellular localization of proteins smaller than two TM (transmembrane) helices was predicted on PSORTb, CELLO and Gneg-mPLoc 3 predicted websites, respectively. The results are shown in FIG. 1: the outer membrane region proteins of 624 aeromonas hydrophila were 66 (10.6%), 124 (19.9%) and 83 (13.3%) respectively. To balance the coverage and confidence of predictions, the 66 candidate outer membrane proteins that were finally screened need to meet the conditions of at least two predictive software (FIG. 1D).
S5: antigenicity evaluation: the VaxiJen software assessed the potential antigenicity of the candidate proteins, with a cutoff value set at 0.4 and 64 out of 66 proteins scored above the threshold (> 0.4), indicating that the identified outer membrane proteins are mostly potential antigenic proteins (table 1).
S6: adhesion evaluation: candidate vaccine antigens were predicted by Vaxign on-line software according to the criteria of adhesion index above 0.5, and 39 out of 66 outer membrane proteins were found to meet the criteria and were selected as candidate antigens for development of subunit vaccines (Table 1).
S7: homology analysis of protein sequences: to analyze the homology of candidate proteins in Aeromonas species, candidate protein sequences were compared with other 37 Aeromonas species, including A.allosaccharophila, A.besiarum, A.caviae, A.dhakensis, A.enchelea, A.eucrenofacilia, A.fluvilis, A.jandaei, A.lusitna, media, A.piscicola, A.popuffii, A.riviposupport, A.salmonicida, the results of sanarelli, a. Sobria, a. Taiwanensis, a. Tecta, a. Veronii, a. Aquatica, a. Aquatilis, a. Australis, a. Bivalviem, a. Castanicola, a. Crassostreae, a. Aversa, a. Enterica, a. Enteropelenies, a. Finlandensis, a. Lacus, a. Guaaghei, a. Hydropathila, a. Intestinalis, a. Moluscorum, a. Rivuli, a. Schubertii, anda. Siae are shown in table 1: 15 of the 66 candidate proteins exhibited high conservation in Aeromonas with an identification rate of greater than 85% and a query coverage of greater than 40%, including BamA, bamE and BamC (AJE 37434.1, AJE 37120.1), ompK (AJE 37487.1) (AJE 37342.1), tolC family outer membrane proteins, immunosuppressive protein A (AJE 37629.1), AJE 3858.1), and carbohydrate porins (AJE 35595. 1) Type IV bacteria Mao Fenbi, pilQ (AJE 35401.1), porin (AJE 38163.1). These proteins play an important role in protein secretion, transport and signal transduction and are involved in iron absorption by bacteria, thus meeting the survival and propagation demands of bacteria.
Table 1: identification of candidate proteins for development of Aeromonas subunit vaccine
S8: identification of Aeromonas hydrophila 16S rDNA: the conserved region of the 16SrDNA gene was amplified by PCR using the 16S rDNA gene specific primer and bacterial genomic DNA of 10 suspected aeromonas hydrophila strains collected in the laboratory as a template, followed by sequencing. Finally, full-length sequences were assembled using software such as DNAStar software, and sequence alignment was performed by BLAST. Results: the 9 strains have higher sequence similarity (99%) with aeromonas hydrophila, the 1 strains have higher sequence similarity (99%) with aeromonas veronii, and the results are consistent with Table 2.
Table 2: information of 10 strains
S9: ERIC-PCR typing of Aeromonas hydrophila: ERIC-PCR was performed using 9 Aeromonas hydrophila genomic DNA as a template with ERIC universal primers. The reaction system is as follows: the final volume of ERIC-PCR mixture was 20. Mu.L, including: mu.L of 10 Xbuffer, 0.5. Mu.L of dNTP mix, 1. Mu.L of each primer, 0.3. Mu.L of TaqDNA polymerase, 2. Mu.L of template DNA, and ddH 2 O adjusts the final volume to 20. Mu.L. The reaction procedure was as follows: pre-denaturation at 94 ℃ for 5min, denaturation, annealing, extension for 30 cycles: denaturation at 94℃for 1min, annealing at 52℃for 1min and extension at 68℃for 8min. Finally, the mixture is extended for 16min at 65 ℃. And detecting the amplified ERIC-PCR product by 1.5% agarose gel electrophoresis, and photographing to obtain the aeromonas hydrophila ERIC fingerprint. As shown in FIG. 2A, lanes 1-10 are respectively 10 strain fingerprints, and 3-5 bands with the molecular weight of 300-5000bp can be obtained from 10 strains through ERIC-PCR amplification. Most strains have obvious main bands at 1000-1500bp and 2000-3000 bp. Analyzing aeromonas hydrophila ERIC fingerprint by using quality One v4.6.0 software, calculating and recording the molecular weight of each strip according to a Marker, and generating an electrophoresis schematic diagram, wherein the analysis parameters are as follows: sensitivity 8.0,Width2.5,Min Dens 0.0,Filter4.0,Shoulder1.0,Size 5.0. The amplified bands were recorded as "1" when present and "0" when not present. The genetic similarity matrix and the distance matrix are obtained by NTSYS-pc2.1 software, and the fingerprint is subjected to cluster analysis by adopting a non-weighted pairing average method (UPGMA). Similarity is equal to or greater than 0.8, and is the same subtype<0.8 is different genotypes. As shown in FIG. 2B, 10 strains were inoculatedThe genotypes were classified into 7 genotypes. Wherein 9 aeromonas hydrophila strains are divided into 6 genotypes, and aeromonas veronii is an independent subtype.
S10: PCR amplification of candidate protein CDS region: PCR amplification of the target protein CDS region: specific primers were designed based on the candidate protein CDS region sequences as shown in table 3 below. 6 strains of aeromonas hydrophila genomic DNA with different gene subtypes are used as templates. The reaction system is as follows: the final volume of the PCR mixture was 25. Mu.L, including: 2. Mu.L of 10 Xbuffer, 0.5. Mu.L of dNTP mixture, 0.5. Mu.L of each primer, 0.3. Mu.L of Ex TaqDNA polymerase, 1. Mu.L of template DNA, and ddH 2 O adjusts the final volume to 25. Mu.L. The reaction procedure was as follows: pre-denaturation at 94 ℃ for 5min, denaturation, annealing, extension for 30 cycles: denaturation at 94℃for 1min, annealing at 56℃for 1min, extension at 72℃for 2min. Finally, the temperature is further increased by 72 ℃ for 10min. The PCR products were detected by 1.5% agarose gel electrophoresis and then sequenced.
Table 3:39 candidate protein primer sequences
S11: blast sequence alignment: the full length sequence of the sequenced strain was analyzed using DNAStar software and the ORF was found. The 39 candidate proteins of the aeromonas hydrophila J-1 strain were BLAST protein sequence aligned with the sequencing strains and aeromonas bacteria in the NR database (table 3). Only the identity data with protein sequence coverage greater than 80% were retained and recorded (fig. 3). The results show that 39 outer membrane proteins are screened out as vaccine candidate antigens based on reverse vaccinology, and all the candidate proteins exist in aeromonas hydrophila and aeromonas bacteria and have higher protein sequence consistency. Wherein 16 proteins have better conservation,
these are OmpA, ompK, tonB-dependent transferrin family receptor, outer membrane protein transporter, tolC family outer membrane protein, peptidoglycan dd-metallopeptidase family protein, TIGR04219 family outer membrane barrel protein, immunosuppressant A and DUF2860 family proteins, respectively. These results will provide a rich candidate antigen material for subsequent vaccine preparation.
Table 4: NCBI NR database 24 information on Aeromonas strains
Table 5: detection rate of 39 proteins in 30 Aeromonas bacteria (%)
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Note that: the values represent positive duty ratios of 39 proteins with protein sequence coverage of greater than 80% in 4 aeromonas bacteria (6 guinea pig aeromonas, 6 salmonicida aeromonas, 6 aeromonas veronii and 6 aeromonas hydrophila) and 6 sequencing strains of the NR database.
The invention is based on reverse vaccinology, uses a computer to carry out bioinformatics analysis on 4268 protein sequences coded by aeromonas hydrophila J-1 strain, can rapidly and efficiently screen out all candidate antigens possibly used as aeromonas hydrophila subunit vaccine of aeromonas hydrophila J-1 outer membrane protein, avoids the defects of time and labor consumption in traditional vaccine antigen screening, and can only study one and two antigens in general. The 39 vaccine candidate proteins are screened out totally by carrying out signal peptide prediction, transmembrane structure prediction, subcellular localization, antigenicity evaluation, adhesion evaluation and homology alignment on the protein sequence through an online website. Traditional vaccine antigen screening generally considers the surface immunogenicity of clinically prevalent strains, but differences between the different subtypes may lead to reduced vaccine accuracy. Aiming at the high interspecific heterogeneity of aeromonas hydrophila, the invention provides abundant alternative materials for preparing subunit vaccine with broad-spectrum immune protection effect, and the invention verifies the conservation of 39 proteins among different strains of aeromonas hydrophila and aeromonas (guinea pig aeromonas, salmon killing aeromonas, veroniona veronii) in different subtypes by PCR and sequencing, screens out the protein with high conservation, covers various epidemic strain subtypes, and is used for solving the problems that the traditional aeromonas hydrophila vaccine can only have immune protection effect on 1 or a few aeromonas hydrophila strains and has non-ideal immune protection effect on partial regions or fish-origin aeromonas hydrophila. The results showed that 39 candidate proteins were widely present in aeromonas hydrophila and aeromonas bacteria. Of these 16 proteins are highly conserved in Aeromonas hydrophila, which are OmpA, ompK, tonB dependent transferrin family receptor, outer membrane protein transporter, tolC family outer membrane protein, peptidoglycan dd-metallopeptidase family protein, TIGR04219 family outer membrane barrel protein, immunosuppressant A and DUF2860 family proteins, respectively. These results will provide a rich candidate antigen material for subsequent vaccine preparation.
Aeromonas hydrophila (Aeromonas hydrophila) is widely existing in fresh water, soil and silt, has a very wide pathogenic range, can infect different aquatic organisms such as pelteobagrus fulvidraco, crucian carp, megalobrama amblycephala, carp, chinese soft-shelled turtle and shrimp and the like, and causes septicemia, skin ulceration, enteritis, gill rot, histopathology and the like of the aquatic animals, so that the high pathogenicity and high death rate of the infected animals are caused, and the economic benefit of the aquaculture industry in China is seriously influenced. The application of the aeromonas hydrophila vaccine effectively prevents the large-scale epidemic of diseases caused by aeromonas hydrophila. However, the serotypes of Aeromonas hydrophila are numerous, and the immunoprotection effect of the existing inactivated vaccine on 1 or a few Aeromonas hydrophila strains is not ideal on partial areas or fish-origin Aeromonas hydrophila, and the broad-spectrum immunoprotection effect is yet to be examined. Therefore, development of broad-spectrum subunit vaccines applicable to aeromonas hydrophila of different serotypes is needed to reduce economic losses caused by diseases caused by aeromonas hydrophila infection, and screening of antigens with cross-protection is a key to the preparation of broad-spectrum protective vaccines. The invention screens 39 candidate proteins with higher homology in aeromonas based on reverse vaccinology, verifies the conservation of the candidate proteins among different subtype aeromonas hydrophila and main pathogenic bacteria of aeromonas hydrophila through PCR and sequencing, and provides materials for preparing broad-spectrum subunit vaccines capable of providing cross protection for various serotypes of aeromonas hydrophila. The invention starts from the whole genome sequence of aeromonas hydrophila J-1 strain, is quite different from the traditional vaccine development thought, does not need to culture pathogens in vitro, and avoids the diffusion of the pathogens; all outer membrane proteins of the pathogen are analyzed and predicted, so that the screening range is increased, and candidate antigens with partial neglect can be screened; the vaccine target of the pathogen can be directly predicted, the screening time of the optimal antigen is greatly shortened, the subsequent targeted experiment is convenient, and the time, the labor and the cost are saved.
The foregoing examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (10)
1. The method for screening the aeromonas hydrophila protective antigen based on the reverse vaccinology technology is characterized by comprising the following steps of: the method comprises the following specific steps:
s1: acquiring a whole genome nucleotide coding sequence of pathogenic aeromonas hydrophila, screening and analyzing genome coding information of a target aeromonas hydrophila J-1 strain, and finding out genes which can be coded into potential antigens in pathogens through a bioinformatics analysis algorithm; the signal peptide prediction, the transmembrane helix structure prediction and the subcellular localization of the protein are carried out by utilizing online software to screen out outer membrane proteins;
s2: predicting the adhesiveness and antigenicity of outer membrane proteins by using online software, screening out proteins exceeding a specified threshold, and then carrying out inter-species homology comparison between proteins, and screening out outer membrane proteins with good antigenicity, adhesiveness and high inter-species conservation as candidate antigens;
s3: and (3) performing outer membrane protein conservation verification, namely detecting the distribution condition of main outer membrane protein coding genes in aeromonas hydrophila of different genotypes by adopting PCR and sequencing, and detecting the sequence similarity of the main outer membrane protein coding genes.
2. The method for screening for protective antigens of aeromonas hydrophila based on reverse vaccinology techniques of claim 1, wherein: signal peptide prediction of the protein in S1: signal peptide predictions were performed on the collected nucleotide-encoded protein sequences using SignalP 5.0 (http:// www.cbs.dtu.dk/services/SignalP) online websites, only proteins with signal peptides remaining were recorded.
3. The method for screening for protective antigens of aeromonas hydrophila based on reverse vaccinology techniques of claim 1, wherein: the transmembrane helix structure prediction in S1: TMHMM 2.0 (http:// www.cbs.dtu.dk/services/TMHMM /) and HMMTOP are used
(http:// www.enzim.hu/hmmtop /) predicting the protein transmembrane helix structure screened in the signal peptide of the protein; the protein with the number less than or equal to 2 of the transmembrane helix structure is recorded and reserved, and the transmembrane helix of the protein is predicted by using two pieces of software, so that the result is more accurate, and the smaller the number of transmembrane helices in the protein is, the smaller the expression and preparation difficulty of the recombinant protein is.
4. The method for screening for protective antigens of aeromonas hydrophila based on reverse vaccinology techniques of claim 1, wherein: subcellular localization in S1: subcellular localization of proteins screened in transmembrane helices was predicted using PSORTb (https:// www.psort.org /), CELLO (http:// CELLO. Life. Nctu. Edu. Tw /) and Gneg-mPLOC (http:// www.csbio.sjtu.edu.cn/bionf/Gneg-multi /), and the record was kept of outer membrane proteins.
5. The method for screening for protective antigens of aeromonas hydrophila based on reverse vaccinology techniques of claim 1, wherein: in the S2, the adhesiveness and antigenicity of the outer membrane proteins are predicted by using online software, wherein the antigenicity is evaluated by adopting VaxiJen software to evaluate the potential antigenicity of the outer membrane proteins screened by subcellular localization, the cut-off value is set to be 0.4, and the proteins with the antigenicity more than or equal to 0.4 are recorded.
6. The method for screening for protective antigens of aeromonas hydrophila based on reverse vaccinology techniques of claim 1, wherein: and in the step S2, the adhesion and antigenicity of the outer membrane proteins are predicted by using online software, wherein the adhesion is evaluated by using Vaxign online software, and the adhesion of the outer membrane proteins is predicted according to the standard that the adhesion index is more than 0.5.
7. The method for screening for protective antigens of aeromonas hydrophila based on reverse vaccinology techniques of claim 1, wherein: the S2 is used for the inter-species homology comparison of the protein: the final screened proteins were subjected to intraspecies BLAST sequence alignment.
8. The method for screening for protective antigens of aeromonas hydrophila based on reverse vaccinology techniques of claim 1, wherein: the specific steps of the S3 for carrying out the outer membrane protein conservation verification are as follows:
s31: designing corresponding primers according to the screened outer membrane protein sequences, and carrying out PCR amplification and sequencing by taking genome DNA of aeromonas hydrophila strains with different genotypes as templates;
s32: and (3) analyzing the sequencing result, and keeping the similarity value of the protein sequence coverage rate exceeding 80%, and judging the conservation of the protein on the epidemic aeromonas hydrophila strains and the aeromonas bacteria in the database according to the value, wherein the higher the value is, the better the conservation of the outer membrane protein is.
9. The method of screening for protective antigens of aeromonas hydrophila based on reverse vaccinology techniques of claim 8, wherein: PCR amplification of candidate protein CDS region in S31: and designing a specific primer according to the CDS region sequence of the candidate protein, taking the genome DNA of the aeromonas hydrophila of different gene subtypes as templates, adjusting a PCR reaction system and a reaction program to obtain clear electrophoresis strips, detecting the PCR products by 1.5% agarose gel electrophoresis, and then sequencing.
10. The method of screening for protective antigens of aeromonas hydrophila based on reverse vaccinology techniques of claim 8, wherein: and the analysis sequencing result in the S32 is that BLAST protein sequence comparison is adopted, DNAStar software is utilized to analyze the full-length sequence of the sequencing strain and find out ORFs, 39 candidate protein sequences of the reference strain are compared with the BLAST protein sequences of the same genus bacteria in the sequencing strain and NR database, and the consistency data with the protein sequence coverage rate of more than 80% are reserved and recorded.
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