Cryptocaryon irritans inhibition antigen bigeminy recombinant protein as well as preparation method and application thereof

Abstract

The invention discloses cryptocaryon irritans inhibition antigen bigeminy recombinant protein as well as a preparation method and application thereof. The amino acid sequence of the cryptocaryon irritans inhibition antigen bigeminy recombinant protein is as shown in SEQ ID NO: 1. By means of terminator replacement and codon optimization and insertion of the Linker protein of the 15 peptide composed of Gly and Ser between the two activity-inhibiting antigen proteins, the situation that the two activity-inhibiting antigen recombinant proteins possibly interfere with each other in the spatial structure, and the integrity of the antigen epitopes of the two activity-inhibiting antigen recombinant proteins is affected is avoided; the defect that a single inhibition antigen provides immune protection for cryptocaryon irritans infection is overcome. The cryptocaryon irritans inhibition antigen bigeminy recombinant protein can be used for preparing vaccines, medicines or immune preparations for preventing and treating cryptocaryon irritans diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Cryptocaryon irritans inhibitory antigen binary recombinant protein and a preparation method and application thereof. Background Art

[0002] Cryptocaryon irritans (commonly known as marine ichthyophthirius) can infect and parasitize the epithelial tissues of the skin, fins, eyes and tongue of most marine cultured fish, such as grouper, golden pomfret, American red fish, large yellow croaker, Atlantic salmon, sharp nose perch, purple snapper, oriental pufferfish, sea bass, bluefish, white pepper snapper, red sea bream, black sea bream, flat sea bream, red snapper, etc., and can cause damage and dysfunction of fish organs, abnormal fish activities, epithelial hyperplasia, breathing difficulties and mechanical damage secondary to bacterial infection, causing great harm to the marine aquaculture industry. After being infected with Cryptocaryon irritans, the body surface of the fish shows obvious "white spot" symptoms, so it is commonly known as "white spot disease". At present, the prevention and control measures for Cryptocaryon irritans mainly adopt chemical methods and physical methods. The chemical method mainly uses copper sulfate and ferrous sulfate mixture, formaldehyde, antiprotozoal drugs such as quinoline derivatives, potassium permanganate, sodium hypochlorite, sulfamethoxazole, penicillin, etc. Physical methods mainly include freshwater immersion, temperature control, drying, rotational breeding, long-term sedimentation or the use of ultraviolet rays and ozone to treat aquaculture water, etc. These methods can slow down the progression of Cryptocaryon irritans to a certain extent and play a certain role in prevention and treatment, but in actual production, they also face problems such as unsuitability for large water bodies, uncertain effects, toxicity of chemical drugs to fish, pollution to the environment, and human health caused by drug residues.

[0003] Vaccination has become one of the most important methods for the prevention and treatment of "white spot disease" due to its high safety and few side effects. At present, there are two main aspects of "white spot disease" vaccine research: whole worm vaccine and recombinant vaccine. Since the in vitro culture method of the worm has not yet been established, the cost of obtaining the worm body through artificial infection of the host is high, and it is difficult to prepare the whole worm vaccine of Cryptocaryon irritans in batches. Therefore, the development of a subunit vaccine for the worm is a good choice for achieving immune prevention and control of Cryptocaryon irritans disease. The screening of antigens is the key to the development of subunit vaccines for Cryptocaryon irritans. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art, provide a Cryptocaryon irritans inhibitory antigen dual recombinant protein and its preparation method and application, and solve the problems in the above-mentioned background technology.

[0005] One of the technical solutions adopted by the present invention to solve the technical problem is:

[0006] Provided is a Cryptocaryon irritans inhibitory antigen binary recombinant protein, the amino acid sequence of the Cryptocaryon irritans inhibitory antigen binary recombinant protein is shown in SEQ ID NO:1.

[0007] SEQ ID NO: 1:

[0008] MAWAPKTAAADWKGTFVVTKSSCLATCGWKIGTTIVIADKTGDSSKVTWMGTVHTTDSTNVDVVSGSCKYVSVVATAGTAGTPAEVLKNSDECAFATGTCGIMGRKQSTPGTVTFNRDTTLDTKPLQILYKQFAMVQKTSTTQKATATDQAAN CDTQASFVDTTTDAKAIVGSLKLSKASCDKCSWDTTKDLKITQDASKKYMVTLAGTIKETATGDCNNKLTASEACYVTKKDDKTYVLVSCATLDTTSKGIPIAITTANSKTTLTMTWTDSASKACSVVGEVTSSSNSVKLISGFSAMLILSLAL LFKGGGGSGGGGSGGGGSNWVEKTAIADWKGTFVVTKSSCLATCGWKIGSTIVIAEKTGDNTKVTWVGTVHTTDATNVDVASGSCKYVSVVAAAGTPGTAAEVLKNDDECVFATGMCTIMGQKQTKPAKITFNRDTTLDTKPLQVLYKQFEMVQ KSSTTQKAAATDQAADCDTQASMVDTTTDAKAIVGTLKLSKAVCNKCSWDTTKDLKITQDATNKYMVTLAGTIKETTSGDCKNKLTASEACYVTKKDDKTFILVSCTLDTTNKGIPIAITTANSKTTLTLTRTDSSSQACNVVGEITHHHHHH

[0009] The second technical solution adopted by the present invention to solve its technical problem is: to provide a gene encoding a binary recombinant protein of Cryptocaryon irritans inhibitory antigen, and the nucleotide sequence of the gene encoding a binary recombinant protein of Cryptocaryon irritans inhibitory antigen is shown in SEQ ID NO: 2.

[0010] SEQ ID NO: 2:

[0011]

[0012] The third technical solution adopted by the present invention to solve its technical problem is:

[0013] A method for preparing the above Cryptocaryon irritans inhibitory antigen dual recombinant protein is provided, comprising the following steps:

[0014] S1. extracting the mRNA of the ciliate stage of Cryptocaryon irritans, reverse-transcribing and synthesizing cDNA templates, screening and obtaining two Cryptocaryon irritans inhibitory antigen coding sequences with conserved antigenic epitopes, designing specific primers, cloning and sequencing using cDNA as a template to obtain the coding sequences of two inhibitory antigen genes;

[0015] S2. Analyze the coding sequences of the two inhibitory antigen genes obtained, find out the stop codons TAA and TAG contained in the coding sequences, replace them with codons CAA and CAG that can encode glutamine, and optimize the codons suitable for prokaryotic cell expression, design a linker protein polypeptide composed of glycine and serine, optimize the coding sequences for prokaryotic cell expression codons, and then connect the two inhibitory antigen molecules;

[0016] S3. The coding sequence of Cryptocaryon irritans binary inhibitory antigen connected by Linker protein molecules, replaced by terminators and optimized by codons was synthesized by whole gene, and the coding sequence encoding 6 His amino acids was added to the end, and the optimized Cryptocaryon irritans inhibitory antigen binary recombinant protein coding sequence was inserted into the expression vector to construct a recombinant expression vector, and the accuracy of the final expression vector was confirmed by enzyme digestion and sequencing;

[0017] S4. After the recombinant expression vector is transformed into the Escherichia coli expression strain, it is cultured and placed under conditions not exceeding 15°C and 37°C to induce expression, protein gel electrophoresis and decolorization are performed to obtain the Cryptocaryon irritans inhibitory antigen dual recombinant protein.

[0018] Preferably, the expression vector in step S3 is any one of the pET series, PGEX series or pMAL series.

[0019] Furthermore, the expression vector in step S3 is any one of pET-28a, pET-30a, and pET-32a.

[0020] Furthermore, the expression vector in step S3 is pGEX-4T or pGEX-6P.

[0021] Furthermore, the expression vector in step S3 is pMAL-p2x or pMAL-c2X.

[0022] Preferably, the E. coli expression strain in step S4 is BL21 or DE3.

[0023] The fourth technical solution adopted by the present invention to solve its technical problem is: providing the above-mentioned Cryptocaryon irritans inhibitory antigen binary recombinant protein for preparing vaccines, drugs or immune preparations for preventing and treating Cryptocaryon irritans disease.

[0024] The life cycle of Cryptocaryon irritans can be divided into the trophozoite stage, cyst stage, and ciliated larval stage. We have constructed a transcriptome database of the trophozoite stage, cyst stage, and ciliated larval stage of Cryptocaryon irritans, and obtained the inhibitory antigen encoding gene library of the larval stage with the strongest infectivity through bioinformatics analysis. Through antigenic determinant immunogenicity analysis, multiple inhibitory antigen encoding sequences with conserved antigenic epitopes have been screened. Interestingly, the inhibitory antigen encoding gene of Cryptocaryon irritans is extremely special, using the common stop codons TAA and TAG to encode glutamine, and the inhibitory antigen encoding gene of Cryptocaryon irritans generally has multiple TAA or TAG. Therefore, the commonly used prokaryotic system cannot directly express the inhibitory antigen gene of Cryptocaryon irritans, and the redundant stop codon TAA needs to be replaced for successful expression. Some scholars have replaced the stop codons TAA and TAG of glutamine encoding the inhibitory antigen by the traditional PCR cloning method for recombinant expression in Escherichia coli, but the method is time-consuming and laborious, and the coding sequence of the inhibitory antigen of Cryptocaryon irritans has not been optimized for prokaryotic expression, so the expression level of the recombinant protein is limited. Therefore, we optimized the codons and replaced the stop codon of an inhibitory antigen with a conservative antigen epitope that we screened in the early stage, and then directly performed gene synthesis to construct a recombinant expression vector. We successfully expressed the inhibitory antigen of Cryptocaryon irritans in Escherichia coli, filling the gap in the genetic recombinant subunit vaccine for Cryptocaryon irritans infection in marine fish. This research result has been authorized by the national invention patent "Zhang Ziping, Wang Yilei, Feng Jianjun, Zou Zhihua, Zhang Xin, He Lianghua. Recombinant protein vaccine of Cryptocaryon irritans and preparation method [P]. Patent number: ZL20171094268.5. Authorization date: 2020.12.04".

[0025] However, given that there are multiple inhibitory antigen molecules with conserved antigenic epitopes on the surface of Cryptocaryon irritans, the immune effect of a recombinant protein vaccine composed of a single inhibitory antigen is limited to a certain extent. In order to prepare a recombinant protein vaccine of inhibitory antigen of Cryptocaryon irritans with a higher immune protection rate, we prepared a dual recombinant protein of inhibitory antigen of Cryptocaryon irritans based on our previous work. In order to overcome the possible mutual interference of the two inhibitory antigen recombinant proteins in spatial structure and affect the integrity of their antigenic epitopes, in addition to replacing the stop codons TAA and TAG of the inhibitory antigen coding sequences with conserved antigenic epitopes and optimizing the codons for prokaryotic expression, we also added a linker protein polypeptide chain "GGGGSGGGGSGGGGS" composed of glycine (Gly, G) and serine (Ser, S) between the two inhibitory antigens, and optimized the codons of its coding sequence for prokaryotic cell expression, and connected the two inhibitory antigen coding sequences. Glycine (Gly) is the amino acid with the smallest molecular weight and the shortest side chain, and has the characteristics of low steric hindrance, which promotes the stability of the spatial structure of the inhibitory antigen. Serine (Ser) has a strong polarity, which helps to increase the hydrophilicity of the inhibitory antigen molecule and maintain the integrity of the inhibitory antigen protein spatial structure in aqueous solution. In addition, adding a coding sequence encoding 6 His amino acids at the end is conducive to the convenient and efficient purification of the Cryptocaryon irritans inhibitory antigen binary recombinant protein.

[0026] The implementation of the present invention has the following beneficial effects:

[0027] 1. The Cryptocaryon irritans inhibitory antigen dual recombinant protein of the present invention avoids the possible mutual interference of the two inhibitory antigen recombinant proteins in spatial structure and affects the integrity of their antigenic epitopes through terminator replacement and codon optimization, as well as the insertion of a linker protein consisting of 15 peptides composed of Gly and Ser between the two inhibitory antigen proteins, thereby overcoming the limitation of a single inhibitory antigen in providing immune protection against Cryptocaryon irritans infection and the disadvantage that there is no dual vaccine recombinant antigen on the market that can effectively prevent Cryptocaryon irritans infection in marine fish;

[0028] 2. The preparation method of the Cryptocaryon irritans inhibitory antigen binary recombinant protein of the present invention overcomes the disadvantage that the binary inhibitory antigen is difficult to express in bacteria, and realizes the efficient expression of the Cryptocaryon irritans inhibitory antigen binary recombinant protein in Escherichia coli. At the same time, a coding sequence encoding 6 His amino acids is added at the end, so that the Cryptocaryon irritans inhibitory antigen binary recombinant protein has a good purification effect and is convenient and simple. It can be used as an important candidate molecule of the Cryptocaryon irritans inhibitory antigen binary vaccine to prevent Cryptocaryon irritans infection in marine fish;

[0029] 3. The Cryptocaryon irritans inhibitory antigen dual recombinant protein of the present invention can be used to prepare vaccines, drugs or immune preparations for preventing and treating Cryptocaryon irritans, and is used in a variety of fish farming, with good market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a comparison diagram of the original gene coding sequence and the optimized coding sequence.

[0031] The yellow mark indicates the stop codon replacement site, the green mark indicates the codon optimization site, and the blue mark indicates the Linker coding sequence.

[0032] Figure 2 The following is a comparison chart of the original gene coding sequence and the optimized coding sequence.

[0033] The yellow mark indicates the stop codon replacement site, the green mark indicates the codon optimization site, and the blue mark indicates the Linker coding sequence.

[0034] Figure 3 The expression of Cryptocaryon stimulating inhibitory antigen dual recombinant protein in BL21 (DE3) was analyzed by SDS-PAGE.

[0035] Lane M is SDS-PAGE Protein Marker,

[0036] Lane 0 is the control group (without IPTG).

[0037] Lane 1 was induced at 15°C for 16 hours.

[0038] Lane 2 was induced at 37°C for 16 hours.

[0039] Figure 4 The solubility of the recombinant protein of Cryptocaryon stimulating inhibitory antigen was analyzed by SDS-PAGE.

[0040] Lane M is SDS-PAGE Protein Marker,

[0041] Lane 1 is the supernatant after the whole bacteria are broken.

[0042] Lane 2 is the precipitate after the whole bacteria were broken.

[0043] Figure 5 The results of the expression and purification of the recombinant protein of Cryptocaryon irritans inhibitory antigen in the inclusion body were analyzed by SDS-PAGE.

[0044] Lane M is SDS-PAGE Protein Marker,

[0045] Lane 1 is the supernatant after Ni-IDA incubation.

[0046] Lane 2-5 is the elution fraction of 100mM Imidazole.

[0047] Lane 6-9 are the fractions eluted with 300 mM Imidazole. DETAILED DESCRIPTION

[0048] In order to better understand the present invention, the present invention is further described in detail below in conjunction with the embodiments and drawings. However, those skilled in the art understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0050] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0051] Example 1 Preparation and verification of Cryptocaryon stimulatory inhibitory antigen dual recombinant protein

[0052] Step S1. MiniBEST Universal RNA Extraction Kit (TaKaRa) was used to extract the mRNA of Cryptocaryon irritans ciliate stage, and PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa) was used to reverse transcribe and synthesize cDNA templates. Based on the transcriptome database of the ciliate stage of Cryptocaryon irritans obtained by the research group, two Cryptocaryon irritans inhibitory antigen coding sequences with conserved antigenic epitopes were screened for specific primer design, and the coding sequences of two inhibitory antigen genes were obtained by cloning and sequencing using cDNA as a template;

[0053] First inhibitory antigen specific primer sequence:

[0054] Upstream primer: 5'-GCATGGGCTCCAAAAACTGCAGCTGC-3' (as shown in SEQ ID NO: 3)

[0055] Downstream primer: 5'-TTTGAATAAAAGAGCTAAAGAGAGGA-3' (as shown in SEQ ID NO: 4)

[0056] The coding sequence of the first inhibitory antigen gene is (as shown in SEQ ID NO: 5):

[0057] 5'-GCATGGGCTCCAAAAACTGCAGCTGCAGATTGGAAAGGTACATTTGTTGTTAC AAAAAGTAGTTGTTTGGCAACTTGTGGTTGGAAAATTGGTACTACTATTGTTATTGCTGATAAAACAGGAGATAGCTCAAAAGTAACTTGGATGGGAACAGTACATACAACTGATTCTACAAATGTTGATGTTGTAAGCGGAAGTTGTAAATATGTTAGTGTTGTAGCAACTGCAGGTACTGCTGGAACACCAGCTGAAGTTTTGAAAAACAGTGATGAATGTGCTTTTGCAACTGGAACTTGCGGTATTATGGGAAGAAAATAATCAACTCCTGGAACAGTAACTTTCAATAGGGATACGACTTTAGATACTAAGCCTTTGCAAATTCTTTATAAATAATTTGCAATGGTATAAAAAACATCAACCACCTAAAAAGCCACAGCTACTGACTAAGCAGCAAACTGTGATACTTAAGCTTCATTCGTAGATACTACAACTGATGCTAAAGCAATTGTAGGAT CATTAAAACTATCTAAAGCTAGTTGTGATAAATGCTCTTGGGATACAACTAAAGATTTAAAAATTACATAAGATGCTTCAAAAAAATATATGGTCACATTGGCAGGAACTATCAAAGAAACTGCAACTGGTGATTGTAATAATAAACTTACAGCTTCTGAAGCATGTTATGTCACAAAAAAAGATGATAAGACTTATGTTCTCGTTAGTTGTGCTACTCTTGACACAACTAGTAAAGGTATTCCAATTGCCATTACAACAGCAAATTCAAAAACCACTTTAACCATGACATGGACTGATTCAGCTTCTAAAGCATGTAGTGTTGTTGGAGAAGTAACTTCTAGTTCTAATAGTGTAAAACTTATTTCAGGATTCAGTGCTATGTTAATCCTCTCTTTAGCTCTTTTATTCAAA-3'

[0058] The second suppressor antigen-specific primer sequence:

[0059] Upstream primer: 5'-AATTGGGTTGAAAAAACCGCGATTGC-3' (as shown in SEQ ID NO: 6)

[0060] Downstream primer: 5'-AGTAATTTCTCCTACAACATTACAT-3' (as shown in SEQ ID NO: 7) The coding sequence of the second inhibitory antigen gene is (as shown in SEQ ID NO: 8):

[0061] 5'-AATTGGGTTGAAAAAACCGCGATTGCGGATTGGAAAGGTACATTTGTTGTTAC AAAAAGTAGTTGTTTGGCTACTTGTGGTTGGAAAATTGGTAGTACAATTGTTATTGCTGAAAAAACAGGAGATAATACAAAAGTTACATGGGTTGGTACAGTACATACAACTGATGCTACAAATGTTGATGTTGCAAGTGGAAGCTGTAAATATGTTAGTGTTGTAGCAGCTGCTGGTACACCTGGTACAGCTGAAGTTTTGAAAAACGATGATGAATGTGTATTTGCAACTGGTATGTGCACTATTATGGGTCAAAAATAAACAAAACCTGCAAAAATTACTTTCAATAGAGATACAACATTAGATACTAAGCCTTTACAAGTCCTTTATAAATAATTTGAAATGGTACAAAAATCATCAACAACTCAAA AAGCTGCTGCTACTGATTAAGCTGCAGATTGTGATACTTAAGCTTCAATGGTAGATACTACTACTGATGCTAAAGCAATTGTAGGAACATTGAAACTTTCTAAAGCTGTTTGTAATAAATGTTCATGGGATACAACCAAAGATTTAAAAATTACATAAGATGCTACAAATAAATATATGGTCACACTTGCAGGAACTATCAAAGAAACTACTAGTGGTGATTGTAAAAATAAACTCACAGCATCTGAAGCATGCTATGTAACAAAAAAAGATGATAAAACATTTATTCTCGTTAGCTGTACTACTCTTGATACAACCAATAAGGGTATTCCAATCGCCATAACAACAGCAAATTCAAAAACCACTTTAACCTTGA CACGGACTGATTCATCTTCTTAAGCATGTAATGTTGTAGGAGAAATTACT-3'

[0062] Step S2: After analyzing the coding sequences of the two inhibitory antigen genes obtained, the stop codons TAA and TAG contained in the coding sequences were found and replaced with the codons CAA and CAG that can encode glutamine, and the codons were optimized for prokaryotic cell expression. In order to ensure the independence and integrity of the spatial structure of the two inhibitory antigen proteins in the Cryptocaryon stimulating inhibitory antigen binary recombinant expression protein, we designed a linker protein polypeptide "GGGGSGGGGSGGGGS" composed of glycine (Gly, G) and serine (Ser, S), and after optimizing its coding sequence for prokaryotic cell expression codons, the two inhibitory antigen molecules were connected;

[0063] Comparison of the original gene coding sequence and the optimized coding sequence Figure 1 , Figure 2 shown.

[0064] Step S3: entrusting a merchant to use whole gene synthesis to obtain a Cryptocaryon irritans binary inhibitory antigen coding sequence connected by Linker protein molecules, replaced by terminators and optimized by codons, and adding a coding sequence encoding 6 His amino acids at the end; the optimized Cryptocaryon irritans inhibitory antigen binary recombinant protein cDNA nucleotide sequence synthesized by the merchant is (as shown in SEQ ID NO: 2):

[0065] SEQ ID NO: 2

[0066]

[0067] Using the optimized Cryptocaryon irritans inhibitory antigen binary recombinant protein cDNA nucleotide sequence as a template, the primers were designed to add restriction enzyme sites NdeI and HindIII at both ends. After PCR amplification, the optimized Cryptocaryon irritans inhibitory antigen binary recombinant protein coding sequence was inserted into the expression vector pET30a vector that was also double-digested by enzyme digestion, and the recombinant expression vector pET30a-binary inhibitory antigen was obtained after ligation using T4 ligase. The pET30a-binary inhibitory antigen recombinant expression vector was transformed into the competent cells of the Escherichia coli DH5α cloning strain, evenly spread on the LB plate (containing 50 μg / mL of kanamycin sulfate), and then inverted at 37°C incubator overnight. Single clones were selected from the transformed plates, inoculated into 4 mL of LB medium (containing 50 μg / mL of kanamycin sulfate), and cultured overnight at 37°C in a shaker at 200 rpm; the bacteria were collected, and the plasmids were extracted and sequenced to confirm the accuracy of the final expression vector;

[0068] Step S4: After the recombinant expression vector pET30a-bivalent inhibitory antigen with correct sequencing was transformed into the E. coli BL21 (DE3) expression strain, it was evenly spread on the LB plate (containing 50 μg / mL of kanamycin sulfate), and then inverted in a 37°C incubator overnight. A single clone was selected from the transformed plate and inoculated into 4 mL of LB medium (containing 50 μg / mL of kanamycin sulfate) and cultured until OD 600 The final concentration of IPTG was 0.2 mM, and the culture medium was placed at 15°C and 37°C to induce expression.

[0069] The amino acid sequence of the Cryptocaryon irritans inhibitory antigen dual recombinant protein is (as shown in SEQ ID NO: 1):

[0070] SEQ ID NO: 1

[0071] MAWAPKTAAADWKGTFVVTKSSCLATCGWKIGTTIVIADKTGDSSKVTWMGTVHTTDSTNVDVVSGSCKYVSVVATAGTAGTPAEVLKNSDECAFATGTCGIMGRKQSTPGTVTFNRDTTLDTKPLQILYKQFAMVQKTSTTQKATATDQAAN CDTQASFVDTTTDAKAIVGSLKLSKASCDKCSWDTTKDLKITQDASKKYMVTLAGTIKETATGDCNNKLTASEACYVTKKDDKTYVLVSCATLDTTSKGIPIAITTANSKTTLTMTWTDSASKACSVVGEVTSSSNSVKLISGFSAMLILSLAL LFKGGGGSGGGGSGGGGSNWVEKTAIADWKGTFVVTKSSCLATCGWKIGSTIVIAEKTGDNTKVTWVGTVHTTDATNVDVASGSCKYVSVVAAAGTPGTAAEVLKNDDECVFATGMCTIMGQKQTKPAKITFNRDTTLDTKPLQVLYKQFEMVQ KSSTTQKAAATDQAADCDTQASMVDTTTDAKAIVGTLKLSKAVCNKCSWDTTKDLKITQDATNKYMVTLAGTIKETTSGDCKNKLTASEACYVTKKDDKTFILVSCTLDTTNKGIPIAITTANSKTTLTLTRTDSSSQACNVVGEITHHHHHH

[0072] Take the induced culture medium and centrifuge it at 12000rpm for 5min, remove the supernatant, add PBS to resuspend the precipitate, and finally add SDS-PAGE loading buffer to heat the sample at 100℃ for 10min, then centrifuge and take the supernatant for electrophoresis. Run electrophoresis at 160V until the bromophenol blue band migrates to 1cm from the bottom of the gel, take out the gel and use the protein gel rapid processing system to stain and decolorize, take pictures and save. The results are as follows Figure 3 As shown, compared with the control, there is an obvious new protein band at the arrow mark, indicating that the Cryptocaryon stimulating nucleus inhibitory antigen dual recombinant protein was correctly expressed in Escherichia coli;

[0073] Step S5: The specific steps of stimulating the expansion of the Cryptocaryon fasciatus inhibitory antigen dual recombinant protein are as follows: expand the culture and grow to OD 600=0.8, add IPTG at a final concentration of 0.2 mM, induce at 15°C for 16 h, collect the whole bacteria, and use 50 mM Tris (pH 8.0), 300 mM NaCl, 20 mM Imidazole containing 1% Triton X-100, 1 mM DTT, 1 mM PMSF for ultrasonic lysis, take the supernatant and precipitate for SDS-PAGE analysis, and take pictures for preservation;

[0074] like Figure 4 As shown, the Cryptocaryon irritans inhibitory antigen binary recombinant protein was mainly expressed in Escherichia coli in the form of inclusion bodies.

[0075] Step S6: Purification of Cryptocaryon stimulatory inhibitory antigen dual recombinant protein, the specific steps are: perform the following operations at low temperature, wash the inclusion body with 50mM Tris (pH8.0), 300mM NaCl containing 1% Triton X-100, 2mM EDTA, 5mM DTT, dissolve the inclusion body with 50mM Tris (pH8.0), 300mM NaCl, 8M Urea, 20mM Imidazole buffer and balance the Ni-IDA column, finally elute the target protein with balancing buffer with different concentrations of imidazole, and collect each elution fraction for SDS-PAGE analysis and detection. Figure 5 As shown, after Ni-IDA affinity chromatography, the elution effect through 100mM Imidazole was the best and the purity was high, so the Lane 3-5 eluate was collected and added to the treated dialysis bag. At 4°C, it was dialyzed into a buffer [1×PBS (pH7.4), 4mM GSH, 0.4mM GSSG, 0.4M L-Arginine, 1M Urea, 5% Glycerol] for renaturation. After renaturation, the Cryptocaryon spp. inhibitory antigen dual recombinant protein was finally dialyzed into a storage solution 1×PBS (pH7.4), 5% Glycerol solution for about 6-8 hours. After the dialysis renaturation was completed, the supernatant was filtered with a 0.22μm filter and then aliquoted and frozen at -80°C.

[0076] In summary, the Cryptocaryon irritans inhibitory antigen binary recombinant protein of the present invention avoids the possible mutual interference of the two inhibitory antigen recombinant proteins in spatial structure, affects the integrity of their antigenic epitopes, overcomes the limitation of a single inhibitory antigen in providing immune protection against Cryptocaryon irritans infection, and the shortcomings of no binary vaccine recombinant antigens on the market that effectively prevent Cryptocaryon irritans infection in marine fish. The preparation method of the Cryptocaryon irritans inhibitory antigen binary recombinant protein of the present invention overcomes the disadvantage that the binary inhibitory antigen is difficult to express in bacteria, realizes the efficient expression of the Cryptocaryon irritans inhibitory antigen binary recombinant protein in Escherichia coli, and at the same time, adds a coding sequence encoding 6 His amino acids at the end, so that the purification effect of the Cryptocaryon irritans inhibitory antigen binary recombinant protein is good and convenient and simple, and can be used as an important candidate molecule of the Cryptocaryon irritans inhibitory antigen binary vaccine to prevent Cryptocaryon irritans infection in marine fish. The Cryptocaryon irritans inhibitory antigen dual recombinant protein of the present invention can be used to prepare vaccines, medicines or immune preparations for preventing and treating Cryptocaryon irritans, and is applied in a variety of fish breeding, and has good market prospects.

[0077] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A Cryptocaryon stimulatory inhibitory antigen dual recombinant protein, characterized in that: The amino acid sequence of the Cryptocaryon irritans inhibitory antigen dual recombinant protein is shown in SEQ ID NO:

1.

2. A gene encoding a dual recombinant protein of Cryptocaryon irritans inhibitory antigen, characterized in that: The nucleotide sequence of the gene encoding the Cryptocaryon stimulatory inhibitory antigen dual recombinant protein is shown in SEQ ID NO:

2.

3. A method for preparing the Cryptocaryon irritans inhibitory antigen dual recombinant protein according to claim 1, characterized in that: The preparation method comprises the following steps: S1. extracting the mRNA of the ciliate stage of Cryptocaryon irritans, reverse-transcribing and synthesizing cDNA templates, screening and obtaining two Cryptocaryon irritans inhibitory antigen coding sequences with conserved antigenic epitopes, designing specific primers, cloning and sequencing using cDNA as a template to obtain the coding sequences of two inhibitory antigen genes; S2. Analyze the coding sequences of the two inhibitory antigen genes obtained, find out the stop codons TAA and TAG contained in the coding sequences, replace them with codons CAA and CAG that can encode glutamine, and optimize the codons suitable for prokaryotic cell expression, design a linker protein polypeptide composed of glycine and serine, optimize the coding sequences for prokaryotic cell expression codons, and then connect the two inhibitory antigen molecules; S3. The coding sequence of Cryptocaryon irritans binary inhibitory antigen connected by Linker protein molecules, replaced by terminators and optimized by codons was synthesized by whole gene, and the coding sequence encoding 6 His amino acids was added to the end, and the optimized Cryptocaryon irritans inhibitory antigen binary recombinant protein coding sequence was inserted into the expression vector to construct a recombinant expression vector, and the accuracy of the final expression vector was confirmed by enzyme digestion and sequencing; S4. After the recombinant expression vector is transformed into the Escherichia coli expression strain, it is cultured and placed under conditions not exceeding 15°C and 37°C to induce expression, protein gel electrophoresis and decolorization are performed to obtain the Cryptocaryon irritans inhibitory antigen dual recombinant protein.

4. The method for preparing the Cryptocaryon stimulatory inhibitory antigen dual recombinant protein as claimed in claim 3, characterized in that: The expression vector in step S3 is any one of the pET series, PGEX series or pMAL series.

5. The method for preparing the Cryptocaryon stimulatory inhibitory antigen dual recombinant protein as claimed in claim 4, characterized in that: The expression vector in step S3 is any one of pET-28a, pET-30a, and pET-32a.

6. The method for preparing the Cryptocaryon stimulatory inhibitory antigen dual recombinant protein as claimed in claim 3, characterized in that: The expression vector in step S3 is pGEX-4T or pGEX-6P.

7. The method for preparing the Cryptocaryon stimulatory inhibitory antigen dual recombinant protein as claimed in claim 3, characterized in that: The expression vector in step S3 is pMAL-p2x or pMAL-c2X.

8. The method for preparing the Cryptocaryon stimulatory inhibitory antigen dual recombinant protein as claimed in claim 3, characterized in that: The E. coli expression strain in step S4 is BL21 or DE3.

9. The use of the Cryptocaryon irritans inhibitory antigen dual recombinant protein as claimed in claim 1, characterized in that: The Cryptocaryon irritans inhibitory antigen dual recombinant protein is used for preparing vaccines, medicines or immune preparations for preventing and treating Cryptocaryon irritans disease.

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