Oral broad-spectrum vibrio subunit microalgae vaccine for aquatic products and preparation method thereof

Abstract

The invention belongs to the field of microorganisms, and particularly relates to an oral broad-spectrum vibrio subunit microalgae vaccine for aquatic products and a preparation method of the oral broad-spectrum vibrio subunit microalgae vaccine. The invention provides an aquatic oral broad-spectrum vibrio subunit microalgae vaccine which expresses vibrio membrane protein OmpK by taking microalgae as a vaccine carrier. The gene expression cassette of the vibrio membrane protein OmpK is inserted into a genome of microalgae, and the sequence of the gene expression cassette of the vibrio membrane protein OmpK is as shown in SEQ ID NO: 4 after sequence optimization. When the microalgae vaccine is used for feeding cultured animals such as fishes and shrimps, the vibrio resistance can be remarkably improved, the environmental safety is high, and the large-scale production cost is low.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to an aquatic oral broad-spectrum Vibrio subunit microalgae vaccine and a preparation method thereof. Background Art

[0002] Vibrio disease is one of the most common bacterial diseases affecting marine aquaculture, which has a great impact not only on fish, but also on other organisms such as shrimp and shellfish. Long-term or excessive use of antibiotics not only creates the risk of antibiotic resistance, but also has an impact on human health and the environment. The development and use of vaccines is the safest and most effective way to prevent aquatic animal diseases and protect the health and sustainability of aquaculture. Currently, several types of vaccines have been developed, including live attenuated vaccines, inactivated vaccine subunit vaccines, DNA vaccines, etc., which have good effects on the prevention and treatment of vibriosis in fish and other organisms. However, most vaccines lack operability in actual application, and individual immunization of fish is labor-intensive, costly, and harmful. Currently, the types of commercialized Vibrio vaccines are still limited.

[0003] In recent years, biotechnology has been shown to play a driving role in the development of new vaccines and new delivery systems. Among them, oral vaccines have always been the demand of the aquaculture industry because of their convenience and safety. They can enhance immunity through mucosal immune response and systemic immune response. Among all types of new vaccines, Vibrio subunit vaccines based on outer membrane proteins (OMPs) have gradually received more attention in the protection of vibriosis due to their role in the interaction between bacteria and hosts. For example, CN106924725A obtained the Vibrio outer membrane protein Ompk by high-density culture and induced expression of Escherichia coli (engineered bacteria) carrying a plasmid containing the Vibrio outer membrane protein Ompk gene in a 500L fermenter, and extracted and purified it. CN102512674B constructed flaA-ompK-pET-28a recombinant plasmid, and obtained a high-purity fusion protein FlaA-OmpK through exogenous induced expression and purification.

[0004] The key to using Vibrio outer membrane protein as an oral vaccine is to avoid the degradation of antigens by the digestive system. Encapsulating recombinant proteins with various biological materials, or expressing antigen proteins in probiotics and tobacco, can effectively alleviate the degradation of antigens by the digestive system and improve the effect of oral use.

[0005] Microalgae are natural food for fish, shrimp and shellfish larvae, and many components in microalgae can be used as immunostimulants to enhance nonspecific immunity and promote growth. Therefore, microalgae is a promising vaccine carrier. At present, there is no literature report on the preparation of a broad-spectrum Vibrio subunit microalgae vaccine. Summary of the invention

[0006] In order to solve the above technical problems, the present invention uses microalgae as a vaccine carrier to express Vibrio membrane protein as a subunit vaccine for oral administration in aquatic products.

[0007] The present invention provides an aquatic oral broad-spectrum Vibrio subunit microalgae vaccine, which is a microalgae expressing the Vibrio membrane protein OmpK. Preferably, the amino acid sequence of the Vibrio membrane protein OmpK is shown in SEQ ID NO: 5. The sequences of other pathogenic microorganisms are added to the Vibrio membrane protein OmpK to achieve multiple immune effects.

[0008] MRKSLSHLSLLHADYSDGDIHKNDYKWMQFNLMGAFDEKGAGPESSHDYLEMEFGRSGIFDLYGYVDVFNLASDPGSDKAGAEKIFMKFAPRMSLDALTGKDLSFGPVQELYVATLMEWGGNSGVNNQKIGLGSDVMVPWLGKVGLNLYGSYYGNNKDWNVTAEVGS GYFKMTDVSFDSDTLGKIKIRNGKSDAQMKEEDADLVITPVEYQFGMEDKEGNKFNTNTSNGGAMFNGIYWHSDRFAVGYGLKLYKDVYGFEDGKALPWTQTVDSSGVAHAAEIYNKDGNKLDLYGKVDGLHYFSQDHNKDGDQSYVRFGFKGETQINDQLTGYG(SEQ ID NO: 5).

[0009] In some embodiments, the microalgae is a cyanobacterium, more preferably Synechococcus.

[0010] In some embodiments, the microalgae contains a gene expression cassette for the Vibrio membrane protein OmpK.

[0011] In some embodiments, the gene expression cassette of the Vibrio membrane protein OmpK comprises a cyanobacterial rbcL promoter, a Vibrio membrane protein ompK gene, and a terminator.

[0012] In some embodiments, the nucleotide sequence of the rbcL promoter is shown in SEQ ID NO: 1, the nucleotide sequence of the Vibrio membrane protein ompK gene is shown in SEQ ID NO: 2, and the nucleotide sequence of the terminator is shown in SEQ ID NO: 3. The sequence of the gene expression cassette of the Vibrio membrane protein OmpK is shown in SEQ ID NO: 4.

[0013] In some embodiments, the gene expression cassette of the Vibrio membrane protein OmpK is inserted into the genome of the microalgae, preferably the Synechococcus sp. 7942 genome.

[0014] The present invention also provides a method for preparing the aquatic oral broad-spectrum Vibrio subunit microalgae vaccine, comprising:

[0015] (1) cloning the microalgae genome fragment, inserting the gene expression cassette of the Vibrio membrane protein OmpK and the antibiotic resistance gene into the genome fragment, and constructing a homologous recombination expression vector;

[0016] (2) adding the homologous recombination expression vector to the microalgae suspension, transforming the microalgae by natural transformation, performing homologous recombination, and screening the successfully transformed microalgae by antibiotics;

[0017] (3) using the homologous recombination expression vector of step (1) as a template, amplifying and deleting the antibiotic resistance gene by the inverse PCR method, and constructing a homologous recombination vector without the antibiotic resistance gene;

[0018] (4) adding the homologous recombination vector without the antibiotic resistance gene to the microalgae suspension successfully transformed in step (2), transforming the microalgae by natural transformation, and screening the microalgae with the antibiotic resistance gene removed by photocopying to obtain the oral broad-spectrum Vibrio subunit microalgae vaccine for aquatic products.

[0019] The present invention also provides application of the aquatic oral broad-spectrum Vibrio subunit microalgae vaccine in preventing and treating Vibrio diseases in aquaculture.

[0020] The present invention also provides an antibacterial agent, comprising the aquatic oral broad-spectrum Vibrio subunit microalgae vaccine.

[0021] The present invention also provides an aquatic feed, comprising the aquatic oral broad-spectrum Vibrio subunit microalgae vaccine.

[0022] Compared with the prior art, the beneficial effects of the present invention include at least:

[0023] 1. The present invention constructs an oral broad-spectrum Vibrio subunit vaccine for aquaculture using microalgae as a carrier, which contains a gene expression cassette of Vibrio membrane protein OmpK. Through amino acid sequence modification and gene sequence optimization, Vibrio membrane protein OmpK is efficiently expressed in microalgae, with high protein yield, which effectively improves the preventive effect of aquaculture against Vibrio infection and other pathogenic microorganisms.

[0024] 2. Vibrio membrane protein OmpK has good immune effect as a subunit vaccine, and is highly conserved among multiple pathogenic Vibrio, and can produce cross-immunity effects.

[0025] 3. The present invention combines homologous recombination and photocopying to construct transgenic algae without antibiotic markers, which is environmentally safe; the subunit vaccine with Synechococcus as the host has low cost for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The expression results of OmpK detected by RT-PCR, where EG is the experimental group, AC is the positive control, NC is the negative control, WL refers to wild algae; TL refers to transgenic algae.

[0027] Figure 2 The expression results of OmpK detected by SDS-PAGE, where WL refers to wild algae and TL refers to transgenic algae.

[0028] Figure 3 The survival rate of zebrafish fed with wild Synechococcus 7942 (control group) and transgenic algae (experimental group) 30 days after Vibrio infection.

[0029] Figure 4 The survival rate of goby fed with wild Synechococcus 7942 (control group) and transgenic algae (experimental group) 30 days after Vibrio infection. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0031] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0032] Example 1

[0033] 1. Antigen selection

[0034] Vibrio membrane protein OmpK has good immune effect as a subunit vaccine and is highly conserved among various pathogenic Vibrio. Therefore, using it as a subunit vaccine can produce cross-immunity effect.

[0035] 2. Design of OmpK gene expression cassette

[0036] The cyanobacterial intrinsic promoter rbcL promoter sequence, the Vibrio ompK gene and the terminator sequence of the pQE-T7 vector were selected to form the OmpK gene expression cassette, wherein the ompK gene sequence was codon optimized using the NewPu Bio online tool to facilitate expression in Synechococcus 7942. The above sequence was synthesized by Shanghai Bioengineering Co., Ltd. and connected to the T vector.

[0037] rbcL promoter sequence:

[0038] CAACGGCTCACAAGCCCAACTAATCACCATTTGGACAAAACATCAGGAATTCTAATTAGAAAGTCCAAAAATTGTAATTTAAAAAACAGTCAATGGAGAGCATTGCCATAAGTAAAGGCATCCCCTGCGTGATAAGATTACCTTCAGAAAACAGATAGTTGCTGGGTTATCGCAGATTTTTCTCGCAACCAAATAACTGTAAATAATAACTGTCTCTGGGGCGACGGTAGGCTTTATATTGCCAAATTTCGCCCGTGGGAGAAAGCTAGGCTATTCAATGTTTATGGAGGACTGACCTAG(SEQ ID NO: 1);

[0039] Optimized OmpK gene:

[0040]

[0041] Terminator sequence of pQE-T7 vector:

[0042] GATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCG CTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTT TG (SEQ ID NO: 3);

[0043] Sequence of OmpK gene expression cassette:

[0044]

[0045] 3. Construction of OmpK gene homologous recombination expression vector

[0046] A fragment with a length of more than 2000 bp between Synpcc7942_0050 and Synpcc7942_0064 of the Synechococcus 7942 genome was cloned, and the OmpK gene expression cassette and the kanamycin resistance gene were respectively inserted into the middle of the fragment by the reverse PCR method, thereby constructing a homologous recombination expression vector.

[0047] 4. Transformation of Synechococcus 7942 by natural transformation

[0048] 2 μg of homologous recombination vector was added to 200 μl of algal cell suspension. After incubation at 28°C in the dark for 18 hours, the suspension was spread on a BG-11 solid culture medium filter containing 5 μg / ml kanamycin and cultured at 28°C in the light for 2-4 weeks. Algae grew and were selected and inoculated in BG-11 culture medium containing 5 μg / ml kanamycin for expansion.

[0049] 5. Knockout of kanamycin resistance gene

[0050] Using the homologous recombination vector as a template, the inverse PCR method was used to amplify and delete the kanamycin resistance gene, and the PCR product was self-ligated to construct a homologous recombination vector without the kanamycin resistance gene. 2 μg of the homologous recombination vector without the kanamycin resistance gene was added to 200 μl of transgenic algae cell suspension, and the transgenic algae with the kanamycin resistance gene removed was screened by the photocopy method, which was the aquatic oral broad-spectrum Vibrio subunit microalgae vaccine.

[0051] Example 2

[0052] 1. Verify the expression of OmpK by RT-PCR and SDS-PAGE. Figure 1 As shown in the figure, EG is the experimental group; AC is the positive control; NC is the negative control; WL is wild algae; TL is transgenic algae. The transgenic algae in the experimental group have amplified bands. The SDS-PAGE test results are shown in the figure. Figure 2 As shown in the figure, WL refers to wild algae and TL refers to transgenic algae. It can be found that the transgenic algae has an expression band of the OmpK gene. Therefore, it can be known that the transgenic algae has correctly expressed the OmpK gene.

[0053] 2. Wild Synechococcus 7942 (control group) and transgenic algae (experimental group) were used to feed zebrafish and goby to test the preventive effect of the transgenic genome as an oral vaccine against Vibrio anguillarum infection. Figure 3 As shown in Figure 2, the survival rate of zebrafish is still above 90%, significantly higher than that of the control group (about 50%). Figure 4As shown, the lanceolatus goby also showed a significant protective effect (experimental group 60%: control 30%).

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. An oral broad-spectrum Vibrio subunit microalgae vaccine for aquatic products, characterized in that: The microalgae expresses the Vibrio membrane protein OmpK; preferably, the amino acid sequence of the Vibrio membrane protein OmpK is shown in SEQ ID NO:

5.

2. The aquatic oral broad-spectrum Vibrio subunit microalgae vaccine according to claim 1, characterized in that: The microalgae is cyanobacteria, more preferably Synechococcus.

3. The aquatic oral broad-spectrum Vibrio subunit microalgae vaccine according to claim 1, characterized in that: The microalgae contains a gene expression cassette of Vibrio membrane protein OmpK.

4. The aquatic oral broad-spectrum Vibrio subunit microalgae vaccine according to claim 3, characterized in that: The gene expression box of the Vibrio membrane protein OmpK comprises a cyanobacterium rbcL promoter, a Vibrio membrane protein ompK gene and a terminator.

5. The aquatic oral broad-spectrum Vibrio subunit microalgae vaccine according to claim 4, characterized in that: The nucleotide sequence of the rbcL promoter is shown in SEQ ID NO: 1, the nucleotide sequence of the Vibrio membrane protein ompK gene is shown in SEQ ID NO: 2, and the nucleotide sequence of the terminator is shown in SEQ ID NO: 3; Preferably, the nucleotide sequence of the gene expression cassette of the Vibrio membrane protein OmpK is shown in SEQ ID NO:

4.

6. The aquatic oral broad-spectrum Vibrio subunit microalgae vaccine according to any one of claims 3 to 5, characterized in that: The gene expression cassette of the Vibrio membrane protein OmpK is inserted into the genome of microalgae, preferably the Synechococcus 7942 genome.

7. The method for preparing the aquatic oral broad-spectrum Vibrio subunit microalgae vaccine according to any one of claims 1 to 6, characterized in that: include: (1) cloning the microalgae genome fragment, inserting the gene expression cassette of the Vibrio membrane protein OmpK and the antibiotic resistance gene into the genome fragment, and constructing a homologous recombination expression vector; (2) adding the homologous recombination expression vector to the microalgae suspension, transforming the microalgae by natural transformation, performing homologous recombination, and screening the successfully transformed microalgae by antibiotics; (3) using the homologous recombination expression vector of step (1) as a template, amplifying and deleting the antibiotic resistance gene by the inverse PCR method, and constructing a homologous recombination vector without the antibiotic resistance gene; (4) adding the homologous recombination vector without the antibiotic resistance gene to the microalgae suspension successfully transformed in step (2), transforming the microalgae by natural transformation, and screening the microalgae with the antibiotic resistance gene removed by photocopying to obtain the oral broad-spectrum Vibrio subunit microalgae vaccine for aquatic products.

8. Use of the aquatic oral broad-spectrum Vibrio subunit microalgae vaccine according to any one of claims 1 to 6 in preventing and treating Vibrio diseases in aquaculture.

9. An antibacterial agent, characterized in that The invention comprises the aquatic oral broad-spectrum Vibrio subunit microalgae vaccine as described in any one of claims 1 to 6.

10. An aquatic feed, characterized in that: The invention comprises the aquatic oral broad-spectrum Vibrio subunit microalgae vaccine as described in any one of claims 1 to 6.

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