CN116496367A

CN116496367A - Streptococcus equi Ma Yachong SE071780-01939 protein, vaccine, preparation method and application thereof

Info

Publication number: CN116496367A
Application number: CN202310400082.8A
Authority: CN
Inventors: 马勋; 王静; 薄新文; 孔翠莲; 平丽莹; 刘彩霞; 寇丽君; 吕双飞; 史唯地; 任慧杰
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2023-04-14
Filing date: 2023-04-14
Publication date: 2023-07-28

Abstract

The invention provides streptococcus equi Ma Yachong SE071780-01939 protein and a vaccine as well as a preparation method and application thereof, and belongs to the technical field of antigens. The SE071780-01939 protein provided by the invention can cause an immune response reaction of streptococcus equi Ma Yachong, and is a good antigen molecule. The SE071780-01939 protein is prepared into a vaccine, which has important significance for preventing the spread of the infection of the equine adenosis.

Description

Streptococcus equi Ma Yachong SE071780-01939 protein, vaccine, preparation method and application thereof

Technical Field

The invention belongs to the technical field of antigens, and particularly relates to streptococcus equi Ma Yachong SE071780-01939 protein and a preparation method and application thereof.

Background

In addition to the natural immune region iceland, equine epidemic disease is developed worldwide, and the animals are not only limited to horses, but also more equine infections are gradually reported. The animals with disease are mainly foal with the age of 1 year old, and the foal with disease is more in spring and autumn. The infectious disease is extremely strong, so that the foal is greatly damaged, and once the foal is infected, pathogens are difficult to thoroughly remove, so that huge economic loss is caused for the equine animal breeding industry.

Streptococcus equi Ma Yachong can be ingested as contaminated food or water or contacted with other contaminants by contact with the nasal or oral cavity of a diseased animal. Once in the mouth or nose, streptococcus equi Ma Yachong adheres to and invades the tongue and palate tonsils through a range of cell surface receptors and then migrates to the lymph nodes of the head and infects the neck within hours after infection. Within the lymph nodes, streptococcus equi subspecies use a variety of immune evasion strategies to neutralize the effects of the innate immune system and establish infection. Active recruitment of neutrophils to the affected lymph nodes and killing of streptococcus equi by the failing immune system leads to lymphadenectasis and abscess formation, which can lead to ineffective antibiotic treatment. Treatment of cases with antibiotics may also hinder the development of humoral immune responses and become resistant strains. So vaccine immunization is the most effective method for protecting horses from pathogenic bacteria, and can effectively prevent and treat equine adenosis.

The animal infectious disease prevention and treatment work in China is kept in advance, the susceptible animals can be protected from being infected by pathogens by effective vaccination, and the subunit vaccine has high safety and good treatment effect, becomes a hotspot of current research, is not mature and perfect enough for the research, and has the problem of selecting protective antigens.

Disclosure of Invention

Therefore, the invention aims to provide streptococcus equi Ma Yachong SE071780-01939 protein which can be used as an antigen molecule to stimulate an organism to generate an immune response, and provides a new material for developing recombinant protein subunit vaccines for resisting the equine epidemic disease.

The invention provides streptococcus equi Ma Yachong SE071780-01939 protein, and the amino acid sequence of the SE071780-01939 protein is shown as SEQ ID NO. 1.

The invention provides a coding gene of streptococcus equi Ma Yachong SE071780-01939 protein, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

The invention provides a recombinant vector containing the coding gene.

The invention provides a recombinant strain comprising the coding gene or the recombinant vector.

The invention provides a equine adenophora vaccine, which comprises streptococcus equi Ma Yachong SE071780-01939 protein and an adjuvant.

Preferably, the volume ratio of the streptococcus equi Ma Yachong SE071780-01939 protein and the adjuvant is 1:1.

Preferably, the concentration of the protein in the vaccine is 300-500 μg/mL.

Preferably, the adjuvant comprises Freund's complete adjuvant or Freund's incomplete adjuvant.

The invention provides application of streptococcus equi Ma Yachong SE071780-01939 protein, the coding gene, the recombinant vector or the recombinant strain in preparing vaccine for preventing and controlling horse adenosis.

The invention provides application of streptococcus equi Ma Yachong SE071780-01939 protein in preparation of a reagent or a kit for evaluating natural immunity and/or artificial immunity effects.

The invention provides streptococcus equi Ma Yachong SE071780-01939 protein, and the amino acid sequence of the SE071780-01939 protein is shown as SEQ ID NO. 1. After the SE071780-01939 protein is obtained through recombinant expression and mixed with an adjuvant to stimulate a mouse, the mouse can generate immune response reaction to streptococcus equi Ma Yachong, is a good antigen molecule, provides antigen materials for preparing a streptococcus equi Ma Yachong-induced equine vaccine, and has important significance for preventing and controlling the spread of equine epidemic diseases. Meanwhile, the SE071780-01939 protein has good safety, and the prepared protein subunit is ensured to have good safety.

Drawings

FIG. 1 shows the results of PCR amplification of SE071780-01939 gene, note: m:2000DNAMarker;1, the PCR amplification result of the SE071780-01939 gene; 2, seM gene identification; 3: a negative control;

FIG. 2 shows the results of double digestion and identification of pMD19T-01939 recombinant plasmid, annotated: m5000 DNA Marker;1: pMD19-T plasmid; 2: pMD19T-01939 double cleavage product;

FIG. 3 shows the results of double digestion and identification of pET32a-01939 recombinant plasmid, and is given by: m5000 DNAMarker;1: pET32a-2193 double cleavage product; 2: pET32a plasmid;

FIG. 4 shows the result of SDS-PAGE analysis of the expression product of recombinant expression plasmid pET32a-01939 in BL21 (DE 3), notes: m: protein molecular weight standard; 1-4: expression products of recombinant plasmids p ET32a-01939 at 2, 4, 6 and 8h respectively;

FIG. 5 is a Western Blot analysis of recombinant expression plasmid pET32a-01939 expression products, notes: m is a protein molecular weight standard; 1: inducing an expression product by the recombinant plasmid pET32 a-01939;

FIG. 6 is 100LD ₅₀ Survival rate of mice after challenge;

FIG. 7 shows the results of detection of specific antibody levels in mouse serum.

Detailed Description

The invention provides streptococcus equi Ma Yachong SE071780-01939 protein, and the amino acid sequence of the SE071780-01939 protein is shown as SEQ ID NO. 1 (MKKLLLILTVFVTAFTLVACSSQSKQQESTSKASLSHKPKIAGVTYYGDIPVKPKRVVSLAATYTGYLAKLDLHLVGVTSYDKKNPVLTKSITQAKQVAATDLEAITALEPDLIVVGSTEENIDQLAKIAPLISIEYRKRDYLQVFSDFGRIFNKEKETDQWLADWKQKTSDYEKEIKQVTGSAATFTIMGLYEKEIYLFGKDWGRGGEIIHQALHYDAPEKVKKEVFKQGYLSLSQEVLPDYIGDYVIVAAEDDQTGSALYESDLWQQIPAVTNNHVIKVNANVFYFTDPLSLEHQLEVLKKAILSTGASQ).

In the present invention, the SE071780-01939 protein is derived from streptococcus Ma Yachong XJ201902 strain. The streptococcus Ma Yachong XJ201902 strain was isolated from horse adenosis dead donkey lung pus. The streptococcus Ma Yachong XJ201902 strain has a genotype of seM-136.

In the present invention, the preparation method of the SE071780-01939 protein preferably comprises the following steps:

amplifying the encoding gene of SE071780-01939 protein, cloning the encoding gene into an expression vector, obtaining a recombinant vector, then converting the recombinant vector into host bacterium competence, screening, culturing, inducing expression, culturing, separating protein and purifying to obtain recombinant protein.

In the present invention, the screening culture preferably includes culture in LB medium containing Amp. In the method for induction expression culture, preferably, an IPTG inducer is added to the bacterial liquid for induction culture. The induction time of the IPTG inducer is preferably 8h.

In the present invention, the purification method preferably includes a nickel column affinity chromatography for purifying a large amount of protein. Preferably, the purification includes identification of the protein. The identification method is preferably WesternBlot identification, and the obtained protein with expected size shows that the SE071780-01939 protein is obtained by successful recombinant expression of the invention.

The invention provides a coding gene of streptococcus equi Ma Yachong SE071780-01939 protein, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2 (ATGAAAAAACTACTGCTTATTTTAACTGTTTTTGTGACAGCTTTTACATTAGTCGCCTGCTCAAGTCAAAGCAAGCAACAAGAAAGCACCAGCAAAGCAAGCCTATCCCACAAGCCAAAAATTGCTGGAGTGACCTATTATGGTGACATTCCTGTCAAGCCAAAGCGCGTGGTTAGCTTAGCAGCTACATATACTGGTTACTTGGCAAAATTAGACCTTCATCTGGTCGGTGTCACCTCCTACGACAAAAAGAACCCTGTTTTAACAAAAAGCATTACTCAAGCCAAGCAGGTGGCAGCTACTGACCTAGAGGCTATTACAGCACTAGAGCCTGATCTCATTGTTGTTGGCTCAACAGAAGAAAACATTGACCAGCTAGCAAAAATCGCACCACTAATCTCCATTGAATACCGCAAGCGTGATTATTTGCAGGTGTTTTCTGATTTTGGCCGTATTTTTAACAAGGAAAAGGAAACTGACCAATGGTTGGCCGACTGGAAACAAAAAACAAGTGACTATGAAAAAGAAATAAAACAAGTCACCGGATCAGCTGCTACCTTTACAATCATGGGGCTCTATGAAAAGGAAATTTACCTTTTTGGTAAGGACTGGGGAAGAGGTGGTGAAATTATTCATCAGGCCTTACATTATGATGCTCCCGAAAAGGTGAAAAAAGAGGTGTTTAAGCAGGGCTATCTGTCCTTATCACAGGAGGTTCTACCAGATTATATTGGTGATTATGTGATTGTTGCTGCTGAGGATGACCAAACCGGCTCAGCCTTATATGAGAGCGACCTTTGGCAGCAGATCCCTGCTGTGACCAACAATCATGTCATCAAGGTCAACGCTAATGTCTTTTACTTCACAGACCCACTGTCACTGGAGCACCAGCTTGAGGTTCTAAAAAAGGCTATCCTCTCAACTGGTGCAAGTCAATAA).

In the present invention, the amplification primers of the coding gene preferably include a forward primer shown in nucleotide sequence SEQ ID NO. 3 and a reverse primer shown in nucleotide sequence SEQ ID NO. 4. The reaction conditions of the amplification primers are preferably shown in Table 1. The reaction procedure for the amplification primers is preferably as shown in Table 2.

The invention provides a recombinant vector containing the coding gene.

The invention is not particularly limited in the kind of the backbone vector of the recombinant vector, and expression vectors known in the art, such as pET32a, may be used. The cloning site of the coding gene in the framework vector is preferably BamHI and XhoI. The method of constructing the recombinant vector is not particularly limited, and methods of constructing recombinant vectors known in the art, such as restriction enzyme digestion, ligation and verification, may be employed.

In the present invention, the host bacteria of the recombinant strain preferably include E.coli. The strain of E.coli is preferably DH 5. Alpha. The method for preparing the recombinant strain is not particularly limited, and methods well known in the art, such as transformation of a recombinant vector into a host bacterium, may be employed. The transformation method is not particularly limited, and transformation methods well known in the art, such as a heat shock method, may be employed.

In the present invention, the volume ratio of streptococcus equi Ma Yachong SE071780-01939 protein and adjuvant is preferably 1:1. In the vaccine, the concentration of the protein is preferably 300 to 500. Mu.g/mL, more preferably 400. Mu.g/mL. The adjuvant preferably includes Freund's complete adjuvant or Freund's incomplete adjuvant. The method of preparing the vaccine is not particularly limited, and the method of preparing the protein subunit vaccine known in the art may be adopted.

In the embodiment of the invention, the safety of the vaccine is verified, and the result shows that no vaccinated mice die, thus the vaccine has better safety; the inoculation site and systemic reaction were examined, and all mice had no obvious local and systemic reaction, and were normal in body temperature, appetite, spirit, respiration, and pulse.

The invention also verifies animal immune experiments of the vaccine, and the results show that mice in a control group die completely, and the immune protection rate of the vaccine group is 50%.

In addition, the invention also verifies the titer of the vaccine, and the highest titer of the vaccine prepared by the invention is 1.28X10 ⁵ . At the same time, the vaccine also participates in the process of cellular immunity, thereby improving the anti-infection process.

The invention also provides application of the streptococcus equi Ma Yachong SE071780-01939 protein in preparing a reagent or a kit for evaluating natural immunity and/or artificial immunity effects.

The streptococcus equi Ma Yachong SE071780-01939 protein, the vaccine, the preparation method and the application thereof provided by the invention are described in detail below with reference to examples, but are not to be construed as limiting the scope of the invention.

Example 1

Cloning and expression of SE071780-01939 antigen protein

The streptococcus equi Ma Yachong XJ201902 strain is separated from the lung pus with the death rate of the equine epidemic disease, and has the culture characteristics that: the bacterial colony inoculated on the BHI solid culture medium added with 5% defibrinated sheep blood is round, smooth, orderly in edge, semitransparent or opaque, typical in beta hemolysis, dew-like and 0.8-1.2 mm in size, does not grow on the LB culture medium, and is round, smooth, orderly in edge, milky white and dew-like on the BHI solid culture medium.

The bacteria liquid of streptococcus equi Ma Yachong XJ201902 strain is used as a template, and primers are designed by referring to the technical Specification for quarantine of Margaritism (SN/T2977-2011): 5'-CAGAAAACTAAGTGCCGG TG-3' (SEQ ID NO: 5), downstream primer: 5'-ATTCGGTAAGAGCTTGACGC-3' (SEQ ID NO: 6).

10. Mu.L of LPremix Taq, 2. Mu.L of DNA template, 2. Mu.L of each of the upstream and downstream primers, and 20. Mu.L of ultrapure water were added to make up the reaction system. PCR reaction conditions: pre-denaturation at 95 ℃, annealing reaction at 61.7 ℃ for 40s after denaturation, and extension at 72 ℃ for 1min. After a total of 35 cycles, the PCR amplification of seM gene was performed by extension at 72℃for 10min, and the resulting PCR product was electrophoresed as a band (542 bp). The amplified bands were subjected to band recovery, and the recovered products were sent to Beijing Hua big Gene technologies Co., ltd for sequencing, and the isolate was identified as Streptococcus equi Ma Yachong and genotype seM-136 by the Streptococcus database PubMLST-seM based on the sequencing gene sequences.

The bacterial liquid of streptococcus equi Ma Yachong XJ201902 strain is used as a template, the nucleotide sequence of the SE071780-01939 gene is searched on NCBI according to the gene accession number GenBank: CP021972.1, SE071780-01939, and PrimerPrimer5 software is adopted to design a specific primer, F:5' -CGGGATCCATGAAAAAACTACTGCTT-3’(SEQ ID NO:3)；R:5’-GCTCGAGTTATTGACTTGCACCAGT-3’(SEQ ID NO:4)。

The gene sequence of SE071780-01939 was amplified by PCR technique, wherein the reaction system is shown in Table 1 and the reaction procedure is shown in Table 2. The PCR product is purified to construct a recombinant expression vector pET32a-01939.

TABLE 1PCR reaction System

TABLE 2PCR reaction conditions

And (3) carrying out double enzyme digestion on the purified PCR product and the pET32a expression plasmid by BamHI and XhoI, and then carrying out gel digestion recovery on the digested product, and connecting the recovered product with a linearization vector under the action of T4 DNA ligase. The obtained ligation product is transformed into DH5 alpha competent cells, and is subjected to screening culture and colony PCR amplification. The strain with pET32a-01939 positive is inoculated into LB medium containing Amp, and shake culture is carried out overnight. Culturing to OD ₆₀₀ And (3) when the bacterial liquid is approximately equal to 0.8, adding 1 mu L of prepared IPTG inducer into each 1mL of bacterial liquid to perform induction culture for 8 hours, and obtaining a culture liquid.

Example 2

Preparation of inactivated antigen

Collecting the protein in the culture solution in example 1, checking that the target protein exists in inclusion bodies through SDS-PAGE electrophoresis, purifying the protein by adopting a nickel column affinity chromatography, eluting the target protein by adopting imidazole solutions with different concentrations, and renaturating the protein eluent by urea solutions of 8M, 6M, 4M, 2M and 1M, wherein the renaturation is carried out for 8 hours at each urea concentration. The resulting protein was concentrated using sucrose. After concentration, the protein concentration was determined and then identified by Western Blot. Protein with the size meeting the expected requirement is judged as qualified protein, the concentration of the recombinant protein is adjusted to 800 mug/mL, and the recombinant protein is fully emulsified with Freund's complete adjuvant or incomplete adjuvant or complete emulsification to prepare a protein subunit vaccine (see figure 5).

Example 3

Safety inspection

8 Kunming mice, 6-8 weeks old, were divided into 2 groups of 4 mice each. The first group was PBS control group, the second group was prepared vaccine group, and each of the i.p. injections of 0.3mL of the vaccine prepared in example 2 was observed for 10d. None of the vaccinated mice died, which indicates that the vaccine has better safety; the inoculation site and systemic reaction were examined, and all mice had no obvious local and systemic reaction, and were normal in body temperature, appetite, spirit, respiration, and pulse.

Example 4

Mouse immunity test

Immunizing 8 Kunming mice 6-8 weeks old with the vaccine prepared in example 3 by intramuscular injection, wherein the number of the mice is 0.1 mL/mouse, 2 groups of mice with the same number are additionally arranged, one group of mice is intramuscular injection of PBS buffer solution as a control group, the other group of mice is intramuscular injection of Freund's adjuvant as an adjuvant control group, each group is subjected to secondary immunization after the first 14 days, the living strain is used for carrying out abdominal cavity toxicity attack test after the second 15 days, and the toxicity attack dose is 4 multiplied by 10 ⁵ CFU, view 10d. Results the control mice died entirely, the immunized mice died 4, and the immunoprotection rate of the immunized group could reach 50% (see fig. 6).

Example 5

Vaccine potency detection

Blood from the orbital venous plexus of mice was collected by glass capillary, and blood from the immunized and control mice 7d, 14d, 21d, 28d, 42d, 56d, 63d after the first immunization of mice, 3 mice each were collected. The collected blood was centrifuged at 3000r/min for 10min and the separated serum was collected. The method for detecting the neutralizing serum (IgG) by adopting a whole cell coating ELISA comprises the following specific steps:

(1) 2.5% glutaraldehyde treatment elisa plate: 2.5% glutaraldehyde solution was added and reacted at 200. Mu.L/well at 37℃for 2 hours.

(2) And (3) whole thallus coating: the glutaraldehyde solution was discarded, and the ELISA plate was washed with PBST, allowed to stand for 3min, the liquid was discarded, and the drying was repeated three times. The prepared concentration is 10 ⁹ CFU/mL inactivated whole cells were added to the ELISA plate at 100. Mu.L/well, and reacted overnight at 4 ℃.

(3) Closing: the coating solution was discarded, and the ELISA plate was washed with PBST, allowed to stand for 3min, the solution was discarded, and the drying was performed three times. 200. Mu.L of 5% nonfat dry milk solution was added to each well as a blocking solution and allowed to act at 37℃for 2 hours.

(4) Combining an antibody: the blocking solution was discarded, and the ELISA plate was washed with PBST, allowed to stand for 3min, the liquid was discarded, and the drying was performed three times. Mice serum to be tested was added at 100 μl/well dilution, with serum dilution from 1: starting at 100, increasing 2 times of the progressive mode; the reaction was carried out at 37℃for 1h.

(5) Binding a secondary antibody: discarding the primary antibody, washing the ELISA plate with PBST, standing for 3min, discarding the liquid, and drying by beating repeatedly for three times. Add 1 per well: 2000 dilution (PBST dilution) of goat anti-mouse HRP-IgG 100. Mu.L, 37℃for 1h.

(6) Color development: discarding the secondary antibody, washing the ELISA plate with PBST, standing for 3min, discarding the liquid, and drying by beating repeatedly for three times. Adding TMB color development solution, 100 mu L of each hole, and standing at 37 ℃ in dark for 15min.

(7) Terminating the reaction: the reaction was terminated by adding 100. Mu.L of a stop solution. OD determination Using ELISA plates ₄₅₀ Values.

(8) And (3) result judgment: serum OD to be detected ₄₅₀ Negative OD ₄₅₀ And if the ratio is more than or equal to 2.1, the result is positive, otherwise, the result is negative.

The detection result shows that after 2 times of immunization, the mice reach the highest value after 14d after the second immunization, and the titer reaches 1.28X10 ⁵ (see FIG. 7).

Example 6

Cytokine detection

The collected serum from mice 1 week, 2 weeks, 1 week and 2 weeks after the first and second immunization was subjected to cytokine concentration detection according to the instructions of IL-2, IL-4, IL-10 and IFN-gamma cytokine kit. The detection result shows that the content of cell factors IL-4 and IL-10 can be obviously improved after the vaccine is used for immunizing mice, and the cell immunity can be participated in the anti-infection process in the invasion process of the organism for resisting streptococcus equi Ma Yachong.

The experimental result shows that the vaccine prepared from the protein is safe to experimental animals, has little toxicity and no obvious side effect, can generate higher immune titer after 2 times of immunization of mice, and can reach ELISA titer of 1.28 multiplied by 10 after two times of immunization ⁵ The immune protection rate against mice was 50%.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The streptococcus equi Ma Yachong SE071780-01939 protein is characterized in that the amino acid sequence of the SE071780-01939 protein is shown as SEQ ID NO. 1.

2. A gene for encoding streptococcus equi Ma Yachong SE071780-01939 protein as defined in claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2.

3. A recombinant vector comprising the coding gene of claim 2.

4. A recombinant strain comprising the coding gene of claim 2 or the recombinant vector of claim 3.

5. An equine epidemic vaccine comprising the streptococcus equi Ma Yachong SE071780-01939 protein according to claim 1 and an adjuvant.

6. The equine adenomatosis vaccine of claim 5, wherein the streptococcus equi Ma Yachong SE071780-01939 protein and adjuvant are present in a 1:1 ratio by volume.

7. The equine adenomatosis vaccine of claim 5, wherein the concentration of said protein in said vaccine is 300 to 500 μg/mL.

8. The Maradeno vaccine according to any one of claims 5-7, characterized in that the adjuvant comprises Freund's complete adjuvant or Freund's incomplete adjuvant.

9. Use of streptococcus equi Ma Yachong SE071780-01939 protein according to claim 1, the coding gene according to claim 2, the recombinant vector according to claim 3 or the recombinant strain according to claim 4 for the preparation of a vaccine for controlling equine epidemic.

10. Use of streptococcus equi Ma Yachong SE071780-01939 protein according to claim 1 for the preparation of a reagent or kit for assessing the effect of natural immunity and/or artificial immunity.