CN113980942A - Streptococcus suis recombinant protein antigen Pul and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a streptococcus suis recombinant protein antigen Pul and application thereof. The invention analyzes the secondary structure, the hydrophilicity and the hydrophobicity, the epitope and the like of the streptococcus suis ApuA protein amino acid sequence, selects 4 segments with higher scores as immunogen, designs a specific primer, PCR amplifies a target strip, constructs an expression vector, induces 4 recombinant proteins to express and purify; 4 kinds of recombinant protein antigens are used for respectively immunizing SPF-level Kunming mice to prepare polyclonal antibodies, the antibodies are detected, and the recombinant protein antigen Pul is screened out. The prepared polyclonal antibody has no specificity to the ApuA protein of the streptococcus suis, and can identify the natural structure of ApuA.

Description

Streptococcus suis recombinant protein antigen Pul and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a streptococcus suis recombinant protein antigen Pul and application thereof.

Background

Streptococcus suis is a gram-positive ellipsoid bacterium with a membrane, and the natural colonizing part of the Streptococcus suis is located in the upper respiratory tract of pigs, particularly the nasal cavity and tonsil, and also comprises the alimentary canal and the reproductive tract, and belongs to conditional pathogenic bacteria. Infected, rehabilitated or healthy pigs may carry pathogens. The pathogens can be infected through mouth, nose and wound, symptoms such as dyspnea, cyanosis or reduction of weaned pigs or fattening pigs are often caused, and meningitis, pneumonia, arthritis, septicemia and the like are taken as main clinical characteristics after the disease course is developed seriously, so that huge economic loss is caused to the pig industry all over the world. Streptococcus suis can also infect humans through wounds or the digestive tract, resulting in meningitis, endocarditis, suppurative arthritis, or toxic shock syndrome, etc., which are harmful to public health.

ApuA is the only bifunctional amylase (amylomululanase) anchored outside the cell wall of streptococcus suis, has double activities of cutting alpha-1, 4-glycosidic bonds and alpha-1, 6-glycosidic bonds, can hydrolyze extracellular alpha-glucan, and provides the streptococcus suis with carbohydrates such as maltodextrin and the like including glucose, maltose, maltotriose and the like. ApuA is encoded by the ApuA gene of Streptococcus suis, contains 2094 amino acids (aa), and has a molecular weight of approximately 230 kDa. The N end of the protein has a typical alpha-amylase structural domain which can recognize and cut alpha-1, 4-glycosidic bonds; the middle section is provided with a pullulanase structural domain which can recognize and cut alpha-1, 6-glycosidic bond; the protein has a conserved LPNTG motif at the C-terminus, which is a cell wall anchoring sequence. Experiments show that ApuA can promote the adhesion of streptococcus suis to human laryngeal carcinoma epithelial cells (Hep-2), porcine tracheal epithelial cells (NPTr) and mucosa (Ferrando et al, Microbiology,2010), and is one of important adhesins of streptococcus suis (Marielae et al, FEBS Letters, 2016). ApuA can interact with human complement protein C3b to interfere the function of the host complement system, thereby inhibiting the elimination of pathogenic bacteria by the host and realizing immune escape. In the research of the inventor of the application, ApuA is also an important virulence factor of streptococcus suis, and the pathological damage condition of tissues of the brain and the lung of a mouse caused by the deletion of the ApuA gene of the streptococcus suis type 2 strain is obviously reduced compared with that of a wild strain (a streptococcus suis type 2 apuA gene knockout mutant strain and application thereof, patent application number: 201910227948.3).

ApuA mediates the adhesion and invasion of Streptococcus suis into a host, helps bacteria acquire carbohydrates in vivo, and is used as an immune escape factor and a virulence factor, and the action mechanism of the ApuA is yet to be deeply researched. ApuA is a functional protein anchored outside the cell wall of Streptococcus suis and is also one of subunit vaccine candidate proteins. In addition, as a few bifunctional amylases, the function and application potential of ApuA in enzymology are worthy of being deeply explored. Therefore, the antibody for preparing the ApuA protein has great significance, not only can provide support for functional research of the ApuA in the aspect of a bacterial pathogenic mechanism, but also lays a foundation for developing the enzyme activity function of the ApuA protein and researching and developing subunit vaccines. However, the ApuA gene has 6285 base pairs (bp), and the sequence length is too long, about 900bp longer than pET series expression plasmid. Therefore, if an ApuA full-length protein is used for immunizing an animal to obtain an antibody, the problems of high PCR amplification condition, low vector construction efficiency, low protein purity and the like caused by overlong gene segments must be faced in the processes of constructing an expression plasmid, inducing protein expression and purifying, time and labor are consumed, and the cost is increased; the polypeptide antigen designed aiming at ApuA has good specificity, but the polyclonal antibody prepared by the polypeptide antigen is mainly applied to ELISA and Western Blot, has generally not ideal effect in the aspect of recognizing the natural structure of protein, and is not suitable for the application of downstream multiple molecular technologies.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a streptococcus suis recombinant protein antigen Pul and application thereof, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.

The invention designs, implements and selects the recombinant protein antigen aiming at the ApuA protein according to the advantages that the recombinant protein antigen is easy to express and purify, the generated antibody is easy to identify natural protein and the like, so as to solve the difficult problems.

The streptococcus suis recombinant protein antigen Pul is realized in such a way that the amino acid sequence of the streptococcus suis recombinant protein antigen Pul is shown in SEQ ID No. 1.

The invention also provides application of the streptococcus suis recombinant protein antigen Pul in preparation of a polyclonal antibody.

The invention also provides application of the streptococcus suis recombinant protein antigen Pul in preparation of a reagent capable of identifying a natural ApuA structure.

The invention also provides application of the streptococcus suis recombinant protein antigen Pul in preparation of ELISA, WesternBlot and immunofluorescence detection reagents for detecting ApuA protein.

Further, firstly, an antibody is prepared by using a streptococcus suis recombinant protein antigen Pul as an antigen, and the obtained antibody is used for ELISA, Western Blot and an immunofluorescence detection reagent.

Further, the obtained antibody is a polyclonal antibody.

The invention also provides application of the polyclonal antibody in preparation of ELISA, Western Blot and immunofluorescence detection reagents for detecting ApuA protein.

The primer sequences for amplifying the recombinant protein antigen Pul of the streptococcus suis are shown as SEQ ID NO.2 and SEQ ID NO. 3.

The invention is realized by the following technical scheme:

prediction and acquisition of recombinant protein antigens

Analyzing the amino acid sequence of the streptococcus suis ApuA protein by using a bioinformatics method, predicting epitope, selecting 4 segments with higher scores as immunogens, designing specific primers, amplifying target bands by PCR, constructing expression vectors, inducing expression of 4 recombinant proteins and purifying.

Second, preparation of polyclonal antibody

The SPF Kunming mice are immunized by 4 recombinant protein antigens respectively, and the immunization program comprises primary immunization and secondary boosting immunization. Whole blood of an immune mouse and a negative mouse is obtained by a method of orbital blood collection, and serum is prepared and purified.

Thirdly, comparing the immunogenicity of recombinant protein antigens

The sensitivity of the 4 polyclonal antibodies to the full-length ApuA protein was tested by ELISA. According to the protein purification effect and ELISA titer detection result, the fragment of 885-1262aa (Pro1) is finally selected as the optimal recombinant protein immunogen and is named as Pul.

Fourthly, further detection of the antibody

WesternBlot is used for further determining whether the polyclonal antibody of the recombinant protein antigen Pul can identify the full-length ApuA and the recombinant protein Pul, and an immunofluorescence test is used for detecting whether the antibody can identify a natural ApuA structure, realize the positioning of ApuA on bacteria and mark streptococcus suis. The test result shows that the polyclonal antibody prepared by the recombinant protein Pul can be used for Westernbolt identification of the full-length ApuA protein or the recombinant protein containing the region, and can be used for immunofluorescence tests such as ApuA or streptococcus suis labeling.

In summary, the advantages and positive effects of the invention are:

(1) according to the invention, through analyzing the secondary structure, the hydrophilicity and the hydrophobicity, the epitope and the like of the streptococcus suis ApuA protein amino acid sequence, and through testing, the screened recombinant protein antigen Pul has the characteristics of easiness in obtaining, low cost, high purity, excellent phytophthora property, capability of identifying the ApuA natural structure by an antibody and the like.

(2) The polyclonal antibody prepared according to the streptococcus suis recombinant protein antigen Pul provided by the invention can be used for biological experiments such as ELISA, Western Blot, immunofluorescence and the like, provides technical support for further researching the functions of the streptococcus suis ApuA protein in the aspects of host adhesion, pathogenicity, immune escape, enzyme activity and the like, and lays a foundation for researching and developing subunit vaccines of the ApuA protein.

(3) The polyclonal antibody prepared according to the streptococcus suis recombinant protein antigen Pul provided by the invention can be used for identifying streptococcus suis thalli and other related biological tests.

(4) The polyclonal antibody prepared according to the streptococcus suis recombinant protein antigen Pul provided by the invention has no specificity on the streptococcus suis ApuA protein, and can be also suitable for detecting homologous higher pullulanase in various bacterial species, particularly streptococcus suis and other streptococcus.

Drawings

FIG. 1 shows the results of the analysis of the secondary structure, hydrophilicity and hydrophobicity, and antigenicity of ApuA protein;

FIG. 2 shows SDS-PAGE detection of purified recombinant protein;

FIG. 3 is a photograph of SDS-PAGE detecting purified ApuA protein;

FIG. 4 is a Westernblot assay result;

FIG. 5 shows the results of immunofluorescence (200-fold) observed by confocal laser microscopy;

FIG. 6 is a negative control (200-fold);

FIG. 7 shows the results of immunofluorescence observed with N-SIM (white scale: 2 μm).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

In the following examples of the present invention, the temperature is not particularly limited, and all of the conditions are normal temperature conditions. The normal temperature refers to the natural room temperature condition in four seasons, no additional cooling or heating treatment is carried out, and the normal temperature is generally controlled to be 10-30 ℃, preferably 15-25 ℃.

The genes, proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques.

The invention discloses a streptococcus suis recombinant protein antigen Pul and application thereof. The specific manipulations performed in the molecular biological experiments are carried out using conventional techniques in the art, except where specifically indicated.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Example 1 design and acquisition of Streptococcus suis ApuA recombinant protein antigen

1. The amino acid sequence (accession number: CAZ52647.1) of the streptococcus suis type 2 ApuA protein in a GenBank database and related information are obtained, the length of the protein is 2094 amino acid residues, and the length of the coding gene is 6285 base pairs.

2. The amino acid sequence of the ApuA protein is analyzed by IEDB software for secondary structure, hydrophilicity and hydrophobicity, antigenicity and the like, 4 region fragments with higher comprehensive evaluation are selected (figure 1), and the region fragments are respectively named as: pro1, Pro2, Pro3 and Pro4, the details of which are shown in Table 1.

TABLE 1 information of 4 predicted recombinant proteins

Recombinant protein name	Amino acid region	Coding sequence region	Length of amino acid sequence	Prediction of recombinant protein quality
					Pro1	885-1262aa	2653-3786bp	378aa	42kDa
Pro2	338-571aa	1012-1713bp	234aa	26kDa
					Pro3	728-870aa	2182-2610bp	143aa	16kDa
Pro4	1583-1922aa	4747-5766bp	340aa	38kDa

3. 4 pairs of amplification primers (Table 2; Tm values are all 56 ℃) were designed based on the gene coding sequence region corresponding to the recombinant protein antigen, and the primers were synthesized by Biotechnology engineering (Shanghai) GmbH.

TABLE 2 primers

4. Fresh bacterial solutions of S.suis type 2 SC19 strain were prepared, and bacterial genomes were extracted using the bacterial DNA kit from OMEGA bio-tek according to the procedures described in the specification. The SC19 genome is used as a template, and 4 pairs of primers in the table 2 are used for PCR amplification of corresponding gene coding regions of 4 recombinant proteins, and a PCR amplification preparation system is shown in the table 3. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 1 min; extension at 72 ℃ for 5 min. The PCR product was detected by 1% agarose electrophoresis, and the amplified product was recovered using Gel Extraction Kit (Trans) according to the protocol.

Note: streptococcus suis SC19 is a virulent strain of Streptococcus suis type 2, and has been isolated from infected pigs in SS2 epidemic area of Sichuan province in 2005 and published in various relevant research literatures (Tan MF et al, 2017, microbiology Open; Tan MF et al, 2015, Plos one; Zhang TF et al, 2016, Scientific Reports, etc.).

TABLE 3 PCR amplification System configuration Table (50. mu.L)

Name of reagent	Sample application volume
		2×Taq PCR buffer(Mg2+plus)	25.0μL
dNTP Mixture(2.50mM each)	4.0μL
		Upstream primer (10.00. mu.M)	1.0μL
Downstream primer (10.00. mu.M)	1.0μL
		DNA template	2.0μL
Taq DNA Polymerase(5U/μL)	0.5μL
		ddH2O	16.5μL

5. The PCR product and the pET-28a plasmid were subjected to double digestion with EcoRI and XhoI, respectively, in accordance with the Takara restriction enzyme instructions. The digested PCR product was recovered with Gel Extraction Kit, ligated to pET-28a vector using Takara T4 DNA Ligase, and then transferred to E.coli DH 5. alpha. strain and identified. The positive recombinant plasmid was sent to Biotechnology engineering (Shanghai) GmbH for sequencing.

6. The identified positive recombinant plasmid was transformed into BL21(DE3) E.coli competence. Positive colonies were picked and transferred to 5mL LB liquid medium, kanamycin (final concentration 50. mu.g/mL) was added, and shake culture was performed overnight at 37 ℃. Then, the bacterial solution 1:100 was transferred to 100mL of LB liquid medium, kanamycin was added, and when the mixture was shake-cultured at 37 ℃ until OD600 became about 0.6 to 0.8, 100. mu.L of 10.0mg/mL IPTG was added. The bacterial suspension was divided into two groups, one group was shake-cultured at 37 ℃ for 4h, and the other group was shake-cultured at 18 ℃ overnight. Two groups of thalli are respectively collected, bacteria are cracked by a high-pressure crusher, and then the whole mycoprotein, the supernatant, the precipitate and the like are subjected to SDS-PAGE electrophoresis detection. The results showed that 4 recombinant proteins were soluble expressed at both 18 ℃ and 37 ℃.

7. Each of 4 recombinant proteins was purified in large amounts according to the soluble protein purification protocol described in the specification of Ni-NTA purification system (Invitrogen). Protein samples were collected and examined by SDS-PAGE. The results are shown in FIG. 2, the Pro1 and Pro3 protein after purification has clear bands and no impurity band; pro2 and Pro4 proteins are distinct, but both contain a hetero-band. The sequence of purities of the 4 recombinant proteins after purification was: pro1 ═ Pro 3> Pro4 > Pro 2.

8. As a control, the full-length ApuA protein was also subjected to vector construction, inducible expression and purification, and the primers and the operation steps were performed with reference to "mechanism of escape complement monitoring by interaction of surface protein ApuA of streptococcus suis with C3 b" (2018) in zhaji major graduation paper. Because the ApuA full-length gene is too long, PCR amplification needs to be completed by Primerstar HS DNA Polymerase, and the cost is high; in the process of cloning the gene fragment after enzyme digestion to an expression vector, the positive cloning detection rate is low; when the correctness of the amplified product is detected by sequencing, a primer needs to be specially designed to complete the sequencing of the cloned gene segment. In addition, SDS-PAGE detection is carried out on the purified ApuA, and the result is shown in figure 3, so that the molecular weight of the purified ApuA is different from the predicted result (230kDa), and the purified ApuA has more protein impurity bands and lower purity.

EXAMPLE 2 preparation of murine polyclonal antibodies

1. Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma. The experimental animals were 25 female SPF-grade Kunming mice 6 weeks old, randomly divided into 5 groups of 5 animals each. Taking 1 group of non-immunized mice whole blood to prepare negative serum; an additional 4 groups of mice were used to prepare 4 polyclonal antibodies.

2. Primary immunization: the 4 recombinant proteins were diluted to 300. mu.g/mL with PBS, 1.5mL of each protein was removed and mixed with 1.5mL of Freund's complete adjuvant at a ratio of 1:1, and the mixture was emulsified thoroughly. Each mouse was injected subcutaneously with 300. mu.l of antigen at multiple points, i.e., each mouse was immunized with 45. mu.g of protein.

3. First booster immunization: 14 days after the initial immunization, the recombinant protein was diluted to 300. mu.g/mL with PBS, 1.5mL was taken out and mixed with 1.5mL of Freund's incomplete adjuvant at a ratio of 1:1, and the mixture was emulsified well. Each mouse was injected subcutaneously with 300. mu.l of antigen at multiple points, i.e., each mouse was immunized with 45. mu.g of protein.

4. Second booster immunization: 14 days after the first booster immunization, each mouse was injected intraperitoneally with 200. mu.g of protein solution without adjuvant.

5. 7 days after the second booster immunization, the mouse orbit was bled and positive sera were isolated.

6. Mouse positive sera were purified using Protein a affinity chromatography packing (Merck Millipore) according to the procedures provided in the instructions.

Example 3 ELISA identification

1. Coating: ApuA full-length protein was diluted with coating solution (sodium carbonate-sodium bicarbonate buffer, pH 9.6) to a final concentration of 2. mu.g/mL, and added to an ELISA plate at 100. mu.L/well overnight at 4 ℃.

2. And (3) sealing: washed 3 times with washing solution PBS-T (PBS buffer containing 0.05% Tween), blocked by adding PBS buffer containing 1% skim milk powder, 200. mu.L per well, and incubated at 37 ℃ for 2 hours.

3. Washing: the blocking solution was discarded, washed 3 times with washing solution and patted dry.

4. Primary antibody incubation: respectively diluting 4 sera from 200 times to 102400 times in gradient with blocking solution, wherein the blank control is blocking solution, and the negative mouse serum diluted by 200 times in blocking solution is used as negative control; add to the wells of an enzyme-labeled plate, 100. mu.L per well, incubate for 1 hour at 37 ℃.

5. Washing: the primary antibody was discarded, washed 3 times with washing solution and patted dry.

6. And (3) secondary antibody incubation: goat anti-mouse IgG/HRP (purchased from Abcam) was diluted 20000-fold with blocking solution and added to wells of an enzyme-labeled plate at 100. mu.L/well and incubated at 37 ℃ for 1 hour.

7. Washing: the secondary antibody was discarded, washed 3 times with washing solution and patted dry.

8. Color development: a developing solution (1% solution A + 10% solution B) was added thereto in an amount of 100. mu.L per well, and the mixture was left at 37 ℃ for 15 minutes. Solution A: DMSO solution containing 1% TMB; and B, liquid B: citrate buffer containing 0.1% H2O 2.

9. And (4) terminating: the reaction was stopped by adding 50. mu.L of stop solution (2M sulfuric acid) to each well.

10. Reading: the absorbance was measured using a microplate reader at a wavelength of 450nm, and the data was recorded and saved, the results are shown in Table 4.

TABLE 4 ELISA test results (OD)₄₅₀Value)

Dilution ratio	200	400	800	1600	3200	6400	12800	25600	51200	102400	Blank space	Negative of	Potency of the drug
														Pro1	2.608	2.615	2.470	2.349	2.106	1.954	1.625	1.367	0.973	0.430	0.105	0.161	25600
Pro2	2.440	2.327	2.014	1.893	1.428	1.031	0.875	0.398	0.187	0.133	0.081	0.120	3200
														Pro3	1.672	1.291	1.073	0.982	0.749	0.353	0.210	0.124	0.104	0.107	0.094	0.137	1600
Pro4	2.543	2.485	2.354	2.161	1.875	1.242	0.947	0.431	0.276	0.192	0.089	0.118	3200

According to the results of ELISA test, the sensitivity sequence of the antibody prepared by 4 recombinant protein antigens is as follows: pro1 > Pro2 > Pro4 > Pro3, and the potency of Pro1 is significantly superior to the other three recombinant protein antigens.

Pro1 was used as the best recombinant protein immunogen based on the overall evaluation of protein purity and ELISA titers after purification. The peptide fragment is named Pul according to the Pullulanase (Pullulanase) activity of the region, and the specific amino acid sequence is shown as follows:

VVQVELPTIGQGGTMLLQTEAGDIVNASVQGATEEPIEAGYFRVHFKTLPSDNLSSLGLWTWDDVEKPSSDLGAWPTGATNFSTAKQDDYGYYLDIKMKDETASKISLLINNTSGDNITGDKTIERISTKMNEAWFDENYQLSLYQPLKEGYIRINYFRTDGNYDQKGLWIWGDVTDLTLGDWPNGIDFENQGKYGAYIDVKLTDLPSSIGFLLLDESKSGDDVKIQQKDYSFKDLKNQTQIFLKDDDATIYTNPYFVNNVRATGVSHVSLTALEAAFTTLEGADKDSILEKLSVTDKNGQTVAVTDLVLDLTSNKVRVLGDFNQENAGYTLKYGNDSFTTTMSWQLKDELYAYDGELGARVRQAGSVVDMTLWSPSA, see SEQ ID NO. 1.

Example 4 Western blot identification

1. Electrophoresis: the full-length ApuA and Pul proteins purified in example 1 were added to the protein loading buffer, and the samples were prepared by boiling in water for 5 minutes. Preparing 10% SDS-PAGE gel, and carrying out SDS-PAGE electrophoresis according to the sample adding sequence of the protein marker, ApuA and Pul.

2. Transfer printing: after SDS-PAGE electrophoresis is finished, preparing cut PAGE gel, filter paper and a PVDF membrane, and performing protein transfer printing operation by a semi-dry transfer method by using a Bio-Rad Western Blot semi-dry transfer membrane instrument.

3. Rinsing: the electroporated PVDF membrane was washed 3 times for 5 minutes each with TBS-T buffer (0.01M TBS, pH 7.5).

4. And (3) sealing: blocking solution (TBS-T buffer containing 5% skimmed milk powder) was added to the PVDF membrane at an area of 0.1-0.15mL/cm2, and the mixture was allowed to stand at 4 ℃ overnight.

5. Primary antibody incubation: the purified Pul antibody positive serum was diluted 1000-fold with TBS-T buffer, added to PVDF membrane at 0.1-0.15mL/cm2, and incubated at 37 ℃ for 2 hours with shaking.

6. Rinsing: the primary antibody incubated PVDF membrane was rinsed 4-6 times for 5 minutes each with TBS-T buffer.

7. And (3) secondary antibody incubation: 10mL goat anti-mouse IgG/HRP diluted 5000-fold with TBS-T buffer was added and incubated at 37 ℃ for 1 hour with shaking.

8. Rinsing: the PVDF membrane after the secondary antibody incubation was rinsed 4-6 times for 5 minutes each with TBS-T buffer.

9. Color development: the PVDF membrane was developed using Bio-Rad ECL developing kit, and the results were photographed.

The experimental result is shown in FIG. 4, the detection bands of ApuA protein and Pul protein are clear and definite, which indicates that the polyclonal antibody prepared by using the recombinant protein Pul in the invention can be used for Western bolt identification of full-length ApuA protein or recombinant protein containing the region.

Example 5 immunofluorescence assay

ApuA is a protein anchored to the outer cell wall of Streptococcus suis. In order to identify whether a mouse polyclonal antibody prepared by Pul recombinant protein antigen can be used for positioning ApuA on the thallus of streptococcus suis or whether the antibody can recognize the natural ApuA structure, an immunofluorescence experiment of streptococcus suis is carried out:

1. preparing thalli: culturing the streptococcus suis type 2 SC19 strain to logarithmic growth phase (OD600 value is about 0.6), re-suspending with precooled PBS, and washing the strain for 3 times; incubating the mycelia with 100% formaldehyde at-20 deg.C for 20 min, and incubating with 80% formaldehyde at 28 deg.C for 1 hr; after the cells were washed 1 time with 80% methanol, the cells were washed with PBS.

2. Antibody incubation: after the cells were blocked with 5% BSA at 37 ℃ for 30 minutes, the cells were incubated with purified mouse anti-Pul positive serum (1:100) and allowed to react at 37 ℃ for 2 hours; the cells were washed with PBS and incubated with goat anti-mouse IgG H & L FTTC fluorescent secondary antibody (1:200) for 1 hour at 37 ℃. Mouse negative serum was also used as a test control.

3. Tabletting: the washed cells were transferred to a microscope slide, covered with a melt-cooled 1% agarose gel, and left to stand at room temperature for 10 minutes in the dark.

4. The cells were observed by a confocal laser microscope (Zeiss), and the results of observation of the cells treated with positive serum are shown in FIG. 5, and the results of observation of the cells treated with negative serum are shown in FIG. 6. The thalli were further observed with a Nikon structured illumination ultra-high resolution microscopy imaging system (N-SIM, Nikon), and the results are shown in FIG. 7. The result shows that the mouse polyclonal antibody prepared by the Pul recombinant protein antigen is suitable for identifying a natural ApuA structure, realizing the positioning of ApuA on thalli, marking streptococcus suis and can be used for immunofluorescence tests such as ApuA or streptococcus suis marking.

Example 6 demonstration of homologous proteins

Pul belongs to the 885-1262aa region of the ApuA protein, and the overlapping length of the region and the pullulanase domain (921-1962aa) of the ApuA protein is 342 aa. Pullulanase is found extensively in bacteria, either intracellularly or extracellularly. Therefore, the polyclonal antibody prepared by Pul has non-specificity to the ApuA protein of the streptococcus suis, and can be also suitable for detecting homologous higher pullulanase in various bacterial species, in particular to the streptococcus suis and other streptococcus.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

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<400> 7

cgctcgagtt aataggcatc tgtgacaact a 31

<210> 8

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccgaattcat gtttactgag gctcaaaaac t 31

<210> 9

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cgctcgagtt atgcaagaac aactgagcta t 31

Claims (8)

1. A recombinant protein antigen Pul of streptococcus suis has an amino acid sequence shown in SEQ ID No. 1.

2. Use of the recombinant protein antigen Pul of streptococcus suis according to claim 1 for the preparation of polyclonal antibodies.

3. Use of the recombinant protein antigen Pul of streptococcus suis according to claim 1 for the preparation of a reagent capable of recognizing the native ApuA structure.

4. The use of the recombinant protein antigen Pul of streptococcus suis according to claim 1 in the preparation of reagents for ELISA, Western Blot and immunofluorescence detection of ApuA protein.

5. Use according to claim 4, characterized in that: firstly, an antibody is prepared by using a streptococcus suis recombinant protein antigen Pul as an antigen, and the obtained antibody is used for ELISA, Western Blot and an immunofluorescence detection reagent.

6. Use according to claim 5, characterized in that: the obtained antibody is a polyclonal antibody.

7. Use of a polyclonal antibody as defined in claim 2, for the preparation of an ELISA, Western Blot, immunofluorescence detection reagent for the detection of ApuA protein.

8. The primer sequences for amplifying the recombinant protein antigen Pul of streptococcus suis as claimed in claim 1 are shown in SEQ ID No.2 and SEQ ID No. 3.

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