CN113980942B

CN113980942B - Streptococcus suis recombinant protein antigen Pul and application thereof

Info

Publication number: CN113980942B
Application number: CN202111471013.3A
Authority: CN
Inventors: 谭美芳; 张帆帆; 杨群; 李海琴; 曾艳兵; 谭佳; 方绍培; 吴诚诚; 饶煜玲
Original assignee: Institute Of Animal Husbandry Veterinary Jiangxi Academy Of Agricultural Sciences
Current assignee: Institute Of Animal Husbandry Veterinary Jiangxi Academy Of Agricultural Sciences
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2023-12-08
Anticipated expiration: 2041-12-03
Also published as: CN113980942A

Abstract

The application belongs to the technical field of biology, and particularly relates to a streptococcus suis recombinant protein antigen Pul and application thereof. According to the application, through analyzing the secondary structure, hydrophilicity and hydrophobicity, epitope and the like of the amino acid sequence of the streptococcus suis ApuA protein, 4 fragments with higher scores are selected as immunogens, specific primers are designed, target strips are amplified by PCR, an expression vector is constructed, 4 recombinant proteins are induced to express and purified; the 4 recombinant protein antigens are respectively immunized with SPF-class Kunming mice to prepare polyclonal antibodies, and antibodies are detected to screen recombinant protein antigen Pul. The prepared polyclonal antibody has no specificity to the streptococcus suis ApuA protein and can recognize the natural structure of the ApuA.

Description

Streptococcus suis recombinant protein antigen Pul and application thereof

Technical Field

The application 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 elliptic coccus with a capsule, the natural site of colonization is located in the upper respiratory tract of pigs, particularly the nasal cavity and tonsils, and also comprises the digestive tract and the genital tract, and belongs to conditional pathogenic bacteria. Pathogen can be carried by infected pigs, recovered pigs or healthy pigs. The pathogen can be transmitted through mouth, nose and wound, often causes symptoms such as dyspnea, cyanosis or reduction of weaned pigs or fattening pigs, and after serious disease course development, the disease course is mainly clinical characteristics such as meningitis, pneumonia, arthritis, septicemia and the like, thus causing huge economic loss for pig industry worldwide. Streptococcus suis can also infect people through wounds or digestive tracts, causing meningitis, endocarditis, suppurative arthritis or toxic shock syndrome and the like, and endangering public health.

ApuA is the only bifunctional amylase (amyloullulanase) 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 glucose, maltose, maltotriose and other maltodextrin and other carbohydrates for streptococcus suis. ApuA is encoded by the apuA gene of Streptococcus suis and contains 2094 amino acids (aa) and has a molecular weight of about 230kDa. The N end of the protein has a typical alpha-amylase structural domain, and 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 C-terminal of the protein has a conserved LPNTG motif, a cell wall anchoring sequence. Experiments have shown that ApuA promotes adhesion of Streptococcus suis to human laryngeal carcinoma epithelial cells (Hep-2), porcine tracheal epithelial cells (NPTr) and mucous membranes (Ferrando et al, microbiology, 2010), and is one of the important adhesins of Streptococcus suis (Mariela et al, FEBS Letters, 2016). ApuA can interact with human complement protein C3b to interfere with the function of the host complement system, thereby inhibiting the clearance of pathogenic bacteria by the host and realizing immune escape. In the research of the inventor, the ApuA is also an important virulence factor of streptococcus suis, and the streptococcus suis type 2 strain lacking the apuA gene is found to obviously reduce the pathological damage condition of the brain and lung tissues of mice compared with a wild strain (a type 2 streptococcus suis apuA gene knockout mutant strain and application thereof, patent application number: 201910227948.3).

ApuA mediate adhesion and invasion of streptococcus suis into hosts, help bacteria acquire carbohydrates in vivo, and act as immune escape factors, virulence factors, which have yet to be studied intensively. ApuA is also one of subunit vaccine candidate proteins as a functional protein anchored outside the streptococcus suis cell wall. In addition, as few bifunctional amylases, apuA also deserves deep development of its enzymatic functions and application potential. Therefore, the preparation of the ApuA protein antibody has great significance, not only can provide support for the functional research of the ApuA on the aspect of bacterial pathogenicity mechanism, but also lays a foundation for developing the enzyme activity function of the ApuA protein and developing subunit vaccine. However, the ApuA gene has 6285 base pairs (bp), and the sequence length is too long, which is about 900bp longer than that of pET series expression plasmids. Therefore, if the ApuA full-length protein is used for immunizing animals to obtain antibodies, the problems of high PCR amplification conditions, low vector construction efficiency, low protein purity and the like caused by overlong gene fragments must be faced in the processes of constructing expression plasmids, inducing protein expression and purifying, time and labor are consumed, and cost is increased; the polypeptide antigen designed for ApuA has good specificity, but the polyclonal antibody prepared by the polypeptide antigen is mainly applied to ELISA and Western Blot, has a generally less ideal effect in the aspect of recognizing the natural structure of protein, and is not suitable for being applied to a plurality of molecular techniques at the downstream.

Disclosure of Invention

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

According to the advantages of easy expression and purification of recombinant protein antigen, easy recognition of natural protein by the produced antibody and the like, the application designs, implements and selects the recombinant protein antigen aiming at ApuA protein so as to solve the problems.

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

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

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

The application 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 streptococcus suis recombinant protein antigen Pul as an antigen, and the obtained antibody is used for ELISA, western Blot and immunofluorescence detection reagents.

Further, the obtained antibody is a polyclonal antibody.

The application 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 streptococcus suis recombinant protein antigen Pul are shown as SEQ ID NO.2 and SEQ ID NO. 3.

The application is realized by the following technical scheme:

1. prediction and acquisition of recombinant protein antigens

Analyzing the amino acid sequence of the streptococcus suis ApuA protein by using a bioinformatics method, predicting antigen epitopes, selecting 4 fragments with higher scores as immunogens, designing specific primers, amplifying target strips by PCR, constructing an expression vector, inducing the expression of 4 recombinant proteins and purifying.

2. Preparation of polyclonal antibodies

SPF-grade Kunming mice were immunized with 4 recombinant protein antigens, respectively, and the immunization procedure included primary immunization and secondary booster immunization. Whole blood of immunized mice and negative mice was obtained by the orbital blood collection method, and serum was prepared and purified.

3. Comparison of immunogenicity of recombinant protein antigens

The sensitivity of the 4 polyclonal antibodies to the full length ApuA protein was detected by ELISA. Based on the protein purification effect and ELISA titer detection results, the 885-1262aa fragment (Pro 1) was finally selected as the optimal recombinant protein immunogen, designated Pul.

4. Further detection of antibodies

The WesternBlot is used for further determining whether the polyclonal antibody of the recombinant protein antigen Pul can recognize the full-length ApuA and the recombinant protein Pul, and an immunofluorescence test is used for detecting whether the polyclonal antibody can recognize the natural ApuA structure, realize the positioning of the ApuA on thalli and label the streptococcus suis. The test result shows that the polyclonal antibody prepared by the recombinant protein Pul can be used for identifying 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 markers.

In summary, the application has the advantages and positive effects that:

(1) The application analyzes the secondary structure, hydrophilicity and hydrophobicity, epitope and the like of the amino acid sequence of the streptococcus suis ApuA protein, and the recombinant protein antigen Pul screened by the test has the characteristics of easy acquisition, low cost, high purity, good epidemic property, capability of recognizing the natural structure of the ApuA by the antibody and the like.

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

(3) The polyclonal antibody prepared by the streptococcus suis recombinant protein antigen Pul provided by the application 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 application has no specificity to streptococcus suis ApuA protein, and can be also suitable for detecting pullulanase with higher homology in various bacterial species, in particular streptococcus suis and other streptococcus.

Drawings

FIG. 1 is a graph showing the results of analysis of the secondary structure, hydrophilicity and hydrophobicity, and antigenicity of the ApuA protein;

FIG. 2 shows the recombinant protein after SDS-PAGE detection purification;

FIG. 3 shows SDS-PAGE detection of purified ApuA protein;

FIG. 4 is a Westernblot detection result;

FIG. 5 shows immunofluorescence results (200 times) of laser confocal microscopy;

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

FIG. 7 is the result of N-SIM observation immunofluorescence (white scale: 2 μm).

Detailed Description

The present application will be described in further detail with reference to the following examples, in which the apparatus and reagents used in the respective examples and test examples are commercially available unless otherwise specified, in order to make the objects, technical schemes and advantages of the present application more apparent. The specific embodiments described herein are to be considered in an illustrative sense only and are not intended to limit the application.

Various modifications to the precise description of the application will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the application is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the application. Indeed, various modifications of the embodiments of the application which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.

For a better understanding of the present application, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless 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. 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 application, "about" means within 10%, preferably within 5% of a given value or range.

In the following examples of the present application, the temperature is not particularly limited, but is a normal temperature condition. The normal temperature is natural room temperature in four seasons, and is generally controlled at 10-30 ℃, preferably 15-25 ℃ without additional cooling or heating treatment.

The genes, proteins or fragments thereof involved in the present application 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 application discloses a streptococcus suis recombinant protein antigen Pul and application thereof. The specific procedures for molecular biology experiments are carried out using conventional techniques in the art, except as specifically described.

The technical scheme of the application will be clearly and completely described in the following in connection with the embodiments of the application.

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

1. The amino acid sequence (accession number: CAZ 52647.1) and related information of the Streptococcus suis type 2 ApuA protein in the GenBank database were obtained, the protein was 2094 amino acid residues in length, and the encoding gene was 6285 base pairs in length.

2. The amino acid sequence of the ApuA protein was analyzed for secondary structure, hydrophilicity and hydrophobicity, antigenicity, etc. using IEDB software, and 4 region fragments with higher comprehensive evaluation (fig. 1) were selected and named: pro1, pro2, pro3 and Pro4, the details of which are shown in Table 1.

TABLE 1 information on 4 recombinant proteins predicted

Recombinant protein designation	Amino acid region	Coding sequence region	Amino acid sequence Length	Predicting 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. According to the gene coding sequence region corresponding to the recombinant protein antigen, 4 pairs of amplification primers (Table 2; tm values are 56 ℃) are designed, and the primers are synthesized by the division of biological engineering (Shanghai).

TABLE 2 primers

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

Note that: streptococcus suis SC19 is a virulent strain of Streptococcus suis type 2, and has been isolated in 2005 from infected pigs in SS2 epidemic areas of Sichuan province, and a number of related research documents have been published (Tan MF et al, 2017,MicrobiologyOpen;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)

Reagent name	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
		ddH ₂ O	16.5μL

5. The PCR product and pET-28a plasmid were double digested with EcoRI and XhoI, respectively, and the digestion system and conditions were referred to the Takara restriction enzyme instructions. The digested PCR product was recovered by Gel Extraction Kit, ligated to pET-28a vector using Takara T4 DNA Ligase, and transferred into E.coli DH 5. Alpha. Strain for identification. The positive recombinant plasmid was sent to the engineering (Shanghai) stock for sequencing.

6. The identified positive recombinant plasmid was transformed into BL21 (DE 3) 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 cultured overnight at 37 ℃. Then, the bacterial liquid 1:100 is transferred to 100mL of LB liquid medium, kanamycin is added, and when the bacterial liquid is shake-cultured at 37 ℃ until the OD600 is about 0.6-0.8, 100 mu L of 10.0mg/mL of IPTG is added. Bacterial solutions were divided into two groups, one group was shake-cultured at 37℃for 4 hours and the other group was shake-cultured at 18℃overnight. Two groups of thalli are respectively collected, after bacteria are cracked by a high-pressure crusher, SDS-PAGE electrophoresis detection is carried out on whole mycoprotein, supernatant, sediment and the like. The results showed that 4 recombinant proteins were soluble expressed at 18℃and 37 ℃.

7. The 4 recombinant proteins were purified in large amounts according to the soluble protein purification protocol described in the Ni-NTA purification system (Invitrogen) specification. Protein samples were collected and subjected to SDS-PAGE detection. The results are shown in FIG. 2, the Pro1 and Pro3 protein bands after purification are clear and have no impurity bands; pro2 and Pro4 proteins are well-defined but both contain bands. The purity sequence after purification of the 4 recombinant proteins is as follows: pro1 = Pro 3> Pro4 > Pro2.

8. As a control, the full length ApuA protein was also vector constructed, induced expressed and purified, and primers and procedures were performed with reference to the Zhu Jiaqi master graduation paper, "mechanism of interaction of streptococcus suis surface protein ApuA with C3b to escape complement monitoring (2018)". Because the ApuA full-length gene is overlong, primerstar HS DNA Polymerase is required to complete PCR amplification, and the cost is high; in the process of cloning the gene fragments after enzyme digestion to an expression vector, the positive cloning detection rate is low; when detecting the correctness of amplified products by sequencing, special primers are required to complete sequencing of cloned gene fragments. In addition, SDS-PAGE detection is carried out on the purified ApuA, and the result is shown in FIG. 3, so that the molecular weight of the purified ApuA is different from the predicted result (230 kDa), and the protein has more 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 company. The experimental animals were 25 female SPF-grade Kunming mice of 6 weeks of age, randomly divided into 5 groups of 5 animals each. Taking whole blood of 1 group of mice which are not immunized, and preparing 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 taken out and mixed with 1.5mL of Freund's complete adjuvant at 1:1, and the mixture was fully emulsified. Each mouse was subcutaneously injected in multiple spots with 300. Mu.l of antigen, i.e.45. Mu.g of protein was immunized per mouse.

3. First boost: after 14 days of primary immunization, the recombinant protein was diluted to 300. Mu.g/mL with PBS, 1.5mL was removed and mixed with 1.5mL of Freund's incomplete adjuvant at 1:1, and the mixture was fully emulsified. Each mouse was subcutaneously injected in multiple spots with 300. Mu.l of antigen, i.e.45. Mu.g of protein was immunized per mouse.

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

5. 7 days after the second boost, the mice were bled from their orbitals and positive serum was isolated.

6. The mouse positive serum was purified according to the procedure provided in the description using Protein a affinity chromatography packing (Merck Millipore).

Example 3 ELISA identification

1. Coating: the 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, added to the ELISA plate at 100. Mu.L per well overnight at 4 ℃.

2. Closing: washing 3 times with PBS-T (PBS buffer containing 0.05% Tween), adding PBS buffer containing 1% skimmed milk powder, sealing, and incubating at 37deg.C for 2 hr.

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

4. Incubation resistance: starting from 200 times of 4 kinds of serum respectively by using a blocking solution, diluting the serum from 2 times to 102400 times in a gradient way, taking blank control as the blocking solution, and taking negative mouse serum diluted by 200 times of the blocking solution as a negative control; add to the wells of the microplate, incubate at 37 ℃ for 1 hour at 100 μl per well.

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

6. Secondary antibody incubation: sheep anti-mouse IgG/HRP (from Abcam) was diluted 20000 fold with blocking solution and added to wells of enzyme-labeled plate, 100. Mu.L per well, incubated for 1 hour at 37 ℃.

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

8. Color development: the color development solution (1%A solution +10% B solution) was added thereto, and the mixture was left at 37℃for 15 minutes in 100. Mu.L per well. And (3) solution A: DMSO solution containing 1% tmb; and (2) liquid B: a citrate buffer containing 0.1% H2O 2.

9. And (3) 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 at a wavelength of 450nm using an enzyme-labeled instrument, and the data were recorded and saved, and the results are shown in Table 4.

TABLE 4 ELISA test results (OD ₄₅₀ Value of

Dilution ratio	200	400	800	1600	3200	6400	12800	25600	51200	102400	Blank space	Negative of	Valency of
														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

As shown by ELISA test results, the sensitivity sequence of the antibodies prepared by the 4 recombinant protein antigens is as follows: pro1 > Pro 2=Pro 4 > Pro3, and the potency of Pro1 is significantly better than the other three recombinant protein antigens.

Pro1 can be used as the optimal recombinant protein immunogen according to comprehensive evaluation of the purity and ELISA titers of the purified protein. Based on the Pullulanase (Pullulanase) activity of this region, this peptide fragment was designated Pul and the specific amino acid sequence is shown below:

VVQVELPTIGQGGTMLLQTEAGDIVNASVQGATEEPIEAGYFRVHFKTLPSDNLSSLGLWTWDDVEKPSSDLGAWPTGATNFSTAKQDDYGYYLDIKMKDETASKISLLINNTSGDNITGDKTIERISTKMNEAWFDENYQLSLYQPLKEGYIRINYFRTDGNYDQKGLWIWGDVTDLTLGDWPNGIDFENQGKYGAYIDVKLTDLPSSIGFLLLDESKSGDDVKIQQKDYSFKDLKNQTQIFLKDDDATIYTNPYFVNNVRATGVSHVSLTALEAAFTTLEGADKDSILEKLSVTDKNGQTVAVTDLVLDLTSNKVRVLGDFNQENAGYTLKYGNDSFTTTMSWQLKDELYAYDGELGARVRQAGSVVDMTLWSPSA, SEQ ID NO.1.

Example 4 Western blot identification

1. Electrophoresis: the purified full length ApuA and Pul proteins of example 1 were taken, added to protein loading buffer, and boiled in boiling water for 5 minutes, respectively, to prepare samples. 10% SDS-PAGE gel was prepared and subjected to SDS-PAGE according to the loading sequence of protein markers, apuA and Pul.

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

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

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

5. Incubation resistance: purified Pul antibody positive serum was diluted 1000-fold with TBS-T buffer, added at a PVDF membrane area of 0.1-0.15mL/cm2, and incubated with shaking at 37℃for 2 hours.

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

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

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

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

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

Example 5 immunofluorescence assay

ApuA is a protein anchored to the extracellular wall of Streptococcus suis. To identify whether a mouse polyclonal antibody prepared with the Pul recombinant protein antigen can be used for localization of ApuA on streptococcus suis thalli or whether the antibody can recognize the native ApuA structure, immunofluorescence experiments of streptococcus suis were performed:

1. preparation of a bacterial strain: streptococcus suis type 2 SC19 strain was grown to log phase (OD 600 value of about 0.6), resuspended in pre-chilled PBS and the cells washed 3 times; incubating the thalli with 100% formaldehyde at-20 ℃ for 20 minutes, and then incubating with 80% formaldehyde at 28 ℃ for 1 hour; after washing the cells 1 time with 80% methanol, the cells were washed with PBS.

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

3. And (3) tabletting: the washed cells were transferred to a microscope slide, covered with a thawed and cooled 1% agarose gel, and allowed to stand at room temperature for 10 minutes under light-shielding conditions.

4. The cells were observed with a confocal laser microscope (Zeiss), the results of the positive serum-treated cells are shown in FIG. 5, and the results of the negative serum-treated cells are shown in FIG. 6. The cells 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 using the Pul recombinant protein antigen is suitable for recognizing the natural ApuA structure, realizing the positioning of the ApuA on thalli, marking streptococcus suis and the like, and can be used for immunofluorescence experiments such as ApuA or streptococcus suis marking and the like.

EXAMPLE 6 description of homologous proteins

Pul belongs to the 885-1262aa region of the ApuA protein, which overlaps with the pullulanase domain (921-1962 aa) of the ApuA protein by a length of 342aa. Pullulanase is widely found in bacteria, either located intracellularly or located outside the cell wall. Therefore, the polyclonal antibody prepared by Pul has non-specificity to the ApuA protein of streptococcus suis, and can be also suitable for detecting the pullulanase with higher homology in various bacterial species, in particular to streptococcus suis and other streptococcus.

The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.

Sequence listing

<110> institute of livestock and veterinary at academy of agricultural sciences in Jiangxi province

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<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<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

1. The application of streptococcus suis recombinant protein antigen Pul with the amino acid sequence shown as SEQ ID NO.1 in preparing polyclonal antibodies is characterized in that: the primer sequences for amplifying the streptococcus suis recombinant protein antigen Pul gene are shown as SEQ ID NO.2 and SEQ ID NO. 3.