CN102940875B - Paa albumen and antibody prepared therefrom and application - Google Patents

Abstract

The invention discloses Paa albumen and antibody prepared therefrom and application.The invention provides Paa albumen and have following 1 in preparation)-3) at least one function product in application: 1) prevent and/or treat by pathogen EHEC diseases induced; 2) pathogen EHEC intestinal infection damage is reduced; 3) pathogen EHEC is reduced in intestinal definite value power; The aminoacid sequence of described Paa albumen is the sequence 2 in sequence table.Experiment of the present invention proves, the present invention obtains encoding gene, the recombinant expressed and purification Paa albumen of Paa albumen by technique for gene engineering clone, the experimentatioies such as the immunologic function of inside and outside assessment Paa albumen, Paa albumen can by O157:H7 infect serum special, identify efficiently, be the important cAg of EHEC pathogen, can be used as antigenic agents for Infect And Diagnose; The mice of Paa protein immunization can resist O157:H7 counteracting toxic substances, shows that this albumen can as the diseases induced vaccine of EHEC.

Description

Paa protein and antibody prepared therefrom and application

technical field

The invention relates to the field of biotechnology, in particular to Paa protein, antibody prepared therefrom and application thereof.

Background technique

Enterohemorrhagic Escherichia coli (enterohemorrhage E.coli, EHEC) is the most virulent and most pathogenic Escherichia coli, which has received extensive attention. EHEC infection can make people suffer from diarrhea, hemorrhagic colitis (HC), and can also cause serious complications such as hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). 5% to 10%. In April 2011, an outbreak of enterohaemorrhagic Escherichia coli O104:H4 infection and hemolytic uremic syndrome (HUS) broke out in Germany, spreading to 16 countries in Europe and North America. In April and May of the same year, a new infection broke out in the Toyama area of Japan. The serotype was O111. Some cases developed neurological symptoms after causing HUS, complicated by disseminated intravascular coagulation and encephalopathy. In EHEC, O157:H7 is highly pathogenic and harmful. In the past 20 years since it was first isolated in 1975 and identified as a pathogenic bacteria in 1982, there have been outbreaks of different scales all over the world, including China. In 1999, there were large-scale outbreaks in Jiangsu and Anhui provinces in my country, with more than 20,000 patients and 177 deaths. EHEC infection has become the first cause of renal failure in children in European and American countries, posing a huge threat to people's health. In addition, as an important zoonotic pathogen, EHEC pathogenic bacteria can reside in the intestines of livestock and poultry such as cattle, sheep, pigs, and chickens for a long time, causing diarrhea in animals and contaminating meat, milk and egg products of livestock and poultry. Water sources and crops have caused huge losses to agricultural and animal husbandry production. Therefore, EHEC infection has become a global public health problem.

At present, clinical treatment of EHEC infection mainly adopts symptomatic treatment, because antibiotic treatment has the risk of exacerbating the disease and is used with caution, and there is no vaccine or specific treatment drug available. Vaccine is considered to be the simplest, cheapest and quickest way to deal with EHEC outbreaks and sporadic infections. If the core antigen or dominant protective antigen can be discovered and obtained, it can be further developed into a new type of vaccine.

Adhesive colonization is a key link in bacterial infection and injury. Bacteria adhere to host epithelial cells and cause A/E (Attaching and effacing, A/E) lesions. Tir, Intimin, Map, EspF, EspG, EspH, SepZ, and EspB are recognized effector molecules encoded by LEE, and they all participate in the A/E injury of EHEC. But the researchers found that there were still other effector molecules not encoded by LEEs.

Paa (porcine attaching and effacing associated) is a 27.6kDa protein encoded by a 753bp gene. Sequence analysis found that the amino acid sequence of the protein encoded by the paa gene was completely consistent between O157:H7EDL933 and Saikai. Chromosomal mapping technology found that the region containing the paa gene was located on the chromosome, between yciD and yciE. Theresa et al. found that EHEC deletion mutants of paa gene can reduce the adhesion to HeLa cells, and also reduce the secretion of certain cell surface proteins, some of which are indispensable for the secretion system of EHEC, and these secretion systems are EHEC An important guarantee for adherent cells, so Paa has some relationship with the adhesion of EHEC. At present, scientists from many countries are conducting research on the adhesion function and molecular mechanism of Paa protein, but so far, the role and function of Paa molecule in bacterial infection detection and immune protection have not been confirmed from an immunological point of view.

Contents of the invention

One object of the present invention is to provide a new application of Paa protein.

The application of the Paa protein provided by the present invention in the preparation of products having at least one function in the following 1)-3):

1) Prevention and/or treatment of diseases infected or caused by pathogenic bacteria EHEC;

2) Reduce the damage of pathogenic bacteria EHEC to intestinal infection;

3) Reduce the value of pathogenic bacteria EHEC in the intestinal tract;

The amino acid sequence of the Paa protein is sequence 2 in the sequence list.

In the above application, the product is a vaccine or a medicine.

In the above application, the intestinal tract is human or animal intestinal tract, and in the embodiment of the present invention, the animal intestinal tract is mouse intestinal tract; the pathogen EHEC is EHEC O157:H7.

The application of Paa protein as a target or detection marker in the development or preparation of products for diagnosing infection with pathogenic bacteria EHEC is also within the protection scope of the present invention; in the above application, the amino acid sequence of the Paa protein is sequence 2 in the sequence listing.

Another object of the invention is to provide a product.

The product provided by the invention has an active ingredient of Paa protein;

The product mentioned is a product with at least one function in the following 1)-3):

1) Prevention and/or treatment of diseases caused by pathogenic bacteria EHEC;

2) Reduce the damage of pathogenic bacteria EHEC to intestinal infection;

3) Reduce the value of pathogenic bacteria EHEC in the intestinal tract.

4) Detect the infection of pathogenic bacteria EHEC.

Antibodies prepared from the Paa protein as an antigen are also within the protection scope of the present invention; the amino acid sequence of the Paa protein is sequence 2 in the sequence listing.

The above-mentioned antibodies are polyclonal antibodies or monoclonal antibodies.

The application of the product for detecting Paa protein in the preparation of EHEC products for diagnosing pathogenic bacteria is also within the protection scope of the present invention; the pathogenic bacteria EHEC is specifically EHEC O157:H7; the above-mentioned product for detecting Paa protein is specifically the above-mentioned antibody.

The application of the above-mentioned antibodies in the preparation of products to improve the host’s resistance to pathogenic bacteria EHEC infection or to improve the host’s resistance to pathogenic bacteria EHEC adhesion is also within the protection scope of the present invention;

In the above application, the host is specifically a human or an animal; the host is specifically a cell in a human or an animal; the pathogen EHEC is specifically EHEC O157:H7; in the embodiments of the present invention, it is used to prove that the host resists the adhesion of the pathogen EHEC The experiment was carried out with HeLa cells as the host.

A third object of the present invention is to provide a product.

The product provided by the present invention, its active ingredient is the above-mentioned antibody;

The product mentioned is a product with at least one function in the following 1)-3):

1) For the detection of pathogenic bacteria EHEC;

2) Improve host resistance to pathogenic bacteria EHEC infection;

3) Improve the adhesion of host anti-pathogen EHEC;

Wherein the host is specifically a human or an animal; the host is particularly specifically a cell in a human or an animal; the pathogenic bacteria EHEC is specifically EHEC O157: H7; in the embodiments of the present invention, it is used to prove that the adhesion of the host anti-pathogenic bacteria EHEC is improved through hela cells as The host was tested.

The experiment of the present invention proves that the present invention clones and obtains the coding gene of Paa protein through genetic engineering technology, recombines expression and purification of Paa protein, evaluates the immunological function of Paa protein in vivo and in vitro and other experimental studies, and confirms that (1) Paa protein can be suppressed by O157: The specific and efficient recognition of H7 infection serum is an important core antigen of EHEC pathogenic bacteria, which can be used as an antigen reagent for infection diagnosis; (2) Paa immunized mice can reduce the colonization of EHEC in the intestine; (3) Paa protein immunization Mice can resist O157:H7 challenge, indicating that this protein can be used as a vaccine against EHEC-induced diseases; (4) Paa protein can be used to prepare antibodies or antiserum, which can specifically recognize EHEC bacterial proteins and can As an antibody reagent to detect EHEC pathogenic bacteria; (5) Paa protein antibody or antiserum can antagonize the adhesion effect of EHEC pathogenic bacteria to target cells, effectively intervene in the colonization of pathogenic bacteria in the intestinal tract, thereby significantly reducing or almost completely inhibiting the effects of pathogen infection on the body (6) Paa protein can stimulate the body to produce a specific response and has a high level of immunogenicity. By inducing a high-efficiency immune effect, it can resist damage or death caused by pathogenic bacteria infection.

Description of drawings

Figure 1 is the purified protein and WB blot of Paa

Figure 2 is the monitoring of antibody titer changes with the number of immunizations and interval time

Figure 3 is the detection of IgA and IgG subtypes in serum of immunized mice

Figure 4 is the detection of the amount of bacteria excreted in the excrement of mice after challenge

Figure 5 is the WB test of anti-Paa antiserum

Figure 6 is the anti-adhesion rate of Paa antiserum to cells

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Some materials are as follows:

1. Vector and strain

EHEC O157: The H7EDL933 strain can be provided by ATCC, catalog number: 700927; the public can also obtain it from the Institute of Microbial Epidemiology, Academy of Military Medical Sciences of the Chinese People's Liberation Army, and the bacterial strain EDL933 strain is recorded in L.W.Riley, R.S.Remis, S.D. Wells, B.R. Davis, R.J. Hebert, E.S. Olcott, L.M. Johnson, N.T. Hargrett, P.A. Blake, M.L. Cohen, Hemorrhagic colitis associated with a rare Escherichia coli serotype, N. Engl, J. Med. 308(1983) 681685. The pEASY-T1 vector, E.coli DH5α, E.coli BL21 (DE3) competent cells were purchased from TransGen, and pET-22b(+) was purchased from Novagen.

2. Reagents and media

NdeI and XhoI restriction endonucleases and T4 ligase were purchased from NEB Company; horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody and HRP-labeled histidine antibody were purchased from Pierce; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG1 antibody, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG2a, Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG2b antibody, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG3 antibody and horseradish peroxidase (HRP)-labeled goat anti-mouse IgG Mouse IgA antibody was purchased from Sigma Company; Taq enzyme and DNAmarker (DL2000) used in PCR were purchased from TransGen Company; plasmid mini-prep kit and agarose gel recovery kit were purchased from Beijing Biomed Company; other chemical reagents were purchased from It is chemically pure or analytically pure; the nickel column was purchased from GE.

EHEC O157: H7 bacteria were cultured using Luria-Bertani (LB, OxoidLTD, Basingstok, Hampshire, England) liquid medium and LB plates. Engineering bacteria were cultured using LB liquid medium and LB plates containing ampicillin at a final concentration of 100 μg/ml.

3. Biochemical and molecular biology reagents

Sorbitol MacConkey Basic Agar and Sorbitol MacConkey Agar Additive were purchased from Qingdao Hi-Tech Park Haibo Biotechnology Co., Ltd.; DEME medium was purchased from GIBCO Company; standard fetal bovine serum was purchased from Biochrom Company; double antibodies were purchased from Hyclone Company.

4. Preparation method

Preparation method of LB medium (1L): Nacl 10g; yeast powder 5g; peptone 10g.

The preparation method of table 1 SDS-PAGE separating gel and stacking gel

5. Experimental animals

Big-eared white rabbits (male, 2.5kg) and BALB/c mice (female, 14-16g) were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences.

Embodiment 1, the recognition of Paa protein to the serum of infection EHEC

1. Purification of Paa protein

1. Fishing of Paa protein coding gene

Design paa primers according to GenBank (GeneID: AE005174), and introduce restriction sites NdeI and XhoI (underlined part), that is, P1: 5'- catatg aggaacataatggcaggtt-3'; P2: 5'- ctcgag tcaagtgcctttcctggtcca-3'.

Using EHEC O157:H7 genomic DNA as a template, the PCR conditions were: 94°C for 5min, 94°C for 30s, 60°C for 30s, 72°C for 1min, 72°C for 10min, 25 cycles.

50 μl PCR reaction system: 1 μl genomic DNA template, 0.5 μl upstream primer, 0.5 μl downstream primer, 4 μl dNTP, 1 μl Taq polymerase, 5 μl 10×buffer, add H ₂ O to 50 μl.

A PCR product of 768bp was obtained, and after sequencing, the nucleotide sequence of the PCR product was sequence 1 in the sequence listing, wherein the sequence 1 in the sequence listing was the coding gene of Paa protein from the 4th to 762nd nucleotides at the 5' end , the amino acid sequence of the Paa protein encoded by the coding gene is sequence 2 in the sequence list.

Gel recovery of the above PCR product was connected to the cloning vector pEASY-T1, transformed into E.coli DH5α competent cells, and colony PCR identification was performed (primers were P1 and P2), and the 768bp PCR product was obtained as a positive colony; the above positive colonies were extracted with plasmids and used Digested with NdeI and XhoI, the digested product of about 768bp was a positive plasmid, which was named pEASY-T1-paa.

2. Construction of Paa protein recombinant expression plasmid and expression engineering bacteria

The cloned plasmid pEASY-T1-paa and the expression vector pET-22(+) were digested with NdeI and XhoI respectively, and the about 768bp fragment was recovered and ligated with the pET-22(+) vector fragment, and the ligated product was transferred into E.coli DH5α sensory State cells, get the transformant, extract the plasmid of the transformant and send it for sequencing. The plasmid is the NdeI and XhoI enzymes that insert the nucleotides 4-763 from the 5' end of the sequence 1 in the sequence listing into the pET-22 (+) vector The plasmid obtained between the cutting sites was named pET-22(+)-paa, and the transformant containing the plasmid was named DH5α/pET-22(+)-paa.

3. Expression and identification of Paa protein

Introduce DH5α/pET-22(+)-paa into fresh LB medium. When the OD ₆₀₀ reaches the logarithmic growth phase, take part of the bacterial liquid as the uninduced control, and add IPTG to the rest of the bacterial liquid to a final concentration of 1mmol/ L, induced expression for 4 hours; induced and uninduced bacterial fluids were obtained.

Centrifuge the induced and uninduced bacteria solution at 5000g for 25min to collect the bacteria; ultrasonically disrupt the bacteria at 400W (working for 5s, interval of 3s, cycle 99 times), collect the whole bacterial lysate, and collect the precipitate by centrifuging at 5000g for 20min and supernatant.

Take the whole bacterial lysate of the uninduced bacterial solution, the whole bacterial lysate of the induced bacterial solution, the supernatant of the induced bacterial solution, and the precipitate of the induced bacterial solution for SDS-PAGE respectively, select 5% concentrated gel and 15% separating gel, The results showed that compared with the uninduced whole bacterium lysate, the induced whole bacterium lysate showed a clear band at 27Kd, which was consistent with the theoretical Paa protein size, indicating that the protein was successfully expressed, and the supernatant of the induced bacterium There were also obvious bands in the precipitation of the solution and the induced bacteria, indicating that the Paa protein was expressed in the form of soluble protein and inclusion body, respectively.

4. Purification of Paa protein

The whole bacterium lysate of above-mentioned induced bacterium liquid is collected supernatant with 5000g, 20min centrifugation, passes through nickel column and purifies; Dehydration is the purification of Paa protein.

The purified Paa protein was subjected to SDS-PAGE electrophoresis, and the results are shown in lane 2 in Figure 1. Lane 1 is a protein 12-80kd marker. It can be seen that a protein fragment of 27KD appears in lane 2, which is consistent with the expected size, indicating that Purified Paa protein was obtained. The concentration of purified Paa protein was measured to be 3.5 mg/ml.

The purified Paa was subjected to Western blot (the WB probe of lane 3 is HRP-labeled anti-His tag antibody), the result is shown in lane 3 in Figure 1, and expression bands and imprinted bands appeared at about 27kd, which can be Recognized by a specific tag antibody, the size is consistent with the predicted molecular weight of the fusion protein.

5. Recognition of purified Paa protein to EHEC infected serum

The purified Paa was subjected to Western blot, the probe was mouse anti-EHEC O157:H7 whole bacterial protein polyclonal antibody (antiserum obtained from mice infected with EHECO157:H7), the secondary antibody was anti-mouse IgG antibody (Sigma), and the result As shown in lane 4 in Figure 1, a specific band also appeared around 27DK, indicating that Paa can be recognized by the mouse anti-O157:H7 polyclonal antibody and has good immunoreactivity, indicating that it is EHEC O157 : H7 antigen, can be used to diagnose EHEC infection.

Embodiment 2, the immunogenicity and protective effect of Paa protein

Female BALB/c mice 14-16g were randomly divided into 2 groups, 10 in each group, and the following experiments were repeated three times:

Paa immunization group: the purified Paa protein obtained in Example 1 was immunized by intraperitoneal route as antigen;

BSA control group: intraperitoneal route immunization with BSA as antigen.

Immunization method: For the first immunization, an equal volume of Freund's complete adjuvant (total volume 100 μl) was used, and for the booster immunization, an equal volume of Freund's incomplete adjuvant was used to mix with the antigen. The interval between each immunization was 2 weeks, and a total of three immunizations were performed. The final concentration of antigen for the first immunization was 25 μg, and the final concentration of antigen for booster immunization was 50 μg. Ten days after the last immunization, the mice in the Paa immunization group and the BSA control group were given 5g/L streptomycin in drinking water for 3 days, and then were given oral administration of 10 ⁹ CFU (10MLD) EHEC O157:H7EDL933 after fasting for 24 hours. The poison was challenged by intragastric administration, and the state and survival of the mice were observed.

1. Immunogenicity detection of Paa protein

The mice in the above BSA control group and Paa immunized group were collected from the tail vein on days 0, 10, 24, 46, and 82, and the serum was separated. ELISA was used to detect serum IgG levels, Paa was used as the antigen coating, BSA was used as the control, the collected mouse serum was used as the antibody to be tested, and HRP-labeled goat anti-mouse IgG antibody was used as the secondary antibody.

The antibody monitoring results are shown in Figure 2. The mouse immunization time points are the 1st, 14th, and 28th days in turn, and the arrows indicate the immunization time points; the ordinate indicates the logarithmic value of the ELISA titer of IgG in the serum; the abscissa indicates the distance The number of days on the first day of immunization; it can be seen that compared with the BSA control group, the mice in the Paa immunization group can obtain a high level of titer after the second immunization, and the IgG level reaches the peak after the third immunization, and reaches the peak after 80 days. During the monitoring process, the Paa antibody titer did not decrease significantly, and the Paa immunized group was at a high antibody titer level of 10 ⁷ throughout the immunization process. It shows that Paa is a significant dominant immune antigen with very strong immunogenicity.

The mice in the BSA control group and Paa immunized group were collected blood from the tail vein on the 14th day after the last immunization, and the serum IgA, IgG subtype IgG1, IgG2a, IgG2b, IgG3 titers were detected by ELISA method, Paa was the antigen coating, BSA was used as a control, and the collected mouse serum was used as the antibody to be tested. HRP-labeled goat anti-mouse IgA antibody, HRP-labeled goat anti-mouse IgG1 antibody, HRP-labeled goat anti-mouse IgG2a, HRP-labeled goat anti-mouse IgG2a, and HRP-labeled goat anti-mouse IgG2a were respectively used as antibodies to be tested. Anti-mouse IgG2b antibody and HRP-labeled goat anti-mouse IgG3 antibody were used as secondary antibodies.

The results are shown in Figure 3, with 10 mice in each group, and the results in the diagram were measured three times, and the ordinate represents the geometric mean ± standard deviation of the ELISA titer in each group; , IgG2a, IgG2b, IgG3 titers were 1.3×10 ⁴ , 1×10 ⁵ , 8.6×10 ³ , 1×10 ⁴ and 1×10 ³ respectively; in the BSA control group, IgA, IgG subtype IgG1, IgG2a, IgG2b, IgG3 titers were both 1×10 ² ; it can be seen that Paa immunization resulted in significantly higher titers of IgA, IgG and its subtypes IgG1, IgG2a, IgG2b compared with BSA immunized group, while IgG3 had no obvious raised.

2. Monitoring of the amount of bacteria excreted by the tested animals

After challenge, the feces of the mice in the Paa immunized group and the BSA control group were collected every 2 days, and 0.2 g was weighed and dissolved in 100 ml of LB medium, and incubated at 4°C for 4 hours until the feces softened and could be shaken into a cloudy liquid. After centrifuging the feces, serially dilute the bacterial solution 10 times, spread the diluted bacteria, apply the Sorbitol MacConkey identification plate and count.

The monitoring results of the amount of bacteria excreted in the tested animals are shown in Figure 4. The vertical axis is the counting result of gradient dilution plate per 0.1g of feces, and below 100CFU/ml is an invalid value, and the horizontal axis is the number of days after the challenge. The error bar is Geometric mean ± standard deviation of CFU in each group;

The bacteria excreted by the Paa immunized group at 2, 4, 6, 8, 10, 12, and 14 days after challenge were 2×10 ⁴ , 9×10 ³ , 1.2×10 ³ , 4.5×10 ² , and 2×10 ² , 2×10 ² , 2×10 ² , bacteria discharge has stopped on the 10th day;

BSA control group had 6.3×10 ⁵ , 1×10 ⁵ , 2×10 ⁵ , 1.3×10 ⁵ , 1.6×10 ⁵ , 4×10 ⁴ , 6.3×10 ⁴ ;

It can be seen that the control group showed a sustained high amount of bacteria excretion throughout the 14-day monitoring process, while the immunized group showed a gradual and sharp decrease in the amount of bacteria excretion, and basically stopped expelling bacteria on the 10th day. It shows that Paa-immunized mice can reduce the colonization of EHEC in the intestine, reduce the amount of excreted bacteria, and reduce the damage to the intestine; it can also reduce the repeated infection of the food chain.

3. Survival rate statistics

In the large amount of bacteria (lethal dose) challenge test to the tested animals, when the 10MLD dose of pathogenic bacteria challenged the immunized mice three times, on the 10th day after the challenge, the survival rate of the tested animals in the Paa immunized group was 100% (10 mice/ 10) were protected, while all mice in the BSA control group died on the seventh day (0/10).

In the same way as above, the challenge dose was further increased, and 50MLD and 100MLD doses were used to test the twice-immunized mice in the Paa immunization group. As a result, the mice in the Paa immunization group could still significantly protect the 50MLD live bacteria attack, and the survival rate was 70% ( 7/10); and the challenge of 100MLD live bacteria can also show a certain degree of protection (2/10).

The above experiments show that Paa protein can be used as a vaccine to prevent and/or treat diseases caused by pathogenic bacteria EHEC.

Embodiment 3, preparation and application of anti-Paa antibody

1. Preparation and identification of anti-Paa antibody (anti-Paa antiserum)

The Paa purified protein obtained in Example 1 with a concentration of 1 mg/ml and a purity of more than 95% was used as an antigen to immunize big-eared white rabbits (male, 2.5 kg). Respectively in the fourth week (25μg/monkey) and eighth week (50μg/bird) booster immunization. Take ear vein blood one week after each immunization, and measure the serum antibody titer to be 10 ³ and 10 ⁶ (ELISA test), when the titer reaches and stabilizes above 1:10 ⁶ , take blood from the heart of the big ear white, 5000g The serum was separated by centrifugation for 10 min, and the serum was filtered with a 2 μm disposable filter (Millipore). The filtered serum was purified by affinity chromatography to obtain anti-Paa antibody (polyclonal antibody), which was stored at -80°C after purification.

Cultivate EHEC O157:H7, collect the bacteria, ultrasonically disrupt, and collect the whole bacterial protein. SDS-PAGE and Western Blot were performed on EHEC O157:H7 whole bacterial protein to detect the ability of anti-Paa antibody to recognize bacterial protein and detect pathogenic bacteria. In Western Blot detection, rabbit serum before Paa immunization and anti-Paa antibody were used as primary antibodies, and HRP Anti-rabbit IgG was labeled as the secondary antibody.

The results are shown in Figure 5, M is protein 12-80kd marker; 1 is EHEC O157:H7 whole bacterial protein; 2 is WB detection of rabbit serum before Paa immunization as primary antibody; 3 is WB detection of anti-Paa antibody as primary antibody It can be seen that the anti-Paa antibody can recognize EHEC O157:H7 whole bacterial protein, and has the ability to recognize pathogenic bacteria antigens; it shows that the anti-Paa antiserum is the antibody of O157:H7, which can be used to diagnose EHEC O157:H7 pathogenic bacteria.

2. Anti-Paa antibody anti-EHEC pathogen cell adhesion in vitro

The wild-type EHEC O157:H7 was inoculated in 5ml LB liquid medium, and cultured with shaking at 37°C for 16h.

In each well of a 96-well cell culture plate, add Hela cells (Shanghai Chuanxiang Biotechnology Co., Ltd., CH01050XI) 10 ⁵ /well and DMEM culture solution, and keep at 37°C, 5% CO ₂ to 80%-90 per well. % of the area is covered with a single cell layer, absorb the old nutrient solution, replace it with fresh DMEM medium, and add fetal bovine serum to the nutrient solution to a final concentration of 0.5%; then add the freshly cultivated wild-type EHEC O157:H7 to In the culture wells, the amount of bacteria was 10 ⁶ bacteria (quantified by colony counting method), and cultured at 37°C for 3h. Aspirate off the nutrient solution, and carefully wash the culture plate with phosphate buffered saline (PBS) with a pH value of 7.0 for 5 times, each time for 3 minutes, to wash off the unadhered bacteria, digest with trypsin after washing, and wash the cells. Wash the cells with residual digestive fluid, lyse the cells with 1% Triton X-100, serially dilute the bacterial solution 10 times after centrifugation, spread the diluted bacteria, apply the Sorbitol MacConkey identification plate and count.

The results are shown in Figure 6, the abscissa is the dilution of the serum, the ordinate is the ratio of the anti-adhesion rate = non-adhered bacteria to the total number of bacteria, and the value is the geometric mean ± standard deviation of the protection rate of each group;

The anti-adhesion rates of the immunized group at 0, 1, 2, 4, 8, 16, 32, 64, 128, and 256 serum dilutions were 90%, 89%, 85%, 75%, 65%, 55%, 39%, 27%, 9% and 0%;

The anti-adhesion rates of the control group at 0, 1, 2, 4, 8, 16, 32, 64, 128, and 256 serum dilutions were all 0%;

It can be seen that in the in vitro anti-bacterial adhesion experiment, the anti-Paa serum does have a significant anti-adhesion effect. With the increase of the anti-Paa serum concentration, the anti-adhesion effect is more obvious, and there is a dose-dependent positive correlation. It shows that anti-Paa antibody can interfere with the adhesion and damage of pathogenic bacteria to target cells, and has a protective effect.

Claims (1)

1. the antibody prepared as antigen by Paa albumen improves host's anti-microbial pathogen EHEC O157:H7 EDL933 in preparation to be infected product or improves host's anti-microbial pathogen EHEC O157:H7 EDL933 and stick application in product; Described antibody is polyclonal antibody;

The aminoacid sequence of described Paa albumen is the sequence 2 in sequence table;

Described polyclonal antibody is prepared as follows: concentration is reached 1mg/ml, purity reaches the Paa purifying protein of more than 95% as antigen, male, the 2.5kg large ear rabbit of immunity; Respectively at 4th week and the 8th week booster immunization, immunizing dose is 4th week 25 μ g/, and the 8th week 50 μ g/ only; Within after each immunity one week, get auricular vein blood, ELISA detects serum antibody titer and is respectively 10 ³with 10 ⁶, reach when tiring and be stabilized in 1:10 ⁶above, to large ear rabbit heart extracting blood, the centrifugal 10min separation of serum of 5000g, and with 2 μm of disposable filter filtering serum, the method for the serum affinity chromatograph after filtration is carried out purification and is obtained anti-Paa polyclonal antibody.