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 albumen and antibody prepared therefrom and application

Technical field

The present invention relates to biological technical field, particularly relate to Paa albumen and antibody prepared therefrom and application.

Background technology

Enterohemorrhagic Escherichia coli (enterohemorrhage E.coli, EHEC) is virulence also the strongest the strongest, pathogenic escherichia coli, is paid close attention to widely.EHEC infects and people can be made to suffer from diarrhoea, hemorrhagic colitis (HC), and also can cause the severe complication such as hemolytic uremic syndrome (HUS) and Thrombotic Thrombocytopenic minimizing purpura (TTP), severe patient can cause death, and fatality rate reaches 5% ~ 10%.In April, 2011, Germany has broken out enterohemorrhagic Escherichia coli O104:H4 and has infected and hemolytic uremic syndrome (HUS) epidemic situation, spreads to 16 countries in Europe and North America.Between the same year four, May, new infeetioa epidemic situation has been broken out in Japan folic hill area, and serotype is O111, and some case, after causing HUS, has occurred nervous symptoms, concurrent diffusion-type intravascular coagulation and encephalopathy.In EHEC, pathogenic strong, endanger and large also have O157:H7.Be separated first from 1975, nineteen eighty-two be confirmed to be pathogenic bacterium since more than 20 year in, comprising China all over the world has the outbreak of epidemic of different scales.1999 in China Jiangsu, there was extensive outbreak of epidemic in Anhui two province, patient more than 20,000 examples, dead 177 examples.EHEC infects the first cause of disease having become American-European countries child renal failure, constitutes grave danger to the health of people.In addition, as a kind of important Zoonosis encephalapthy agent, EHEC pathogen can reside in the intestinal of the domestic animals and fowls such as cattle, sheep, pig, chicken for a long time, causes the diarrhoea of animal, and pollute meat milk egg products and water source and the crops etc. of poultry, cause huge loss to agriculture-stock production.Therefore, EHEC infects has become global public health problem.

At present, clinical infection for EHEC mainly takes symptomatic treatment, by careful use, does not have operable vaccine and specific therapy medicine because antibiotic therapy has the danger that aggravates one's illness.Vaccine is considered to reply EHEC outbreak of epidemic and distributes infection the most simply, economical, means efficiently.If can find and obtain cAg or advantage protective antigen, just new generation vaccine can be further development of.

Stick the key link that field planting is bacteriological infection damage, bacterial adhesion causes A/E(Attaching and effacing, A/E in host epithelial cells) pathological changes.Tir, Intimin, Map, EspF, EspG, EspH, SepZ, EspB are putative effector molecules of being encoded by LEE, all participate in the A/E damage of EHEC.But research worker finds the effector molecule that still there are other non-LEE codings.

Paa(porcine attaching and effacing associated) be the 27.6kDa albumen of 753bp gene code, sequence analysis finds that the aminoacid sequence of paa gene coded protein is completely the same in O157:H7EDL933 and Saikai.The region that chromosome mapping scientific discovery comprises paa gene is positioned on chromosome, between yciD and yciE.Theresa etc. find that the EHEC deletion mutation strain of paa gene can reduce sticking hela cell, also can reduce the secretion of some cell surface protein simultaneously, these albumen some be indispensable to the excretory system of EHEC, and these excretory systems are important leverages of EHEC adherent cell, so sticking of Paa and EHEC has some relation.At present, multinational scientist is just launching research for the adhesive function of Paa albumen and molecular mechanism, but up to now, does not also confirm that Paa molecule detects at bacteriological infection from immunology angle, effect in immune protection and function.

Summary of the invention

An object of the present invention is to provide the novelty teabag of Paa albumen.

Paa albumen provided by the invention has following 1 in preparation)-3) at least one function product in application:

1) disease being infected by pathogen EHEC or caused is prevented and/or treated;

2) reduce pathogen EHEC to damage intestinal infection;

3) pathogen EHEC is reduced in intestinal definite value power;

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

In above-mentioned application, described product is vaccine or medicine.

In above-mentioned application, described intestinal is behaved or animal intestinal, and animal intestinal is mouse intestinal in an embodiment of the present invention; Described pathogen EHEC is EHEC O157:H7.

Paa albumen as target or detect mark exploitation or the application prepared in the product of diagnose infections pathogen EHEC be also the scope of protection of the invention; In above-mentioned application, the aminoacid sequence of described Paa albumen is the sequence 2 in sequence table.

Another object of the present invention is to provide a kind of product.

Product provided by the invention, its active component is Paa albumen;

Described product is for having following 1)-3) in the product of at least one function:

1) prevent and/or treat by pathogen EHEC diseases induced;

2) reduce pathogen EHEC to damage intestinal infection;

3) pathogen EHEC is reduced in intestinal definite value power.

4) infection of pathogen EHEC is detected.

Also be the scope of protection of the invention by Paa albumen as antibody prepared by antigen; The aminoacid sequence of described Paa albumen is the sequence 2 in sequence table.

Above-mentioned antibody is polyclonal antibody or monoclonal antibody.

The product detecting Paa albumen is also being the scope of protection of the invention for the preparation of the application in diagnosis pathogen EHEC product; Described pathogen EHEC is specially EHEC O157:H7; The product of above-mentioned detection Paa albumen is specially above-mentioned antibody.

Above-mentioned antibody improves host's anti-microbial pathogen EHEC infection product in preparation or improves host's anti-microbial pathogen EHEC application of sticking in product is also the scope of protection of the invention;

In above-mentioned application, described host is specially human or animal; Host is especially specially the cell in human or animal; Described pathogen EHEC is specially EHEC O157:H7; In an embodiment of the present invention, be used for proving that raising host anti-microbial pathogen EHEC sticks to be tested as host by hela cell.

3rd object of the present invention is to provide a kind of product.

Product provided by the invention, its active component is above-mentioned antibody;

Described product is for having following 1)-3) in the product of at least one function:

1) for detecting pathogen EHEC;

2) improve host's anti-microbial pathogen EHEC to infect;

3) improve host's anti-microbial pathogen EHEC to stick;

Wherein host is specially human or animal; Host is especially specially the cell in human or animal; Described pathogen EHEC is specially EHEC O157:H7; In an embodiment of the present invention, be used for proving that raising host anti-microbial pathogen EHEC sticks to be tested as host by hela cell.

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, confirm (1) 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; (2) mice of Paa immunity can reduce the field planting of EHEC in intestinal; (3) mice of Paa protein immunization can resist O157:H7 counteracting toxic substances, shows that this albumen can as the diseases induced vaccine of EHEC; (4) Paa albumen can be used to Dispersal risk or antiserum, and this antibody or antiserum can specific recognition EHEC tropinas, can detect EHEC pathogen as antibody reagent; (5) antibody of Paa albumen or antiserum can stick effect to target cell by antagonism EHEC pathogen, and the damage that pathogenic bacterial infection causes body is significantly lowered or almost suppressed completely to effective prevention pathogen in the field planting of intestinal, thus; (6) Paa albumen can produce special response by excitating organism, has high-caliber immunogenicity, by inducement efficient immunological effect, and the damage that opposing pathogenic bacterial infection causes or death.

Accompanying drawing explanation

Fig. 1 is purifying protein and the WB trace of Paa

Fig. 2 be antibody titer with immune time and interval time variation monitoring

Fig. 3 is the detection of IgA and IgG hypotype in immune serum

Fig. 4 is the detection of discharge of bacteria amount in mice Excreta after counteracting toxic substances

Fig. 5 is the sero-fast WB inspection of anti-Paa

Fig. 6 is the anti-adherence rate of Paa antiserum to cell

Detailed description of the invention

The experimental technique used in following embodiment if no special instructions, is conventional method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Portion of material is as follows:

1, carrier and bacterial strain

EHEC O157:H7EDL933 strain can be provided by ATCC, catalog number (Cat.No.): 700927; The public also can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL, this bacterial strain EDL933 strain is documented in L.W.Riley, R.S.Remis, S.D.Helgerson, H.B.McGee, J.G.Wells, B.R.Davis, R.J.Hebert, E.S.Olcott, L.M.Johnson, N.T.Hargrett, P.A.Blake, M.L.Cohen, Hemorrhagic colitisassociated with a rare Escherichia coli serotype, in N.Engl, J.Med.308 (1983) 681685..PEASY-T1 carrier, E.coli DH5 α, E.coli BL21(DE3) competent cell is purchased from TransGen company, and pET-22b (+) is purchased from Novagen.

2, reagent and culture medium

NdeI and XhoI restriction endonuclease and T4 ligase are purchased from NEB company; The polyhistidine antibody of the goat anti-rabbit igg antibody of horseradish peroxidase (HRP) labelling, the goat anti-mouse igg antibody of horseradish peroxidase (HRP) labelling and HRP labelling is purchased from Pierce company; The equal available from Sigma of sheep anti-Mouse IgA antibody of goat anti-mouse igg 1 antibody of horseradish peroxidase (HRP) labelling, the goat anti-mouse igg 2a of horseradish peroxidase (HRP) labelling, the goat anti-mouse igg 2b antibody of horseradish peroxidase (HRP) labelling, goat anti-mouse igg 3 antibody of horseradish peroxidase (HRP) labelling and horseradish peroxidase (HRP) labelling; The Taq enzyme that PCR is used and DNAmarker(DL2000) purchased from TransGen company; The little extraction reagent kit of plasmid and agarose gel reclaim test kit all purchased from Beijing Bo Maide company; Other chemical reagent are chemical pure or analytical pure; Nickel post is purchased from GE company.

EHEC O157:H7 antibacterial culturing adopts Luria-Bertani(LB, OxoidLTD, Basingstok, Hampshire, England) fluid medium and LB be dull and stereotyped.Recombinant all adopt containing the LB fluid medium of ampicillin final concentration 100 μ g/ml and LB dull and stereotyped.

3, biochemistry and molecular biology reagent

Sorbitol Mai Kangkai base agar and sorbitol-MacConkey agar additive are purchased from Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd; DEME culture medium is purchased from GIBCO company; Standard hyclone is purchased from Biochrom company; Dual anti-purchased from Hyclone company.

4, compound method

The compound method (1L) of LB culture medium: Nacl 10g; Yeast powder 5g; Peptone 10g.

The compound method of table 1SDS-PAGE separation gel and concentrated glue

5, laboratory animal

Large ear rabbit (male, 2.5kg), BALB/c mouse (female, 14-16g) are all purchased from Military Medical Science Institute's Experimental Animal Center.

Embodiment 1, Paa albumen are to the identification infecting EHEC serum

One, purification Paa albumen

1, the fishing of Paa protein coding gene is got

Design paa primer according to GenBank (GeneID:AE005174), introduce restriction enzyme site NdeI and XhoI(dashed part), i.e. P1:5 '- catatgaggaacataatggcaggtt-3 '; P2:5 '- ctcgagtcaagtgcctttcctggtcca-3 '.

With EHEC O157:H7 genomic DNA for template, PCR condition is: 94 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 72 DEG C of 10min, 25 circulations.

The system that 50 μ l PCR react: genomic DNA template 1 μ l, forward primer 0.5 μ l, downstream primer 0.5 μ l, dNTP4 μ l, Taq polymerase 1 μ l, 10 × buffer5 μ l, adds H ₂o to 50 μ l.

Obtain the PCR primer of 768bp, through order-checking, the nucleotides sequence of this PCR primer is classified as the sequence 1 in sequence table, sequence 1 wherein in sequence table is the encoding gene of Paa albumen from 5 ' end 4-762 position nucleotide, and the aminoacid sequence of the Paa albumen of this encoding gene encodes is the sequence 2 in sequence table.

Glue reclaims above-mentioned PCR primer and connects cloning vehicle pEASY-T1, and be converted into E.coli DH5 α competent cell, carry out bacterium colony PCR qualification (primer is P1 and P2), obtaining 768bp PCR primer is positive bacterium colony; Above-mentioned positive bacterium colony is extracted plasmid, and with NdeI and XhoI enzyme action, what can obtain the digestion products of about 768bp is positive plasmid, by this positive plasmid called after pEASY-T1-paa.

2, the structure of Paa Protein reconstitution expression plasmid and expression engineering bacteria

Respectively with NdeI and XhoI double digestion cloned plasmids pEASY-T1-paa and expression vector pET-22(+), reclaiming about 768bp fragment and pET-22(+) carrier segments is connected, connection product is proceeded to E.coli DH5 α competent cell, obtain transformant, the plasmid extracting transformant sends to order-checking, this plasmid is that sequence in sequence table 1 is inserted pET-22(+ from 5 ' end 4-763 position nucleotide) plasmid that obtains between NdeI and the XhoI restriction enzyme site of carrier, called after pET-22(+)-paa, transformant called after DH5 α/pET-22(+ by containing this plasmid)-paa.

3, the Expression and Identification of Paa albumen

By DH5 α/pET-22(+)-paa accesses fresh LB, works as OD ₆₀₀when reaching exponential phase, get after part bacterium liquid does and do not induce contrast, it is 1mmol/L that remainder bacterium liquid adds IPTG to final concentration, abduction delivering 4h; Obtain induction bacterium liquid and do not induce bacterium liquid.

By induction bacterium liquid and do not induce bacterium liquid respectively the centrifugal 25min of 5000g collect thalline; Thalline to be worked 5s, interval 3s at 400W(, circulates 99 times) under ultrasonication, collect full bacterium lysate, with 5000g, 20min centrifugal collecting precipitation and supernatant.

Get and do not induce the supernatant of the full bacterium lysate of the full bacterium lysate of bacterium liquid, induction bacterium liquid, induction bacterium liquid, induce the precipitation of bacterium liquid to carry out SDS-PAGE respectively, select 5% concentrated glue and 15% separation gel, result shows, compare the full bacterium lysate of not inducing, band is clearly there is in the full bacterium lysate of induction at 27Kd place, in the same size with theoretic Paa albumen, albumen successful expression is described, and the supernatant of induction thalline also occurs obvious band herein with being deposited in of thalline of induction, show that Paa albumen is expressed with soluble protein and inclusion bodies respectively.

4, the purification of Paa albumen

By full bacterium lysate 5000g, 20min collected by centrifugation supernatant of above-mentioned induction bacterium liquid, cross nickel post and carry out purification; The concentration of balance liquid imidazoles is 40mmol/L, and the imidazole concentration of elution buffer is 400mmol/L; Collect eluent and be purification Paa albumen.

By purification Paa albumen through SDS-PAGE electrophoresis, result is as shown in the swimming lane 2 in Fig. 1, and swimming lane 1 is protein 12-80kd marker, can find out, occurs the protein fragments of 27KD at swimming lane 2, with expection in the same size, the Paa albumen obtaining purification is described.The Paa protein concentration recording purification is 3.5mg/ml.

Probe purification Paa being carried out the WB of Western blot(swimming lane 3 is the anti-His tag antibody of HRP labelling), result is as shown in the swimming lane 3 in Fig. 1, band of expression and trace band is there is greatly about about 27kd place, can by special tag antibody identification, size conforms to the fusion protein molecule amount of prediction.

5, purification Paa albumen infects the identification of serum to EHEC

Purification Paa is carried out Western blot, probe is mouse-anti-EHEC O157:H7 whole bacterial protein multi-resistance (antiserum that EHECO157:H7 infecting mouse obtains), two resist for dynamics is two anti-(Sigma), result is as shown in the swimming lane 4 in Fig. 1, also there is a specific band at about 27DK, illustrated that Paa by the identification of mouse-anti-O157:H7 whole bacterial protein multi-resistance, can have good immunoreactivity, illustrate that it is EHEC O157:H7 antigen, can be used for diagnosing EHEC to infect.

The immunogenicity of embodiment 2, Paa albumen and protective effect

Female BAl BIc/c mice 14-16g is divided into 2 groups at random, often organize 10, following experiment all in triplicate:

Paa immune group: the purification Paa albumen that abdominal channels immunity is obtained by embodiment 1 is as antigen;

BSA matched group: BSA is as antigen in abdominal channels immunity.

Immunization ways: first immunisation adopts isopyknic Freund's complete adjuvant (cumulative volume 100 μ l), booster immunization adopts incomplete Freund's adjuvant to mix with antigen equal-volume, each immunization interval 2 weeks, altogether immunity three times.First immunisation antigen final concentration is 25 μ g, and booster immunization antigen final concentration is 50 μ g.After final immunization 10 days, Paa immune group and BSA control group mice all gave the drinking-water of 5g/L streptomycin, drink 3 days, then after the jejunitas 24h that cuts off the water supply respectively with 10 ⁹cFU (10MLD) EHEC O157:H7EDL933 carries out per os gavage counteracting toxic substances, observes mice state and survival condition.

1, the immunogenicity of Paa albumen detects

Above-mentioned BSA matched group and Paa immune group mice are collected and separation of serum in the 0th, 10,24,46,82 day tail venous blood sampling.Adopt ELISA method to detect Serological IgG level, Paa is antigen coated, compares with BSA, and the mice serum of collection is antibody to be checked, and the goat anti-mouse igg antibody of HRP labelling resists as two.

As shown in Figure 2, mouse immune time point is followed successively by the 1st, 14,28 day to antibody detection result, and arrow is depicted as immunization time point; Vertical coordinate represents the logarithm value that in serum, the ELISA of IgG tires; Abscissa is the natural law apart from immune first day; Can find out, compared with BSA matched group, Paa immune group mice secondary immunity can obtain high level and tire, IgG level reaches peak value after the third immunization, reaching in the observation process of 80 days, the antibody titer of Paa does not all significantly decrease, and Paa immune group is in 10 in whole immunologic process ⁷high antibody titer level.Illustrate, Paa is a kind of significant advantage immunizing antigen, has very strong immunogenicity.

By BSA matched group and Paa immune group mice, the 14th day tail venous blood sampling after final immunization collects serum, employing ELISA method detection serum IgA, IgG hypotype IgG1, IgG2a, IgG2b, IgG3 tire, Paa is antigen coated, compare with BSA, the mice serum collected is antibody to be checked, resists respectively using goat anti-mouse igg 3 antibody of the goat anti-mouse igg 2b antibody of goat anti-mouse igg 2a, HRP labelling of goat anti-mouse igg 1 antibody of the sheep anti-Mouse IgA antibody of HRP labelling, HRP labelling, HRP labelling, HRP labelling as two.

Result as shown in Figure 3, often organizes 10, graphical results repeated measure 3 times, and vertical coordinate represents the geometrical mean ± standard deviation often organized ELISA and tire; Can find out, in Paa immune group, IgA, IgG hypotype IgG1, IgG2a, IgG2b, IgG3 tire and are respectively 1.3 × 10 ⁴, 1 × 10 ⁵, 8.6 × 10 ³, 1 × 10 ⁴with 1 × 10 ³; In BSA matched group, IgA, IgG hypotype IgG1, IgG2a, IgG2b, IgG3 tire to be respectively and are 1 × 10 ²; Can find out, the immunity of Paa causes IgA, IgG and hypotype IgG1 thereof, IgG2a, IgG2b tire all has remarkable rising compared with BSA immune group, and IgG3 is without obvious rising.

2, the discharge of bacteria amount monitoring of animal subject

Carried out the feces collection of Paa immune group, BSA control group mice after counteracting toxic substances every 2 days, take 0.2g and be dissolved in 100mlLB culture medium, hatch 4h for 4 DEG C, treat that feces softens, can vibrate for till turbid solution.By centrifugal for feces rear 10 times of serial dilution bacterium liquid, coating dilution gained antibacterial, is coated with sorbitol Mai Kangkai qualification flat board and counts.

Animal subject discharge of bacteria amount monitoring result as shown in Figure 4, vertical coordinate is every 0.1g feces gradient dilution coated plate count results, and below 100CFU/ml is invalid value, and abscissa is the natural law after counteracting toxic substances, and error bar is the geometrical mean ± standard deviation often organizing CFU;

Paa immune group discharge of bacteria amount of 2,4,6,8,10,12,14 days after counteracting toxic substances is respectively 2 × 10 ⁴, 9 × 10 ³, 1.2 × 10 ³, 4.5 × 10 ², 2 × 10 ², 2 × 10 ², 2 × 10 ², stopped discharge of bacteria at the 10th day;

BSA matched group discharge of bacteria amount of 2,4,6,8,10,12,14 days after counteracting toxic substances is respectively 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;

Can see that matched group showed lasting higher discharge of bacteria amount at whole 14 days in observation process, immune group in showing the discharge of bacteria amount fallen sharply gradually, and stopped discharge of bacteria substantially at the 10th day.Illustrate that the mice of Paa immunity can reduce the field planting of EHEC in intestinal, reduce discharge of bacteria amount, reduce the damage to intestinal; Also the repeated infection of food chain can be reduced.

3, survival rate statistics

To in large bacterium amount (fatal dose) challenge test of animal subject; when 10MLD dosage pathogen attacks three immune mouses; after counteracting toxic substances the 10th day, Paa immune group animal subject survival rate be 100%(10 only/10) protection, and BSA matched group is the 7th day mice all dead (0/10).

Identical with said method, improve counteracting toxic substances dosage further, adopt twice immune mouse of 50MLD and 100MLD dosage to Paa immune group to test respectively, result Paa immune group mice still can obviously protect 50MLD live bacterial challenge, survival rate 70%(7/10); And also can show protective effect (2/10) to a certain degree to 100MLD live bacterial challenge.

Above-mentioned experiment shows, Paa albumen can as vaccine prevention and/or treatment pathogen EHEC diseases induced.

The preparations and applicatio of embodiment 3, anti-Paa antibody

1, the preparation of anti-Paa antibody (anti-Paa antiserum) and qualification

Concentration is reached 1mg/ml, purity reaches more than the 95% Paa purifying protein obtained by embodiment 1 as antigen, immune large ear rabbit (male, 2.5kg).Respectively at 4th week (25 μ g/ only) and the 8th week (50 μ g/ only) booster immunization.Within after each immunity one week, get auricular vein blood, survey serum antibody titer and be respectively 10 ³with 10 ⁶(ELISA detection), reaches when tiring and is stabilized in 1:10 ⁶above, to the white heart extracting blood of large ear, the centrifugal 10min separation of serum of 5000g, and with 2 μm of disposable filter (Millipore) filtering serum, the method of the serum affinity chromatograph after filtration is carried out purification and is obtained anti-Paa antibody (polyclonal antibody) ,-80 DEG C of preservations after purification.

Cultivate EHEC O157:H7, collect thalline, ultrasonication, collect whole bacterial protein.EHEC O157:H7 whole bacterial protein is carried out SDS-PAGE and Western Blot to detect anti-Paa antibody recognition tropina, detect the ability of pathogen, when Western Blot detects respectively with rabbit anteserum before Paa immunity and anti-Paa antibody for primary antibodie, be two to resist with the sub-IgG of HRP labelling anti-rabbit.

As shown in Figure 5, M is protein 12-80kd marker to result; 1 is EHEC O157:H7 whole bacterial protein; 2 is that before Paa immunity, rabbit anteserum is the WB detection of primary antibodie; 3 for anti-Paa antibody be primary antibodie WB detect; Can find out that anti-Paa antibody can identify EHEC O157:H7 whole bacterial protein, there is pathogen antigen binding ability; Illustrate that anti-Paa antiserum is the antibody of O157:H7, can be used for diagnosis EHEC O157:H7 pathogen.

2, anti-Paa antibody anti-EHEC pathogen cell adhesion effect in vitro

Wild type EHEC O157:H7 is inoculated in 5ml LB fluid medium, cultivates 16h in 37 DEG C of concussions.

In each hole of 96 porocyte culture plates, add hela cell (Chuan Xiang bio tech ltd, Shanghai, CH01050XI) 10 ⁵/ hole and DMEM culture fluid, at 37 DEG C, 5%CO ₂on every hole, the region of 80%-90% covers with monolayer, draws outmoded nutritional solution, renews fresh DMEM culture fluid, and in nutritional solution, add hyclone to final concentration be 0.5%; Then add the wild type EHEC O157:H7 of above-mentioned fresh cultured in culture hole, amount of bacteria is 10 ⁶individual antibacterial (colony counting standard measure), cultivates 3h at 37 DEG C.Suck nutritional solution, and carefully clean culture plate 5 times with the phosphate buffer (PBS) that pH value is 7.0, each 3min, washes off the antibacterial of not sticking, with trypsinization after washed, cell is washed down, PBS cleans cell residue Digestive system, 1%Triton X-100 cell lysis, centrifugal rear 10 times of serial dilution bacterium liquid, coating dilution gained antibacterial, is coated with sorbitol Mai Kangkai qualification flat board and counts.

As shown in Figure 6, abscissa is the dilution factor of serum to result, and vertical coordinate is the ratio of anti-adherence rate=non-adherent bacteria and total number of bacteria, and numerical value is the geometrical mean ± standard deviation often organizing protective rate;

Immune group is respectively 90%, 89%, 85%, 75%, 65%, 55%, 39%, 27%, 9% and 0% in the dilution anti-adherence rate of 0,1,2,4,8,16,32,64,128,256 serum;

Matched group is all 0% in the dilution anti-adherence rate of 0,1,2,4,8,16,32,64,128,256 serum;

Can find out, antibacterium is sticked in experiment in vitro, and anti-Paa serum really exists and significantly anti-ly sticks effect, and along with the raising of anti-Paa serum-concentration, its anti-adhesion effect is more obvious, there is dose-dependent positive correlation.Show, anti-Paa antibody can intervene pathogen sticking and damaging target cell, has 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.