CN104231076B - The therapeutic monoclonal antibodies of anti-coli-infection, produce hybridoma cell strain of the monoclonal antibody and application thereof - Google Patents

Abstract

The invention discloses a kind of therapeutic monoclonal antibodies of anti-coli-infection, producing hybridoma cell strain of the monoclonal antibody and application thereof, the invention also discloses the epitope polypeptide of the MraY albumen combined with the monoclonal antibody specificity.Heretofore described monoclonal antibody is produced by the hybridoma for being named as M H11, and there is specificity to nucleotide sequence coded polypeptide shown in SEQ ID No.1.The beneficial effects are mainly as follows：The invention provides a kind of new anti-Escherichia coli clones antibody, and produce the hybridoma cell strain of the monoclonal antibody, and its epi-position and In Vitro Bacteriostasis characteristic are identified with cylinder therapeutic effect, to applying therapeutic monoclonal antibodies preventing and treating coli-infection to be significant, while being also that the treatment of gram positive bacterial infection and the further investigation of bacterium shadow vaccine are laid a good foundation.

Description

The therapeutic monoclonal antibodies of anti-coli-infection, produce the monoclonal antibody Hybridoma cell strain and application thereof

Technical field

The present invention relates to a kind of therapeutic monoclonal antibodies of anti-coli-infection, producing the hybridization of the monoclonal antibody Tumor cell strain, further relates to the epitope polypeptide of Escherichia coli MraY albumen combined with monoclonal antibody specificity.The present invention Belong to biological technical field.

Background technology

(MraY, i.e. translocase are I) by mraY bases for gram-negative bacteria phosphoric acid-N-acetylmuramate-Thymopentin translocase Because of coding, it is one of biosynthetic key enzyme of bacteria cell wall, is catalyzed the formation of first step lipid intermediate, is UDP- A member of GlcNAc/MurNAc enzyme families：Polyisoprenyl-P GlcNAc/MurNAc 1-P indexing enzyme families, are called GPT enzyme families.MraY is the attack target that some antibacterials such as tunicamycin, kytoplasm mycin A and fat match mycin B, kytoplasm mycin The cracking of the gram-negative bacterias such as Pseudomonas aeruginosa can be caused by suppressing MraY activity.This shows MraY as novel antibacterial The important potentiality of target position researched and developed by preparation.

Therapeutic antibodies have the magnetic target therapy medicine of unique advantage as a class, have become global medicament research and development now One of focus.To the end of the year 2012, the therapeutic antibodies of U.S. FDA approval listing have 32, are just separately having 350 Multiple Antibodies products In different clinical experimental stages, plant wherein 30 more and enter phase iii clinical trial.The full people Dan Ke for the treatment of infectious diseases In grand antibody, have and be directed to bacterial antigens, have directed toward bacteria endotoxin (such as by Merck ＆ Co., Inc. develop for clostridium difficile The MK-3415A of enterotoxin), for mouse antibodies 14B7, ABthrax and raxibacumab of the anti-PA of bacillus anthracis.In addition Also include by the KB001 for Pseudomonas aeruginosa of Sai Nuofei/Kalobios joint developments, developed by Kenta Biotech companies Panobacumab, the Aurograb for methicillin-resistant staphylococcus aureus developed by Novartis Co., Ltd, public by Novartis Efungumab of department's exploitation for Candida heat shock protein 90.But the therapeutic monoclonal antibody medicine with MraY as target spot is at present not Appear in the newspapers.

Relative to small-molecule drug, the maximum advantage of monoclonal antibody product is exactly " accurate ", can enter for specific target spot Row treatment；And traditional medicine is usually broad-spectrum curing, normal and pathological tissues, " accurate " of monoclonal antibody product can be simultaneously acted on Action effect is enhanced while lowering side reaction over the course for the treatment of.Monoclonal antibody is a kind of " biological missile " simultaneously, can be independent Using or carry medicine, toxin or radioactive substance attacking target cell.A kind for the treatment of of the anti-coli-infection of the present invention Property monoclonal antibody can specifically bind colibacillary MraY albumen, can be used for treat coli-infection.

In addition, MraY albumen is the target spot of bacterial virus bacteriolysis effect, including the multiple bacteriophages including phiX174 bacteriophages Suppress the activity of MraY albumen bacterium being cracked so as to discharging progeny virus by synthesizing lysis proteins during infection development. The E genes of manual control cloning are expressed in Gram-negative bacteria, meeting producing strains shadow, and which is used as a kind of new vaccine and assistant Dosage form formula has been subjected to the extensive concern of domestic and international researcher.But slow with regard to bacteriolyze Recent Advances in Mechanism, arguement is constantly.This The therapeutic monoclonal antibodies of a kind of anti-coli-infection of bright proposition can apply to MraY albumen during bacteria lysis Expression and localization research, so as to the Forming Mechanism for fundamentally illustrating bacterium shadow lays the foundation.

Content of the invention

An object of the present invention is to provide a kind of therapeutic monoclonal antibodies of anti-Escherichia coli MraY albumen.

The therapeutic monoclonal antibodies of a kind of anti-Escherichia coli MraY albumen of the present invention, it is characterised in that the monoclonal Hybridoma cell strain secretion of the antibody by deposit number for CGMCC No.9241 is produced.

Identified described monoclonal antibody is IgG2b types, and light chain belongs to κ types.Described monoclonal antibody can be special Property combine the B cell antigen epi-position polypeptide of Escherichia coli MraY, the amino acid sequence of described epitope polypeptide is PESHFSKRGTPT (shown in SEQ ID NO.1).

It is demonstrated experimentally that described monoclonal antibody can suppress colibacillary growth in vitro, suppress large intestine in vivo The propagation of bacillus simultaneously treats the infection caused by Escherichia coli.

On this basis, the present invention proposes described monoclonal antibody in the reagent for preparing detection coli-infection Application.Described detection, including the detection to Escherichia coli MraY expressions, may also comprise and Escherichia coli MraY is determined Position detection.The Position Research that the monoclonal antibody is applied to Escherichia coli MraY, is to find and confirmation Escherichia coli MraY Powerful measure is provided, is also laid a good foundation for disclosing the bacteriolyze mechanism of phage E albumen；And

Application of the described monoclonal antibody in the reagent for suppressing colibacillary propagation is prepared.Described suppression large intestine The propagation of bacillus includes suppressing colibacillary propagation in vivo and suppresses colibacillary propagation in vitro；And

Application of the described monoclonal antibody in the reagent for preparing treatment coli-infection.

The second object of the present invention is to provide a kind of hybridoma cell strain for producing the monoclonal antibody.

A kind of hybridoma cell strain of the present invention, is that the splenocyte of mouse is produced through cell fusion with SP2/0 cells Hybridoma cell strain, is named as M-H11, and Classification And Nomenclature is hybridoma cell strain, is deposited in Chinese microorganism strain preservation management Committee's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its bacterial classification Deposit number is：CGMCC No.9241, preservation date are on May 28th, 2014.

The third object of the present invention is to provide what a kind of monoclonal antibody specificity with anti-Escherichia coli MraY was combined One epitope polypeptide of MraY albumen.

A kind of B cell antigen epi-position polypeptide of Escherichia coli MraY of the present invention, it is characterised in that described epitope polypeptide Combined with the monoclonal antibody specificity described in any of the above item, the amino acid sequence of described epitope polypeptide (Mpep) is PESHFSKRGTPT (shown in SEQ ID NO.1).

Present invention also offers the nucleotide sequence of the described B cell antigen epi-position polypeptide of coding.

In a particular embodiment of the present invention, described nucleotide sequence is as shown in SEQ ID NO.2.

Further, present invention also offers described epitope polypeptide is in the antibody for preparing the anti-Escherichia coli MraY of detection Application in reagent.The polypeptide Mpep energy Competitive assays monoclonal antibody M-H11 is combined with M protein moleculars, can be used for M protein functions Research.And described nucleotides sequence is listed in the application in the reagent of the antibody for preparing the anti-Escherichia coli MraY of detection.

In order to achieve the above object, technical scheme below present invention employs：

The present invention has obtained one plant by hybridoma cell technology screening and has been capable of the miscellaneous of monoclonal antibody described in stably excreting Tumor cell strain, its preparation method is handed over to include：Little with the fusion protein of GST (GST-M-ab) immunity with two hydrophilic area fragments of MraY Mouse, takes Mouse spleen cells and myeloma cell fusion, carried out with GST-M-ab fusion proteins and GST albumen respectively positive with negative To screening, the hybridoma for being capable of monoclonal antibody described in stably excreting is obtained, from hybridoma supernatant or from note Penetrate in the animal ascites of hybridoma, obtain the monoclonal antibody.Wherein in GST-M-ab fusion proteins, MraY albumen parent The amino acid sequence and nucleotide sequence of pool a sections as shown in SEQ ID NO.3, in GST-M-ab fusion proteins, MraY albumen The amino acid sequence and nucleotide sequence of hydrophilic area b sections is as shown in SEQ ID NO.4.

The fusion protein is obtained by following methods：Will be containing two hydrophilic areas fusion fragment (a fragments and b fragments) of MraY The pGEX-6p-M-ab plasmid vectors conversion Escherichia coli of related nucleotide sequences, IPTG inductions are lower to obtain inclusion body, to forgiving Body is purified, and obtains described GST-M-ab fusion proteins, the IPTG (Isopropy1- β-D- of indication of the present invention Thiogalactopyranoside, isopropyl-β-D thiogalactosides) can be expressed with inducible protein, can be to lactose operon Extremely strong inductive effect is produced, is strong inducer.

Specifically, the monoclonal antibody is prepared by the following method：

(1) immunogenic preparation：PGEX-6p-M-ab recombinant vectors are converted BL21 (DE3) Escherichia coli, is lured in IPTG Lead down, the GST-M-ab of molecular weight about 32ku is present with inclusion bodies.In an optimization experiment scheme of the present invention, with letter Just method is purified to above-mentioned inclusion body, and using the albumen that purified as mice immunized with antigen.

(2) animal immune：With the above-mentioned GST-M-ab fusion protein immunization BALB/c mouses for purifying, it is directed to producing The monoclonal antibody specific of MraY albumen.Every mouse carries out dorsal sc injection with the amount of 100 μ g, and once every two weeks totally three Secondary, the 4th booster immunization once, to be fused after 3 days, adaptive immune mouse.

(3) foundation of hybridoma cell line：The spleen cell of immune mouse is taken as the thick liquid cell that can produce antibody, with The myeloma cell (SP2/0) of syngeneic animal is merged with polyethylene glycol (PEG3350) mediation.After fusion, cell is trained through HAT Foster based selective culture, respectively with GST-M-ab and GST as envelope antigen, using indirect enzyme-linked immunosorbent assay (ELISA) Carry out positive and negative sense screening positive clone.The hybridoma in positive hole is carried out with limiting dilution assay colonized culture until Cloning cell antibody positive rate is 100%.By through serial passages in vitro and repeatedly frozen for hybridoma, recovery, Zhi Daoxi Born of the same parents system can stably excreting monoclonal antibody.

(4) preparation of monoclonal antibody and purifying：By build the hybridoma that is be expelled to through paraffin oil pre-process little Mouse abdominal cavity, induces ascites.The ascites for inducing obtains the monoclonal antibody of purifying with affinity chromatography.

The invention further relates to the screening of the specific peptide fragment of the targeted Escherichia coli MraY of described monoclonal antibody.Utilize The display technique of bacteriophage in random dodecapeptides storehouse, using table of the indirect enzyme-linked immunosorbent assay (ELISA) to monoclonal antibody Position carries out 3 wheel elutriations, titer determination, positive phage clones sequencing and Competitive assays test, it was demonstrated that Mpep：PESHFSKRGTPT (shown in SEQ ID NO.1) is the specific peptide fragment for described monoclonal antibody.

The beneficial effects are mainly as follows：The invention provides a kind of new anti-Escherichia coli clones are anti- Body, and the hybridoma cell strain of the monoclonal antibody is produced, and its antagonistic property and therapeutic effect are identified, corresponding It is significant with mab treatment coli-infection, while the also treatment for gram positive bacterial infection and bacterium The further investigation of shadow vaccine lays the foundation.

Description of the drawings

Fig. 1 is the SDS-PAGE figures after GST-M-ab fusion protein purifications；

1 is the blank of sample-loading buffer；2 are purifying GST-M-ab fusion proteins；M is marker；

Fig. 2 is anti-Escherichia coli MraY monoclonal antibodies M-H11 SDS-PAGE after purification；

1 is the M-H11 monoclonal antibodies of purifying；M is marker；

Fig. 3 is identified for M-H11 monoclonal antibodies hypotype；

Fig. 4 is M-H11 and colibacillary immunofluorescence figure；

Fig. 5 is the result that western blot hybridization detects M-H11 monoclonal antibody specificities；

1 is GST-M-ab；2 is GST；M is marker；

Fig. 6 is analyzed for bacteriophage positive colony sequencing；

Fig. 7 is the ELISA detections combined with monoclonal antibody by synthetic peptide Mpep；

Fig. 8 is tested for the Competitive assays of synthetic peptide Mpep；

Fig. 9 is the growth curve and inhibiting rate that M-H11 suppresses e. coli bl21 (DE3) plysS in vitro；

Result of the tests of the Figure 10 for M-H11 interior therapeutic coli-infections.

Specific embodiment

Below by experiment and the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiments The purpose of illustration is only used for, protection scope of the present invention is never limited in.

Embodiment 1：The structure of MraY hydrophilic areas fusion protein prokaryotic expression carrier

With bacillus coli DH 5 alpha genomic DNA as template, a fragments (SEQ ID that 96bp is amplified using PCR method Shown in NO.3) and 99bp b fragments (shown in SEQ ID NO.4), two fragments are merged by overlapping PCR method, pass through After restriction enzyme (Bam H I and Sal I) is cut, it is connected with pGEX-6P-1 (purchased from Amersham companies), is transformed into large intestine In bacillus BL21 (DE3) (purchased from Merck companies), and recombinant plasmid pGEX-6p-M-ab is obtained after digestion and PCR identifications.

The expression and purification of 2 fusion protein of embodiment

(1) induction of recombinant protein

Picking contains the monoclonal bacterium colony of plasmid, is inoculated in the LB culture mediums that 5ml contains ampicillin respectively, 37 DEG C 220rpm shaken overnights.Next day, with 1:100 are diluted in the 500ml LB culture mediums containing corresponding antibiotic, 37 DEG C cultivate to OD₆₀₀About 0.6, add IPTG to make its concentration for 1mM, 37 DEG C induce 4 hours, and precipitation is collected by centrifugation.

(2) inclusion body is extracted in urea deformation

1) in 4ml/100ml bacterium solutions (bacterium solution volume is bacterium solution volume when IPTG is induced) ratio 12mL re-suspension liquids (20mM Tris, 150mM NaCl) will be precipitated and be hanged；

2) ultrasound 30 minutes, per ultrasonic 5 seconds, are spaced 15 seconds, and amplitude 30% is operated on ice；

3) 4 DEG C of 12000 × g are centrifuged 20 minutes, collect precipitation 12mL cleaning solutions (20mM Tris, 150mM NaCl, 2M Urea, 0.5%NP₄₀) washing precipitation；

4) 3) repeat step, collects the dissolving of precipitation 12ml 8M urea and ultrasound is limpid to solution；

5) 4 DEG C of 12000 × g are centrifuged 20 minutes, collect supernatant protein sample, and -80 DEG C frozen.

(3) purifying of GST-M-ab fusion proteins

1) SDS-PAGE concentration glue and separation gel is prepared, sample comb loading is not inserted, according to a conventional method electrophoresis；

2) glue is put in 4mol/L NaAc solution after electrophoresis terminates and shakes on decolorization swinging table 1 hour, destination protein can be presented One white band；

3) destination protein is cut down to be placed in distilled water to shake on decolorization swinging table 20 minutes and sloughs NaAc；

4) and then by the glue for scaling off (also filling electrophoresis liquid in bag filter) is placed in the bag filter of sealing；

5) again bag filter is placed in Horizontal electrophoresis tank, effect under the voltage of 120V can make destination protein dissociate for 30 minutes Out；

6) bag filter is directly placed in the beaker for filling PBS solution, 4 DEG C of dialysed overnights；

7) bag filter is embedded with PEG20000, is concentrated into suitable volumes, suction out protein liquid.

SDS-PAGE identifications purity of protein (shown in Fig. 1), and protein concentration is determined with Coomassie Brilliant Blue.

3 sero-fast preparation of embodiment

The GST-M-ab fusion proteins of purifying are mixed with isopyknic Freund's complete adjuvant (purchased from Sigma companies) and breast After change, subcutaneous multiple spot immunity 6-8 week old female BAl BIcs/c mouse (are tested purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture Animal center), injection dosage is 100 μ g/；Altogether immunity three times, after (be purchased from isopyknic incomplete Freund's adjuvant twice Sigma companies), immunizing dose is also 100 μ g/.7 days after third time immunity, tail vein is taken a blood sample, and separates serum as the positive Serum.Potency is determined with indirect elisa method, when reaching 10^-4Prepare fusion afterwards.Merge first three day with the albumen abdominal cavity for being not added with adjuvant Injection carries out booster immunization, and dosage is also 100 μ g/.

The preparation of 4 monoclonal antibody of embodiment

(1) feeder cells bed board

In fusion the previous day, an aphylactic BALB/c mouse is taken, take off neck and put to death, and be completely soaked in 75% About 5 minutes in alcohol.With the careful abdominal cut skin of scissors, but peritonaeum should not be cut off.Cotton ball soaked in alcohol carries out disinfection to peritonaeum, The syringe of 20ml draws 5ml HAT screening and culturing mediums, carefully pushes mouse peritoneal, takes feeder cells, is then injected into cultivating In ware.The mixing of about 36ml complete mediums is added, is added in four piece of 96 well culture plate with the volley of rifle fire, 100 μ l/ holes.Observation growth, Prevent from polluting.

(2) splenocyte after immunity is merged with murine myeloma cell

The BALB/c mouse spleen of booster immunization is taken out under aseptic condition, washes connective tissue in basic culture solution off.Again Spleen is moved into another filling and carry out in basal medium secondary cleaning, then crossed 100 mesh steel wires, smashed to pieces, by suspension Proceed in centrifuge tube, 1000 × g is centrifuged 10 minutes, has hanged splenocyte with basic culture solution, has been counted.Collect growth conditions SP2/0 cells that are good and being in exponential phase are (in purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's technological service The heart), counted.It is 1 in proportion:10 splenocyte and myeloma cell are merged, and 1000 × g is centrifuged 7 minutes, abandon on to the greatest extent Clearly.Centrifuge tube is shaken gently, the suspension that cell is slight is made, is easy to fusion agent to penetrate between cell.Centrifuge tube is put into 37 In DEG C water, 1ml PEG3350 are slowly added to, and centrifuge tube are rocked in drop.1 minute is stood, 1ml/ minutes, 3mL/ is then pressed and is divided Clock, 6ml/ minutes speed substep are added dropwise culture medium, are finally slowly added drop-wise to 45ml again, play thorough termination.1000 × g from The heart 10 minutes, exhausts supernatant, preheats the ready HAT Selective agar mediums containing 20% hyclone with 37 DEG C, gently hangs Sedimentation cell, and constant volume is to 40ml, is then transferred in sterilizing plates, adds 96 porocyte culture plates by the amount of 100 μ l of every hole In, it is put into cell culture incubator and is cultivated.

The compound method of HT culture mediums：The HT liquid (1ml) of 100 times of concentrations is put into the DMEM containing 20% hyclone In 99ml fluid nutrient mediums.

The compound method of HAT culture mediums：The HAT liquid (1ml) of 100 times of concentrations is put into containing 20% hyclone In DMEM 99ml fluid nutrient mediums.

(3) screening of hybridoma

With the growing state of cell after the fusion of micro- sem observation, the cell of pollution is removed in time with Solid NaOH pellets.Per 3 Once, used medium is different according to the liquid time is changed, and is cultivated with HAT within the after fusion the 3rd, 6 days for its replacing culture medium Base is partly changed liquid, is used HT culture mediums instead within the 9th day and is partly changed liquid, is changed full liquid with HT culture mediums within the 12nd day.When at 15 days The cell of Shi Ronghe forms microcolony, reach about plate floor space 1/4 when, can use 100 μ l of supernatant, using indirect ELISA side Method, detects to the level of cell secretory antibody.Positive hole is carefully blown and beaten with pipettor, is mixed cell and is suspended.Then take In the basic culture solution of the DMEM that 100 μ l are put into 2ml, fully mix, counted.The mixed liquor of about 100 cells is taken, is put into In 10ml HT nutrient solutions, and it is drawn onto with the volley of rifle fire of 100 μ l and is covered with 96 orifice plates of feeder cells.Often observation of cell growth State, nutrient solution turns yellow to carry out changing liquid.With GST-M-ab and GST as envelope antigen, (5 μ g/ml of coated concentration, per 50 μ of hole respectively L), indirect ELISA is carried out, OD is surveyed₄₅₀Value, screens positive single hybridoma group by positive with negative sense.Same procedure is used again The clone for carrying out positive hybridoma cell, until obtained cell line energy stably excreting monoclonal antibody.One plant of final acquisition is miscellaneous Tumor cell strain is handed over, M-H11 is named as, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its bacterium Planting deposit number is：CGMCC No.9241, preservation date are on May 28th, 2014.

The preparation of the ascites of 5 monoclonal antibody of embodiment and purifying

The BALB/c mouse lumbar injection atoleine 0.5ml/ of 8-10 week old is taken only, after one week, inoculation growth conditions are good Monoclonal hybridoma M-H11, about 0.5 × 10⁶Individual cell, treats that one week or so mouse web portion expands, and extracts ascites, 3000 × g is centrifuged 10 minutes, takes supernatant, is distributed into 1.5ml centrifuge tubes, and -20 DEG C standby.With saturated ammonium sulphate method by ascites Carry out preliminary purification, be further purified with Protein A affinity chromatographies afterwards, monoclonal antibody-purified after SDS- PAGE is shown in Fig. 2.The figure illustrates the light chain and heavy chain condensate of monoclonal antibody, it was demonstrated that the purity of monoclonal antibody is higher.

Embodiment 6M-H11 monoclonal antibody titer of ascites and hypotype identification

With indirect ELISA method, detect that the potency of antibody in M-H11 monoclonal antibody ascites is more than 100,000, be shown in Table 1. Subclass of antibody is carried out using Southern Biotech Subclass of antibody kit to the monoclonal antibody M-H11 for obtaining to identify, as a result： Monoclonal antibody M-H11 heavy chains are IgG2b, and light chain is κ chains, sees Fig. 3.

Antibody titer testing result in table 1M-H11 monoclonal antibody ascites

The atopic of 7 monoclonal antibody of embodiment

Take the fresh bacterium solution of bacillus coli DH 5 alpha and be fixed on slide, anti-as one with purified monoclonal antibody M-H11, carry out immunofluorescence Test.As a result as Fig. 4 shows that M-H11 monoclonal antibodies are incorporated into Bacillus coli cells shell, illustrate that this antibody is to react with Escherichia coli Specific antibody.

Purifying MraY-AB protein samples are carried out SDS-PAGE, NC films is transferred, is carried out Western-Blot detections, use GST Albumen does negative control, and the monoclonal antibody M-H11 of purifying is anti-for one.Show occur specific band at 32KD such as Fig. 5, and GST pair According to without band, showing that the monoclonal antibody is the specific antibody for MraY albumen, the MraY-AB areas of specific recognition fusion protein Section.

Embodiment 8M-H11 monoclonal antibody recognizes the identification of epitope

Special using New England Biolabs phage display random dodecapeptides storehouse kit identification of M-H11 monoclonal antibodies Property combine Escherichia coli MraY epitope polypeptide.Eluriated by three-wheel, carry out titer determination, ELISA determines high reaction signal Bacteriophage, choosing 15 positive phage clones carries out sequencing analysis, and as a result 15 phage clones are measured and derive institute The amino acid sequence of displaying, is shown in Fig. 6.Compared by the integration of amino acid sequence and finally determine a concensus sequence Mpep： PESHFSKRGTPT (shown in SEQ ID NO.1), thus it is speculated that polypeptide PESHFSKRGTPT is linear B cell epitopes, by SEQ Coded by nucleotide sequence shown in ID NO.2.

The ELISA detections combined with monoclonal antibody M-H11 by 9 synthetic peptide of embodiment

The elisa plate that synthetic peptide Mpep is pressed 10 μ g/mL coatings, 96 holes, per 50 μ L of hole, 4 DEG C overnight.Next day removes coating Liquid, is closed with the mixed liquor of 10 × BSA, 10 × CBS and water, 200 μ l/ holes, and 4 DEG C are closed 2 hours, and PBST washes 3 times, 3 points Clock/time.Add 50 μ L antibody (by 8 gradient dilutions such as 2,4,6,8,10,12,14,16 μ g/ml, dilution is PBST) per hole, 37 DEG C incubation 1 hour, PBST is washed 3 times, 3 minutes/time.Plus the goat anti-mouse IgG two of HRP marks resists, and carries out 1 with PBST:10000 Dilute again.37 DEG C are incubated 1 hour, and PBST is washed 3 times, 3 minutes/time.Tmb substrate nitrite ion, 50 μ L/ holes, 37 DEG C of lucifuges is added to show Color 10 minutes.Add terminate liquid 2M H₂SO₄, with ELIASA detection sample 450nm absorbance (OD₄₅₀).Sun is set up simultaneously Property control and blank.As a result show that Mpep can be specifically bound with monoclonal antibody M-H11, see Fig. 7.

The Competitive assays test combined with GST-M-ab fusion proteins to monoclonal antibody M-H11 by 10 synthetic peptide of embodiment

GST-M-ab fusion proteins are coated with 5 μ g/ml, 100 μ l/ holes, and 4 DEG C are overnight, with 10 × BSA, 10 × CBS and The mixed liquor of water is closed, 200 μ l/ holes, and 4 DEG C are closed 2 hours.1 × PBST is washed 3 times, 3 minutes/time.Add variable concentrations (5,10,15,25,50,75,100 μ g/mL) synthetic peptide Mpep is (advance with the mixed liquor of final concentration of 0.2 μ g/ml monoclonal antibody M-H11 Act on 2 hours at 4 DEG C), 37 DEG C are incubated 1 hour, discard mixed liquor, and PBST is washed 3 times, 3 minutes/time.Add the goat of HRP marks Anti-mouse IgG, 37 DEG C are incubated 1 hour, and PBST is washed 3 times, 3 minutes/time.If Control is compareed for unrelated small peptide.Add TMB bottoms Thing nitrite ion, 50 μ L/ holes, 37 DEG C of lucifuges develop the color 10 minutes.Add terminate liquid 2M H₂SO₄, existed with ELIASA detection sample Absorbance (the OD of 450nm₄₅₀).As a result show：As the increase inhibiting rate of peptide concentration also increases, Fig. 8 is seen.

11 monoclonal antibody M-H11 of embodiment is to colibacillary extracorporeal bacteria inhibitor test

Frozen e. coli bl21 (DE3) plysS streak inoculations are taken in the solid LB media containing Cm (34 μ g/ml) On, 37 DEG C of incubated overnights of insulating box choose single bacterium colony, are inoculated in the LB liquid medium of Cm (34 μ g/ml), constant-temperature table 37 DEG C, 220rpm culture 8-12h.Take above-mentioned bacterium solution 1:100 transfer in the LB liquid medium of Cm (34 μ g/ml), constant-temperature table 37 DEG C, 220rpm cultivate to OD590 values be approximately equal to 0.6.With aseptic 96 porocyte culture plates, 100 μ l bacterium solutions are added per hole.So Afterwards respectively plus the M-H11 monoclonal antibodies 0 of purifying, 1 μ g, 5 μ g, 7 μ g, control group adds aseptic PBS and anti-phiX174 phage Es egg respectively Bai Dankang, reaction system are 200 μ l with aseptic PBS polishings.Repeat in per group of three holes.After with ELIASA readings, constant-temperature table 37 DEG C, 220rpm culture.Every 20min, growth curve of bacteria, such as Fig. 9 are drawn to 96 orifice plate readings OD590 with ELIASA.Will be upper State the bacterium solution difference 10 after effect³、10⁴、10⁵、10⁶、10⁸After dilution, the solid LB media containing Cm (34 μ g/ml) is coated On, 37 DEG C of incubated overnights of insulating box.Colony counting, calculates inhibiting rate.Inhibiting rate computing formula：(1- monoclonal antibodies group bacterium number/control group Bacterium number) × 100%.As a result show, the bacterium that with the addition of M-H11 monoclonal antibodies starts growth inhibition occur in 100min, and PBS pair There is no growth inhibition phenomenon according to group and the control group bacterium that with the addition of anti-phiX174 phage Es albumen monoclonal antibody, illustrate that M-H11 is mono- Anti- play the role of Developing restraint to e. coli bl21 (DE3) pLysS bacterial strains.The M-H11 monoclonal antibody incubation groups of 7 μ g, bacterial growth Inhibiting rate is 100%, the M-H11 monoclonal antibody incubation groups of 5 μ g, and bacterial growth inhibiting rate is 93.5%, and the M-H11 monoclonal antibodies of 1 μ g are incubated Group, bacterial growth inhibiting rate are 88.4%, illustrate that M-H11 monoclonal antibodies assume concentration dependent to the suppression of bacterial growth.

12 monoclonal antibody M-H11 interior therapeutic coli-infections of embodiment

First, determine the infection time of e. coli bl21 (DE3) pLysS：Single bacterium colony is chosen, is inoculated in containing Cm (34 μ g/ Ml in LB liquid medium), 37 DEG C of constant-temperature table, 220rpm culture 8-12h.Take above-mentioned bacterium solution 1:100 transfer in chloride mould In the LB liquid medium of plain (34 μ g/ml), 37 DEG C of constant-temperature table, 220rpm are cultivated to OD₅₉₀Value 0.6 or so.Take 6 week old left Right mouse 12, the aseptic PBS of+100 μ l of 100 μ l bacterium solutions of tail vein injection, takes three mouse solutions respectively in 3h, 5h, 7h, 9h Cut open.Liver, lung, spleen, nephridial tissue respectively about 0.1g, plus the aseptic PBS of 1ml is taken, is ground with aseptic copper mesh, is taken 100 μ l, coat chloride mould On the solid LB media of plain (34 μ g/ml), 37 DEG C of incubated overnights of insulating box carry out colony counting.As a result show little during 3-5h Mouse liver spleen lotus bacterium number at most, determines that 3.5h is the cut open inspection time.

Secondly, Inhibition test in monoclonal antibody body is carried out：Single bacterium colony is chosen, the LB liquid medium containing Cm (34 μ g/ml) is inoculated in In, 37 DEG C of constant-temperature table, 220rpm culture 8-12h.Take above-mentioned bacterium solution 1:100 transfer trains in the liquid LB containing Cm (34 μ g/ml) In foster base, 37 DEG C of constant-temperature table, 220rpm are cultivated to OD₅₉₀Value 0.6 or so.100 μ l bacterium of every mouse tail vein injection of experimental group The aseptic PBS of+100 μ l M-H11 of liquid ,+100 μ l of 100 μ l bacterium solutions of every mouse tail vein injection of control group.3.5h dissects mouse, takes Liver, spleen organize each about 0.1g, plus the aseptic PBS of 1ml, are ground with aseptic copper mesh, grind bacterium solution 10³Coated plate after diluting again, insulating box 37 DEG C of incubated overnights, carry out colony counting.As a result see Figure 10, it can be seen from fig. 10 that compared to control group.Experimental mice Escherichia coli quantity in liver and spleen tissue is remarkably decreased, and shows that monoclonal antibody M-H11 has treatment Escherichia coli in vivo The effect of infection.

The preferred embodiments of the present invention are the foregoing is only, is merely illustrative for the purpose of the present invention, and nonrestrictive； Those of ordinary skill in the art understand, can carry out many to which and change in the spirit and scope limited by the claims in the present invention Become, modification, or even equivalent change, but fall within protection scope of the present invention.

Claims (5)

1. a kind of B cell antigen epi-position polypeptide of Escherichia coli MraY albumen, it is characterised in that described epitope polypeptide with by protecting Hide the monoclonal antibody specificity combination that the hybridoma cell strain secretion that numbering is CGMCC No.9241 is produced, described epi-position The amino acid sequence of polypeptide is as shown in SEQ ID NO.1.

2. the nucleotide sequence of the B cell antigen epi-position polypeptide described in claim 1 is encoded.

3. nucleotide sequence as claimed in claim 2, it is characterised in that the nucleotide sequence is as shown in SEQ ID NO.2.

4. application of the epitope polypeptide described in claim 1 in the antibody reagent for preparing detection Escherichia coli MraY.

5. the nucleotides sequence described in Claims 2 or 3 is listed in the application in the antibody reagent for preparing detection Escherichia coli MraY.