CN100441689C - EHEC 0157 shiga-like toxin IIA resisting subunit monoclonal antibody 5F3 light and heavy chain variable region gene and its application - Google Patents

Abstract

The present invention belongs to the field of biological medicine preparation, which relates to an anti-enterohemorrhagic Escherichia coli (EHEC)O157 Shiga-like toxin II toxin subunit Stx2A monoclonal antibody 5F3 heavy chain and light chain variable region gene, polypeptide encoded by the gene and an application of the gene and the polypeptide in the process of preparing medicines for diagnosing and treating EHECO157 infection and complications thereof. The inventor adopts universal degenerate primers to successfully clone the light chain and heavy chain variable region gene of the corresponding antibody from hybridoma cell strains of a strain of cultivated anti-Stx2A specific monoclonal antibody 5F3, various forms of small molecule gene engineering antibodies are constructed and expressed by a gene engineering method on the basis of the light chain and heavy chain variable region gene of the monoclonal antibody 5F3, such as a ScFv antibody, a chimeric antibody, an antibody fusion protein, etc. The present invention is used for preparing medicines for diagnosing and treating EHECO157 infection and complications thereof and provides a new path for diagnosing and treating EHECO157 infection and complications thereof.

Description

Anti-EHEC O157 shiga-like toxin II A subunit monoclonal antibody 5F3 is light, heavy chain variable region gene and application

Technical field

The invention belongs to field of biological pharmacy, relate to anti-enterohemorrhagic Escherichia coli (EHEC) the O157 shiga-like toxin II toxin Stx2A of subunit monoclonal antibody 5F3 heavy chain and chain variable region gene and polypeptide, and described gene and polypeptide preparation be used for diagnosing and treat that EHEC O157 infects and complication aspect the application of medicine.

Background technology

Since EHEC O157 was confirmed to be pathogenic bacterium in nineteen eighty-two, the food poisoning of its initiation comprised that all over the world outbreak of epidemic has all taken place in China.The infection of this bacterium has become a global public health problem, no matter in developed country or developing country, all is subjected to extensive concern.China has classified enterohemorrhagic Escherichia coli as 21 century may one of 12 kinds of pathogenic micro-organisms of great effect to compatriots' hygiene and health.O157:H7 infects can cause severe complication such as hemolytic uremic syndrome (Hemolyticuremicsyndrom, HUS), thrombocytopenic purpura,thrombotic (Thromboricthromobocytopenicporpura, TTP), severe patient can cause death, and lethality rate can reach 5-10%.The O157 bacterium is cultivated easily, breeding is rapid, infectivity is strong, route of infection is extensive, makes it very likely as bacterial warfare agent in the following military struggle and bio-terrorism war agent.The Center for Disease Control (CDC) is classified EHEC O157 bacterium as category-B bio-terrorism pathogenic agent and is strictly taken precautions against.Yet at present its infection is still lacked effective methods of treatment, mainly adopt antibiotic therapy and corresponding symptomatic treatment at the infection of O157 bacterium clinically.Discovering recently, microbiotic breaks thalline, cause O157 bacterium shiga toxin (Shiga toxin, emission levels ST) improves greatly, make microbiotic not have obvious influence even cause the prolongation of the course of disease, the dangerous of complication taken place and cause death thereby may increase to the course of disease." enterorrhagia Bacillus coil 0157: H7 infectious diarrhea plan for emergency handling (trying) " the 5th patient's that health ministry in 2002 is formulated the regulation of isolating for treatment: to enterorrhagia Bacillus coil 0157: patient H7 and patient suspected isolate for treatment.Patient's treatment can be used probiotics based on the supportive treatment of suiting the medicine to the illness, in principle without diarrhea and the medicine that suppresses intestinal peristalsis.Enterorrhagia Bacillus coil 0157 patient and patient suspected (comprising that stool sample O157 antigen colloidal gold method detects male diarrhoea patient) ban use of microbiotic, the general diarrhoea of other in epidemic-stricken area patient Ying Shen microbiotic.Therefore, still be the needs of biological anti-terrorism no matter from public health, the treatment research of strengthening EHEC O157:H7 has seemed very urgent.

EHEC O157 can produce two kinds of toxin, is called shiga toxin I (Stx1) and shiga toxin II (Stx2).In bacterial body, Stx2 is a secretion type expression, and Stx1 expresses in the born of the same parents.Two kinds of toxin are formed by 1 A subunit and 5 B subunits, and A subunit has toxicity in the cell, can cause protein synthesis to stop with the 28SrRNA effect, and be Escherichia coli O 157: H7 causes the pathologic basis of clinical manifestation; B subunit has the cell binding characteristic, can combine with the cell with special receptor (Gb3), thereby guiding A subunit plays a role.Most O157:H7 bacteriums produce Stx2, and Stx2 is closer with the relation of severe complication clinically than Stx1, and Stx2 is bigger 1000 times than the toxicity of Stx1.Stx2 can enter blood circulation, causes the destination organization organ, as the damage of renal glomerulus and gi tract endotheliocyte.Therefore Stx2 is the important substance basis of this bacteria pathogenic.

Before HUS takes place, the prodrome of the hemorrhagic colitis (HC) of 3～5d is generally arranged, behind the generation HC, before Stx is attached to the kidney endotheliocyte, there is one section to stop the Stx/Gb3 bonded time.Studies have shown that in the use energy and the specific antibody of Stx2 can prevent the appearance of complication such as HUS and thrombotic thrombopenic purpura.Therefore required in the practice before complication does not take place EHEC infected and make rapid diagnosis, in time use Antybody therapy.Since Kohler in 1975 and Milstein create the B lymphocyte hybridoma technology, various monoclonal antibodies occur in succession, these monoclonal antibodies are not only being brought into play positive effect aspect the fundamental research of disease, also brought new hope for the diagnosis and treatment of multiple disease simultaneously.Up to the present, still do not have both at home and abroad and can be used for treating the antibody class medicine that O157 infects, we have obtained a strain at the high-affinity of Stx2A and the hybridoma cell strain of high specific through secular a large amount of colony screening, and it has higher using value and DEVELOPMENT PROSPECT.

Summary of the invention

The present invention aims to provide extremely encoded polypeptides of the heavy chain of anti-shiga-like toxin II (the Shiga like toxin 2) Stx2A of toxin subunit monoclonal antibody 5F3 and chain variable region gene, can give expression to specific recognition after its reorganization and in conjunction with the antibody activity fragment of Stx2A.

Another object of the present invention is to the gene of described antibody 5F3 or polypeptide preparation be used for diagnosing and treat that EHEC O157 infects and complication aspect the application of medicine.

According to an aspect of the present invention, the present invention relates to heavy chain and the chain variable region gene of a kind of anti-shiga-like toxin II toxin Stx2A of subunit monoclonal antibody 5F3.The heavy chain of anti-Stx2A monoclonal antibody 5F3 of the present invention and chain variable region gene be from can the stably excreting high-affinity and the hybridoma cell strain 5F3 of the monoclonal antibody of the anti-Stx2A of mouse source property of high specific the clone obtain.The acquisition mode is: extract total RNA from the hybridoma cell strain 5F3 of anti-Stx2A, with oligo (dT) 15 is random primer, reverse transcription generates cDNA, antibody is light, heavy chain variable region gene to adopt general merger primer specificity to increase respectively then, through sequencing with in NCBI after the BLAST comparative analysis, confirm that 5F3 weight chain variable region nucleotide sequence is sequence table＜400〉nucleotide sequence shown in 1, its amino acid sequence coded is sequence table＜400〉aminoacid sequence shown in 3.The chain variable region gene gene order is sequence table＜400〉nucleotide sequence shown in 2, its amino acid sequence coded is sequence table＜400〉aminoacid sequence shown in 4.Above-mentioned two genes can give expression to specific recognition and in conjunction with the proteic antibody ScFv of the anti-shiga-like toxin II toxin Stx2A of subunit active fragments after reorganization.

Light, the heavy chain variable region gene of the anti-Stx2A monoclonal antibody specific 5F3 that successfully clones for the inventor, use professional known antibodies geneseq database (IMGT) respectively and (NCBI) institute's calling sequence is carried out the homology comparison and show with its germline gene source analysis, the gene order that is obtained is all not quite identical from the various antibody gene sequences of mouse germline gene and existing report really.The mouse antibodies variable region that gained VH and VL gene codified are correct.

According to another aspect of the present invention, the invention still further relates to the gene of described monoclonal antibody 5F3 and encoded polypeptides thereof preparation be used for diagnosing and treat that EHEC O157 infects and complication aspect the application of medicine.

The inventor adopts general merger primer to clone from the hybridoma cell strain of the anti-Stx2A monoclonal antibody specific of the strain 5F3 that cultivates successfully that its corresponding antibody is light, heavy chain variable region gene.Light, heavy chain variable region gene based on above-mentioned monoclonal antibody 5F3, can adopt gene engineering method, make up and express the small molecules genetic engineering antibody of various ways, as ScFv antibody, chimeric antibody, antibody fusion protein etc., preparation is used for diagnosis and treatment EHEC O157 infects and complication aspect medicine, will to diagnosis and treatment EHEC O157 infects and complication provides new approach.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure that extracts total RNA from the hybridoma cell strain 5F3 of anti-Stx2A.

Fig. 2 extracts the agarose gel electrophoresis figure that total RNA reverse transcription generates cDNA among the 5F3.

Fig. 3 is that the anti-Stx2A monoclonal antibody 5F3 of pcr amplification is light, the agarose gel electrophoresis figure of heavy chain variable region gene.

Fig. 4 is that the anti-Stx2A monoclonal antibody 5F3 of pcr amplification is light, heavy chain variable region gene mat LinkerDNA connects back agarose gel electrophoresis figure.

Fig. 5 is for utilizing the order-checking collection of illustrative plates of the ScFv antibody expression vector that 5F3 is light, heavy chain variable region gene is constructed that obtains.

Fig. 6 combines with antigen for Western blot detects ScFv antibody.

Specific embodiments

1. mouse-anti Stx2A MONOCLONAL ANTIBODIES SPECIFIC FOR

2. anti-Stx2A monoclonal antibody 5F3 is light, the clone of heavy chain variable region gene

Extracting total RNA from anti-Stx2A hybridoma cell strain 5F3, is with power traction with oligo (dT) 15, and the RT-PCR reverse transcription generates cDNA, and antibody is light, heavy chain variable region gene to adopt general merger primer specificity to increase respectively then.

3. structure, expression and the purifying of the ScFv form antibody of anti-Stx2A monoclonal antibody 5F3

Antibody is light, heavy chain variable region gene is with (the G that encodes ₄S) ₃The dna fragmentation of small peptide connects into 5 ' VH-Linker-VL3 ' fragment, this fragment of pcr amplification.ScFv fragment behind the purifying links to each other transformed into escherichia coli HB2151 with the carrier pCANTAB6His that has transformed.The IPTG abduction delivering adopts the solid phase nickel ion affinity chromatograph that ScFv is carried out preliminary purification.

4. anti-Stx2A monoclonal antibody 5F3 is light, heavy chain variable region gene and encoded polypeptides thereof preparation be used for diagnosing and treat that EHEC O157 infects and complication aspect the application of medicine

Embodiment 1 anti-enterohemorrhagic Escherichia coli (EHEC) the O157 shiga-like toxin II toxin Stx2A of subunit MONOCLONAL ANTIBODIES SPECIFIC FOR

1. immune Balb/c mouse

Immune Balb/c mouse (mouse 6～8 weeks of age) behind the target protein Stx2A process purifying, 100ug/, first immunisation, 100 μ L antigens mix with the complete freund adjuvant of equivalent, injection mouse web portion and the subcutaneous immunity of inguinal region.Immunity once more behind the 14d, the same first immunisation of immunizing dose and approach adopts incomplete freund adjuvant.In the 28d supplementary immunization, 150ug/, abdominal injection.

2. the preparation of hybridoma

1) collect bone-marrow-derived lymphocyte: behind the supplementary immunization 3 days, extract the eyeball of mouse bloodletting, serum gives over to positive control.Spleen is taken out in aseptic technique, and spleen is placed in the incomplete substratum of the pre-temperature of 10ml, peels off reticular tissue on every side, puts in the 100 order stainless (steel) wires, uses the inner core of syringe to grind, and drips incomplete substratum flushing while grinding.Cell suspension after collection is filtered is in centrifuge tube, and counting gets 1 * 10 ⁸Individual cell, centrifugal, abandon supernatant, it is stand-by to put room temperature.

2) preparation of murine myeloma cell: merged preceding 10 days, the myeloma cell is taken out from liquid nitrogen, put into 37 ℃ of water-baths rapidly, constantly rock, dissolve fully until refrigerating fulid.Centrifugal, abandon supernatant, be suspended in mid-37 ℃ of 8-AG substratum, 5%CO ₂Cultivate in the incubator.According to cell growth condition, change liquid, abandon a part of cell.Merged preceding 2～3 days, one bottle of cell is reached 4 bottles, and use perfect medium instead and continue to cultivate.

3) preparation of nurse cell; Get the healthy BALB/c mouse in one 6～8 age in week, dislocation is put to death.By the aseptic technique rules, cut off skin (noting not breaking peritonaeum), draw the incomplete substratum of 5ml with disposable 5ml syringe, inject mouse peritoneal, gently rub belly with aseptic cotton balls, slowly draw back twice so repeatedly.The cell suspension of sucking-off places centrifuge tube, and is centrifugal, abandons supernatant.Add 20ml HAT substratum, mixing is seeded to two 96 porocyte culture plates, 100 μ l/ holes.Put 37 ℃, 5%CO ₂The incubator overnight incubation if nurse cell is adherent good and pollution-free, promptly can be used for inoculating fused cell.

4) cytogamy: fusion is put in CO with PEG/DMSO the day before yesterday ₂Adjust pH value and temperature in the incubator.Ready myeloma cell and spleen bone-marrow-derived lymphocyte are moved in the 50ml centrifuge tube, add the incomplete substratum of 30ml, centrifugal, inhale and remove supernatant.Centrifuge tube is placed on the centre of the palm shakes, make cell mass become loose pasty state.Centrifuge tube is placed 37 ℃ of water-baths, draw the PEG/DMSO solution of the pre-temperature of 0.7ml, slowly adding in the cell along tube wall from the pipe about 2cm in end place, the limit edged rotates centrifuge tube, and 1min adds.Leave standstill 1min, draw the incomplete substratum of 25ml again, slowly add.The limit edged slowly stirs, and slow earlier back is fast, and preceding 2min adds 2ml, and back 2min adds remaining substratum.Centrifugal, inhale and remove supernatant.Cell mass is shaken diffusing, add 10ml HAT substratum, piping and druming gently, mixing.Fused cell is seeded to the 96 porocyte culture plates that are covered with feeder cell, and 100 μ l/ holes are put 37 ℃, 5%CO ₂Cultivate in the incubator.

5) selectivity of hybridoma is cultivated: inoculate back first day, add HAT substratum 100 μ l/ holes, the 2nd, 3,5,8 and 11 days, culture supernatant was partly measured in each hole sucking-off, and then added fresh HAT substratum 100 μ l/ holes.Observation of cell clonal growth situation under the while mirror.

6) hybridoma screening: merge 2 weeks of back, the ELISA method detects each cell cultures hole supernatant liquor, the same Serum Antibody Detection of method.The positive cell clone of screening secretion purpose antibody is to carry out cloning.

7) cloning of positive hybridoma cell: after filtering out positive colony, collecting cell and counting, the full substratum that toos many or too much for use is by 10 ⁵, 10 ⁴, 10 ³, 10 ²Pair cell is made serial dilution, gets the latter then and is diluted to 10/ml with the HT substratum.Get one 96 porocyte culture plate, in advance by every hole 10 ⁵～10 ⁶Individual/100 μ l HT substratum have added nurse cell, add the cell suspension of 10/ml then, 100 μ l/ holes.Put 37 ℃, 5%CO ₂Incubator was cultivated 7～10 days.Added 2/hole of HT substratum respectively at the 2nd day and the 5th day, and under inverted microscope the observation of cell growing state, to really performing mark for the hole of having only a cell clone growth.Antagonist male monoclonal cell through 3～4 unicellular separation and Culture, reaches 100% until the monoclonal cell culture hole of antibody positive, can tentatively decide strain and name.

Embodiment 2 anti-Stx2A monoclonal antibody 5F3 are light, the clone of heavy chain variable region gene

1. used cell strain adopts a plant height avidity of aforesaid method acquisition, the anti-Stx2A monoclonal antibody 5F3 hybridoma cell strain of high specific for the inventor, its excretory antibody does not combine with multiple enterotoxins of Escherichia coli antigens such as CT, LT, and its excretory antibody molecule hypotype is IgG1 _k

2. the 5F3 hybridoma (2 * 10 of taking the logarithm vegetative period ⁶), adopt guanidinium isothiocyanate single stage method (TaKaRa company) to extract total RNA, take a morsel and carry out the quantitative and 1% agarose gel electrophoresis detection of ultraviolet spectrophotometer.Be random primer with oligo (dT) 15 (TaKaRa company) subsequently, reverse transcription generates cDNA.Adopt general merger primer (Amersham company) specificity increase respectively light, the heavy chain variable region gene of monoclonal antibody 5F3 then.(VH, amplified production VL) cut glue and separate the purpose fragment behind 1% agarose gel electrophoresis will to contain variable region gene.After glue recovery test kit (Promega company) purifying purpose fragment, 1% agarose gel electrophoresis is identified, referring to accompanying drawing 1-3.Afterwards, antibody is light, heavy chain variable region gene is cloned into pMD-18T carrier, sequencing analysis.

Relevant operation concrete steps are as follows:

1. total RNA extracts (using the TaKaRa RNA of company to extract test kit)

1) gets 2 * 10 ⁶Hybridoma, the Catrimox-14 of adding 1ml ^TMSolution, fully mixing.Centrifugal, 3000rpm, 5min.

2) remove solution, the DEPC that adds 1ml handles H ₂O, centrifugal after the mixing for several times of turning upside down, 12,000rpm, 2min.

3) remove solution, fierce vibration makes precipitation loosening, adds the 2M LiCl solution of 1ml, and is centrifugal after the fierce vibration, and 12,000rpm, 5min.

4) remove solution, precipitation is washed once with 70% cold ethanol, and the DEPC that is dissolved in 1ml handles H ₂Among the O.

2.RT-PCR amplification VH and VL

1) be template with the total RNA that extracts, oligo (dT) 15 (TaKaRa company) is a random primer, and the scheme of synthetic cDNA first chain of reverse transcription is as follows:

Total RNA 2 μ l

10 * RT-PCR damping fluid, 1 μ l

dNTPs(10mmol/L)1μl

MgCl ₂(0.025mmol/L)4μl

oligo(dT)15Primer(2.5pmol/μl)0.5μl

AMV?Reverse?Transcriptase(5U/μl)0.5μl

RNase?Inhibitor(40U/μl)25μl

RNase Free dH ₂O mixes the back and adds 3 of paraffin oils to final volume 11 μ l.

Reaction conditions: 30 ℃, 1min; 42 ℃, 0.5min; 99 ℃, 5min; 5 ℃, 5min; 1 loop cycle.

2) be that template adopts following PCR system and program amplification V H and V L with first chain:

Template cDNA 11 μ l

5 * PCR damping fluid (containing magnesium chloride), 10 μ l

Gently, each 1 μ l of heavy chain primer (0.025mmol/L)

Ex?Taq?HS(5u/μl)0.25μl

Add deionized water to final volume 50 μ l, mix the back and add 3 in mineral oil.

Reaction conditions: behind 94 ℃ of pre-sex change 2min, 94 ℃, 0.5min; 55 ℃, 0.5min; 72 ℃, 1min; 30 loop cycles.

3.PCR the clone of product

Adopt TA cloning process clone PCR products, method is seen document, and (Yang Guizhen: TA clone and double-stranded DNA check order, and introduce the method for a kind of quick clone and analysis PCR product, Chinese Journal of Immunology, 1994,10 (1): 5) for Yu Yongli, numb red brightness.

4.PCR the sequential analysis of product

The TA clone is transformed bacterial strain deliver to company, (J.Sambrook according to a conventional method, Polyacrylamide gel electrophoresis1.21-1.32, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989) extract plasmid, adopt the terminal cessation method of two deoxidations, carry out sequencing inserting fragment.Sequencing result is referring to accompanying drawing 5.

Further as follows to the anti-Stx2A monoclonal antibody 5F3 that cloned is light, heavy chain variable region gene carries out sequential analysis:

Use following two databases respectively:

http://imgt.cines.fr：8104/cgi-bin/IMGTnap.jv

http://www.ncbi.nim.nih.gov/blast/Blast.cgi

Institute's calling sequence and existing other various antibody genes of having reported are carried out homology relatively, and analyze its germline gene source, the result is as follows:

1) germline gene of 5F3 heavy chain source:

V-GENE：AF304558IGHV1S137*01；

D-GENE：IGHD-SP2.8*01；

J-GENE：IGHJ4*01；

Analyze demonstration by FR-IGMT and CDR-IGMT:

CDR1：GGT?TGC?TAC?ATG?CAC

CDR2：TAT?ATT?AGT?TGT?TAC?AAT?GGT?GCT?ACT?AGC?TAC?AAC?CAG?AAG?TTCAAG?GGC?AAG

CDR3：AAG?GGC?TAC?GGT?AGT?AGT?CAT?TAC?TAT?GCT?ATG?GAC?TAC

The homology comparative result shows among the NCBI:

Request?ID?1132707495-31830-19491971776.BLASTQ1

Sequence?producing?significant?alinments：

gi|11611970|gb|AF303832.1|AF303832 540?1e-150

gi|38348519|ref|NM_198640.1| 433?3e-118

gi|430841|gb|S66092.1| 408?1e-110

2) germline gene of 5F3 light chain source:

V-GENE：AJ235950IGKV12-89*01；

J-GENE：V00777IGKJ2*01；

Analyze demonstration by FR-IGMT and CDR-IGMT:

CDR1：AGG?GCC?AGC?CAA?AGT?ATT?AGC?AAC?AAC?CTA

CDR2：TAT?GCT?TCC?CAG?TCC?ATC?TCT

CDR3：CAA?CAG?AGT?AAC?AGC?TGG?CCT?CTC?ACG

The homology comparative result shows among the NCBI:

Request?ID?Request?ID?1132709610-9086-134297927386.BLASTQ4

Database：ALL?GenBank+EMBL+DDBJ+PDB?sequences(but?no?EST，STS，GSS，or?phase0，1or2?HTGS?sequences)

gi|7529622|emb|AJ277215.1|MMU277215 4e-164 585

gi|63543416|ref|XM_620323.1| 9e-143 514

gi|809057|emb|X86544.1|MMVL1B10 1e-112 414

Above-mentioned analytical results shows: the anti-Stx2A monoclonal antibody 5F3 gene order that is obtained is really from the mouse germline gene, all not quite identical with various other known antibodies gene orders of existing report, be to belong to a kind of new antibody gene that function is arranged at Stx2A.

The structure of the SeFv form antibody of embodiment 3 anti-Stx2A monoclonal antibodies and expression (using the Pharmacia scFv of company to express test kit)

With VH, VL and Linker (G ₄S) ₃After mole such as primer mixes, carry out polymerization and fill reaction, VH is connected together with VL mat LinkerDNA, form ScFvDNA.Add the restriction site primer then, amplification polymeric ScFv and 5 ' end is introduced Sfi I restriction site, and 3 ' end is introduced Not I restriction site.Be cloned among secretion expression's carrier pCANTAB6His of transformation, be transformed among the intestinal bacteria H B2151 and carry out abduction delivering.Identify and the dna sequencing analysis through restriction enzyme digestion, confirm gene constructed correct.Detect expression product with methods analysts such as SDS-PAGE electrophoresis, Western-blot behind the affinitive layer purification expressing protein, referring to accompanying drawing 6.SDS-PAGE electrophoresis and Western-blot analysis revealed ScFv gene successful expression in intestinal bacteria.The relative molecular mass of expression product (Mr) is 27KD, conform to theoretical expected value, and can with the corresponding antigen specific combination, successfully realized the prokaryotic secretion expression of anti-Stx2 single-chain antibody, for its further application in diagnosis and treatment EHEC O157 infection and complication thereof is laid a good foundation.

Relevant operation concrete steps are as follows:

1. plasmid DNA extracting (using Omega company plasmid extraction test kit)

1) separates good bacterium colony transferred species on the picking flat board in being with corresponding antibiotic LB nutrient solution, 37 ℃ of shaking table overnight incubation.

2) get bacterium liquid and be sub-packed in the 1.5mL centrifuge tube, the centrifugal 3min of 12000g leaves and takes precipitation.

3) every pipe adds 100 μ L Solution I suspension, and mixing fully vibrates.

4) add 100 μ L SolutionII, soft mixing, ice-water bath 5min.

5) add 250 μ L SolutionIII, the mixing that gently shakes, room temperature is placed 10min.

6) 4 ℃, the centrifugal 10min of 12000g move to supernatant in the separator tube.

7) the centrifugal 1min of 12000g topples over the waste liquid in the collection tube.

8) add 500 μ L washing buffer in separator tube, the same centrifugal and discard waste liquid in the collection tube.Repeated washing once.

9) the centrifugal 1min of 12000g volatilizees ethanol fully.

10) separator tube is placed another clean EP pipe and add a certain amount of TE buffer, 65 ℃ of water-bath 5min, the centrifugal 1min of 12000g.

11) get a certain amount of elutriant and carry out electrophoresis, all the other place-20 ℃ of preservations standby.

2. agarose gel electrophoresis

1.0% sepharose, 1 * TAE damping fluid, 120-150mA, electrophoresis 20-40 minute.

50 * TAE storage liquid prescription: 2.0mol/L Tris base, 1.0mol/L NaAc, 0.1mol/L Na ₂EDTA; Regulate pH8.3 with Glacial acetic acid.

3. the endonuclease reaction of plasmid DNA

1) 1 μ g plasmid DNA

1 μ L, 10 * M damping fluid (seeing Takara company product description)

1 μ L restriction enzyme Sfi I (10u/ μ L)

With distilled water polishing to 10 μ L

Reaction conditions: 50 ℃ of reaction 4h.

1) in above-mentioned reaction system, add successively:

2 μ L, 10 * H damping fluid (seeing Takara company product description)

1 μ L restriction enzyme NotI (10u/ μ L)

With distilled water polishing to 20 μ L

Reaction conditions: 37 ℃ of reaction 4h.

4. the target DNA of agarose electrophoresis glue reclaims purifying (Promega company)

Under ultraviolet lamp, observe and downcut the target DNA electrophoresis band on the sepharose, move into 1.5mL EP pipe.Add Omega company glue and reclaim the DNA binding buffer of test kit, 65 ℃ of water-baths are dissolved gel fully and are kept pH value of solution between 5.0～6.0.Sol solutions is moved into separator tube, and the centrifugal 1min of 12000g discards the liquid in the collection tube.Add supporting Washing buffer, the centrifugal 1min of 12000g discards the liquid in the collection tube.Repeated washing 1 time.The centrifugal 1min of 12000g, another clean 1.5mL EP pipe of separator tube dislocation, the TE buffer of adding certain volume is hatched 10min for 65 ℃, and the centrifugal 1min of 12000g gets a certain amount of electrophoresis, and the UVP ultraviolet scanner detects and reclaims purification effect.

5. ligation (using Takara company to connect test kit)

By the concentration of UV spectrophotometer measuring purpose D N A fragment and carrier segments, be generally 1: 2～10 principle according to external source fragment and carrier mole ratio, design ligation system is as follows:

Target DNA 1 μ L

Plasmid vector 1～2 μ L

ligation?solution 5μL

ddH ₂O 2～3μL

Total?volume 10μL

16 ℃ connect 12-16h.

6. preparation (the CaCl of competence bacteria ₂Method)

1) the aseptic inoculation ring dips in and gets-70 ℃ of frozen bacteriums guarantor kind of liquid, and the trilinear method streak inoculation was cultivated 12～16 hours for 37 ℃ in the LB flat board.

2) the single colony inoculation of picking is in 2mL LB nutrient solution, and 37 ℃ of shaking tables are cultivated 12～16h.

3) with the DH5a of incubated overnight in 1% ratio transferred species to the LB nutrient solution, 37 ℃ of shaking tables are cultured to OD ₆₀₀Be 0.2～0.4 o'clock, the centrifugal 5min of 8000g collects bacterium.

4) the 0.1M CaCl of adding 1mL precooling ₂Resuspended precipitation, ice-water bath 3h.4 ℃ of centrifugal 5min of 8000g abandon supernatant.The 0.1M CaCl that adds 100 μ L precoolings ₂Suspend and precipitate, ice-water bath 1h, standby.

7. connecting product transforms

1) gets competence bacteria liquid 100 μ L, add the ligation product; Ice-water bath 60min, 42 ℃ of water-bath heat-shocked 100s place ice-water bath 1～2min rapidly.

20 add 100 μ LLB nutrient solutions, and 37 ℃ of shaking tables are cultivated 1h.

3) with the centrifugal 10min of 8000g, mixing precipitated after 100 μ L supernatants were abandoned in suction, respectively got 50 μ L spread plates, 37 ℃ of incubator overnight incubation.

2. efficiently express the structure and the screening of fusion rotein engineering bacteria

To contain the recombinant plasmid pCANTAB6His transformed into escherichia coli HB2151 of ScFv gene and extract the plasmid enzyme restriction evaluation.The plasmid extraction enzyme of the competence bacteria preparation of gene engineering colibacillus HB2151, conversion and reorganization bacterium is cut and is identified ditto.

Get and identify that errorless reorganization bacterium is inoculated in 3mL and contains in the LB nutrient solution of Amp 30 ℃ of shaking table overnight incubation.Contain the recombinant bacterial strain of incubated overnight in the LB nutrient solution of Amp in 20mL in 1% ratio transferred species next day, and 30 ℃ of shaking tables were cultivated 2.5 hours, induced 24h with IPTG, and SDS-PAGE detects Expression of Fusion Protein form and expression amount, the screening efficient expression strain.

Embodiment 4 anti-Stx2A monoclonal antibody 5F3 are light, heavy chain variable region gene and encoded polypeptides thereof preparation be used for diagnosing and treat that EHEC O157 infects and complication aspect the application of medicine.

1. set out with light, weight chain variable gene of the present invention, can reconstruct and the antibody drug of expressing various ways, can be directly used in diagnosis and treatment EHEC O157 infects and complication.For example:

1) chimeric antibody: be that C district with the V district of mouse monoclonal antibody and human IgG is formed by connecting and is people-mouse chimeric antibody.Because it has intactly kept the specificity and the avidity of mouse monoclonal antibody, has reduced untoward reactions such as HAMA simultaneously.

2) humanized antibody: be humanization modified at the variable region gene structure, thereby comprise that CDR transplants, surface amino groups acid residue is modified, specificity and avidity that the exchange of skeleton district, the location keeps and the epi-position guiding selects etc. to have reduced the variable region mouse source property have kept the mouse monoclonal antibody simultaneously again.

3) small molecular antibody: mainly contain VH-CH1 and VL-C1 form Fab antibody, with a polypeptide (Gly4Ser) ₃The single domain antibody that joint connects single-chain antibody that VH gene and VL gene form, be made up of with non covalent bond be combined into Fv fragment antibody, by VH or functional domain of VL VH gene and VL gene, the atom that constitutes by single CDR etc.

Multivalence miniantibody: mainly contain double-stranded antibody, (ScFv) ₂, (ScFv) ₄Deng.Because the polyvalent antigen binding site is arranged, avidity height, characteristics such as molecular size is moderate and have high clinical value.

5) bi-specific antibody: be the antibody that a class has dual specificity and dual-use function, claim bifunctional antibody again.

6) recombinant antibody fusion proteins: the ScFv gene fragment, a kind of recombinant protein that is connected to form with other protein genes such as the toxin of non-antibody or enzymes with specific biological activity guiding target site.

7) phage antibody: after the V district gene of Ig and filobactivirus DNA gone up gene III or gene VIII and is connected transfecting host bacterium, make its fusion protein product at the surperficial outer casing protein expression ScFv of film.By this product being taken turns more the affine absorption of related antigen, therefrom elutriation goes out required more high-affinity and more specific new antibodies.

2. with anti-Stx2A monoclonal antibody 5F3 of the present invention, can be used for:

1) the ultrasonic supernatant of O157 bacterium is attacked among poison-anti-Stx2A monoclonal antibody 5F3 and the protection experiment

With lethal dose (LD ₁₀₀) the ultrasonic supernatant liquor of O157 bacterium (total protein 0.05mg) inject mouse peritoneal, every treated animal gives the antibody neutralizer of various dose as follows through abdominal injection simultaneously, every mouse of control group gives 10mmol/L PBS, total amount of liquid of last every injected in mice is 0.5ml, and animal divides into groups as table 1.Observe the death condition of respectively organizing mouse, after 10 days observation period, calculate death/survival rate such as the table 2 of mouse.

Table 1

The ultrasonic supernatant of table 2O157 bacterium is attacked among poison-anti-Stx2A monoclonal antibody 5F3 and the protection effect

The result shows: the anti-Stx2A monoclonal antibody 5F3 behind the 0.15mg purifying attack against each other malicious mouse in and protection ratio reach 80%.

Conclusion: anti-Stx2A monoclonal antibody 5F3 can attack in the malicious mouse generation effectively and provide protection at the ultrasonic supernatant toxin of O157 bacterium.

2) fundamental research of enterohemorrhagic Escherichia coli (EHEC) O157 shiga-like toxin related antigen, or be used to find new shiga-like toxin related antigen or the antigenic new epi-position of known shiga-like toxin.These antigens all can be further used for clinical Serological testing, have certain clinical diagnosis meaning.

3) because monoclonal antibody has the characteristic of synantigen specific combination, can carry out separation and purification to corresponding antigens.There are defectives such as method is loaded down with trivial details, yield is low in the existing method natural shiga-like toxin of purifying from EHEC O157 wild strain.Can with the affinity chromatography method shiga-like toxin Stx2A effectively be separated and purifying resisting Stx2A monoclonal antibody 5F3 to be directly connected on the solid phase carrier.

Sequence table

＜110〉Military Medical Univ No.3, P.L.A

＜120〉light, heavy chain variable region gene of anti-EHEC O157 shiga-like toxin II toxin subunit monoclonal antibody 5F3 and application

<160>4

<210>1

<211>378

<212>DNA

＜213〉mouse

<220>

<221>V-region

<222>(1)...(378)

<400>1

CCAGCCATGG?CCCAGGTCCA?ACTGCAGCAG?TCAGGACCTG?AGCTAGTGAA?GACTGGGGCT 60

TCAGTGAAGG?TATCCTGCAA?GGCTTCTGGT?TACTCATTCA?CTGGTTGCTA?CATGCACTGG 120

GTCAAGCAGA?GCCATGGAAA?GAGCCTTGAG?TGGATTGGAT?ATATTAGTTG?TTACAATGGT 180

GCTACTAGCT?ACAACCAGAA?GTTCAAGGGC?AAGGCCACAT?TTACTGTAGA?CACATCCTCC 240

AGCACAGCCT?ACATGCAGTT?CAACAGCCTG?ACATCTGAAG?ACTCTGCGGT?CTATTACTGT 300

GCAAGAAAGG?GCTACGGTAG?TAGTCATTAC?TATGCTATGG?ACTACTGGGG?CCAAGGGACC 360

ACGGTCACCG?TCTCCTCA 378

<210>2

<211>376

<212>PRT

＜213〉mouse

<220>

<221>V-region

<222>(1)...(126)

<400>2

Pro?Ala?Met?Ala?Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Glu?Leu

1 5 10 15

Val?Lys?Thr?Gly?Ala?Ser?Val?Lys?Val?Ser?Cys?Lys?Ala?Ser?Gly

20 25 30

Thr?Ser?Phe?Thr?Gly?Cys?Thr?Met?His?Trp?Val?Lys?Gln?Ser?His

35 40 45

Gly?Lys?Ser?Leu?Glu?Trp?Iie?Gly?Tyr?Iie?Ser?Cys?Tyr?Asn?Gly

50 55 60

Ala?Thr?Ser?Tyr?Asn?Gln?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Phe?Thy

65 70 75

Val?Asp?Thr?Ser?Ser?Ser?Thr?Ala?Tyr?Met?Gln?Phe?Asn?Ser?Leu

80 85 90

Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Lys?Gly?Tyr

95 100 105

Gly?Ser?Ser?His?Tyr?Tyr?Ala?Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr

110 115 120

Thr?Val?Thr?Val?Ser?Ser

125

<210>3

<211>327

<212>DNA

＜213〉mouse

<220>

<221>V-region

<222>(1)...(327)

<400>3

GACATCGAGC?TCACTCAGTC?TCCAGCCACC?CTGTCTGTGA?CTCCAGGAGA?TAGCGTCAGT 60

CTTTCCTGCA?GGGCCAGCCA?AAGTATTAGC?AACAACCTAC?ACTGGTATCA?ACAAAAATCA 120

CATGAGTCTC?CAAGGCTTCT?CATCAAGTAT?GCTTCCCAGT?CCATCTCTGG?GATCCCCTCC 180

AGGTTCAGTG?GCAGTGGATC?AGGGACAGAT?TTCACTCTCA?GTATCAACAG?TGTGGAGACT 240

GAAGATTTTG?GAATGTATTT?CTGTCAACAG?AGTAACAGCT?GGCCTCTCAC?GTTCGGTGCT 300

GGGACCAAGC?TGGAGCTGAA?ACGGGCG 327

<210>4

<211>109

<212>PRT

＜213〉mouse

<220>

<221>V-region

<222>(1)...(109)

<400>4

Asp?Iie?Glu?Leu?Tyr?Gln?Ser?Pro?Ala?Tyr?Leu?Ser?Val?Tyr?Pro

1 5 10 15

Gly?Asp?Ser?Val?Ser?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Iie?Ser

20 25 30

Asn?Asn?Leu?His?Trp?Tyr?Gln?Gln?Lys?Ser?His?Glu?Ser?Pro?Arg

35 40 45

Leu?Leu?Iie?Lys?Tyr?Ala?Ser?Gln?Ser?Iie?Ser?Gly?Iie?Pro?Ser

50 55 60

Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Ser?Iie

65 70 75

Asn?Ser?Val?Glu?Thr?Glu?Asp?Phe?Gly?Met?Tyr?Phe?Cys?Gln?Gln

80 85 90

Ser?Asn?Ser?Trp?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu

95 100 105

Leu?Lys?Arg?Ala

Claims (2)

1. the heavy chain variable region gene and the chain variable region gene of the anti-EHEC O157 shiga-like toxin II toxin Stx2A of subunit monoclonal antibody, its gene order is the nucleotide sequence shown in sequence table 1 and the sequence table 3.

The described gene of claim 1 preparation be used for diagnosing or treat that EHEC O157 infects and complication aspect the application of medicine.

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