CN103789270B - The antibody of humanization anti-anthrax protective antigen PA and application - Google Patents

Abstract

The antibody of humanization anti-anthrax protective antigen PA and application, the variable region of heavy chain of described antibody has the amino acid sequence of SEQ ID NO:2, and variable region of light chain has the amino acid sequence of SEQ ID NO:4；CH has the amino acid sequence of SEQ ID NO:6, and constant region of light chain has the amino acid sequence of SEQ ID NO:8.This antibody can specific binding anthrax protective antigen (PA), block lethal factor and close LF and enter intracellular, thus play protective effect.

Description

The antibody of humanization anti-anthrax protective antigen PA and application

Technical field

The present invention relates to the gene of anti-anthrax monoclonal antibody, the polypeptide of gene code, mouse source monoclonal antibody and human mouse chimeric antibody prepare Technology, and the application that described gene and polypeptide are in preparation anthrax treatment, preventive medicine.

Background technology

Anthracnose is the acute deadly infectious disease of a kind of infecting both domestic animals and human caused by Bacillus anthracis, mainly passes through skin Skin, alimentary canal, respiratory tract infection animal and people, the most widely distributed.Bacillus anthracis can produce in blood And discharge three kinds of albumen: protective antigens (protective antigen, PA), lethal factor (lethal factor, LF), Edema factor (edema factor, EF).It is experimentally confirmed that anthrax toxin is a kind of AB(active-binding) pattern.PA During i.e. PA83 is compound " B ", (2 type capillaries form PROTEIN C MG2 or tumour with the anthrax toxin acceptor on cell membrane Endothelial cells mark 8TEM8) combine, by the Furin proteolytic cleavage being positioned on cell membrane except its aminoterminal 20kD fragment PA20, remaining Under 63kD fragment (PA63) occur oligomerization, on film formed heptamer ((PA63)₇).LF and EF is " A ", it is possible to combine seven The compound that aggressiveness is formed enters cell by receptor-mediated endocytosis.In the environment of endosome acid pH, PA heptamer Occurred conformation changes, and forms a pipeline, makes EF and LF enter cell liquid, plays toxic action respectively.These three albumen itself Being nontoxic, but the mixture of PA and LF is referred to as lethal toxin, the mixture of PA and EF is referred to as edema toxin.Experiment is ground in vitro In studying carefully, the highly purified lethal toxin of simple use or edema toxin can make animal dead.Be combined with acceptor by blocking PA or It is and LF and EF, and affects its oligomerization, can effectively prevent the pathogenic effects of anthrax toxin.And PA can induce body Protective immunity, be currently the only to obtain the people of U.S. food Drug Administration approval with anthrax adsorbed vaccine (anthrax Vaccine absorbed, AVA) main active.Therefore, protective antigens is the promising target of anthracnose treatment.

The medical protection measure of anthrax mainly has prevention and treatment two broad aspect.The most effective precautionary measures mainly connect Plant Anthrax vaccine, contact front 1～carry out immunization campaign in 2 months, immune protection can be provided for crowd.Effective remedy measures is main There are two big classes: a class is bacteriostatic agent and the bactericide of bacillus anthracis, including the bacteriolyze composition of antibiotic and bacteriophage；Another Class is the toxin inhibitor according to anthrax pathology modelling, mainly includes the neutralizing antibody of anthrax toxin, soluble recepter, poison Element mutant and small molecular antagonists.

The treatment of anthrax includes high dose intravenous injection and oral antibiotic, such as penicillin, Ciprofloxacin, Fourth Ring Mycin,

Erythromycin and vancomycin.And must be timely with antibiotic therapy, and the toxin for having discharged be not have effect , and have now been found that the drug-fast bacterial strain of tool.

Research both domestic and external shows, uses anti-PA antibody before being exposed to anthrax spore, it is possible to play prevention anthrax sense The effect of dye protection body.And, after attacking poison, certain time administration (24～48h) remains to play a protective role, even It is administered after sign occurs and can also play certain therapeutic action.

There is now many companies or mechanism of research institute carries out the research of anthrax antibody, external existing many companies are carried out Preclinical study.But in Chinese patent about the research display adhesion of antibody of anthrax antibody or animal protection effect also Have greatly improved space.

Summary of the invention

Solve the technical problem that: the present invention provides antibody and the application of a kind of humanization anti-anthrax protective antigen PA, logical Cross monoclonal antibody technique and screened the monoclonal antibody that anthrax protective antigen PA is had high affinity, and obtain Give the heavy chain of the different characteristic of described antibody and the amino acid sequence of variable region of light chain and nucleotide sequence, and the weight to described antibody Chain and constant region of light chain have carried out humanization modified, are finally obtained and have special antigen-binding, the neutralization of excellent toxin Active and good animal protection function.

Technical scheme: the hybridoma cell strain PA6 of anti-anthrax protective antigen (Protective Antigen, PA).

Anti-PA monoclonal antibody PA6, is produced by above-mentioned hybridoma cell strain PA6.

A kind of containing above-mentioned antibody weight, people's mouse chimeric mAb of the anti-PA of variable region of light chain, described monoclonal resists The variable region of heavy chain of body has the amino acid sequence of SEQ ID NO:2, and variable region of light chain has the amino acid sequence of SEQ ID NO:4 Row.

The CH of described monoclonal antibody has the amino acid sequence of SEQ ID NO:6, and constant region of light chain has The amino acid sequence of SEQ ID NO:8.

Described variable region of heavy chain has the DNA sequence dna of SEQ ID NO:1, and variable region of light chain has the DNA of SEQ ID NO:3 Sequence.

Described CH has the DNA sequence dna of SEQ ID NO:5, and constant region of light chain has the DNA of SEQ ID NO:7 Sequence.

A kind of expression vector containing the nucleotide coding encoding monoclonal antibody heavy or light chain, variable region of heavy chain has The DNA sequence dna of SEQ ID NO:1, variable region of light chain has the DNA sequence dna of SEQ ID NO:3, and CH has SEQ ID The DNA sequence dna of NO:5, constant region of light chain has the DNA sequence dna of SEQ ID NO:7.

Described carrier is IgG antibody 1 hypotype secretion type eukaryon expression vector.

A kind of host cell containing above-mentioned expression vector, host cell is FreeStyle^TM293-F cells。

The application in preparation treatment anthracnose medicine of the described monoclonal antibody.

Beneficial effect: the invention provides a kind of have high protectiveness, high specific, the anti-PA of high-affinity people mouse be fitted together to Monoclonal antibody hmPA6.PA is the albumen that Bacillus anthracis discharges in blood, therefore the monoclonal antibody of the present invention HmPA6 can be applicable in the diagnosis about anthracnose, treatment and Prevention Research.

The present invention, with protective antigens PA as target molecule, prepares mouse source monoclonal antibody PA6, prepares people on the basis of PA6 Mouse chimeric antibody hmPA6.The mouse source monoclonal antibody PA6 and human mouse chimeric antibody hmPA6 of preparation is done Function Identification.Immunology detection shows Showing, this mouse source monoclonal antibody and human mouse chimeric antibody can be specific binding with anthrax protective antigen PA63 and PA83, external neutralization Experimental result confirms, PA6 and hmPA6 antibody can be effectively blocked the pathogenic effects of anthrax toxin.With Fischer344 rat For model, in vivo studies confirms that hmPA6 antibody protective rate reaches 100%.

Accompanying drawing explanation

Fig. 1 is the SDS-PAGE testing result of restructuring PA83 albumen, M, marker；1, the ultrasonic supernatant of bacterium；2, purifying- PA83 albumen.Visible purity of protein reaches more than 90%；

Fig. 2 is the SDS-PAGE testing result purifying PA6 antibody, M, marker；1, PA6 antibody；2, cells and supernatant； 3, flow through；The antibody purity of visible purifying is the highest；

Fig. 3 is the EUSA testing result of anti-PA monoclonal antibody PA6, it is seen that PA6 antibody and PA83 egg White binding ability is stronger；

Fig. 4 is the antigen immune trace qualification result of anti-PA monoclonal antibody PA6 and PA63 recombinant protein, M, marker； 1, PA63 expression of recombinant proteins bacteria lysis supernatant；2, unrelated protein expresses bacteria lysis supernatant.Visible PA6 antibody is to PA63 weight Histone has specific binding capacity；

Fig. 5 is the antigen immune trace qualification result of anti-PA monoclonal antibody PA6 and attenuated strain source, M, marker；1, Attenuated strain cracking supernatant；2, anthrax unrelated protein expresses bacteria lysis supernatant.Visible PA6 antibody is to attenuated strain protective antigens PA Albumen has specific binding capacity；

Fig. 6 is the result of the cell in vitro protective effect of anti-PA monoclonal antibody PA6, it is seen that the hybridoma of screening In strain, PA6 antibody protected effect is best, and when LF reaches 10 μ g/mL, PA6 protective rate reaches more than 95%；

Fig. 7 is the nucleic acid electrophoresis result of hmPA6 construction of recombinant expression plasmid, reverse transcription PCR amplification PA6 weight, light chain.M, DL2000；1, PA6 variable region of heavy chain PA6VH；2, PA6 variable region of light chain PA6VK；

Fig. 8 is the nucleic acid electrophoresis result of hmPA6 construction of recombinant expression plasmid, M, DL10000；1, recombinant expressed hmPA6 weight Chain plasmid i.e. pFUSE-CHIg-hG1 hmPA6H；2, linearize heavy chain template plasmid pFUSE-CHIg-hG1；3, hmPA6 heavy chains Variable region i.e. hmPA6VH；4, the recombinant expressed i.e. pFUSE-CLIg-hk-hmPA6K of hmPA6 light chain plasmids；5, linearize light chain mould Plate plasmid pFUSE-CLIg-hk；6, hmPA6 variable region of light chain i.e. hmPA6VK；

Fig. 9 is the SDS-PAGE testing result of antibody purification hmPA6, M, marker；1, hmPA6 antibody purification；2, comparison IgG；3, culture supernatant；4, flow through；5, untransfected plasmid 293F culture supernatant；

Figure 10 is the EUSA testing result of anti-PA monoclonal antibody hmPA6；

Figure 11 is the SDS-PAGE result of anti-PA monoclonal antibody hmPA6 co-immunoprecipitation, M, marker；1, hmPA6 with Attenuated strain bacteria lysis supernatant is hatched altogether；2, hmPA6；3, attenuated strain PA83 albumen；4, negative control (unrelated IgG and attenuated strain Bacteria lysis supernatant is hatched altogether).Visible hmPA6 antibody can be left behind the albumen that albumen size is 83kDa and 63kDa size, Two bands are cut and does mass spectrum.Because not expressing PA63 albumen in attenuated strain bacterium, it produces for PA83 protein cleavage, and amount is relatively Few；

Figure 12 a is the mass spectral results of the co-immunoprecipitation of 83kDa size albumen；

Figure 12 b is the mass spectral results of the co-immunoprecipitation of 63kDa size albumen；

Figure 13 is the result of the cell in vitro protectiveness test of anti-PA monoclonal antibody hmPA6, it is seen that antibody hmPA6 is dense Degree only 4 μ g/mL and toxin LF concentration reach 10 μ g/mL time, cytoprotection rate still is able to reach more than 80%；

Figure 14 is the result of anti-PA monoclonal antibody hmPA6 animal vivo test, and this antibody gives 50ug every rat 100% protective rate can be reached during antibody；

Figure 15 is the protein sequence comparison chart of antibody variable sequences and antibody library.

Detailed description of the invention

The preparation of embodiment 1 mouse source monoclonal antibody PA6 and selective mechanisms

1) application hybridoma technology prepares anti-PA monoclonal antibody, specifically comprises the following steps that

One, antigen (PA83 albumen) is prepared

Build PA83 expression plasmid.

By above-mentioned Plastid transformation Escherichia coli BL-21 competent cell, screening positive transformant carries out prokaryotic expression, to table Reach product and carry out SDS-PAGE detection, it is thus achieved that the recombinant protein of 83kd, be consistent with the molecular size range of PA83 albumen, use His Destination protein is purified by affinity column (Amersham company of the U.S.), obtains the PA83 recombinant protein with His label, Named PA83 albumen.See Fig. 1.

Two, hybridoma cell strain (all of mouse myeloma SP2/0 clone for hybridoma technology of anti-PA is prepared Derive from being sheerly BALB/c mouse):

Immunity inoculation is carried out as immunogene in mouse web portion hypodermic injection with PA83 recombinant protein, each 100 μ g/mL, Totally five times, first three sky of immunity inoculation carries out booster immunization in abdominal cavity the last time.Merge and took mouse spleen the same day, use RPMI- 1640 incomplete culture mediums (GIBCO company of the U.S.) are prepared as single cell suspension, at 50%PEG(pH8.0) in the presence of, spleen is thin Born of the same parents and SP2/0 murine myeloma cell merge, and with HAT selective medium, (RPMI-1640 culture medium 98mL, HT store Liquid 1mL, A store liquid 1mL) cultivate 7 days, use HT culture medium (RPMI-1640 culture medium 99mL, HT1mL) instead and cultivate.

Three, the screening of anti-PA positive monoclonal antibody

According to the detection screening of Growth of Hybridoma Cell situation row routine ELISA, take the cell time cloning again in the positive hole of detection Changing, through 5 time clonings, after in all of hole, monoclonal cell detection is all the anti-PA positive, access hole expands to be cultivated and portion Divide frozen.Finally choose the hybridoma of 4 strain stably excreting anti-PA antibody, be respectively designated as PA5, PA6, PA7 and PA8, it The monoclonal antibody secreted be respectively designated as PA5, PA6, PA7 and PA8.

Four, the hypotype of monoclonal antibody is identified

Monoclonal antibody hypotype identification kit (ISO-2KT) with Sigma Co., USA carries out the hypotype of antibody, result The hypotype showing anti-PA antibody PA5, PA6, PA7 and PA8 is all IgG1.

Five, the mass propgation of monoclonal antibody purifying

With hybridoma special serum free medium (Hybridoma, Gibico) mass propgation four strain of hybridoma strain.5 Collect culture supernatant after it, with Protein G affinity column (Amersham company of the U.S.) antibody purification, divide after measuring concentration Dress ,-70 DEG C frozen.

Purification result antibody purity as shown in Figure 2 reaches more than 95%.

2) functional activity of anti-PA monoclonal antibody PA6 is identified

One, ELISA

With EUSA, the combination of PA6 and the PA83 albumen of embodiment 1 is identified.Method particularly includes: Being coated ELISA96 orifice plate with being coated liquid (0.1M carbonate buffer solution, pH9.6) dilution PA83 albumen to 2 μ g/mL, every hole adds 100 μ L, 4 DEG C overnight；PBST(PBS contains 0.5%Tween20) 5% skim milk-lavation buffer solution closing, hatch 2h for 37 DEG C；PBST After washing 5 times, each hole adds 100 μ L PA6(2 μ g/mL initial concentrations, 15 concentration gradients dilutions) 4 DEG C overnight；With 1: Anti-(Beijing Zhong Shan company) 100 μ L/ hole of sheep anti mouse two of 4000 dilutions joins in hole, hatches 1h for 37 DEG C；At the bottom of peroxidase Thing nitrite ion 100 μ L/ hole, room temperature uses 2M sulfuric acid stopped reaction, upper machine testing colorimetric employing dual wavelength 450nm/ after lower 15 minutes 690nm。

Result is shown in that Fig. 3 shows: monoclonal antibody PA6 of this purifying has with attenuated strain PA83 albumen and is preferably combined activity, and The comformational epitope of proteantigen can be identified.

Two WB

The PA6 of embodiment 1 is carried out Western blotting qualification.Method particularly includes: with the e. coli strains of expression of other proteins For negative control, respectively PA63 expression of recombinant proteins bacterium, attenuated strain and anthrax unrelated protein bacterial strain cracking supernatant is carried out 10%SDS-PAGE electrophoresis electricity forward on nitrocellulose membrane, by this film and 2 μ g/mL PA6 incubated at room 1h, 1:2000 dilution Sheep anti-mouse igg two anti-(Beijing Zhong Shan company) and ECL luminescence reagent box (Pierce company of the U.S.) are exposed to gel imaging system (Bio-Rad company).

Result is as shown in Figure 4, Figure 5: PA6 antibody has specifically with PA83 albumen with PA63 recombinant protein and PA6 antibody In conjunction with.

Three cytoprotective tests

Tested cell (J774A.1) is cultivated at DMEM (the D μ containing 10% calf serum (FBS) and 1% antibiotic (P/S) Lbecco Modified Eagle Medium) in culture medium, cultivate under 37 DEG C of 5% carbon dioxide conditions.Treat that cell is cultivated good Rear transferred species is on 96 micropore Tissue Culture Plates, and incubated overnight is when cell is grown in reaching 70% on 96 micropore Tissue Culture Plates, aseptic Under the conditions of add the anthrax lethal toxin (PA:0.1 μ g/mL, LF:10 μ g/mL) diluted in proportion and PA6 antibody by various dose (50 μ g/mL), continues to cultivate 3 hours, and basis of microscopic observation cell death situation is also taken a picture, and then adds Cell Titer96aqueous nonradioactive cell Proliferation assay (Promega MI) checks LDH, meter Calculation and Analysis cell death percentage, experiment is in triplicate.

Result is as shown in Figure 5: the protective rate of PA6 is high, and LF10 μ g/mL when, protective rate remains to reach more than 80%.

The preparation of embodiment 2hmPA6 antibody

Through the detection of above-mentioned mouse source monoclonal antibody, we select PA6 hybridoma cell strain to prepare human mouse chimeric antibody hmPA6.

1) amplification of antibody variable gene fragment and checking:

PA6 antibody hybridoma cell is cultivated to exponential phase, Trizol-chloroform-isopropanol method extracting cell total rna； Being dissolved in by dried cell total rna in 20 μ L water, measure OD260/OD280, value is 1.9.The RNA taking 14 μ L reverses Record, with the mRNA in total serum IgE as template, with OligodT₁₅For primer, post transcription cloning obtains strand cDNA.

Design 19 VH upstream primers and 17 V κ upstream primers, 4 VH downstream primers and 3 V κ downstream primer:

V κ 5 ' upstream primer:

Vκ-1

5’-GGG CCC AGG CGG CCG AGC TCG AYA TCC AGC TGA CTC AGC C-3’

Vκ-2

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCW CCC AGT C-3’

Vκ-3

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGM TMA CTC AGT C-3’

Vκ-4

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGY TRACAC AGT C-3’

Vκ-5

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TRA TGA CMC AGT C-3’

Vκ-6

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTM AGA TRA MCC AGT C-3’

Vκ-7

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTC AGA TGA YDC AGT C-3’

Vκ-8

5’-GGG CCC AGG CGG CCG AGC TCG AYA TYC AGA TGA CAC AGA C-3’

Vκ-9

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCA WCC AGT C-3’

Vκ-10

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG WGC TSA CCC AAT C-3’

Vκ-11

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTS TRA TGA CCC ART C-3’

Vκ-12

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTK TGA TGA CCC ARA C-3’

Vκ-13

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CBC AGK C-3’

Vκ-14

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TAA CYC AGG A-3’

Vκ-15

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CCC AGW T-3’

Vκ-16

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CAC AAC C-3’

Vκ-17

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTT TGC TGA CTC AGT C-3’

V κ 3 ' downstream primer

VκR1

5’-AGA TGG TGC AGC CAC AGT TCG TTT KAT TTC CAG YTT GGT CCC-3’

VκR2

5’-AGA TGG TGC AGC CAC AGT TCG TTT TAT TTC CAA CTT TGT CCC-3’

VκR3

5’-AGA TGG TGC AGC CAC AGT TCG TTT CAG CTC CAG CTT GGT CCC-3’

VH5 ' upstream primer

VH1

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTR MAG CTT CAG GAG TC-3’

VH2

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTB CAG CTB CAG CAG TC-3’

VH3

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAG CTG AAG SAS TC-3’

VH4

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAR CTG CAA CAR TC-3’

VH5

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTY CAG CTB CAG CAR TC-3’

VH6

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTY CAR CTG CAG CAG TC-3’

VH7

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAC GTG AAG CAG TC-3’

VH8

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAS STG GTG GAA TC-3’

VH9

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AWG YTG GTG GAG TC-3’

VH10

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAG SKG GTG GAG TC-3’

VH11

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAM CTG GTG GAG TC-3’

VH12

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG CTG ATG GAR TC-3’

VH13

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAR CTT GTT GAG TC-3’

VH14

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTR AAG CTT CTC GAG TC-3’

VH15

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAR STT GAG GAG TC-3’

VH16

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTT ACT CTR AAA GWG TST G-3’

VH17

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAA CTV CAG CAR CC-3’

VH18

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAC TTG GAA GTG TC-3’

VH19

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG GTC ATC GAG TC-3’

VH3 ' downstream primer

VH R1

5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC CGT GGT-3’

VH R2

5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC TGT GAG AGT GGT-3’

VH R3

5’-CGA TGG GCC CTT GGT GGA GGC TGC AGA GAC AGT GAC CAG AGT-3’

VH R4

5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC TGA GGT-3’

By V κ 5 ' upstream primer, V κ 3 ' downstream primer, VH5 ' upstream primer, VH3 ' downstream primer is each molten for final concentration 100pmol/ μ L, the most all with 1:1 volume ratio mix, expand VH, VL gene respectively, amplification condition be 95 DEG C 4 minutes, 95 DEG C 30 seconds, 60 DEG C 40 seconds, 72 DEG C 45 seconds, 30 circulations；Last 72 DEG C extend 10 minutes, and electrophoresis reclaims, purifies the gene sheet of amplification Section (see figure 7), is connected to pMD-18T carrier, converts bacillus coli DH 5 alpha, obtains light chain, weight chain variabl area sequence, sequence after order-checking As follows:

Wherein the nucleotides sequence of VH is classified as:

The nucleotides sequence of VL is classified as

2) optimization of antibody variable sequences

By the protein sequence comparison of antibody variable sequences Yu antibody library, result such as Figure 15.

Because amplimer sequence the initial base sequence not in full conformity with mouse source antibody, so by sequencing result with anti- Body storehouse protein sequence is compared, and makes optimization, and heavy chain initiates the first amino acids E and the 5th bit amino of the i.e. FR1 of framework region Acid V is separately optimized as D and Q, corresponding mutating alkali yl sequence.In like manner optimize the amino acid in FR1 and the FR4 district of light chain.

3) design of the primer of PCR and gene magnification

Heavy chain amplimer:

F:5 '-GGTGTCCACTCGCTAGATGTGCAGCTGCAGGAATCGGGACCT-3 '

R:5 '-GCCCTTGGTGGATGCTGCAGAGACAGTGAC-3 '

Light chain amplimer:

F:5 '-ACAGACGCTCGCTGCCAAATTGTGCTCACTCAGTCTCCAG-3 '

R:5 '-TGCAGCCACCGTACGTTTGATTTCCAGTTTGGTC-3 '

4) amplification hmPA6 heavy chain of antibody, light chain

With above-mentioned heavy, variable region of light chain be connected to pMD18-T carrier sequencing analysis correct as template, relate to above-mentioned respectively And heavy chain and the upstream and downstream primer amplification human mouse chimeric antibody heavy chain of light chain, light chain gene.

(1)PCR

Reaction system:

Reaction condition:

(2) 2% agarose gel electrophoresis, observe purpose band under ultraviolet, cut glue and reclaim.

(3) glue reclaims kits target DNA fragment, and deionized water elutes.

5) double digestion IgG expression plasmid

IgG expression plasmid pFUSE-CHIg-hG1, pFUSE-CLIg-hk (purchased from Invivogen company) comprise IgG1 type The heavy chain in people source and light chain (Kappa) constant region alkali yl coding sequence.

(1) double digestion of pFUSE-CHIg-hG1, pFUSE-CLIg-hk template vector

Reaction system:

Reaction condition: 37 DEG C are digested overnight.

(2) 1% agarose gel electrophoresis, ultraviolet incision glue reclaims.

(3) glue reclaims kits target DNA fragment, and deionized water elutes.

6) Infusion PCR recombinant expression plasmid

Reaction system:

Reaction condition: hatch 15min for 50 DEG C.

Taking 5 μ L reactant liquor transformed competence colibacillus bacteriums, be laid on the flat board of corresponding resistant, next day chooses clone and send order-checking.To survey The clone that sequence result is correct preserves bacterial classification and expands cultivation, extracts plasmid.

Result successfully builds the expression plasmid of hmPA6.See Fig. 8.

7) expression of hmPA6 antibody

(1) take 250 μ L pFUSE-CHIg-hG1-hmPA6H (i.e. 50 μ g) and in the Opti-MEM culture medium of 1mL, take 250 μ L pFUSE-CLIg-hk-hmPA6K (i.e. 50 μ g) in the Opti-MEM culture medium of 1mL, take the 293Fectin of 200 μ L in In the Opti-MEM culture medium of 2.8mL, above-mentioned three kinds of mixed liquor room temperatures are stood 5min.

(2), after then two plasmid mixed liquors being mixed, add after the Opti-MEM culture medium of 500 μ L mixes It is directly added into the mixed liquor of transfection reagent 293Fectin, after mixing, stands 20min.Period processes 293F cell, by 293F Use 293F Expression Medium resuspended after cell centrifugation, then count and calculate cell viability ratio by platform dish indigo plant, inhaling Take 90 × 10⁶Individual cell, in blake bottle, is 94mL with 293F Expression Medium constant volume.

(3) after 20min terminates, the compound of DNA, 293Fectin of 6mL is added in ready 293F cell.

(4) cell is placed in shaking table incubator cultivation, condition of culture 8%CO₂, 120rmp, collects cell by 37 DEG C after 6 days Supernatant.

8) purifying of hmPA6 antibody

The cell conditioned medium membrane filtration of 0.22 μm that will collect, simultaneously by equilibrium liquid and eluent filter membrane.Use AKATA Purify the standard step purifying that instrument purifies according to Protein A, with the speed loading of 1mL/min, wash with the speed of 1.5mL/min De-.

Result successful expression also purifies hmPA6 antibody.SDS-PAGE is shown in Fig. 9.

The functional activity of embodiment 3hmPA6 antibody is identified

1) ELISA

With being coated liquid (0.1M carbonate buffer solution, pH9.6) dilution attenuated strain PA83 albumen (in Disease Control and Prevention in China Heart plague room is given) it is coated ELISA96 orifice plate to 2 μ g/mL, every hole adds 100 μ L, and 4 DEG C are overnight；PBST(PBS contains 0.5% Tween20) 5% skim milk-lavation buffer solution is closed, and hatches 2h for 37 DEG C；After PBST washs 5 times, each hole adds 100 μ L The people mouse of anti-PA is fitted together to full molecule IgG(2 μ g/mL initial concentration, 14 concentration gradients dilutions) 37 DEG C of 2h；With 1:4000 dilution Goat-anti people two resists 100 μ L/ holes to join in hole, hatches 1h for 37 DEG C；Peroxidase substrate nitrite ion 100 μ L/ hole, under room temperature 15 By 2M sulfuric acid stopped reaction after minute, upper machine testing colorimetric uses dual wavelength 450nm/690nm.

Result such as Figure 10 shows, chimeric hmPA6 antibody can play antigen-antibody reaction with attenuated strain PA83 albumen.

2) co-immunoprecipitation

With the mixed liquid of protein containing PA83 antigen and 5 μ g hmPA6 antibody, mixeding liquid volume PBS is adjusted to 300 μ L, After after 4 DEG C of refrigerators rotate and hatch 2h altogether, addition Pro.A immunomagnetic beads continues to hatch 1h altogether, 4000rmp is centrifuged 10min and removes supernatant, After centrifugal process PBST washes 5 times, add 50 μ L citric acid eluents, centrifugal collection supernatant, and add in 10 μ L Tris-base With.3/4 supernatant is run SDS-PAGE, and will examine the blue bands visible that dyes and send mass spectrum.

Co-immunoprecipitation SDS-PAGE result is shown in Figure 11, examines dye band mass spectral results and sees Figure 12 a, Figure 12 b.

3) cell in vitro protective effect

Tested cell (J774A.1) is cultivated at DMEM (the D μ containing 10% calf serum (FBS) and 1% antibiotic (P/S) Lbecco Modified Eagle Medium) in culture medium, cultivate under 37 DEG C of 5% carbon dioxide conditions.Treat that cell is cultivated good Rear transferred species is on 96 micropore Tissue Culture Plates, and incubated overnight is when cell is grown in reaching 70% on 96 micropore Tissue Culture Plates, aseptic Under the conditions of add the anthrax lethal toxin (PA:0.1 μ g/mL, LF:10 μ g/mL) diluted in proportion by various dose and hmPA6 resists Body (4 μ g/mL), continues to cultivate 3 hours, and basis of microscopic observation cell death situation is also taken a picture, and then adds Cell Titer96aqueous nonradioactive cell Proliferation assay (Promega MI) checks LDH, meter Calculation and Analysis cell death percentage, experiment is in triplicate.

Result is shown in Figure 13, shows: the protective rate of hmPA6 is high, is 4 μ g/mL and protection when LF10 μ g/mL at AC Rate remains to reach more than 80%.

4) animal protection experiment

Use the female Fischer344(F344 about body weight 150g) rat, often 6 rats of group, attacking toxic agent amount is 300 μ GPA+300 μ gLF/ rat, first tail vein injection toxin (aseptic PBS constant volume to 300 μ L)；Attacking after poison after 30min, tail vein is noted Penetrating hmPA6 antibody, the dosage of monoclonal antibody is 50 μ g(aseptic PBS constant volume to 300 μ L), observe the survival condition of rat.

Result see Figure 14, hmPA6 antibody when dosage is 50 μ g, survival of rats rate is 100%.This experiment display present invention The monoclonal antibody provided has good animal protection function, can apply to prepare anthrax medicine.

Sequence table

<110>Chinese People's Liberation Army Medical Research Institute Of Nanjing Military Region

Nanjing Medical University

<120>antibody of humanization anti-anthrax protective antigen PA and application

<130>

<160> 55

<170> PatentIn version 3.3

<210> 1

<211> 360

<212> DNA

<213>artificial sequence

<400> 1

gatgtgcagc tgcaggaatc gggacctggc ctggcgaaac cttctcagtc tctgtccctc 60

acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatccggcag 120

tttccaggaa acaaactgga gtggatgggc tacataagct acagtggtag cactagctac 180

aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240

ctgcagttga attctgtgac tactgaggac acagccacat attactgtgc aagacgggac 300

tacggctacc tcacctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 360

<210> 2

<211> 120

<212> PRT

<213>artificial sequence

<400> 2

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ala Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Gly Tyr Leu Thr Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 3

<211> 312

<212> DNA

<213>artificial sequence

<400> 3

caaattgtgc tcactcagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gtgccagctc aagtataagt tacatgcact ggtaccagca gaagccaggc 120

acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc 180

ttcagtggca gtgggtctgg gacctcttat tctctcacaa tcagcagcat ggaggctgaa 240

gatgctgcca cttattactg ccatcagcgg agtagttgga cgttcggtgg agggaccaaa 300

ctggaaatca aa 312

<210> 4

<211> 104

<212> PRT

<213>artificial sequence

<400> 4

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Trp Thr Phe Gly

85 90 95

Gly Gly Thr Lys Leu Glu Ile Lys

100

<210> 5

<211> 993

<212> DNA

<213>artificial sequence

<400> 5

gcatccacca agggcccatc tgtcttcccc ctggccccat cctccaagag cacctctggc 60

ggcacagctg ccctgggctg cctggtgaag gactacttcc ctgagcctgt gacagtgtcc 120

tggaactctg gcgccctgac cagcggcgtg cacaccttcc ctgctgtgct ccagtcctct 180

ggcctgtact ccctgagcag cgtggtgaca gtgccatcca gcagcctggg cacccagacc 240

tacatctgca atgtgaacca caagcccagc aacaccaagg tggacaagcg ggtggagccc 300

aagtcctgtg acaagaccca cacctgcccc ccatgccccg cccctgagct gctgggcggc 360

ccatctgtct tcctgttccc ccccaagccc aaggacaccc tgatgatctc ccggaccccc 420

gaggtgacct gtgtggtggt ggatgtgagc catgaggacc ccgaggtgaa gttcaactgg 480

tatgtggatg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 540

agcacctacc gggtggtgag cgtgctgaca gtgctgcatc aggactggct gaatggcaag 600

gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc ccattgagaa gaccatctcc 660

aaggccaagg gccagccccg ggagccccag gtctacaccc tgcccccctc ccgggaggag 720

atgaccaaga accaggtgag cctgacctgc ctggtgaagg gcttctaccc cagcgacatt 780

gctgtggagt gggagagcaa cggccagcct gagaacaact acaagaccac cccccctgtg 840

ctggactctg atggctcctt cttcctgtac agcaagctga cagtggacaa gagccggtgg 900

cagcagggca atgtcttctc ctgctctgtg atgcatgagg ccctgcacaa ccactacacc 960

cagaagagcc tgtccctgtc ccccggcaag tga 993

<210> 6

<211> 330

<212> PRT

<213>artificial sequence

<400> 6

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 7

<211> 324

<212> DNA

<213>artificial sequence

<400> 7

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg ttag 324

<210> 8

<211> 107

<212> PRT

<213>artificial sequence

<400> 8

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 9

<211> 40

<212> DNA

<213>artificial sequence

<400> 9

gggcccaggc ggccgagctc gayatccagc tgactcagcc 40

<210> 10

<211> 40

<212> DNA

<213>artificial sequence

<400> 10

gggcccaggc ggccgagctc gayattgttc tcwcccagtc 40

<210> 11

<211> 40

<212> DNA

<213>artificial sequence

<400> 11

gggcccaggc ggccgagctc gayattgtgm tmactcagtc 40

<210> 12

<211> 40

<212> DNA

<213>artificial sequence

<400> 12

gggcccaggc ggccgagctc gayattgtgy tracacagtc 40

<210> 13

<211> 40

<212> DNA

<213>artificial sequence

<400> 13

gggcccaggc ggccgagctc gayattgtra tgacmcagtc 40

<210> 14

<211> 40

<212> DNA

<213>artificial sequence

<400> 14

gggcccaggc ggccgagctc gayattmaga tramccagtc 40

<210> 15

<211> 40

<212> DNA

<213>artificial sequence

<400> 15

gggcccaggc ggccgagctc gayattcaga tgaydcagtc 40

<210> 16

<211> 40

<212> DNA

<213>artificial sequence

<400> 16

gggcccaggc ggccgagctc gayatycaga tgacacagac 40

<210> 17

<211> 40

<212> DNA

<213>artificial sequence

<400> 17

gggcccaggc ggccgagctc gayattgttc tcawccagtc 40

<210> 18

<211> 40

<212> DNA

<213>artificial sequence

<400> 18

gggcccaggc ggccgagctc gayattgwgc tsacccaatc 40

<210> 19

<211> 40

<212> DNA

<213>artificial sequence

<400> 19

gggcccaggc ggccgagctc gayattstra tgacccartc 40

<210> 20

<211> 40

<212> DNA

<213>artificial sequence

<400> 20

gggcccaggc ggccgagctc gayattktga tgacccarac 40

<210> 21

<211> 40

<212> DNA

<213>artificial sequence

<400> 21

gggcccaggc ggccgagctc gayattgtga tgacbcagkc 40

<210> 22

<211> 40

<212> DNA

<213>artificial sequence

<400> 22

gggcccaggc ggccgagctc gayattgtga taacycagga 40

<210> 23

<211> 40

<212> DNA

<213>artificial sequence

<400> 23

gggcccaggc ggccgagctc gayattgtga tgacccagwt 40

<210> 24

<211> 40

<212> DNA

<213>artificial sequence

<400> 24

gggcccaggc ggccgagctc gayattgtga tgacacaacc 40

<210> 25

<211> 40

<212> DNA

<213>artificial sequence

<400> 25

gggcccaggc ggccgagctc gayattttgc tgactcagtc 40

<210> 26

<211> 42

<212> DNA

<213>artificial sequence

<400> 26

agatggtgca gccacagttc gtttkatttc cagyttggtc cc 42

<210> 27

<211> 42

<212> DNA

<213>artificial sequence

<400> 27

agatggtgca gccacagttc gttttatttc caactttgtc cc 42

<210> 28

<211> 42

<212> DNA

<213>artificial sequence

<400> 28

agatggtgca gccacagttc gtttcagctc cagcttggtc cc 42

<210> 29

<211> 44

<212> DNA

<213>artificial sequence

<400> 29

gctgcccaac cagccatggc cctcgaggtr magcttcagg agtc 44

<210> 30

<211> 44

<212> DNA

<213>artificial sequence

<400> 30

gctgcccaac cagccatggc cctcgaggtb cagctbcagc agtc 44

<210> 31

<211> 44

<212> DNA

<213>artificial sequence

<400> 31

gctgcccaac cagccatggc cctcgaggtg cagctgaags astc 44

<210> 32

<211> 44

<212> DNA

<213>artificial sequence

<400> 32

gctgcccaac cagccatggc cctcgaggtc carctgcaac artc 44

<210> 33

<211> 44

<212> DNA

<213>artificial sequence

<400> 33

gctgcccaac cagccatggc cctcgaggty cagctbcagc artc 44

<210> 34

<211> 44

<212> DNA

<213>artificial sequence

<400> 34

gctgcccaac cagccatggc cctcgaggty carctgcagc agtc 44

<210> 35

<211> 44

<212> DNA

<213>artificial sequence

<400> 35

gctgcccaac cagccatggc cctcgaggtc cacgtgaagc agtc 44

<210> 36

<211> 44

<212> DNA

<213>artificial sequence

<400> 36

gctgcccaac cagccatggc cctcgaggtg aasstggtgg aatc 44

<210> 37

<211> 44

<212> DNA

<213>artificial sequence

<400> 37

gctgcccaac cagccatggc cctcgaggtg awgytggtgg agtc 44

<210> 38

<211> 44

<212> DNA

<213>artificial sequence

<400> 38

gctgcccaac cagccatggc cctcgaggtg cagskggtgg agtc 44

<210> 39

<211> 44

<212> DNA

<213>artificial sequence

<400> 39

gctgcccaac cagccatggc cctcgaggtg camctggtgg agtc 44

<210> 40

<211> 44

<212> DNA

<213>artificial sequence

<400> 40

gctgcccaac cagccatggc cctcgaggtg aagctgatgg artc 44

<210> 41

<211> 44

<212> DNA

<213>artificial sequence

<400> 41

gctgcccaac cagccatggc cctcgaggtg carcttgttg agtc 44

<210> 42

<211> 44

<212> DNA

<213>artificial sequence

<400> 42

gctgcccaac cagccatggc cctcgaggtr aagcttctcg agtc 44

<210> 43

<211> 44

<212> DNA

<213>artificial sequence

<400> 43

gctgcccaac cagccatggc cctcgaggtg aarsttgagg agtc 44

<210> 44

<211> 46

<212> DNA

<213>artificial sequence

<400> 44

gctgcccaac cagccatggc cctcgaggtt actctraaag wgtstg 46

<210> 45

<211> 44

<212> DNA

<213>artificial sequence

<400> 45

gctgcccaac cagccatggc cctcgaggtc caactvcagc arcc 44

<210> 46

<211> 44

<212> DNA

<213>artificial sequence

<400> 46

gctgcccaac cagccatggc cctcgaggtg aacttggaag tgtc 44

<210> 47

<211> 44

<212> DNA

<213>artificial sequence

<400> 47

gctgcccaac cagccatggc cctcgaggtg aaggtcatcg agtc 44

<210> 48

<211> 42

<212> DNA

<213>artificial sequence

<400> 48

cgatgggccc ttggtggagg ctgaggagac ggtgaccgtg gt 42

<210> 49

<211> 42

<212> DNA

<213>artificial sequence

<400> 49

cgatgggccc ttggtggagg ctgaggagac tgtgagagtg gt 42

<210> 50

<211> 42

<212> DNA

<213>artificial sequence

<400> 50

cgatgggccc ttggtggagg ctgcagagac agtgaccaga gt 42

<210> 51

<211> 42

<212> DNA

<213>artificial sequence

<400> 51

cgatgggccc ttggtggagg ctgaggagac ggtgactgag gt 42

<210> 52

<211> 42

<212> DNA

<213>artificial sequence

<400> 52

ggtgtccact cgctagatgt gcagctgcag gaatcgggac ct 42

<210> 53

<211> 30

<212> DNA

<213>artificial sequence

<400> 53

gcccttggtg gatgctgcag agacagtgac 30

<210> 54

<211> 40

<212> DNA

<213>artificial sequence

<400> 54

acagacgctc gctgccaaat tgtgctcact cagtctccag 40

<210> 55

<211> 34

<212> DNA

<213>artificial sequence

<400> 55

tgcagccacc gtacgtttga tttccagttt ggtc 34

Claims (7)

1. people's mouse chimeric mAb of anti-PA63 and PA83, it is characterised in that the heavy chain of described monoclonal antibody can Becoming district and have the amino acid sequence of SEQ ID NO:2, variable region of light chain has the amino acid sequence of SEQ ID NO:4；Heavy chain is permanent Determining district and have the amino acid sequence of SEQ ID NO:6, constant region of light chain has the amino acid sequence of SEQ ID NO:8.

2. the nucleotide sequence of people's mouse chimeric mAb described in coding claim 1, it is characterised in that described variable region of heavy chain Having the DNA sequence dna of SEQ ID NO:1, variable region of light chain has the DNA sequence dna of SEQ ID NO:3.

3. the nucleotide sequence of people's mouse chimeric mAb described in coding claim 1, it is characterised in that described CH Having the DNA sequence dna of SEQ ID NO:5, constant region of light chain has the DNA sequence dna of SEQ ID NO:7.

4. the expression vector containing the nucleotide coding encoding monoclonal antibody heavy or light chain, it is characterised in that heavy chain can Becoming district and have the DNA sequence dna of SEQ ID NO:1, variable region of light chain has the DNA sequence dna of SEQ ID NO:3, and CH has Having the DNA sequence dna of SEQ ID NO:5, constant region of light chain has the DNA sequence dna of SEQ ID NO:7.

Expression vector the most according to claim 4, it is characterised in that described carrier is IgG antibody 1 hypotype secreting type eucaryon Expression vector.

6. the host cell containing the expression vector described in claim 5, host cell is FreeStyle 293-F cells。

7. the application in preparation treatment anthracnose medicine of the monoclonal antibody described in claim 1.