CN1102660C - Antihumanfibrin monoclonal antibody heavy chain and light chain variable region gene and its application - Google Patents

Abstract

The present invention discloses a heavy chain variable region gene and a light chain variable region gene of an anti-human fibrin monoclonal antibody, polypeptide coded by the genes, a carrier containing the genes, and the application of the genes to the preparation of a thrombus level diagnosis reagent and thrombolytic medicaments.

Description

Anti-human fibrin monoclonal antibody heavy chain and chain variable region gene and application thereof

The present invention relates to anti-human fibrin monoclonal antibody heavy chain and chain variable region gene,, contain the carrier and the application of described gene in preparation thrombus level diagnosis reagent and thrombolytic agent of described gene by the polypeptide of described genes encoding.

Immunoglobulin (Ig) is to reset the glycoprotein macromole that produces by polygene, it is by two heavy chains and two light chain be combined intos, wherein according to the conservative property of the aminoacid sequence of the immunoglobulin (Ig) in its different genera source, heavy chain and light chain can be divided into constant region and variable region again.The antigen bonding unit that heavy chain and variable region of light chain constitute is the key of immunoglobulin (Ig) specific recognition and conjugated antigen, and therefore, the peptide chain that the reorganization variable region gene produces has the title of little antibody.This single-chain antibody molecule of being made up of heavy chain immunoglobulin and variable region of light chain (scFV) has only about 30KDa, can enter the various tissues of body, can from body, remove soon again, and the non-antigen-specific combination that does not have the FC acceptor to cause, be the desirable probe of pathology level diagnosis.Can specific recognition and conjugated antigen by the functional little antibody scFv that heavy chain immunoglobulin and chain variable region gene reorganization produce.But because of lacking the constant region effector molecule, scFv itself can not cause the biological effect of whole antibody.Utilizing the antigen-binding specificity of scFv that other effector molecule is taken to target position, is the purpose of the functional little antibody of artificial preparation.Report with the scFV level diagnosis tumour of radioisotope labeling is existing (Colcher D.et al, J.Natl.Cancer Inst.1990,82:1191-1197).Different scFV molecules can also covalent attachment constitute bifunctional antibody (Gruber M.et al, J.Immun.1994,152:5368-5374; Jonge JD et al, Mole.Immun.1995,32:1405-1412), or combine formation " magic bullet " with other effector molecule such as immunotoxin, cell toxicity medicament, cytokine or radionuclide etc., in oncotherapy, shown good application prospect (Winter G.et al, Nature, 1991,349:293-299; Chaudhary VK et al, Proc.Natl.Acad.Sci.USA, 1990,87:1066-1070; Yang J.et al, Mole.Immun.1995,32:873-881; Wels W.et al, CancerRes.1992 52:6310-6317), has promoted the research of various genetic engineering antibody.Cancer knurl at the serious harm human life, Chaudhqry etc. (document is the same) take immunotoxin to the malignant lymphatic cell with the anti-Tac variable region, (Cancer Biother such as Xiang, 1993,8:327-337) splice at gene level with antitumor cell monoclonal antibody variable region and Gamma Interferon, rabbit or tumour necrosis factor, produce fusant, improved the concentration of these cytokines, reduced their toxic side effect at tumor by local.This is some successful examples in the oncotherapy research.Except that the cancer knurl, the thrombus that is made of fibrin clot is another important goal of this class research, result of study shows that antifibrin scFV can be used for the level diagnosis of dvt, also can combine half clean-up time that prolongs the latter with thrombolytic drug such as urokinase and increase steering capability (Holvoet P.et al, Blood, 1993,81:696-703).Can use lower dosage when above-mentioned fusion molecule treatment thrombus disease is adopted in prompting thus, thereby the serious side effects of thrombolytic drugs such as minimizing urokinase is the approach that tool is wished in the present thromboembolism treatment.The good thrombolytic performance that the antifibrin variable region that the protein level splicing produces and the fusion molecule of urokinase show in experimentation on animals has promoted the research of gene level splicing antifibrin variable region and various thrombolytic drugs.The inventor is a starting point with the hybridoma that can secrete anti-human fibrin monoclonal antibody, utilize polymerase chain reaction from total RNA of hybridoma, to amplify heavy chain immunoglobulin and chain variable region gene, through the recombinant expressed scFV that goes out to discern human fibrin, finished the present invention thus.

Therefore, one object of the present invention is to provide a kind of anti-human fibrin monoclonal antibody heavy chain variable region gene and chain variable region gene, can give expression to specific recognition after both reorganization and in conjunction with the antibody activity fragment of human fibrin.

Another object of the present invention is to provide by described anti-human fibrin monoclonal antibody heavy chain variable region gene and chain variable region gene encoded polypeptides product.

A further object of the present invention is to provide the expression vector that contains heavy chain variable region gene and chain variable region gene.

Another purpose of the present invention is described anti-human fibrin monoclonal antibody heavy chain variable region gene and the application of chain variable region gene in the preparation thrombolytic agent.

According to the present invention, a kind of anti-human fibrin monoclonal antibody heavy chain variable region gene is provided, be called VH-8E5, total length is 375bp, its nucleotide sequence is shown in SEQ ID NO:1 in the sequence table.Described gene comprises IgG2b framework region and three complementary determining regions (CDR), and wherein CDR1 is 15bp, is positioned at the 85-99 position Nucleotide of SEQ ID NO.1; CDR2 is 51bp, is positioned at the 142-192 position Nucleotide of SEQ ID NO.1; CDR3 is 33bp, is positioned at the 289-321 position Nucleotide of SEQ ID NO.1.These three CDR districts are specificity important structure in conjunction with human fibrin.

According to the present invention, a kind of anti-human fibrin monoclonal antibody light chain chain variable region gene is provided, be called VK-8E5, total length is 366bp, its nucleotide sequence is shown in SEQ ID NO:2 in the sequence table.The feature of described gene is to have three complementary determining regions (CDR), wherein CDR1 is 51bp, be positioned at the 70-120 position Nucleotide of SEQ ID NO.2, CDR2 is 21bp, be positioned at the 166-186 position Nucleotide of SEQ ID NO.2, CDR3 is 27bp, is positioned at the 283-309 position Nucleotide of SEQ ID NO.2.Equally, these three CDR districts are specificity important structure in conjunction with human fibrin.

VH-8E5 gene of the present invention and chain variable region gene VK-8E5 coupling and the single-chain antibody molecule scFv-8E5 that expresses can be used for thrombus level diagnosis and preparation thrombus dissolving fusion rotein.

The dna sequence analysis result of VH-8E5 gene of the present invention and the dna sequence dna in the database relatively prove that our isolating immunoglobulin heavy chain variable region gene is unique, do not have identical dna sequence dna with it in the database.The about 30KDa of expression product molecular weight (stating the result of embodiment 3 as follows) after the VK-8E5 gene recombination in VH-8E5 and same source, its specific recognition and to illustrate that in conjunction with the ability of human fibrin this scFv molecule has carried out in the host bacterium correct folding.

The invention still further relates to the recombination expression product scFv-8E5 of heavy chain variable region gene VH-8E5 of the present invention and chain variable region gene VK-8E5, its antigenic determinant is that human fibrin B chain N holds 7 peptides, this 7 peptides only result from after the Fibrinogen hydrolysis, and be exposed to the scleroproein surface, therefore this single-chain antibody molecular energy specific recognition scleroproein.Result of study proves that this scFv molecule truly has the antigen recognition specificity, is the necessary material of doing the molecule splicing with various thrombolytic drugs.The free cysteine of its C-terminal, convenient radioisotope labeling is used for the dvt level diagnosis.Therefore one side more of the present invention is described anti-human fibrin monoclonal antibody heavy chain variable region gene and the application of chain variable region gene in the medicine of diagnosis for preparing thrombus disease and thromboembolism treatment.

Another aspect of the present invention is the expression vector that contains described heavy chain variable region gene VH-8E5 and chain variable region gene VK-8E5, described expression vector can be a prokaryotic expression carrier, it also can be carrier for expression of eukaryon, be preferably protokaryon recombinant expression vector such as pOPE51-8E5, pOPE51-8E5 derives from pOPE51-215 (latter derives from doctor Dubel, German tumor research center).The pOPE51-215 carrier has lac promotor and polygalacturonase leader, regulate and control the expression of the anti-fruit bat melanogaster rna plymerase ii mouse monoclonal antibody scFv in its downstream, its 3 ' terminal c-myc small peptide codon can be used as the mark of expression product, 5 Histidine codons are used for the purifying (people such as Kipriyanov SM of expression product, molecular immunology, 1994,31:1047-1058).POPE51-8E5 is the gene that replaces corresponding anti-fruit bat melanogasterRNA polymerase II mouse monoclonal antibody scFv among the pOPE51-215 with VH-8E5 gene of the present invention and VK-8E5 gene.Utilize described expression vector can express single-chain antibody variable region recombinant protein at an easy rate.

Monoclonal antibody results from hybridoma originally, and early stage genetic engineering antibody also is to express in eukaryotic cell.The breeding that intestinal bacteria are can be in the low consumption substratum quick and a large amount of has become the host of modern many gene engineering products.With the escherichia coli expression engineered antibody also is majority's selection.The preferred expression system pOPE51-8E5 that adopts has made full use of the advantage of bacterium high yield among the present invention.Having remedied bacterium with the polygalacturonase leader can not the proteic defective of processing, as long as just can obtain to account for the functional scFv of total protein content 7.3% by the IPTG abduction delivering of incubated overnight bacterium and 3 hours, this is very beneficial for studying new product.For obtaining high yield, the concentration that can increase inductor IPTG is to 100 μ M, and this moment, expression product can reach 28.9% of host bacterium total protein.But because concentration is too high, surpassed the processing treatment ability of bacterium, formed many undissolved inclusion bodys.Contain 54% expression product in our the inclusion body by centrifugal recovery.

The summary of accompanying drawing

Fig. 1 is the agarose gel electrophoresis figure of the product behind the mRNA of reverse polymerase chain reaction (RT-PCR) amplification 8E5 hybridoma, wherein, the M road illustrates molecular weight marker and (is followed successively by 1631 from top to bottom, 517/506,396,221/220), the 1-2 road is a variable region of light chain VK-8E5 gene of the present invention, and the 3-4 road is a variable region of heavy chain VH-8E5 gene of the present invention.

Fig. 2 illustrates clone's strategy synoptic diagram of variable region of light chain VK-8E5 gene of the present invention and variable region of heavy chain VH-8E5 gene.

Fig. 3 is the synoptic diagram that comprises variable region of light chain VK-8E5 gene of the present invention and variable region of heavy chain VH-8E5 expression carrier pOPE51-8E5.

Fig. 4 for expression vector pOPE51-8E5 transformed into escherichia coli JM109 of the present invention after or the SDS-PAGE electrophorogram (A) and the corresponding proteins matter engram analysis figure (B) of the expression product after inducing without IPTG.Among the figure, 1 road is inductive transformant total protein not, 2 roads are 100 μ M IPTG inductive transformant total proteins, 3 roads are the intermembranous albumen of the wall of not inductive transformant, 4 roads are the intermembranous albumen of wall of 20 μ M IPTG inductive transformants, 5 roads are inductive transformant inclusion body protein not, and 6 roads are 100 μ M IPTG inductive transformant inclusion body proteins.

The invention will be further described hereinafter with reference to embodiment and accompanying drawing, but and unrestricted the present invention.Separation 1, clone and the carrier of embodiment 1 anti-human fibrin monoclonal antibody heavy chain variable region gene VH-8E5 and chain variable region gene VK-8E5

Hybridoma cell line 8E5 provides (Institute of Radiation Medicine, Chinese Academy of Medical Sciences) by professor Lin Han.The 8E5 cell derives from human fibrin β chain N and holds 7 peptide immune mouses, can secrete anti-human fibrin monoclonal antibody, can be used for immune globulin variable region gene and separates.

Taq polysaccharase, archaeal dna polymerase, restriction enzyme and chemical reagent required in gene clone intermediate carrier pUC18 and the gene isolation process are all available from Gibco company.Expression vector pOPE51-215 provides (German tumor research center) by doctor Dubel.This carrier has lac promotor and polygalacturonase leader, regulate and control the expression of the anti-fruit bat melanogaster rna plymerase ii mouse monoclonal antibody scFv in its downstream, its 3 ' terminal c-myc small peptide codon can be used as the mark of expression product, 5 Histidine codons are used for the purifying (people such as Kipriyanov SM of expression product, molecular immunology, 1994,31:1047-1058).2, variable region of heavy chain and variable region of light chain cDNA clone and sequential analysis

Clone's strategy adopts conventional guanidine thiocyanate extraction and cesium chloride centrifugation method (molecular cloning laboratory manual, the 2nd edition) from 10 as shown in Figure 2 ⁷Individual 8E5 hybridoma prepares total RNA, uses oligo-dT post separating mRNA as template again, prepare the first chain cDNA with oligo-dT primer and reversed transcriptive enzyme after, obtain immunoglobulin heavy chain variable region cDNA by polymerase chain reaction (PCR).For the gamma variable region of heavy chain cDNA that increases, VH-5 ' primer (5 ' AGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTG) and VH-3 ' primer (5 ' TGGATAGACAAGCTTGGGTGTCGTTTTGGC) is used in design.Pcr amplification is carried out at 100 μ l reactive systems, and the first chain cDNA20-25ng is wherein arranged, each 25pmol of above-mentioned primer, dNTPs each 200 μ M and 1 Taq of unit archaeal dna polymerase.Change temperature with Hybaid temperature controller (Britain Hook company), 95 ℃ of sex change 1 minute, 50 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, obtained amplified productions through 25 circulations.The PCR product is identified through agarose electrophoresis, detects the amplified band about 380bp, as shown in Figure 1.Amplified production is mended flat with the big fragment of archaeal dna polymerase Klenow behind the agarose electrophoresis purifying, insertion is in the intermediate carrier pUC18 of SmaI enzymic digestion and dephosphorylation, the carrier called after pUC18VH that obtains, with its transformed into escherichia coli JM109, in the LB substratum, screen white positive bacterium colony with XGal and sec.-propyl-B-D-thio-galactose pyran-glucoside (IPTG).Get single bacterium colony amplification back and extract recombinant plasmid pUC18VH, enzyme is cut the back agarose electrophoresis and is identified.For dna sequence analysis, to choose and identify errorless positive colony, plasmid DNA is extracted in the amplification back, downcut the VH gene fragment and mend flat with SmaI with the big fragment of archaeal dna polymerase Klenow, insert the SmaI point of contact of sequencing vector M13mp19 again, transformed into escherichia coli JM109 is again with XGal and IPTG screening.Get positive single bacterium colony amplification, the centrifugation bacterium is collected supernatant and extracts single stranded DNA, makes dna sequence analysis with fluorescein-labeled dideoxy nucleotide and Taq enzyme sequencing kit (American AB I company) on ABI370A type sequence instrument (American AB I company).According to the dna sequence analysis result, select the correct VH cDNA clone of framework, this clone is named as VH-8E5, and its sequence is shown in the SEQ ID NO.1 of sequence table.Dna sequence dna and the dna sequence dna in the Kabat database database of VH-8E5 are compared, 47670 dna sequence dnas have been retrieved, therefrom select 4189 variable region of heavy chain DNA and make one by one base ratio, the result proves that VH-8E5 belongs to the Gamma heavy chain, comprise the variable region, the D district, J district and constant region initial section be 375bp altogether, with the many VH dna sequence dna height homologies in the database, but none is identical.

The separation of chain variable region gene of the present invention is to use identical hybridoma cell line 8E5 and adopts identical RT-PCR method, this gene is named as VK-8E5, different is for the designed primer of amplification chain variable region gene to being VK-5 ' primer: 5 ' GAAGCACGCGTAGATATCG (T) TGA (C) TC (G) ACCCAG (A) TCTCCAVK-3 ' primer: 5 ' GAAGATGGATCCAGCGGCCGCAGCATCAGC

VK-8E5 of the present invention shows through sequential analysis, it has the described nucleotide sequence of SEQ ID NO.2, total length is 366bp, and wherein 70-120 position Nucleotide, 166-186 position Nucleotide and the 283-309 position Nucleotide in the sequence of SEQ ID NO.2 is three complementary determining regions.With the dna sequence dna in this gene order and the Kabat database database relatively, the many VK dna sequence dna height homologies in discovery and the database, but none is identical.The reorganization of embodiment 2 VH and VK cDNA

Dna sequence analysis result according to embodiment 1, select correct VH of skeleton construction and VK cDNA clone and extract plasmid DNA, downcut the VH fragment with pVU II and Hind III, downcut the VK fragment with EcoR V and BamH 1, insert expression vector pOPE51-215 behind the agarose electrophoresis purifying in two steps through same restriction endonuclease digestion and dephosphorylation, be built into recombinant expression vector pOPE51-8E5, be used to express the little antibody scFv of anti-human fibrin strand.The structural representation of recombinant expression vector pOPE51-8E5 as shown in Figure 3.Expression and the purifying of embodiment 3 single-chain antibody scFv-8E5

With the pOPE51-8E5 plasmid DNA transformed into escherichia coli JM109 that embodiment 2 obtains, transformant increases in the LB nutrient solution, is that the IPTG abduction delivering of 20 μ M and 100 μ M was collected thalline after 3 hours with concentration respectively.20 μ M IPTG induce the thalline of group at broken wall damping fluid (50 μ M Tris-HCl, 1mM EDTA and 20% sucrose, pH8.0) ice bath is 1 hour in, centrifugal 40 minutes of 30000g, collect the soluble proteins in the supernatant liquor, after dialysing with the IMAC column purification (people such as Dubel, Cell Biophy.1992,21:69-79).Nickel ion on the IMAC post can combine with the Histidine of expression product end, by the affinity chromatography single step purification.100 μ M IPTG induce the thalline of group to need ultrasonication, centrifugal 30 minutes of 30000g, and collecting precipitation, with 6M guanidine hydrochloride dissolution inclusion body, centrifugal 1 hour of 30000g, collecting expression product is that single-chain antibody scFv-8E5 is used for analyzing.

With the purified product sample collected and control sample (the not total protein of inductive transformant, the intermembranous albumen of wall and inclusion body protein) through dialysis and concentrate the back with 12% polyacrylamide also virgin rubber make electrophoresis, use the protein content of laser scanner analytical electrophoresis district band after Coomassie brilliant blue dyes.Same SDS-PAGE glue is transferred to protein band on the acetate film through electrotransfer, is used for Western hybridization.Based on the c-myc small peptide of expression product end, be one anti-with anti-c-myc monoclonal antibody 9E10, the anti-mouse serum of the rabbit of horseradish peroxidase-labeled is two anti-, with substrate benzidine reaction back colour developing.The result of SDS-PAGE electrophoresis and Western engram analysis as shown in Figure 4, as seen from Figure 4, transformant is through with 20 μ M IPTG abduction deliverings, the intermembranous soluble proteins of bacteria wall demonstrates about 30KDa and expresses band on SDS-PAGE glue, it is intermembranous proteic 7.3% that its protein content accounts for wall, becomes the master tape on the SDS-PAGE glue behind the purifying.Western trace result proves that this 30KDa band is an expression product, and the molecular weight of promptly expressed single-chain antibody scFv-8E5 is about 30KDa.Output obviously increases behind the 100 μ M IPTG abduction deliverings, and the laser scanning result proves that the 30KDa band accounts for 28.9% of total bacterial protein, but the undissolved inclusion body of most formation.Contain expression product 54% through in the inclusion body of centrifugal recovery.Embodiment 4 single-chain antibodies the antigen recognition specificity analyses

Observe the antigen recognition specificity of the purifying scFv-8E5 of embodiment 3 preparations with improved enzyme linked immunoassay (ELISA).In order to prepare antigen, with scleroproein primordial covering 96 orifice plates (U.S. Nune company), dry the back with 15% calf serum DMEM nutrient solution (Gibco) activation for 37 ℃, form homogeneous fibre albumen thin layer, through the sealing of 0.2% gelatin with clean after the different dilution scFv testing samples of adding.With anti-c-myc monoclonal antibody 9E10 is one-level antibody, and the anti-mouse serum of the rabbit of horseradish peroxidase-labeled is secondary antibody, and diaminobenzidine is a substrate, carries out the ELISA test.Detect the color reaction intensity of 492nm with microplate reader (Bio-Rad).Can will be fixed in the hole with antigen bonded scFv, carry out color reaction by ELISA.

ELISA detects with the positive contrast of 8E5 whole antibody, and the average OD number of 4 detected results is 1.435.The irrelevant antibody of negative control group, the average OD number of 4 detections is 0.011.The duplicate detection result of 2 batches of expression product different concns prove the expression product scFv-8E5 after isolating VH and the VK gene recombination specific antigens binding ability is arranged, the results are shown in following table 1.

The antigen-binding specificity of table 1 recombinant single chain antibody scFv

Sample	Consumption (μ g)	OD 492Reading
			8E5 whole antibody (positive control) scFv (negative control) scFv-8E5 that has nothing to do	2.4 24.0	1.435 0.011 0.250 0.834

Sequence table SEQ ID NO.1 sequence length: 375bp chain: single chain molecule type: cDNA feature: 85-99 position Nucleotide, 142-192 position Nucleotide and 289-321 position Nucleotide are

Three complementary determining region sources: people

CAG CTG CAG CAG 12

Q L Q Q____VH-5 ' primer _ _ _ _ _ _ _ _ _ _ TCT GGA GCT GAG CTG GTG AAG CCT GGG GCT TCA GTG AAG ATG TCC TGC 60 S G A E L V K P G A S V K M S C

CDR 1AAG GCT TCT GGC TAC ACC TTC ACC AAC TAT TGG ATG CAC TGG GTG AAG 108 K A S G Y T F T N Y W M H W V KCAG AGG CCT GGA CAA GGC CTT GAG TGG ATC GGA ACG ATT GAT CCT GCA 156 Q R P G Q G L E W I G T I D P A

CDR 2GAT AGT TAT ACT AGT TAC AAT CAA AAC TTC AAG GAC AAG GCC ACA TTG 204 D S Y T S Y N Q N F K D K A T LACT GTA GAC AAA CCC TCC AGC ACA GCC TAC ATG CAG CTC AGC AGC CTG 252 T V D K P S S T A Y M Q L S S LACA TTT GGG GAC TCT GCG GTC TAT TTT TGT GCA AGA GAG GGG GTC TAC 300 T F G D S A V Y F C A R E G V Y CDR 3TAT AGA TAC TAC TTT GAC TAC TGG GGC CAC GGC ACC ACT CTC ACA GTC 348 Y R Y Y F D Y W G H G T T L T V

_ _ _ _ _ _ _ _ _ VH-3 ' primer _ _ _ _ _ _ _ _ _ TCC TCA GCC AAA ACG ACA CCC AAG CTT 375 S S A K T T P K LSEQ ID NO.2 sequence lengths: 366bp chain: single chain molecule type: cDNA feature: 70-120 position Nucleotide, 166-186 position Nucleotide and 283-309 position Nucleotide are

Three complementary determining region sources: people

_ _ _ _ _ _ _ VK-5 ' primer _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

GAT ATC GTG CTG ACC CAG TCT CCA TCC TCC CTG GCT ATG 39

D I V L T Q S P S S L A M

CDR 1TCA GTA GGA CAG AAG GTC ACT ATG AGC TGC AAG TCC AGT CAG AGC CTT 87 S V G Q K V T M S C K S S Q S LTTA AAT AGT AGC AAT CAA AAG AAC TAT TTG GCC TGG TAC CAG CAG AAA 135 L N S S N Q K N Y L A W Y Q Q K

CDR 2CCA GGA CAG TCT CCT AAA CTT CTG GTA TAC TTT GCA TCC ACT AGG GAA 183 P G Q S P K L L V Y F A S T R ETCT GGG GTC CCT GAT CGC TTC ATA GGC AGT GGA TCT GGG ACA GAT TTC 231 S G V P D R F I G S G S G T D FACT CTT ACC ATC AGC AGT GTG CAG GCT GAA GAC CTG GCA GAT TAC TTC 279 T L T I S S V Q A E D L A D Y F

CDR 3TGT CAG CAA CAT TAT AGC ACT CCG CTC ACG TTC GGT GCT GGG ACA AAG 327 C Q Q H Y S T P L T F G A G T K

_ _ _ _ _ _ _ _ VK-3 ' primer _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ CTG GAG CTG AAA CGG GCT GAT GCT GCG GCC GCT GGA TCC 366 L E L K R A D A A A A G S

Claims (6)

1, a kind of anti-human fibrin monoclonal antibody heavy chain variable region gene, but its with chain variable region gene reorganization after expression specificity identification and combine the antibody of human fibrin, described gene has the sequence of SEQ IDNO.1, it is characterized in that 85-99 position Nucleotide, 142-192 position Nucleotide and the 289-321 position Nucleotide at the dna sequence dna of SEQ ID NO.1 is that three specificitys are in conjunction with the required complementary determining region of human fibrin.

2, a kind of by the described anti-human fibrin monoclonal antibody heavy chain variable region gene encoded polypeptides product of claim 1.

3, a kind of expression vector, the anti-human fibrin monoclonal antibody chain variable region gene that it contains the described anti-human fibrin monoclonal antibody heavy chain variable region gene of claim 1 and has sequence shown in the SEQ ID NO.2.

4, expression vector as claimed in claim 3, it is pOPE51-8E5 as shown in Figure 3.

5, as the expression product of claim 3 or 4 described expression vectors.

6, anti-human fibrin monoclonal antibody heavy chain variable region gene as claimed in claim 1 and have sequence shown in the SEQ ID NO.2 anti-people's fiber egg from the application of monoclonal antibody chain variable region gene in preparation thrombus level diagnosis reagent and thrombolytic agent.