CN102153651B - Anti-dextran single-chain antibody and preparation method of anti-dextran single-chain antibody - Google Patents
Anti-dextran single-chain antibody and preparation method of anti-dextran single-chain antibody Download PDFInfo
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- CN102153651B CN102153651B CN2011100239019A CN201110023901A CN102153651B CN 102153651 B CN102153651 B CN 102153651B CN 2011100239019 A CN2011100239019 A CN 2011100239019A CN 201110023901 A CN201110023901 A CN 201110023901A CN 102153651 B CN102153651 B CN 102153651B
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Abstract
The invention discloses an anti-dextran single-chain antibody and a preparation method of the anti-dextran single-chain antibody, relates to the anti-dextran antibody and provides the anti-dextran single-chain antibody and the preparation method of the anti-dextran single-chain antibody. The preparation method comprises the following steps of: cloning a gene of the anti-dextran single-chain antibody and inducing the gene into a vector to construct a recombinant vector; inducing the recombinant vector into host bacteria to construct recombinant engineering bacteria for expressing the anti-dextran single-chain antibody; and carrying out fermentation culture on the recombinant engineering bacteria and separating and purifying fermentation liquor to obtain the anti-dextran single-chain antibody. A heavy and light chain variable region sequence of the antibody is obtained by utilizing the anti-dextran monoclonal antibody hybridoma cell strain D9 through the RT-PCR (reverse transcription-polymerase chain reaction) technology, so that the gene of the anti-dextran single-chain antibody is formed; and a recombinant expression vector pET-22(b+)-scFv is constructed so as to carry out expression, purification and identification on the anti-dextran single-chain antibody. Under the great background of the genetic engineering technology, the obtaining of the anti-dextran single-chain antibody can be beneficial to further development and application of the antibody in the future.
Description
Technical field
The present invention relates to the antibody of anti-VISOSE, particularly anti-VISOSE single-chain antibody and preparation method thereof.
Background technology
VISOSE has another name called Expex, is polymerized by a plurality of glucose molecules, has higher molecular weight, mainly connects with α-1,6 key between the glucosyl residue, and side chain mainly contains 1,2 key, 1,3 key, 1,4 key etc.VISOSE can be produced by the bacterial dextran sucrase catalysis of polluting in the sucrose solution, and VISOSE can be divided into α type and β type on structure.α type VISOSE is produced by bacterial degradation sucrose usually, and β type VISOSE has physiologically active, be present in the fungal cell wall (1, Fan Ruimei, Fan Jiaheng. the immunization of VISOSE and progress. sugarcane sugar industry, 2006, (6): 38-41).
(Single chain antibody is an important function of gene engineered antibody ScFv) to single-chain antibody, is that heavy chain variable region gene is connected with the oligomerization nucleic acid with chain variable region gene; Become single chain polypeptide at expression in escherichia coli; And in bacterial body, be folded into a kind of novel antibody that only constitutes by heavy chain and variable region of light chain (2, Shen doubly puts forth energy Chen Zhinan, Liu Minpei. recombinant antibodies [M]. Beijing: Beijing Science Press; 2005,21-23).The size of single-chain antibody is merely 1/6 of complete antibody, compares with antibody molecule, and ScFv has shown better tissue penetration on pharmacokinetics, when being used to treat, can get into the inaccessiable position of general antibody.Simultaneously, because the antigen bonding surface is constant, so single-chain antibody has whole binding specificities of antibody.The peptide linker of single-chain antibody can be designed to have the site of specific function, like metal-chelating, connection toxin or medicine etc., to be used for image and clinical treatment.Single-chain antibody can pass through the inclusion body great expression; Be easy to genetically engineered operation, especially be easy to make up antibody fusion protein, be the maximum small molecular antibody of research at present (3, Wang Rui. based on the application of hybridoma single-chain antibody technology of preparing; The structure [D] of anti-GPV-NS1 and GoIFN-γ single-chain antibody; Northeast Agricultural University, 2009,1-2).
Summary of the invention
First purpose of the present invention is to provide anti-VISOSE single-chain antibody.
Another object of the present invention is to provide the preparation method of anti-VISOSE single-chain antibody.
The aminoacid sequence of said anti-VISOSE single-chain antibody is:
Met Glu Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
20 25 30
Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp
35 40 45
Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Glu Thr Tyr Tyr Ala
50 55 60
Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
65 70 75 80
Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile
85 90 95
Tyr Tyr Cys His Tyr Asp Val Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Leu
130 135 140
Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln
145 150 155 160
Ser Ile Gly Thr Ser Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser
165 170 175
Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro
180 185 190
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile
195 200 205
Asn Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser
210 215 220
Asn Ser Trp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
225 230 235 240
Arg Leu Glu His His His His His His
245
The gene order of said anti-VISOSE single-chain antibody is:
atggaggtgc aactgcagca gtctggagga ggcttggtgc aacctggagg atccatgaaa 60
ctctcttgtg ctgcctctgg attcactttt agtgacgcct ggatggactg ggtccgccag 120
tctccagaga aggggcttga gtgggttgct gaaattagaa gcaaagctaa taatcatgaa 180
acatactatg ctgagtctgt gaaagggagg ttcaccatct caagagatga ttccaaaagt 240
agtgtctacc tgcaaatgaa cagcttaaga gctgaagaca ctggcattta ttactgtcat 300
tacgacgtgg ctatggacta ctggggccaa gggaccacgg tcaccgtctc ctcaggaggc 360
ggtggttctg gaggcggtgg ttctggaggc ggtggttctg acattgagct cacccagtct 420
ccagccatcc tgtctgtgag tccaggagaa agagtcagtt tctcctgcag ggccagtcag 480
agcattggca caagcataca ctggtatcag caaagaacaa atggttctcc aaggcttctc 540
ataaagtatg cttctgagtc tatctctggg atcccttcca ggtttagtgg cagtggatca 600
gggacagatt ttactcttag catcaacagt gtggagtctg aagatattgc agattattac 660
tgtcaacaaa gtaatagctg gccgtggacg ttcggtggag gcaccaagct ggaaatcaaa 720
cggctcgagc accaccacca ccaccac 747
The 1st～354 heavy chain variable region gene that Nucleotide is anti-VISOSE single-chain antibody wherein, total length is 354bp; The 355th～399 Nucleotide is connection peptides Linker, total length 45bp; The 400th～723 Nucleotide is for to be that the chain variable region gene of anti-VISOSE single-chain antibody, total length are 324bp.
The preparation method of said anti-VISOSE single-chain antibody may further comprise the steps:
1) the anti-VISOSE single-chain antibody gene of clone, and, make up recombinant vectors with its importing carrier;
2) recombinant vectors is imported host bacteria, the recombinant bacterial strain of the anti-VISOSE single-chain antibody of construction expression;
3) with the recombinant bacterial strain fermentation culture, the separation and purification fermented liquid promptly gets and resists the VISOSE single-chain antibody.
In step 1); The template of the anti-VISOSE single-chain antibody gene of said clone is from the hybridoma cell strain D9 of anti-dextran monoclonal antibody; Said hybridoma cell strain D9 is preserved in Chinese typical culture collection center (CCTCC) on December 7th, 2010; Preservation centre address China. Wuhan. Wuhan University, preservation center deposit number is CCTCC NO:C2010107.
The anti-VISOSE single-chain antibody gene of said clone can be cloned anti-VISOSE single-chain antibody heavy chain variable region gene and anti-VISOSE single-chain antibody chain variable region gene respectively, through connection peptides Linker both is connected again.
The primer of the anti-VISOSE single-chain antibody of said clone heavy chain variable region gene is:
5 ' end primer: 5 '-CAT GCC ATG GAG GTS MAR CTG CAG SAG TCW GG-3 ';
3 ' end primer: 5 '-
GCC TCC AGA ACC ACC GCC TCCTGA GGA GAC GGT GAC CGT GG-3 '.
The primer of the anti-VISOSE single-chain antibody of said clone chain variable region gene is:
5 ' end primer: 5 '
GGT GGT TCT GGA GGC GGT GGT TCTGAC ATT GAG CTC ACC-3 ';
3 ' end primer: 5 '-CCGCTCGAG CCG TTT GAT TTC CAG CTT GGT GCC-3 '.
Said carrier can be pET-22b (+) plasmid.
In step 2) in, said host bacteria can be E.coli BL21 (DE3).
In step 3), said purifying can adopt affinity chromatography.
The present invention utilizes the hybridoma cell strain D9 of anti-dextran monoclonal antibody; Through the RT-PCR technology; Weight, the light chain variable region sequence of this antibody have been obtained; Further be assembled into anti-VISOSE single-chain antibody gene, and make up recombinant expression vector pET-22 (b+)-scFv, carry out expression, purifying and the evaluation of anti-VISOSE single-chain antibody.Under the overall background of genetic engineering technique, the acquisition of anti-VISOSE single-chain antibody will help the further development and application of following this antibody.
Description of drawings
Fig. 1 is anti-VISOSE single-chain antibody heavy chain variable region gene (V
H) and chain variable region gene (V
L) PCR product electrophorogram.In Fig. 1,1,2 is chain variable region gene (V
L), 3 is dna molecular amount mark (DNA Marker), 4,5 is heavy chain variable region gene (V
H).
Fig. 2 is the PCR product electrophorogram of anti-VISOSE single-chain antibody gene.In Fig. 2,1 is dna molecular amount mark (DNAMarker), and 2 is anti-VISOSE single-chain antibody gene.1500,1000,700,500,300,100 is the size of dna molecular amount mark.
Fig. 3 is the electrophorogram of the anti-VISOSE single-chain antibody behind the purifying.In Fig. 3,1 is molecular weight of albumen mark (Marker), and 116.0,45.0,35.0,25.0,18.4,14.4 is the molecular weight of albumen size, and 2,3 is the anti-VISOSE single-chain antibody behind the purifying.
Fig. 4 is the electrophorogram behind the anti-VISOSE single-chain antibody prokaryotic expression.In Fig. 4; 1 is without IPTG inductive empty carrier mycoprotein; 2 are the mycoprotein after IPTG induces, and 3 is the UW supernatant of inducing bacterium through IPTG, and 4 for inducing the UW deposition of bacterium through IPTG; The 5th, molecular weight of albumen mark (Marker), 97.4,66.0,42.7,31.0,14.4 is the molecular weight of albumen size.
Fig. 5 is that anti-VISOSE single-chain antibody is identified figure.In Fig. 5, X-coordinate is an extent of dilution, and ordinate zou is OD490nm.
Embodiment
1) used cell and the anti-dextran monoclonal antibody of mouse source property
The hybridoma cell strain D9 of anti-dextran monoclonal antibody is developed by Xiamen University and Guangzhou Inst of Cane Sugar jointly; Be preserved in Chinese typical culture collection center (CCTCC) on December 7th, 2010, preservation center deposit number is CCTCCNO:C2010107.
Hybridoma cell strain D9 is conventional to be cultivated in the RPMI1640 of 10% foetal calf serum substratum, in 37 ℃, and 5%CO
2Cultivate.
2) design of primers
The antibody variable gene sequence that increases, its variable region gene sequence is unknown, so the primer that the present invention uses adopts the primer sequence of promoting in the world.
At V
H5 of primer ' end adds restriction enzyme site Nco I:CATGCCATGG (containing protectiveness base CATG), at V
L3 of primer ' end adds restriction enzyme site Xho I:CCGCTCGAG (containing protectiveness base CCG).With connection peptides Linker (Gly
4Ser)
3Design is at V
H3 ' end and V
L5 ' end underlines expression.
The primer of amplification variable region of heavy chain is:
V
H5 ' end primer: 5 '-CAT GCC ATG GAG GTS MAR CTG CAG SAG TCW GG-3 '; (annotate: S=G, C; M=A, C; R=A, G; W=A, T);
V
H3 ' end primer: 5 '
GCC TCC AGA ACC ACC GCC TCCTGA GGA GAC GGT GAC CGT GG-3 '.
The primer of amplification variable region of light chain is:
V
L5 ' end primer: 5 '
GGT GGT TCT GGA GGC GGT GGT TCTGAC ATT GAG CTC ACC-3 ';
V
L3 ' end primer: 5 '-CCGCTCGAG CCG TTT GAT TTC CAG CTT GGT GCC-3 '.
3) V
HAnd V
LThe clone of gene
Get hybridoma cell strain D9 in good condition, adopt Trizol (available from Invitrogen company) reagent, the method that goes up is to specifications extracted total RNA, is template with total RNA, is random primer with Oligo (dT) 20 (available from TOYOBO), is reversed into cDNA.It is following to be with Oligo (dT) 20 that random primer carries out the rt scheme:
RT-PCR reaction system: 5 * AMV damping fluid, 4.0 μ L; DNTP (10mmol/L) 1.0 μ L; Oligo (dT) 20 random primers (20 μ mol/L) 2.0 μ L; RNase suppressor factor 0.5 μ L; Total RNA 5.0 μ L; AMV reversed transcriptive enzyme 1.0 μ L; DEPC water 6.5 μ L; TV 20 μ L.
The RT-PCR reaction conditions: 50 ℃, 60min; 75 ℃, 15min.-20 ℃ of reaction product preservations are subsequent use.
CDNA with the RT-PCR amplification is a template, amplification heavy chain and chain variable region gene.
The pcr amplification system is: 10 * PCR damping fluid, 2.5 μ L; DNTP (10mmol/L) 0.5 μ L; 5 ' end primer (20 μ mol/L), 1.0 μ L; 3 ' end (20 μ mol/L) 1.0 μ L; CDNA 1.0 μ L; Taq polysaccharase 0.5 μ L; Distilled water 18.5 μ L; TV 25.0 μ L.
The PCR reaction conditions is: 94 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 1min, 30 circulations of increasing, last 72 ℃ are extended 10min.Product is through 1.5% agarose gel electrophoresis observation analysis result (referring to Fig. 1), and blended rubber purifying and recovering test kit (OMEGA) reclaims V
HAnd V
LGene product.
The clone of embodiment 2 anti-VISOSE single-chain antibody genes and the structure of expression vector thereof
With the V that reclaims
HAnd V
LThe gene product mixed solution is a template, through V
H5 ' and V
L3 ' primer amplification adds the segmental anti-VISOSE single-chain antibody gene (V of connection peptides Linker
H-Linker-V
L) (referring to Fig. 2), its sequence is:
atggaggtgc aactgcagca gtctggagga ggcttggtgc aacctggagg atccatgaaa 60
ctctcttgtg ctgcctctgg attcactttt agtgacgcct ggatggactg ggtccgccag 120
tctccagaga aggggcttga gtgggttgct gaaattagaa gcaaagctaa taatcatgaa 180
acatactatg ctgagtctgt gaaagggagg ttcaccatct caagagatga ttccaaaagt 240
agtgtctacc tgcaaatgaa cagcttaaga gctgaagaca ctggcattta ttactgtcat 300
tacgacgtgg ctatggacta ctggggccaa gggaccacgg tcaccgtctc ctcaggaggc 360
ggtggttctg gaggcggtgg ttctggaggc ggtggttctg acattgagct cacccagtct 420
ccagccatcc tgtctgtgag tccaggagaa agagtcagtt tctcctgcag ggccagtcag 480
agcattggca caagcataca ctggtatcag caaagaacaa atggttctcc aaggcttctc 540
ataaagtatg cttctgagtc tatctctggg atcccttcca ggtttagtgg cagtggatca 600
gggacagatt ttactcttag catcaacagt gtggagtctg aagatattgc agattattac 660
tgtcaacaaa gtaatagctg gccgtggacg ttcggtggag gcaccaagct ggaaatcaaa 720
cggctcgagc accaccacca ccaccac 747
The 1st～354 heavy chain variable region gene that Nucleotide is anti-VISOSE single-chain antibody wherein, total length is 354bp; The 355th～399 Nucleotide is connection peptides Linker, total length 45bp; The 400th～723 Nucleotide is for to be that the chain variable region gene of anti-VISOSE single-chain antibody, total length are 324bp.
The PCR system of answering is: 10 * PCR damping fluid, 2.5 μ L; DNTP (10mmol/L) 0.5 μ L; V
H5 ' primer (20 μ mol/L), 1.0 μ L; V
L3 ' primer (20 μ mol/L), 1.0 μ L; V
HAnd V
LEach 1 μ L of gene product; Taq polysaccharase 0.5 μ L; Distilled water 18.5 μ L; TV 25.0 μ L.
The PCR reaction conditions: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 40s, 58 ℃ of annealing 45s, 72 ℃ are extended 1min, 30 circulations of increasing, 72 ℃ are extended 10min.
Anti-VISOSE single-chain antibody gene product and pET-22b (+) plasmid (available from Novagen company) are used restriction enzyme Nco I and Xho I (NEB) to carry out enzyme respectively and are cut, and glue reclaims the product after enzyme is cut.Under the catalysis of T4DNA ligase enzyme; To resist the VISOSE single-chain antibody gene to be cloned among the plasmid pET-22b (+); Construction recombination plasmid pET-22b (+)-scFv, Transformed E .coliBL21 (DE3), screening positive clone is also served the order-checking of extra large handsome Bioisystech Co., Ltd.
The prokaryotic expression and the purifying of embodiment 3 anti-VISOSE single-chain antibody genes
Select the correct single bacterium colony of E.coli BL21 (DE3) that contains pET-22b (+)-scFv recombinant plasmid of order-checking, 37 ℃ of shaking culture are spent the night, and are diluted in the LB nutrient solution by 1: 100, and shaking culture is to OD
600Be worth about 0.6～0.8 o'clock; Adding final concentration is the IPTG abduction delivering 4h of 0.5mmol/L; Collect thalline and carry out the SDS-PAGE electrophoresis,, find that the expression product of E.coliBL21 (DE3) has tangible band at the 28kD place through dyeing, decolouring; Consistent with its intended purposes protein band size, show that anti-VISOSE single-chain antibody gene has obtained expression (referring to Fig. 3).The aminoacid sequence of anti-VISOSE single-chain antibody is:
Met Glu Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
20 25 30
Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp
35 40 45
Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Glu Thr Tyr Tyr Ala
50 55 60
Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
65 70 75 80
Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile
85 90 95
Tyr Tyr Cys His Tyr Asp Val Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Leu
130 135 140
Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln
145 150 155 160
Ser Ile Gly Thr Ser Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser
165 170 175
Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro
180 185 190
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile
195 200 205
Asn Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser
210 215 220
Asn Ser Trp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
225 230 235 240
Arg Leu Glu His His His His His His
245
The SDS-PAGE electrophoresis shows that the prokaryotic expression product overwhelming majority of anti-VISOSE single-chain antibody gene appears in the ultrasound precipitation, explains that mainly there be (referring to Fig. 4) in fusion rotein with the form of inclusion body.Inclusion body is crossed Ni after treatment
2+Affinity column washes rabphilin Rab with elutriant, almost only occurs 1 protein band on the glue, and the fusion rotein that contains 6 * His through affinitive layer purification to higher degree is described.Show that through gel imaging (Quantity One-4.6) analysis its purity reaches 90%.Adopt progressively dialysis method that purified proteins is carried out renaturation, the recombinant protein recovery is 45%, through ultraviolet spectrophotometer scanning, according to protein concn=1.45OD
280-0.74OD
260Formula, recording purification of samples concentration is 0.6mg/mL.
The evaluation of embodiment 3 anti-VISOSE single-chain antibodies
Adopt the ELISA method to measure the BA of anti-VISOSE scFv antibody fragment.VISOSE and ovum gallinaceum serum albumin cross-linking agent are diluted to 5ug/mL with carbonate buffer solution, encapsulate in 96 orifice plates, 100 μ L/ holes, 4 ℃ are spent the night.Gelatin sealing 2h with 1% washes 96 orifice plates 5 times with PBST, and every hole adds the protein 10 0 μ L of doubling dilution.Simultaneously, place 1h for 37 ℃ with the negative contrast of pET-22b (+) empty carrier abduction delivering product, wash 5 times with the PBST washings after; Add mouse-anti 6 * His antibody, place 1h, wash 3 times for 37 ℃ with the PBST washings; Add the sheep anti-mouse igg ELIAS secondary antibody again, place 45min, wash 5 times with PBST again for 37 ℃; Add the OPD substrate solution, 37 ℃ of lucifuges are placed 15min, add stop buffer (2M H
2SO4 solution) termination reaction, ELIASA 490nm reads result (referring to Fig. 5).
Recombinant expressed single-chain antibody is OD when dilution in 1: 1
490Being 2.31, is 0.201 when dilution in 1: 128, along with the decline of AC, OD
490Value reduces gradually, show single-chain antibody can with encapsulate onboard VISOSE and carry out specificity and combine.
Claims (9)
1. anti-VISOSE single-chain antibody, the aminoacid sequence that it is characterized in that said anti-VISOSE single-chain antibody is shown in sequence 2.
2. anti-VISOSE single-chain antibody as claimed in claim 1 is characterized in that its gene order shown in sequence 1, the 1st～354 heavy chain variable region gene that Nucleotide is anti-VISOSE single-chain antibody wherein, and length is 354bp; The 355th～399 Nucleotide is connection peptides Linker, and length is 45bp; The 400th～723 Nucleotide is the chain variable region gene of anti-VISOSE single-chain antibody, and length is 324bp.
3. the preparation method of anti-VISOSE single-chain antibody as claimed in claim 1 is characterized in that may further comprise the steps:
1) the anti-VISOSE single-chain antibody gene of clone, and, make up recombinant vectors with its importing carrier; The template of the anti-VISOSE single-chain antibody gene of said clone is from the hybridoma cell strain D9 of anti-dextran monoclonal antibody; Said hybridoma cell strain D9 is preserved in Chinese typical culture collection center on December 7th, 2010, and preservation center deposit number is CCTCC NO:C2010107;
2) recombinant vectors is imported host bacteria, the recombinant bacterial strain of the anti-VISOSE single-chain antibody of construction expression;
3) with the recombinant bacterial strain fermentation culture, the separation and purification fermented liquid promptly gets and resists the VISOSE single-chain antibody.
4. the preparation method of anti-VISOSE single-chain antibody as claimed in claim 3; It is characterized in that in step 1); The cloning process of the anti-VISOSE single-chain antibody gene of said clone is to clone anti-VISOSE single-chain antibody heavy chain variable region gene and anti-VISOSE single-chain antibody chain variable region gene respectively, through connection peptides Linker both is connected again.
5. the preparation method of anti-VISOSE single-chain antibody as claimed in claim 4 is characterized in that the primer of the anti-VISOSE single-chain antibody of said clone heavy chain variable region gene is:
5' holds primer: 5'-CAT GCC ATG GAG GTS MAR CTG CAG SAG TCW GG-3';
3' holds primer: 5'-
GCC TCC AGA ACC ACC GCC TCCTGA GGA GAC GGT GAC CGT GG-3';
Wherein, S=G, C; M=A, C; R=A, G; W=A, T.
6. the preparation method of anti-VISOSE single-chain antibody as claimed in claim 4 is characterized in that the primer of the anti-VISOSE single-chain antibody of said clone chain variable region gene is:
5' holds primer: 5'-
GGT GGT TCT GGA GGC GGT GGT TCTGAC ATT GAG CTC ACC-3';
3' holds primer: 5'-CCGCTCGAG CCG TTT GAT TTC CAG CTT GGT GCC-3'.
7. the preparation method of anti-VISOSE single-chain antibody as claimed in claim 3 is characterized in that in step 1), and said carrier is pET-22b (+) plasmid.
8. the preparation method of anti-VISOSE single-chain antibody as claimed in claim 3 is characterized in that in step 2) in, said host bacteria is E.coli BL21 (DE3).
9. the preparation method of anti-VISOSE single-chain antibody as claimed in claim 3 is characterized in that in step 3), and said purifying adopts affinity chromatography.
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