CN104017080A - Canine single-chain antibody, and construction method and application thereof - Google Patents
Canine single-chain antibody, and construction method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly discloses a canine single-chain antibody, and a construction method and application thereof. By designing the canine antibody variable region degenerate primers, the canine heavy chain variable region VH and light chain variable region VL genes are successfully amplified. On such basis, a bacteriophage display technique is utilized to successfully construct the canine single-chain antibody scFv library. The scFv single-chain antibody library has diversity and enough storage capacity. A Dot-ELISA method is utilized to screen 3 single chain antibodies capable of being combined with the canine DEA 1.1 blood group antigen from the scFv single-chain antibody library by using the canine DEA 1.1 blood group antigen, wherein Clone 16 has stronger combination characteristic. A pET-32a-Clone16 recombinant protein is constructed according to the Clone 16 gene to perform prokaryotic expression, and the purified antibody has combination activity. The canine single-chain antibody lays solid foundation for preparing canine DEA 1.1 blood grouping reagents.
Description
Technical field
The present invention relates to biological technical field, more specifically, relate to a kind of dog source single-chain antibody and construction process and application.
Background technology
Traditional antibody preparation, comprises hybridoma-monoclonal antibody technique, all needs first with antigen, body to be carried out to immunity, and the performance of the antibody that obtains depends on the validity of immunity in body.Therefore poor antigen, autoantigen, the antibody of the virose antigen of tool and the preparation of human antibody are all more difficult.The phage antibody library technique growing up the nineties is the major progress in antibody engineering field, and it has become the important channel that Human monoclonal antibody is developed.Phage antibody library technique has been simulated internal antibody generative process, for possibility is provided without passing immunization, gradually becomes one of important means of preparing human antibody.
Blood group is the one heredity shape showing with blood antigen form.Blood group system is the system of according to antigen type difference on erythrocyte membrane, blood being carried out somatotype.The dog blood group system having been found that at present exceedes 12, and each blood group system adopts DEA+ numerical approach to name.The blood group of confirming through the world at present has 8 kinds, is respectively DEA1.1, DEA1.2, DEA3, DEA4, DEA5, DEA6, DEA7 and DEA8.Because the information of dog blood group antigen is all unknown, can't obtain relatively large dog blood group antigen by the method for genetic expression, biosynthesizing and antigen purification now.Prepare dog Blood group McAb by the method that routine is prepared monoclonal antibody by the method for hybridoma very difficult, can be because the antibody of more other red cell antigenses of the impure generation of antigen propose larger challenge to preparation and the screening of specific antibody.But the erythrocytic method of mutual immune dog just can be avoided the impact of a large amount of non-blood group antigen between dog.What the polyclonal antiserum that produces was more is aimed at dog blood group.Meanwhile, display technique of bacteriophage can extract antibody gene information by the peripheral blood lymphocyte of animal after immunity.
Summary of the invention
Technical problem to be solved by this invention is the deficiency overcoming in prior art for the antibody investigative technique of dog blood group antigen, and a kind of dog source single-chain antibody is provided.
Second object of the present invention is to provide a kind of for building the primer sets of dog source single-chain antibody library.
The 3rd object of the present invention is to provide a kind of dog source single-chain antibody library.
The 4th object of the present invention is to provide the construction process of described dog source single-chain antibody library.
The 5th object of the present invention is to provide the application in preparation dog bracket for blood grouping reagent of described primer sets or dog source single-chain antibody or single-chain antibody library.
The object of the invention is to be achieved by the following technical programs:
A kind of dog source single-chain antibody Clone 16, is characterized in that, the long 870bp of scFv sequence of described antibody, and sequence is as shown in SEQ ID NO:48; The wherein long 435bp of variable region of heavy chain VH, sequence is as shown in SEQ ID NO:49; The long 390bp of variable region of light chain VK, sequence is as shown in SEQ ID NO:50; The long 45bp of connection peptides sequence, sequence is as shown in SEQ ID NO:51.
The aminoacid sequence of described dog source single-chain antibody Clone 16 is as shown in SEQ ID NO:52.
A kind of primer for described single-chain antibody Clone 16 gene orders that increase is provided, and sequence is as shown in SEQ ID NO:53～54.
Be provided for building the primer sets of dog source single-chain antibody library, comprise antibody heavy chain variable region primer, variable region of light chain primer and connection peptides aligning primer, its primer sequence is as shown in SEQ ID NO:1～47.
A kind of dog source single-chain antibody library is provided, and described antibody library contains dog blood group antibody variable region of heavy chain VH and variable region of light chain VL, and middle joining region linker sequence (Gly4Ser)
3, there is complete antigen-binding site, taking pCANTAB-5E as carrier, storage capacity reaches 7 × 10
5.
The construction process that described dog source single-chain antibody library is provided, comprises the following steps:
S1. total RNA extraction and cDNA's is synthetic: getting the canine peripheral blood after immunity, separate lymphocyte, extract total RNA, is cDNA through reverse transcription PCR reverse transcription;
S2. pcr amplification heavy chain of antibody and chain variable region gene: according to dog antibody gene sequence, design respectively the primer of pcr amplification heavy chain of antibody, variable region of light chain, the sequence of described primer is as shown in SEQ ID NO:1～33; The cDNA obtaining taking S1 is as template, and variable region gene VH and VL increase;
S3. the amplification of single-chain antibody scFv gene: design containing connection peptides sequence linker and
sfiI, NotIthe primer of restriction enzyme site, obtains scFv gene through amplification on the basis of the heavy chain VH being obtained by S2 and light chain VL variable region gene again;
S4. the structure in single-chain antibody scFv storehouse: double digestion scFv gene fragment, and insert pACNTAB-5E construction recombination plasmid, transformed competence colibacillus
e.colitG1, obtains preliminary single-chain antibody library, through repeatedly washing in a pan sieve, obtains dog source single-chain antibody library.
The present invention utilizes the pCANATB-5E carrier of Amerhsma company that scFv antibody is showed on g3 albumen.So most of expressing protein can be readed over, express scFv-g3 fusion rotein, and be showed on phage capsid by the assembling of phage.
The cell derived that builds phage antibody library generally has three aspects: hybridoma, immune body sensitization bone-marrow-derived lymphocyte, non-sensitization bone-marrow-derived lymphocyte.Wherein non-sensitization bone-marrow-derived lymphocyte is the good source of object antibody gene through the patient B lymphocyte of natural immunity.Because the human body endolymph cell of natural immunity is through antigen pressure selection and affinity maturation, the mRNA abundance of its object antibody gene is relatively high, is conducive to the screening in built library, and is beneficial to the antibody (Ichiyoshi that clones high-affinity
et al., 1995; Kasaian
et al., 1994).Therefore, it is immunogenic that dog DEA1.1 blood group antigen red corpuscle is selected in this research, the dog of immune DEA1.1 feminine gender.Can maximum possible reduce other the non-blood group antigen materials participation immunostimulation dog immunity systems on dog red corpuscle like this.And wish that the anti-dog DEA1.1 blood group antibody storehouse building thus can reflect the time of day of blood group antibody in this dog body as far as possible.
Preferably, described in S1, the immunization method of the dog after immunity is: selecting dog DEA1.1 blood group antigen red corpuscle is immunogenic, the dog of immune DEA1.1 feminine gender.In the present invention, select to build antibody gene storehouse through the canine peripheral blood lymphocyte of dog DEA1.1 blood group antigen immunity, be conducive to increase the specific aim of antibody library.When canine peripheral blood lymphocyte mRNA reverse transcription is become to cDNA, what the present invention selected is that Oligo dT18 carries out reverse transcription, and the total length that needs reverse transcription to go out mRNA due to this primer is just effective, and therefore the extraction of the total mRNA of human peripheral blood is had relatively high expectations.
Although design amplification mouse, people, rabbit, chicken and the antibody variable region VH of other species and the degenerated primer of VL gene are all reported (Chiang
et al., 1989; Coloma
et al., 1991; Orlandi
et al., 1989; Wang
et al., 2000), but also do not report about dog antibody variable gene degenerated primer so far.Antibody variable region (V district) is made up of separately 4 skeleton districts (FR) and 3 GeCDR districts, is (N end) " FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4 " (C end) and arranges.And comparatively speaking, FR encoding sequence in skeleton district is relatively conservative.Therefore, design the primer for the relative conserved dna base sequence in antibody variable gene upstream and downstream respectively, just likely obtain a complete set of antibody variable region sequence by PCR.The present invention designs and has verified that VH and VL gene two overlap different degenerated primers, and every primer mates with the end sequence of dog antibody heavy and light chain variable region respectively.In order to increase the diversity of antibody gene, the present invention carries out random combine between two the primer of all designs, and antibody gene fragment increases respectively.Avoid due to situation about mutually suppressing between primer pair or gene template copy is lower.Can amplify respectively the VH of about 430bp and the VL gene of 390bp by pcr amplification.
This research adopts three traditional fragment SOE-PCR method assembling VH, VL and linker.VH and VL are through containing coding (Gly
4ser)
3the Linker of sequence is assembled into scFv Linker total length 45bp, and mate with VH gene 3' end and VL gene 5' end sequence respectively at two ends, middle 45bp coding (Gly
4ser)
3connect tripe.The equivalent of VH and VL gene fragment adds, and is effectively to assemble and the key of pcr amplification.When pcr amplification assembling scFv, introduce respectively at 5' and the 3' end of sFcv
sfiI,
notIrestriction enzyme site, inserts scFv gene in pCANATB-5E carrier by these two restriction enzyme sites.
sfiI,
notIthe frequency that restriction enzyme site occurs in antibody gene is very low, in the time building antibody library, can ensure that most of scFv gene can correctly be cloned.
Preferably, described in S3 connection peptides sequence linker and
sfiI, NotIthe primer sequence of restriction enzyme site is as shown in SEQ ID NO:34～47.
Phage antibody library screening method has a lot, comprises that pure antigen selection, non-pure antigen selection, functionality screening, selectivity infect screening etc.Non-pure antigen selection comprises cell surface screening, tissue screening, the interior screening of living animal etc.For purifying or the uncertain antigen of antigenic property, there are a lot of limitation in traditional immobilization and liquid phase screening method.This research adopts the Dot-ELISA method oneself building, and dog DEA1.1 blood group antigen are fixed on nitrocellulose filter, by the method for immobilization antigen, carries out the screening of antibody.As everyone knows, the former character of dog red corpuscle cell surface is uncertain.Blood group relative specific antigen is not only contained on its surface, also contains a large amount of heterogenetic antigens, as albumen, carbohydrate and lipid etc. simultaneously.Because phage antibody is easily combined with these heterogenetic antigens, while utilizing single dog DEA1.1 positive cell to screen, likely screen a large amount of non-specific antibodies.The phage that simultaneously makes a few surface express antibody with high specificity is lost, and screening efficiency is lower.In this research, utilize the dog red corpuscle antagonist storehouse of dog DEA1.1 feminine gender to carry out negativity screening and dog DEA1.1 blood group positive antigen carries out positive-selecting, screening efficiency is improved greatly.
Preferably, described in S4, washing in a pan the method for sieving is: with the dog blood group antigen positive and negative erythrocyte membrane, be cured on nitrocellulose filter, single-chain antibody scFv storehouse carried out to the screening of " adhesion-wash-out-amplification "; Every wheel after screening carried out titer determination, the enrichment result of detection specificity phage to the phage of amplification.
Phage antibody improves constantly through input/yield ratio of continuous " absorption-wash-out-amplification " process phage, so just can obtain to be combined phage clone more closely with dog DEA1.1 blood group antigen.In pCANATB-5E carrier, scFv gene inserts between g3 signal peptide and g3 albumen, and 3' end adds E-Tag label, and is connected with g3 gene by l succsinic acid terminator codon.TG1 bacterial strain is expressed C-terminal with E-Tag label, makes primary antibodie by anti-E-Tag antibody, the combination activity of checking phage antibody.Take turns 100 clones of random choose in the bacterial colony obtaining screening from the 3rd and carry out Dot-ELISA activity identification, determine the phage that filters out anti-dog DEA1.1 blood group.Merge the antibody gene repeating through gene sequencing, testing sieve is selected the positive colony of the anti-dog DEA1.1 of 3 strain blood group antigen, and through ELISA method validation, Clone16 can be good at the blood group antigen in conjunction with dog DEA1.1, and has very high avidity.The present invention selects carrier pET-32a to build recombinant protein plasmid pET-32a-Clone16, has a His-Tag label in cloning site downstream, is conducive to high expression level and the antibody purification of Clone16.Empirical tests and analysis, the recombinant protein of structure can well solubility expression.The Clone16 single chain antibody protein of purifying carries out clinical sample checking by Dot-ELISA method, has good combination activity.
The application of described primer sets in preparation dog bracket for blood grouping reagent is provided.
The application of described single-chain antibody in preparation dog bracket for blood grouping reagent is provided.
The application of described single-chain antibody library in preparation dog bracket for blood grouping reagent is provided.
Compared with prior art, beneficial effect of the present invention:
The present invention is based on dog DEA1.1 blood group antigen can not get under the prerequisite of purifying antigen, dog with the positive dog red corpuscle of DEA1.1 as immunogen immune DEA1.1 feminine gender, obtain good immune effect through immunity repeatedly, and obtain a large amount of anti-dog DEA1.1 blood group polyclonal antiserums.
The present invention successfully builds the degenerated primer of dog, has successfully set up phage displaying antibody storehouse, dog source by described degenerated primer, finds through qualification, and designed primer can amplify the diversified single-chain antibody scFv assortment of genes.Calculate by library storage capacity, the dog DEA1.1 blood group antigen immunity Kuku capacity that this institute sets up is greater than 7 × 10
5, can think enough large immune storehouse.Show through the random choose sequence obtaining that checks order, constructed dog single-chain antibody library has diversity.
The present invention, taking Dot-ELISA method as basis, is fixed to dog DEA1.1 blood group antigen on nitrocellulose filter, and carries out 3 with phage antibody immunity storehouse and take turns sieve, and finishing screen is chosen the single-chain antibody that 3 strains can combine with dog DEA1.1 blood group antigen.Clinical sample is verified to such an extent that Clone16 has stronger binding characteristic.According to Clone16 gene constructed pET-32a-Clone16 recombinant protein carry out prokaryotic expression, the antibody that purifying obtains has in conjunction with active.For preparation dog DEA1.1 bracket for blood grouping reagent provides solid basis.
Brief description of the drawings
Fig. 1 is dog source VH, V
κand V
λthe PCR gel electrophoresis figure that increases;
Fig. 2 is variable region of heavy chain primer pair amplification; 1～7 upstream primer is JH1-2, and 8～14 upstream primers are JH3;
Fig. 3 is variable region of light chain kappa primer pair amplification checking; 1～9 is respectively kappa For 1～9 combination of primers amplification;
Fig. 4 is variable region of light chain lambda primer pair amplification checking; (A): 1～8 is JL1 and VL1～8; 9～17 is JL2 and VL1～8; (B) 1～8 is JL3 and VL1～8; 9～17 is JL4 and VL1～8; (C): 1～8 is JL5 and VL1～8; 9～13 is JL1～5 and VL9;
Fig. 5 is the fusion pcr amplification gel electrophoresis figure of dog source scFv;
Fig. 6 is the qualification of scFv phage library plasmid enzyme restriction;
Fig. 7 is the elutriation Dot-ELISA qualification in phage scFv storehouse;
Fig. 8 is anti-dog DEA1.1 blood group antigen dog source scFv aminopeptidase gene acid sequence;
Fig. 9 is Clone16 gene clone and qualification; M:Marker DL2000; 1: positive Clone16 bacterium liquid PCR qualification; 2-3:pET-32a-Clone16 positive bacteria PCR qualification; 4: negative control;
Figure 10 is the qualification of recombinant plasmid pET-32a-Clone16 double digestion;
Figure 11 is the SDS-PAGE result of different time IPTG induction expressing fusion protein; M: protein molecule quality standard; 1: before carrier pET-32a induction; 2: 6h after carrier pET-32a induction; Before 3 recombinant plasmid pET-32a-Clone16 inductions; 4: 1h after recombinant plasmid pET-32a-Clone16 induction; 5: 2h after recombinant plasmid pET-32a-Clone16 induction; 6: 3h after recombinant plasmid pET-32a-Clone16 induction; 7: 4h after recombinant plasmid pET-32a-Clone16 induction; 8: 5h after recombinant plasmid pET-32a-Clone16 induction; 9: 6h after recombinant plasmid pET-32a-Clone16 induction;
Figure 12 is recombinant protein soluble analysis; M: molecular weight of albumen standard; 4h cracking precipitation after 1:pET-32a-Clone16 induction; 4h cracking supernatant after 2:pET-32a-Clone16 induction;
Figure 13 is Clone16 single-chain antibody Western-Blot qualification result; M: albumen Marker; 1: cracking precipitation; 2: cracking supernatant; 2: negative control;
Figure 14 is single-chain antibody Clone16 clinical sample Dot-ELISA activation analysis;
Figure 15 is the corresponding collection of illustrative plates of single-chain antibody Clone16 gene order and aminoacid sequence;
Figure 16 is the corresponding collection of illustrative plates of single-chain antibody Clone1 gene order and aminoacid sequence;
Figure 17 is the corresponding collection of illustrative plates of single-chain antibody Clone5 gene order and aminoacid sequence.
Embodiment
Below in conjunction with Figure of description and specific embodiment, further set forth the present invention.These embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in lower routine embodiment, conventionally according to normal condition or according to the condition of manufacturer's suggestion.Unless otherwise defined, in embodiment, molecule clone technology used is openly sufficient routine techniques of this area, the same meaning that all specialties that use in literary composition and scientific words and those skilled in the art are familiar.
the amplification of embodiment 1 scFv gene
(1) the lymphocytic separation of canine peripheral blood
With the negative dog of the positive antigen erythrocyte immune of dog DEA1.1 DEA1.1, through abdominal cavity and vein immunity 5% red corpuscle 5ml, after 5 immunity, gather canine peripheral blood, get fresh anticoagulated whole blood, EDTA(Sodium Citrate or heparin at every turn) antithrombotics all can.With equal-volume PBS or 0.9% NaCl(physiological saline) dilution whole blood.In centrifuge tube, add the parting liquid of certain volume, by tile parting liquid liquid level top of the blood sample after dilution, keep two liquid level interfaces clear.Parting liquid, anti-freezing not diluted whole blood, PBS(or physiological saline) volume ratio is 1:1:1.Room temperature, horizontal rotor 700～800g(2000～2500rpm) centrifugal 20～30min.After centrifugal end, at the bottom of centrifuge tube pipe, be red corpuscle, middle layer is parting liquid, and the superiors are blood plasma/tissue homogenate layers, and plasma layer is the thin and finer and close tunica albuginea of one deck with separating between liquid layer, is mononuclearcell (comprising lymphocyte and monocyte) layer.The careful tunica albuginea layer of drawing is in another centrifuge tube.Be diluted to certain volume with PBS, put upside down and mix.Room temperature, horizontal rotor 250g(1000rpm), centrifugal 10min, abandons supernatant.Repeated washing 1～2 time.
(2) the total RNA of dog lymphocyte extracts
The dog lymphocyte that separation is obtained carries out cell counting, and packing is about 10
7every pipe is put into 1.5mL centrifuge tube, and Xiang Guanzhong adds 750 μ L Trizol Ls Reagent By Direct Pyrolysis cells, acutely puts upside down vibration, and room temperature leaves standstill 5min; Add 200 μ L chloroforms, under room temperature, mix the rear 1～2min of leaving standstill, then at 2～8 DEG C, the centrifugal 15min of 10000r/min, then taking out centrifuge tube carefully draws supernatant liquor 500 μ L and adds another new DEPC(coke diethyl phthalate) in the 1.5mL centrifuge tube processed, and add 500 μ L Virahols (supernatant liquor mixes with Virahol equal proportion) and mix to this pipe; Then at 2～8 DEG C, the centrifugal 10min of 12000g, takes out centrifuge tube and carefully topples over and fall supernatant liquor, then add 1000 μ L 75% ethanol, the then centrifugal 5min of 8000g at 2～8 DEG C, takes out centrifuge tube and carefully outwells supernatant liquor, is inverted centrifuge tube and on filter paper, makes it at room temperature fully dry; Finally add 11.5 μ L DEPC process water, obtain the total RNA of dog lymphocyte, directly carry out reverse transcription or-20 DEG C frozen for subsequent use.
(3) first chain cDNA are synthetic
Following reagent is added in an aseptic Eppendorf pipe: 1 μ L Oligo (dT) 18(0.5 μ g/ μ L), the total RNA of 10 μ L, 1 μ LDEPC water, cumulative volume is 12 μ L.After 70 DEG C, 5min reaction, be placed in immediately on ice.Add following reagent: 4 μ L 5 × buffer (the first chain), 1 μ L RNase inhibitor, 1 μ L 10mM dNTP.Softly mix, hatch 5min for 37 DEG C.Add 1 μ L reversed transcriptive enzyme, softly mix up and down with pipettor, avoid producing bubble.Hatch 60min for 42 DEG C, 70 DEG C, 10min termination reaction, place on ice.Product can synthesize or be stored in-20 DEG C for the second chain.
The total RNA of canine peripheral blood lymphocyte extracting is visible through agarose gel electrophoresis, and two bands of 18s RNA and 25s RNA are high-visible, illustrate that total RNA integrity of gained is good.Become after the first chain cDNA through random primer Oligo (dT) 18 reverse transcriptions, verify with variable region of heavy chain primer whether gene can be used.As shown in Figure 1, increase according to heavy chain primer, can amplify very much 430bp fragment, variable region of light chain primer can amplify 390bp fragment, illustrates that the RNA extracting can carry out next step experiment.
(4) design of primers
The antibody gene sequence of having delivered according to dog, by sequence classification, respectively at conservative region design amplification antibody heavy chain variable region gene primer and chain variable region gene primer.The primer (table 1,2) that contains restriction enzyme site and linker sequence according to the design of weight strand primer.Primer is listed as follows:
Table 1 dog source heavy chain and antibody light chain variable region primer
The gene constructed primer of table 2 dog source scFv
Owing to there is no the primer sets of ripe dog source phage antibody library, therefore the present invention according to the antibody gene sequential analysis of the dog of having delivered, has designed many groups primer pair, variable region of heavy chain primer pair, primer sequence comparison result, divides into 2 districts variable region of heavy chain FR1.FR4 divides into 7 districts, designs respectively primer, combined crosswise checking primer sets, and whether amplification checking primer pair can be used.Empirical tests result as shown in Figure 2.Heavy chain primer upstream and downstream combination of two can amplify respectively the specific band of 430bp.Primer sequence comparison result, divides into 9 districts kappa variable region FR1, and FR4 divides into 1 district, designs respectively primer, combined crosswise checking primer sets.Empirical tests result as shown in Figure 3.Light chain kappa primer upstream and downstream combination of two can amplify respectively the specific band of 390bp.Primer sequence comparison result, divides into 5 districts lambda variable region FR1.FR4 divides into 9 districts, designs respectively primer, combined crosswise checking primer sets.Empirical tests result as shown in Figure 4.Light chain lambda primer upstream and downstream combination of two can amplify respectively the specific band of 390bp.
(5) antibody variable gene amplification
VH and VL gene amplification system are as follows:
VH and VL Gene response program:
(6) amplification of scFv gene
Reclaim test kit according to sky root glue and reclaim VH and VL amplification gene product fragment.Using the fragment of purifying as template, with the primer that is added with linker, the corresponding light heavy chain fragment of amplification.Reaction system and program are as follows:
Reaction system:
Response procedures:
The VLL(light chain having increased is added to the product of linker and restriction enzyme site amplification) and VHH(heavy chain interpolation linker and restriction enzyme site amplified production), building scFv gene through following response procedures, reaction system is as follows:
Response procedures is:
After having reacted, in every pipe, be added with the upstream and downstream primer of restriction enzyme site, each 0.5 μ L;
With variable region gene fragment VH, the V of purifying
λand V
κgene fragment is template, carries out secondary PCR amplification, at 5 ' end and the variable region of light chain V of variable region of heavy chain VH
λ, V
κ3 ' end respectively insert
sfiIwith
notIrestriction enzyme site.And hold and variable region of light chain V at 3 ' of variable region of heavy chain VH
λ, V
κ5 ' end inserted respectively linker fragment, make complementary, middle 45bp coding (Gly in the middle of heavy chain and light chain
4ser)
3connection peptides, the connection peptides of this section of soft easy song, can ensure not affect the activity of single-chain antibody.After merging PCR, as shown in Figure 5, gene fragment is about 850bp left and right to amplification.Conform to calculated value.
the structure of embodiment 2 phage antibody libraries and naughty sieve
(1) scFv is connected with pCANTAB-5E
Carry out according to following operation:
16 DEG C connect 12～16 hours, 70 DEG C, 10min fire extinguishing T4 ligase enzyme.
(2) preparation of electric transformed competence colibacillus bacterium TG1
Connection carrier is resuspended in 1mL LB nutrient solution.37 DEG C of incubated overnight, 250rpm.Be inoculated into mmp(minimal medium plate) substratum, 37 DEG C spend the night (12h) cultivate.Choose single bacterium colony in 5mL LB substratum, 37 DEG C, 250rpm, 10h cultivates.Draw 900 μ L stable growth phases
ecoli. TG1 cell, add 250 μ L sterilizing 80% glycerine, mix preservation-70 DEG C.Specifically comprise the following steps:
A. the single bacterium colony of picking from micro-substratum plate, is inoculated in 10mL 2 × YT substratum, and 37 DEG C of shaking culture are spent the night;
B. get the above-mentioned bacterium 1:100 dilution of spending the night and inoculate in 2 × YT nutrient solution that 1000mL is fresh, 37 DEG C of shaking culture, to OD600=0.5～0.7, are cultivated approximately 2.5～3h;
C. the above-mentioned triangular flask with bacterium liquid is put to ice bath 15～30min;
D. 4 DEG C of centrifugal 20min of 4000g.Remove supernatant, with the resuspended cell precipitation of aseptic 1mmol/L Hepes (pH value 7.0) solution of 1000mL precooling;
E. 4 DEG C of centrifugal 20min of 4000g.Remove supernatant, with the resuspended cell precipitation of aseptic 1mmol/L Hepes (pH value 7.0) solution of 500mL precooling;
F. 4 DEG C of centrifugal 20min of 4000g.1mmol/L Hepes (pH70) the solution re-suspended cell that contains 10% glycerine with 20mL;
G. 4 DEG C of 4000g are centrifugal, finally by 1mL 10% glycerine re-suspended cell precipitation, and the about 2mL of total amount left and right;
H. packing tubule, every pipe 100 μ L, are placed in ice bath stand-by, or freeze immediately in dry ice, deposit in-70 DEG C.
(3) structure in scFv library
ScFv gene after 10 μ L purifying is connected to product and add 40 μ L's
ecoli. in TG1 competence, transform with 25uF, 2.5kV, 200 ohms condition electric shocks, add immediately the 1mL LB of pre-temperature to mix, put the gentle shake of 37 DEG C of shaking tables 1h, bacterium liquid coating SOBAG culture plate, and by bacterium liquid dilution metering storage capacity, 30 DEG C of overnight incubation of all culture plates.Second day with 2 × YT substratum wash-out bacterium colony from flat board, add glycerine to concentration be 20%, 70 DEG C frozen.
(4) detect library storage capacity
The scFv library that the conversion of process electricity is set up, gets respectively 10 μ L and transforms rear bacterium liquid, according to 10 times of doubling dilutions, gets a certain amount of coating SOBAG flat board, does other two groups simultaneously and transforms rear bacterium liquid in contrast.Cultivate 20～24h for 30 DEG C.Calculate and estimate library capacity according to the flat-plate bacterial colony quantity of different extent of dilution coatings: storage capacity (cfu)=colony number × extension rate, preserve dull and stereotyped for 4 DEG C.
Electricity turns 10 times of doubling dilutions of after product, detects transformation efficiency, and result obtains transformation efficiency 10
6.From flat board, 20 bacterium colonies of picking carry out PCR detection at random.There are 17 positives in result, finally calculates the actual storage capacity in constructed scFv library 7 × 10
5.PCR detects positive bacterium colony, after amplification extracting plasmid
noti and
sfithe qualification of I double digestion.Result shows as Fig. 6, and after electricity transforms, positive bacteria extracts plasmid double digestion qualification, and result obtains specific band in the position of 850bp.
Appropriate 2 × YT substratum being added in the flat board of experimental group coating, with spreading rod, bacterium liquid is scraped in substratum, collect substratum and mix, is now the antibody library of preserving with bacterium liquid form.Get part bacterium liquid and carry out phage antibody and save experiment, it is that 30% glycerine is in-70 DEG C of preservations that all the other bacterium liquid add final concentration.
(5) bring back to life phage antibody library
A. get the undiluted electricity of 900 μ L and transform scFv library cell, add 2 × YT-AG substratum of 9.1mL;
B. jolt and cultivate 1h at 30 DEG C, 250rpm,, to OD600=0.5, make scFv library cell in logarithmic phase, can not hypertrophy;
C. add 50 μ L 20mg/mL Amp
+with 4 × 10
10the auxiliary M13K07 of pfu, 37 DEG C of standing 10min, shaking table 250rpm cultivates 1h;
D. the centrifugal 10min of 1500g then, abandons supernatant and stays cell.Add 10mL(200mL) the resuspended thalline of 2 × YT-AK substratum.In shaking table, 200rpm jolts 37 DEG C of incubated overnight;
E. the centrifugal 20min of 1500g, gets supernatant in new centrifuge tube, 4 DEG C of preservations.And supernatant being taken out to the TG1 that infects logarithmic phase, coating SOBAG culture plate, measures the phage titre of preparing.
(6) the enrichment elutriation in single stranded phage storehouse
Coated dog DEA1.1 positive and negative antigen red corpuscle on nitrocellulose filter.Then seal and wash.Specifically comprise the following steps:
A. prepare the phage antibody of purifying, negative ready dog DEA1.1 antigen nitrocellulose filter is immersed in phage antibody solution completely.Cultivate 2h, jolt gently during this time 3～5 times for 37 DEG C;
B. take out the negative antigen nitrocellulose filter of dog DEA1.1, discard.Positive ready dog DEA1.1 antigen nitrocellulose filter is immersed in phage antibody solution completely.Cultivate 2h, jolt gently during this time 3～5 times for 37 DEG C;
C. wash nitrocellulose filter 20 times with PBS, rinse at every turn;
D. use the elutriant wash-out nitrocellulose filter of glycine-hydrochloric acid (pH2.2), incubated at room 10min, the continual nitrocellulose filter of repeatedly blowing and beating, adds Tris-HCl (pH7.0) neutralizer, collects whole liquid;
E. getting 100 μ L phages adds in the logarithmic phase TG1 bacterium of the fresh preparation of 1mL, hatch after 1h for 37 DEG C, get 100 μ L nutrient solutions, with 2 × YT substratum 10 times of dilution bacterium liquid (1:10,1:100,1:1000,1:10000), coating SOBAG flat board, 30 DEG C of overnight incubation, calculate colony-forming unit, be coated with culture plate as negative control with the empty bacterium of logarithmic phase TG1 simultaneously;
F. use 2 × YT substratum by the bacterium colony of SOBAG flat board collect, be resuspended in containing in 2 × YT-AG substratum of 15% glycerine, be the affine phage antibody filtering out of the first round, frozen in-70 DEG C;
G. the affine phage antibody bacterium liquid filtering out of the first round is saved.And do second and take turns screening, second takes turns when screening increases the negative antigen nitrocellulose filter of dog DEA1.1 of 2 times and increases by 2 times of action times.
With dog DEA1.1 antigen positive and negative erythrocyte membrane, be cured on nitrocellulose filter, phage single-chain antibody variable region antibody library is carried out to 3 screenings of taking turns " adhesion-wash-out-amplification ".Every wheel after screening carried out titer determination, the enrichment result (table 3) of detection specificity phage to the phage of amplification.Show that, along with the increase of eluriating number of times, the phage number eluting also increases thereupon, before the 3rd output capacity ratio of taking turns after affine screening, increased approximately 1.125 × 10
3doubly.The phage that the positive antigen erythrocyte binding of energy and DEA1.1 is described has obtained highly enriched.
The enrichment result of the special phage of table 3
the detection of embodiment 3 positive bacteriophage single-chain antibodies
(1) positive bacteriophage antibody gene amplification order-checking
A. add in 400 μ L 2 × YT-AG substratum to 96 pipe, from third round eluriate SOBAG flat board on 96 mono-clonals of random picking in 96 pipes, 200rpm, 30 DEG C of incubated overnight;
B. add 400 μ L 2 × YT-AG substratum in new pipe, get the cell of 40 μ L incubated overnight in new pipe, residue bacterium liquid glycerine saves backup;
C. get new pipe 200rpm, cultivate 2h for 30 DEG C.The centrifugal 1500g of room temperature, 20min, abandons supernatant;
D. centrifugal preparation 50mL 2 × YT-AI substratum, shifts 400 μ L in every pipe;
E. cultivate 3～4h, 200rpm for 30 DEG C.Centrifugal 1500 g of room temperature, 20min;
F. carefully shift 320 μ L supernatants in new pipe.Add the confining liquid of 80 μ L to seal 320 μ L supernatants, incubated at room 10min, detects recombinant phages antibody (HRP/Anti-ETag conjugate) with Dot-ELISA.
From the 3rd phage-infect of taking turns screening
e.coliin TG1, the random single bacterium colony 96 of picking is cloned, phage antibody supernatant is prepared in superingection, detect the combination activity of the anti-DEA1.1 antibody containing in supernatant by Dot-ELISA method, use respectively the positive polyclonal antibody of DEA1.1, positive bacteriophage antigen, the negative antigen of DEA1.1, PBS contrasts as yin and yang attribute.Wherein the detected result color of 12 strain supernatant Dot-ELISA is darker, is judged as positive in (Fig. 7).Show that these phage antibodies have the activity of specific binding DEA1.1 blood group antigen.
Dot-ELISA is detected to positive phage antibody sample, add in 2mL 2 × YT-AG substratum 10mL centrifuge tube 200rpm, 30 DEG C of incubated overnight.Extract plasmid, and sequencing analysis gene order.
The positive colony of gained, through sequence verification, has a few strain clone sequences identical.Wherein 1,13,27,33 is identical with 51 cloned sequences, 5,28,34 is identical with 36 cloned sequences, and 16 is identical with 55 cloned sequences, is divided into 3 kinds of different gene types, their CDR sequence there are differences, by this 3 strain clone called after Clone1, Clone5 and Clone16(Fig. 8 respectively).Clone1 and Clone5 are with VH and V
karray mode, Clone16 is with VH and V
λarray mode.Clone1 order-checking obtains the scFv of 873bp, 291 amino acid of encoding, and variable region of heavy chain VH is long is 429bp, 143 amino acid of encoding, variable region of light chain VK is long is 399bp, 133 amino acid of encoding.Clone5 order-checking obtains the scFv of 858bp, 286 amino acid of encoding, and variable region of heavy chain VH is long is 414bp, 138 amino acid of encoding, variable region of light chain VK is long is 399bp, 133 amino acid of encoding.Clone16 order-checking obtains the scFv of 870bp, 290 amino acid of encoding, and variable region of heavy chain VH is long is 435bp, 145 amino acid of encoding, variable region of light chain V
klong is 390bp, 130 amino acid of encoding, long 45 bp of sequence of connection peptides, 15 amino acid of encoding.The clone strain that utilizes Megalign comparison to obtain, finds that the CDR1 of VH is relative little with the variation of CDR2 district, and CDR3 variation is larger.The VL variation of Clone1 and Clone5 is less.
(2) dog blood group antigen ELISA detects
This research gathers the blood of 56 dogs altogether, and extracts erythrocyte membrane.By antiglobulin experiment and the common DEA1.1 blood group that detects 56 dogs of cohesion amine medium detection reagent.Detected result shows, totally 30 dog blood group DEA1.1 positives, 26 dog blood group DEA1.1 feminine genders.The scFv single-chain antibody obtaining due to this Preliminary Study is univalent antibody, can not make red corpuscle generation aggegation.Therefore, the combination activity of antibody and DEA1.1 positive red blood cell membrane antigen in the induction of the ELISA Preliminary detection taking the dog DEA1.1 red corpuscle that gathers and extract as antigen supernatant.
Find through ELISA detected result the single-chain antibody that three clones obtain, the activity of being combined with 56 dog blood rbc membrane samples has very big-difference.Clone1 is more similar with Clone5 result, detects and is greater than 2 positive criterion with P/N value, and result only detects 28 positive findingses.Sample 3 and sample 17 detect P/N and are all less than 2, and detect the positive that obtains in conjunction with activity all than Clone16 low (table 4).Clone16 clone gained single-chain antibody, detects sample results identical with cohesion amine medium reagent detected result with antiglobulin, has good combination activity.
Table 4 single-chain antibody Clone16 clinical sample ELISA activation analysis
(3) amplification of positive bacteriophage antibody gene and purifying
According to phage single-chain antibody screening, gene amplification sequencing result and ELISA qualification result.Carry out bacterium liquid pcr amplification taking positive sample bacterium liquid as template, PCR reaction amplification Clone16 gene 898bp carries out next step experiment.Upstream primer is: 5 '-AGC
gGATCCaTGGAGTCTGTGCTCAGC-3 ' contains
hindiII site.Downstream primer is: 5 '-ATA
tGTCGAaAGGACGGTCAGTGGGGTTCC-3 ' contains
xhoi restriction enzyme site.
Reaction system is:
Pcr amplification condition is:
PCR product carries out purifying and also detects with 1% agarose gel electrophoresis.There is amplified fragments (Fig. 9) at about 898bp place, conform to expection clip size.
the expression and purification of embodiment 4 dog source single-chain antibody Clone16
(1) expression plasmid pET-32a-Clone16 builds
PCR product obtained above and empty carrier pET-32a are all used
hindiII and
xhoi carries out double digestion reaction.It is as follows that enzyme is cut system:
After mixing, put 37 DEG C of water-baths 4 hours.Afterwards enzyme is cut to product detects on 1.5% agarose gel electrophoresis.Enzyme is cut product DNA purification kit and is carried out purifying.
Use double digestion product to obtained carrier pET-32a of T4 DNA ligase and the double digestion product of Clone16 gene object fragment to carry out ligation.By both concentration of spectrophotometer measurement.In linked system, Clone16 gene fragment is about 3～10:1 with the ratio of the molecule number of pET-32a expression vector.
Reaction system is as follows:
After mixing, more than putting 16 DEG C of effect 15 h of connection instrument.
The positive bacterium colony of recombinant plasmid pET-32a-Clone16 picking is after PCR qualification, and the plasmid that enlarged culturing is extracted carries out double digestion qualification.Enzyme can obtain fragment and carrier segments that size is 892bp after cutting be 5600bp fragment, and the fragment that double digestion obtains is in the same size with expection, as Figure 10.Sequencing result is consistent with expection, does not undergo mutation, and represents that this plasmid has obtained correct structure.
(2) screening of recombinant expression plasmid and preparation
To connect in accordance with the following steps product transformed competence colibacillus DH5 α:
A. under aseptic condition, connection product 5 μ L are joined in 50 μ L DH5 α competent cells, after flicking and mixing, ice bath 30min;
B. transformation system is taken out fast in ice bath, put 42 DEG C of water-bath 90 s, note not shaking centrifuge tube;
C. fast centrifuge tube is placed in to ice bath 5 min;
D. add 700 μ L LB liquid nutrient mediums, with 37 DEG C of 150 r/min shaking culture 45 min;
E. by culture with centrifugal 4 min of 4000g, under aseptic condition, discard part supernatant, mix precipitation, and suspension evenly coated and contained Amp
+on the LB agar plate of (100 μ g/mL);
F. plate is placed in to room temperature until liquid is completely absorbed;
G. plate is put to 37 DEG C of incubators and cultivated 12～18 h, in the time that colony growth is good, plate taking-up is put to 4 DEG C and save backup.
Bacterium colony is carried out to PCR and carry out preliminary evaluation.Identify that positive bacterium liquid carries out enlarged culturing, and carry out plasmid extraction and qualification.Select plasmid PCR and double digestion to identify that all positive recombinant plasmid send the order-checking of Hua Da gene company limited.By this plasmid called after pET-32a-Clone16.
(3) abduction delivering of pET-32a-Clone16 single-chain antibody
To transform and express bacterium BL21(DE3 by above step through the correct plasmid of order-checking qualification) competent cell, 37 DEG C of overnight incubation, the single bacterium liquid of picking is cultivated, and through being accredited as the part bacterium liquid of positive recombinant bacterium liquid, to add final concentration be the sterile glycerol of 10 % ,-80 DEG C of preservations.Do the contrast of pET-32a empty carrier simultaneously.
Positive colony inoculation 5 mL of picking are containing Amp
+lB liquid nutrient medium in, 37 DEG C of shaking culture are spent the night as seed liquor, get seed liquor and are inoculated in 3 mL containing Amp in 1% ratio
+lB liquid nutrient medium in, it is 0.4～0.6 o'clock that 200 37 DEG C of r/min joltings are cultured to OD600, with IPTG induction escherichia coli BL21(DE3) expression fusion rotein.After induction, 4 h get bacterium liquid 1 mL, centrifugal 1 min of 12000 g, abandon supernatant, with the resuspended thalline of 100 μ L 1 × SDS-PAGE sample-loading buffer, in boiling water, boil 5～10 min, centrifugal 2 min of 12000 4 DEG C of g, get supernatant and carry out SDS-PAGE analysis, establish not Induction Transformation bacterium, BL21(DE3 simultaneously) contrast of bacterium, pET-32a empty carrier transformed bacteria sample.
Inductor IPTG final concentration is set and is respectively 2mM, 1mM, 0.8mM, 0.6mM, 0.4mM and 0.2mM and is optimized inductor concentration experiment, be placed in respectively 37 DEG C of induction 1h, 2h, 3h, 4h, 5h, 6h and optimize induction time.
Recombinant plasmid pET-32a-Clone16 transforms after (DE3) competent cell, setting-up time is that 1h, 2h, 3h, 4h, 5h, 6h induce through IPTG, and relatively find after SDS-PAGE electrophoresis with not inducing positive bacteria, there is respectively obvious specific proteins band about 34KDa place, in the same size with expection recombinant protein.Recombinant bacterium liquid is analyzed at the SDS-PAGE of the induced product of different time sections, and selecting the induction time of recombinant protein is 4 h(Figure 11).
Adding respectively final concentration is the IPTG of 0.2,0.4,0.6,0.8,1.0,2.0 mmol/L, 37 DEG C of abduction delivering 4h, and through SDS-PAGE electrophoretic analysis, IPTG concentration does not have significant difference to abduction delivering amount, and selecting the induced concentration of IPTG is 1.0 mmol/L.
(4) antibody expression soluble analysis
Protein purification carries out with reference to the working instructions of the Ni Sepharose 6 Fast Flow of GE Healthcare company.Concrete steps are as follows:
By centrifugal 20 min of 4 DEG C of 12000g of thalline ultrasonication product, collect supernatant, with the rearmounted 4 DEG C of preservations of filtering with microporous membrane of 0.45 μ m; Wash away the ethanol in post with the distilled water of 10 column volumes; With 10 times of volume Binding Buffer balance columns beds; By sample upper prop after the filtering with microporous membrane of 0.45 μ m; With 10 times of volume Binding Buffer flushings; After leaving standstill 10 min with 4 mL Elution Buffer, collect elutriant, after the protein content with ultraviolet spectrophotometer working sample ,-20 DEG C frozen; 10 times of volume Binding Buffer balance columns beds for pillar; Wash post bed with 10 times of volume distilled waters; Fill post bed with 20 % ethanol.
(5) Clone16 antibody Western blot analyzes
Get appropriate protein sample, carry out SDS-PAGE electrophoresis, electrophoresis finishes rear taking-up gel, cut 1 with the nitrocellulose filter of SDS-PAGE formed objects, and 6 3mm filter paper.The first balance 15min in the transfer liquid of precooling by filter paper and nitrocellulose filter, prepares according to following layer of structure: the order of 3 metafiltration paper-nitrocellulose filter-gel-3 metafiltration paper stacks successively from bottom to top, avoids upper and lower filter paper contact.Electrotransfer is installed, with 15V voltage transfer 30min.
After transfer, take off nitrocellulose filter, carry out mark and carry out next step operation:
A. sealing: take out transfer film, with 5% skim-milk TBST damping fluid seal, 4 DEG C spend the night or room temperature under jog 2h.Confining liquid is reusable;
B. wash film: discard confining liquid, 37 DEG C of 56r/min wash film 3 times with TBST damping fluid, each 5min;
C. primary antibodie is hatched: discard TBST damping fluid, add the anti-DEA1.1 blood group antigen of dog polyclonal serum, 37 DEG C of 50r/min are hatched 2h;
D. wash film: discard primary antibodie, 37 DEG C of 56r/min wash film 3 times with TBST damping fluid, each 5min;
E. two anti-hatching: discard TBST damping fluid, add goat-anti dog HRP mark two anti-, 37 DEG C of 50r/min are hatched 1h;
F. wash film: discard two and resist, 37 DEG C of 56r/min wash film 3 times with TBST damping fluid, each 5min;
G. TMB dyeing: add sedimentation type TMB nitrite ion, after at 37 DEG C, react 5～15min.Stopped reaction, uses distilled water flushing plate;
H. record result: Taking Pictures recording experimental result.
By bacterium after best induction time abduction delivering, carry out ultrasonic degradation, get respectively isopyknic cleer and peaceful precipitation and carry out SDS-PAGE electrophoresis detection (as Figure 12), result is presented in supernatant and has more target protein, and illustration purpose albumen has solubility.
Protein delivery on gel after SDS-PAGE electrophoresis, to after on NC film, is carried out to Western blot checking to expressing protein with His tag monoclonal antibody, found that recombinant protein can show specific binding band (Figure 13).In supernatant, expression amount is higher.
(6) dog blood group antigen Dot-ELISA checking antibody activity
With the erythrocyte membrane of 56 dogs that gather and extract.Use Dot-ELISA method, detect the Clone16 single-chain antibody of purifying and the combination activity of DEA1.1 positive red blood cell membrane antigen.Using dog DEA1.1 positive and negative red corpuscle as antigen, with single-chain antibody, as primary antibodie, anti-His antibody is two anti-.Checking detects dog DEA1.1 blood group erythrocyte binding activity.
The Clone16 single-chain antibody that detects purifying with 56 dog blood rbc film clinical samples is in conjunction with activity, use Dot-ELISA method validation, Clone16 single-chain antibody acquired results as shown in figure 14, detects 30 dog blood group DEA1.1 positives altogether, 26 dog blood group DEA1.1 feminine genders.Result is with consistent with antiglobulin experiment and cohesion amine medium detection reagent detected result.Illustrate that Clone16 can well be combined with DEA1.1 blood group antigen, can be for the exploitation of Blood grouping reagent.
SEQUENCE LISTING
<110> Agricultural University Of South China
<120> dog source single-chain antibody and construction process and application
<130>
<160> 54
<170> PatentIn version 3.3
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tgaggagaca gtgaccaggg ttc 23
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aatatggcct ggtcccctct cctcct 26
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aataatatga cctccaccag ggcctggtcc cctc 34
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atgacttcca ctgtgggatg gtcccctct 29
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aaggacggtc agttgggttc 20
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aataatgaag agggcagttg gggg 24
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aatgaggacg gtcagatggg tg 22
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aataatgagg acggccaggc gggtgccttt g 31
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gaggacagtc agaaaagtgc ctccg 25
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tgtgatctct accgtggtcc ct 22
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aactacggcc cagccggcca tggartctgt gctcdgctg 39
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gccagagcca cctccgcctg aaccgcctcc acctgaggac acgaagagt 49
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ggcggaggtg gctctggcgg tggcggatcc atgaggttcc cwtctcagct cct 53
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gagtcattct cgacttgcgg ccgcacgttt gakctccasy ttggtycct 49
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ggcggaggtg gctctggcgg tggcggatcc atggsctggt yccctctcmt 50
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<213> K9 Vl Back linker-2
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ggcggaggtg gctctggcgg tggcggatcc atggcctgga ckstgctyct 50
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ggcggaggtg gctctggcgg tggcggatcc atgacttcca ctrtggsatg gt 52
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ggcggaggtg gctctggcgg tggcggatcc atggactggg ttccctttta c 51
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ataagaatgc ggccgcaagg acggtcagtk gggtt 35
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<213> Clone16 gene order
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atggagtctg tgctcagctg ggtttccctt gtcgctattt taaaaggtgt ccagagtgag 60
gtgcaactgg tggagtctgg gggagacctg gtgaagcctg ggggatccct gagactctcc 120
tgcgtggcct ctggattcaa cttcagtaac tatgacatga actgggtccg ccaggctcca 180
gggaaggggc tgcagtgggt cgcatacatt agcagtggtg ggatcaacac atactatgca 240
gatgttgtgc agggccggtt caccatctcc agagacaacg ccaagagtat gttgtatctt 300
cagatggaca ggctgagagt cgaggacacg gccatgtatt actgtgcggg tgagggccct 360
tattctagag gttcgggctt attcggttac ggtatggact actggggtcc tggcacctca 420
ctcttcgtgt cctcaggtgg aggcggttca ggcggaggtg gctctggcgg tggcggatcc 480
atgggctggt tccctctcct cctcaccctc cttgctcatt tcacagggtc ctgggcccag 540
cctgtgctga ctcagccacc ctccgtgtct gggtccctgg accagagggt caccatttcc 600
tgcactggaa gcagctccaa cgttggctat agcagtagtg tgggctggta ccaacagttt 660
ccaggaagag gccccagaac catcatctat tttgatacta gtcgaccctc gggggtcccc 720
gatcgattct ctggctccaa gtctggcaac acagccaccc tgactatctc tggactccgg 780
actgaggatg gggctcatta ttactgctca tcttgggaca gcggtgtcag agctccggta 840
ttcggctcgg gaaccccact gaccgtcctt 870
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<213> clone16 heavy chain encoding sequence
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atggagtctg tgctcagctg ggtttccctt gtcgctattt taaaaggtgt ccagagtgag 60
gtgcaactgg tggagtctgg gggagacctg gtgaagcctg ggggatccct gagactctcc 120
tgcgtggcct ctggattcaa cttcagtaac tatgacatga actgggtccg ccaggctcca 180
gggaaggggc tgcagtgggt cgcatacatt agcagtggtg ggatcaacac atactatgca 240
gatgttgtgc agggccggtt caccatctcc agagacaacg ccaagagtat gttgtatctt 300
cagatggaca ggctgagagt cgaggacacg gccatgtatt actgtgcggg tgagggccct 360
tattctagag gttcgggctt attcggttac ggtatggact actggggtcc tggcacctca 420
ctcttcgtgt cctca 435
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<213> clone16 light chain encoding sequence
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atgggctggt tccctctcct cctcaccctc cttgctcatt tcacagggtc ctgggcccag 60
cctgtgctga ctcagccacc ctccgtgtct gggtccctgg accagagggt caccatttcc 120
tgcactggaa gcagctccaa cgttggctat agcagtagtg tgggctggta ccaacagttt 180
ccaggaagag gccccagaac catcatctat tttgatacta gtcgaccctc gggggtcccc 240
gatcgattct ctggctccaa gtctggcaac acagccaccc tgactatctc tggactccgg 300
actgaggatg gggctcatta ttactgctca tcttgggaca gcggtgtcag agctccggta 360
ttcggctcgg gaaccccact gaccgtcctt 390
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<213> clone16 connection peptides encoding sequence
<400> 51
ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcc 45
<210> 52
<211> 290
<212> PRT
<213> clone16 aminoacid sequence
<400> 52
Met Glu Ser Val Leu Ser Trp Val Ser Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Asn Phe
35 40 45
Ser Asn Tyr Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Gln Trp Val Ala Tyr Ile Ser Ser Gly Gly Ile Asn Thr Tyr Tyr Ala
65 70 75 80
Asp Val Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser
85 90 95
Met Leu Tyr Leu Gln Met Asp Arg Leu Arg Val Glu Asp Thr Ala Met
100 105 110
Tyr Tyr Cys Ala Gly Glu Gly Pro Tyr Ser Arg Gly Ser Gly Leu Phe
115 120 125
Gly Tyr Gly Met Asp Tyr Trp Gly Pro Gly Thr Ser Leu Phe Val Ser
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Met Gly Trp Phe Pro Leu Leu Leu Thr Leu Leu Ala His Phe Thr Gly
165 170 175
Ser Trp Ala Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ser
180 185 190
Leu Asp Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Val
195 200 205
Gly Tyr Ser Ser Ser Val Gly Trp Tyr Gln Gln Phe Pro Gly Arg Gly
210 215 220
Pro Arg Thr Ile Ile Tyr Phe Asp Thr Ser Arg Pro Ser Gly Val Pro
225 230 235 240
Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile
245 250 255
Ser Gly Leu Arg Thr Glu Asp Gly Ala His Tyr Tyr Cys Ser Ser Trp
260 265 270
Asp Ser Gly Val Arg Ala Pro Val Phe Gly Ser Gly Thr Pro Leu Thr
275 280 285
Val Leu
290
<210> 53
<211> 27
<212> DNA
<213> Clone16 upstream primer
<400> 53
agcggatcca tggagtctgt gctcagc 27
<210> 54
<211> 30
<212> DNA
<213> clone16 downstream primer
<400> 54
atatgtcgaa aggacggtca gtggggttcc 30
Claims (9)
1. a dog source single-chain antibody Clone 16, is characterized in that, the long 870bp of scFv sequence of described antibody, and sequence is as shown in SEQ ID NO:48; The wherein long 435bp of variable region of heavy chain VH, sequence is as shown in SEQ ID NO:49; The long 390bp of variable region of light chain VK, sequence is as shown in SEQ ID NO:50; The long 45bp of connection peptides sequence, sequence is as shown in SEQ ID NO:51.
2. dog source single-chain antibody Clone 16 according to claim 1, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:52.
3. for a primer for single-chain antibody Clone 16 gene orders described in the claim 1 that increases, it is characterized in that, sequence is as shown in SEQ ID NO:53～54.
4. for building the primer sets of dog source single-chain antibody library, it is characterized in that, comprise antibody heavy chain variable region primer, variable region of light chain primer and connection peptides aligning primer, its primer sequence is as shown in SEQ ID NO:1～47.
5. a dog source single-chain antibody library, is characterized in that, described antibody library contains dog blood group antibody variable region of heavy chain VH and variable region of light chain VL, and middle joining region linker sequence (Gly4Ser)
3, there is complete antigen-binding site, taking pCANTAB-5E as carrier, storage capacity reaches 7 × 10
5.
6. a construction process for dog source single-chain antibody library described in claim 5, is characterized in that, comprises the following steps:
S1. total RNA extraction and cDNA's is synthetic: getting the canine peripheral blood after immunity, separate lymphocyte, extract total RNA, is cDNA through reverse transcription PCR reverse transcription;
S2. pcr amplification heavy chain of antibody and chain variable region gene: according to dog antibody gene sequence, design respectively the primer of pcr amplification heavy chain of antibody, variable region of light chain, the sequence of described primer is as shown in SEQ ID NO:1～33; The cDNA obtaining taking S1 is as template, and variable region gene VH and VL increase;
S3. the amplification of single-chain antibody scFv gene: design containing connection peptides sequence linker and
sfiI, NotIthe primer of restriction enzyme site, obtains scFv gene through amplification on the basis of the heavy chain VH being obtained by S2 and light chain VL variable region gene again;
S4. the structure in single-chain antibody scFv storehouse: double digestion scFv gene fragment, and insert pACNTAB-5E construction recombination plasmid, transformed competence colibacillus
e.colitG1, obtains preliminary single-chain antibody library, through repeatedly washing in a pan sieve, obtains dog source single-chain antibody library.
7. the construction process of dog according to claim 6 source single-chain antibody library, is characterized in that, described in S3 connection peptides sequence linker and
sfiI, NotIthe primer sequence of restriction enzyme site is as shown in SEQ ID NO:34～47.
8. the construction process of dog according to claim 6 source single-chain antibody library, it is characterized in that, the method of washing in a pan sieve described in S4 is: the positive and negative erythrocyte membrane with dog blood group antigen, be cured on nitrocellulose filter, and single-chain antibody scFv storehouse is carried out to the screening of " adhesion-wash-out-amplification "; Every wheel after screening carried out titer determination, the enrichment result of detection specificity phage to the phage of amplification.
9. the application of single-chain antibody library in preparation dog bracket for blood grouping reagent described in primer sets or claim 5 described in single-chain antibody or claim 4 described in claim 1 or 2.
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| CN104761639A (en) * | 2015-04-16 | 2015-07-08 | 哈尔滨博翱生物医药技术开发有限公司 | ScFv antibody, encoding gene thereof and application of scFv antibody to preparation of preparation for treating or preventing hepatitis B |
| CN107164399A (en) * | 2017-07-07 | 2017-09-15 | 江苏省疾病预防控制中心 | A kind of high flux expresses the linear expression cassette of monoclonal antibody |
| CN110540998A (en) * | 2018-05-28 | 2019-12-06 | 深圳华大生命科学研究院 | Method and reagent for quickly constructing multivalent antibody expression vector |
| CN110628797A (en) * | 2019-09-16 | 2019-12-31 | 广州艺得诺生物科技有限公司 | Identification method of canine erythrocyte antigen DEA1.1 |
| CN110799532A (en) * | 2017-06-22 | 2020-02-14 | 莫佛塞斯公司 | Canine antibody library |
| CN110964103A (en) * | 2019-11-21 | 2020-04-07 | 青岛农业大学 | Canine-derived anti-canine parvovirus antibody, antibody library and construction method |
| CN111040031A (en) * | 2019-12-13 | 2020-04-21 | 中国农业科学院兰州兽医研究所 | ScFv antibody for resisting African swine fever virus and preparation method thereof |
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| CN117801110A (en) * | 2024-03-01 | 2024-04-02 | 北京纳百生物科技有限公司 | Monoclonal antibody for resisting canine erythrocyte antigen DEA1, detection reagent and application thereof |
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| CN110799532B (en) * | 2017-06-22 | 2023-11-03 | 莫佛塞斯公司 | Canine Antibody Library |
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