CN102094035B - Expression vector for constructing high-quality bacteriophage antibody library - Google Patents
Expression vector for constructing high-quality bacteriophage antibody library Download PDFInfo
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Abstract
The invention discloses an expression vector for constructing a high-quality bacteriophage antibody library. The vector disclosed by the invention is prepared by the following steps: 1) inserting coding genes of a light-chain region SacB in multiple cloning sites of pDF to obtain an intermediate vector PSBX; 2) mutating BssHII enzyme cutting sites of the intermediate vector PSBX obtained in the step 1) into BglII enzyme cutting sites to obtain an intermediate vector PSGX; and 3) inserting coding genes of a heavy-chain region SacB in Nhe I and Nco I enzyme cutting sites of the intermediate vector PSGX obtained in the step 2) to obtain a vector pDF-D-SacB. Experiment disclosed by the invention proves that the obtained phagemid vector pDF-D-SacB containing suicide genes is applicable to the construction of a high-capacity bacteriophage antibody library.
Description
Technical field
The present invention relates to the expression vector that a kind of structure is applied to the high quality phage antibody library.
Background technology
The development of phage antibody library technique and be applied as the antibody technique field and brought huge variation; Each research fields such as biology, medical science have been widely used in; Combination through genetically engineered and display technique of bacteriophage; Can obtain humanized antibody to nearly all epitope, this has greatly promoted the development and application of various excellent property antibody and multipurpose antibody fusion rotein.Phage antibody library has been simulated the natural antibody storehouse, makes people to become possibility but directly utilize antigen just can from antibody library, filter out specific antibody without the complex immunological process.It had both solved the difficult problem such as animal derived of poor efficiency and mouse monoclonal antibody of source difficulty, the human body hybridoma system of humanized's monoclonal antibody, also made MONOCLONAL ANTIBODIES SPECIFIC FOR become simple, and was effectively stable, made the preparation of human monoclonal antibodies that breakthrough arranged.But; This technology also has many problems to wait to solve at present; As the variety that guarantees antibody library with present efficient in, how to improve the usable storage amount, how when making up antibody library, to improve on the problem such as antibody gene joint efficiency, be still waiting further solution.
Summary of the invention
An object of the present invention is to provide a kind of carrier.
Carrier provided by the invention prepares according to following method:
1) encoding sox 1 with SacB is inserted between the MCS of pDF, obtains intermediate carrier PSBX;
2) the BssHII restriction enzyme site of the intermediate carrier PSBX that step 1) is obtained changes the BglII restriction enzyme site into, obtains intermediate carrier PSGX;
3) encoding sox 2 with SacB is inserted into step 2) between the Nhe I and NcoI restriction enzyme site of the intermediate carrier PSGX that obtains, obtain carrier pDF-D-SacB;
The encoding sox 1 of said SacB for the sequence 1 of sequence table from 5 ' terminal 30-1908 position Nucleotide;
The encoding sox 2 of said SacB for the sequence 2 in the sequence table from 5 ' terminal 7-1857 position Nucleotide.
In the step 1), the MCS that said encoding sox 1 with SacB is inserted into pDF is inserted between the BssHII and Xba I restriction enzyme site of pDF for the encoding sox 1 with SacB.
Another object of the present invention provides a kind of carrier.
Carrier provided by the invention prepares according to following method:
1) with puc19-SacB is template, carries out pcr amplification to 1, obtain PCR product 1, PCR product 1 is inserted between the BssHII and Xba I restriction enzyme site of pDF, obtain intermediate carrier PSBX with primer; Said PCR product 1 is the encoding sox 1 of SacB, the encoding sox of said SacB 1 for the sequence 1 of sequence table from 5 ' terminal 30-1908 position Nucleotide;
Said primer is classified the sequence 5 in the sequence table as to the nucleotides sequence of a primer in 1, and said primer is classified the sequence 6 in the sequence table as to the nucleotides sequence of another primer in 1;
2) the BssHII restriction enzyme site on the intermediate carrier PSBX that step 1) is obtained is changed into the BglII restriction enzyme site, obtains intermediate carrier PSGX;
3) with pUC19-SacB be template, carry out pcr amplification to 2, obtain PCR product 2, the PCR product 2 that obtains is inserted into step 2 with primer) between the Nhe I and Nco I site of the intermediate carrier PSGX that obtains, obtain recombinant vectors pDF-D-SacB; Said PCR product 2 is the encoding sox 2 of SacB, the encoding sox of said SacB 2 for the sequence 2 in the sequence table from 5 ' terminal 7-1857 position Nucleotide;
Said primer is classified the sequence 7 in the sequence table as to the nucleotides sequence of a primer in 2, and said primer is classified the sequence 8 in the sequence table as to the nucleotides sequence of another primer in 2.
Above-mentionedly change the BssHII restriction enzyme site into the BglII restriction enzyme site and carry out according to following method:
A: after intermediate carrier PSBX cut with the BssHII enzyme, add the SEAP dephosphorylation again, obtain dephosphorylized PSBX linear fragment; B: the primer that will contain BglII is to annealing at 98 ℃-20 ℃, and it is right to obtain the annealed primer, handles carrying out phosphorylation to the annealed primer again, and the annealing primer that obtains phosphorylation is right;
C: the annealing primer of phosphorylation to being connected with dephosphorylized PSBX linear fragment, is obtained intermediate carrier PSGX;
A primer of the primer centering of the said BglII of containing is the sequence 3 in the sequence table, and another primer is the sequence 4 in the sequence table.
The said primer that will contain BglII is specially the primer that contains BglII annealing at 98 ℃, 90 ℃, 80 ℃, 70 ℃, 60 ℃, 50 ℃, 40 ℃, 30 ℃ and 20 ℃ annealing at 98 ℃-20 ℃.
The transgenic cell line or reorganization bacterium or the recombinant virus that contain described carrier also are the scopes that the present invention protects.
The application in the preparation phage antibody library of described carrier or said transgenic cell line or reorganization bacterium or recombinant virus also is the scope that the present invention protects.
In the said application, said antibody is the anti-hepatitis b surface antigen antibody.
Experiment of the present invention proves; After antibody gene inserted suicide vector (containing suicide gene SacB), suicide gene lost function, and does not insert the suicide vector of antibody gene; Suicide gene is expressed suicide albumen; Express this proteic Gram-negative bacteria when in containing the substratum of sucrose, growing, can be to the deleterious product of bacterium with sucrose decomposition, thereby kill this proteic bacterium of expression.Be specially through, light chain regional insertion SacB gene heavy to pDF, and engineered antibody gene clone site, made up the pDF-D-SacB carrier; Through detecting, pDF-D-SacB can express the Fab phage antibody with function, and the fixed point reorganization of the Cre-Loxp mediation of expection can take place in the bacterium born of the same parents of secretion Cre proteolytic enzyme; Selected non-existent restriction enzyme site in the antibody embryonal system variable region gene among the pDF-D-SacB for use; To guarantee that the restriction endonuclease sequence can not have influence on the N-terminal sequence of ripe antibody molecule; BssHII changes BglII into restriction enzyme site; Thereby greatly improved the joint efficiency of antibody gene, reduced workload.Therefore, use suicide gene as forward screening-gene associatings such as anti-sieve gene and resistant genes, be used to make up the seamless deletion mutantion strain of bacterium, the antibody library quality and the stability that make up in this way are improved; Phagemid carrier pDF-D-SacB of the present invention provides effective tool for making up the large vol phage antibody library, has increased the variety of antibody to antigen recognition simultaneously.The antibody library that phagemid carrier pDF-D-SacB makes up, not only capacity is big, and quality is higher, can obtain being bordering on effective clone of 100% in theory; For the various types of antigenic phage antibodies of elutriation are laid solid research basis.
Description of drawings
Fig. 1 is the suicide function of checking PUC19-SacB
Fig. 2 is that pcr amplification SacB isogeneity is reclaimed
Fig. 3 cuts for EcoR I enzyme and identifies 1% gel electrophoresis figure
Fig. 4 cuts for Dra I enzyme and identifies 1% gel electrophoresis figure (light chain)
Fig. 5 cuts for Dra I enzyme and identifies 1% gel electrophoresis figure (heavy chain)
Fig. 6 is the ELISA detected result of the expressed phage antibody of pDF-D-SacB
Fig. 7 is the structural representation of pDF-D-SacB
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The acquisition of embodiment 1, carrier PDF-D-SacB
Intestinal bacteria trans1-Blue is available from the biological ltd of the full formula gold in Beijing; Phage VCSM13, BS1365 bacterium, expression vector pDF are presented by Navy General Hospital professor Zhou Lijun; The Puc19-SacB plasmid is presented by the Huang Liuyu researcher of prevention and control of diseases institute of Military Medical Science Institute; The plasmid B4-HFLF that has anti-hepatitis B virus surface antigen (HBsAg) human Fab fragment gene is preserved by this chamber.
Phusion, BssHII, Xba I, Nco I, Nhe I, BglII, SEAP is available from New EnglandBiolabs (NEB) company; EcoR I, Dra I, T4 DNA Ligase, T4 Plolynucleotide Kinnase (PNK) is available from TaKaRa (Dalian) company; EasyPure Plasmid MiniPrep Kit, EasyPure Quick Gel ExtractionKit is available from the biological ltd of the full formula gold in Beijing; 5000DNA Marker, DL2000DNA Marker is available from Beijing Bo Maide company;
1, the suicide function of checking pUC19-SacB
1) pUC19-SacB (Wang Yufei, Chen Zeliang, Qiao Feng, Wang Zhoujia, Du Xinying, Yuan Xitong, Huang Liuyu; The structure of brucella suicide vector and the application in mutant strain makes up thereof; World Chinese digests magazine, on September 28th, 2007,15 (27): 2934-2937; The public can obtain from the microorganism of military medical sciences academy epidemic research.) change in the intestinal bacteria; Draw 100 μ l and join in the 5ml non-resistant liquid LB substratum, the 220r/min jolting is dipped in this bacterium liquid that takes a morsel to saturated with choosing the bacterium rod on 37 ℃ of shaking tables; Rule containing on the solid LB substratum of ammonia Bian resistance, put into 37 ℃ of incubators and spend the night.
The mono-clonal that picking carries the pUC19-SacB plasmid from flat board contains the ammonia Bian resistance liquid LB substratum to 3ml, and 220r/min jolting 10h is to logarithmic phase in 37 ℃ of shaking tables; Get above-mentioned bacterium liquid 200 μ l join respectively the 5ml that is prepared in advance contain ammonia Bian resistance liquid LB substratum with contain in ammonia Bian resistance and two test tubes of 5% liquid sucrose LB substratum; 220r/min jolting 5h on 37 ℃ of shaking tables, result see shown in Figure 1A, from figure, find out; Bacteria growing in only containing ammonia Bian resistance liquid LB substratum; But bacterium does not grow in the substratum that contains ammonia Bian resistance and 5% sucrose, shows, pUC19-SacB has the suicide function under the sucrose environment.
With the bacterium liquid of two test tubes respectively from 10
-1Be diluted to 10
-9, respectively get the bacterium liquid 5 μ l of each dilution gradient, join pre-prepd containing on the ammonia Bian resistance solid LB culture medium flat plate in the corresponding grid, put into 37 ℃ of incubators, next day observations.The result sees shown in Figure 1B, can be found out by Figure 1B: the intestinal bacteria (left figure) that in containing Amp resistance liquid LB substratum, grow, and 10
-1To 10
-5The clone is all arranged, and 10
-1-10
-3Extension rate the clone can't count, 10
-486 mono-clonals are arranged, 10
-511 mono-clonals are arranged; And the intestinal bacteria 10 that (right figure) grows in containing Amp resistance and 5% liquid sucrose LB substratum
-1To 10
-5All there is not the clone.Show whether intestinal bacteria contain in the media environment of sucrose, growth differs 5 one magnitude, explains that the PUC19-SacB plasmid has the suicide function, further proves the function of suicide gene SacB.
2, design primer
In order to improve light chain antibody gene joint efficiency; Design two primers and transformed original phagemid carrier pDF (Qiao Yuanyuan, Wang Yan, Chen Xiaosui, Wang Yuxiao, change ice; The structure of large vol phage antibody library and evaluation, Chinese microbiology and Journal of Immunology, the 24th the 3rd phase of volume of March in 2004; 194-197, the public can obtain from the microorganism of military medical sciences academy epidemic research.) go up in order to connect the restriction enzyme site BssHII of light chain antibody gene; Its new restriction enzyme site is BglII; The primer sequence that is designed for clone SacB suicide gene simultaneously makes up the antibody library carrier, and concrete primer sequence is seen table 1-1, and the primer sequence that is used to clone the anti-hepatitis b surface antigen antibody; Concrete primer is seen table 1-2, and all primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Table 1-1 primer sequence
Annotate: 1:BssHII:gcgcgc Xba I:tctaga BglII:agatct Nco I:ccatgg NheI:gctagc 2: all primers are (5 '-3 '), and underscore is a restriction enzyme site
Table 1-2 primer sequence
Annotate: 1:Xba I:tctaga Bgl II:agatct Nco I:ccatgg Nhe I:gctagc
2: all primers are (5 '-3 '), and underscore is a restriction enzyme site
3, the structure of expression vector pDF-D-SacB
1) makes up phasmid carrier PSBX
With Puc19-SacB is template, and pDF-BssH and pDF-R-Xba are primer, carries out pcr amplification and obtains SacB encoding sox 1 and purifying and recovering; The result sees shown in Figure 2, wherein, and M:DNA Marker DL20001:SacB gene; Obtain the fragment of 1882bp; Through order-checking, the Nucleotide of SacB encoding sox 1 has the sequence 1 of sequence table from 5 ' terminal 30-1908 position Nucleotide, and the coding region is that sequence 1 is from 5 ' terminal 30-1880 position Nucleotide.But sequence 1 is synthetic also.
With the PCR product after BssHII and the Xba I enzyme switchback receipts; Be connected for 16 ℃ with the T4DNA ligase enzyme with the pDF carrier of cutting through electrophoretic separation, purifying, enzyme equally, connector transforms DH5 α competence bacterium, bed board picking list colony; Extract DNA; Identify with the HindIII restriction endonuclease, obtain the positive clone of fragment of about 1800bp, called after PSBX (PSBX last have only a BssH II site);
2) make up phasmid carrier PSGX
After using 50 ℃ of digested plasmid PSBX of BssHII 3.5h again, add 1 μ l SEAP CIP, temperature is reduced to and is reclaimed the 7787bp fragment after 37 ℃ of enzymes are cut 1h.
Primer DDTB-F (sequence 3) and DDTB-R (sequence 4) are respectively got 2 μ l, add H
2O is 20 μ l to TV, puts into the PCR appearance and anneals by 98 ℃-20 ℃, puts into the PCR appearance and anneals, and condition is following:
98 ℃ 90 ℃ 80 ℃ 70 ℃ 60 ℃ 50 ℃ 40 ℃ 30 ℃ 20 ℃ of temperature
Degree
The time 3min 1min 1min 1min 1min 1min 1min 1min 1min
Between
From the PCR appearance, take out the annealed primer; 37 ℃ leave standstill 30min, take out 17 μ l and mix with 10 * T4DNALigaseBuffer, 2 μ l, add 1 μ l T4 Plolynucleotide Kinnase again; 37 ℃ of reaction 30min, then 70 ℃ of reaction 10min are with all enzyme-deactivatings.The phasmid carrier PDF-SacB (PSBX) after enzyme cut and the annealing primer fragment of phosphorylation are carried out after routine is connected, transforms, chooses the clone; Cut evaluation with BglII and EcoRV enzyme; Obtained the purpose fragment of 2 4562bp that conform to theoretical value, 1843bp, 1410bp; Sequencing analysis, the positive clone that forward inserts, called after pDF-SacB-GX (PSGX).
3)pDF-D-SacB
Be template once more with pUC19-SacB; Primer pDF-Nco and pDF-R-Nhe carry out pcr amplification and obtain SacB encoding sox 2 and purifying and recovering; Through order-checking; The Nucleotide of SacB encoding sox 2 has the sequence 2 of sequence table, and (SacB encoding sox 1 is than SacB encoding sox more than 2 several bases from 5 ' terminal 7-1882 position Nucleotide; Additional base be added the base of several restriction enzyme sites and for preventing the reading frame added base that is shifted), the coding region is that sequence 2 is from 5 ' terminal 7-1857 position Nucleotide.But sequence 2 is synthetic also.
Carry out the SacB encoding sox 2 that enzyme is cut purifying and recovering with Nhe I and Nco I, with pDF-SacB-GX (PSGX) carrier of cutting through electrophoretic separation, purifying, enzyme equally, with 16 ℃ of connections of T4DNA ligase enzyme; Conventional conversion, bed board, picking list colony; With EcoR I recon is carried out enzyme behind the extraction plasmid and cut evaluation, the result is as shown in Figure 3, and wherein M:5000DNA Marker 1-10 is the recon plasmid; Can find out by Fig. 3: the recon plasmid is carried out enzyme with restriction endonuclease EcoR I cut evaluation; Obtained 5 (1,3,4,5 and 8) conform to the theoretical value purpose fragment of (3804bp, 2653bp, 2025bp), promptly positive, be recombinant phage vector.
The recombinant phage vector of above-mentioned structure is carried out complete sequence determination; The result for this carrier for to be inserted into sequence 1 between the BssHII and Xba I restriction enzyme site of pDF from 5 ' terminal 30-1908 position; Sequence 2 all has been inserted into the carrier that obtains between Nhe I and the Nco I restriction enzyme site of pDF from 5 ' terminal 7-1882 position; And the BssHII among the pDF sports BglII, and does not find to have phenomenons such as base mutation or decoding displacement, the reorganization phasmid carrier called after pDF-D-SacB that therefore obtains; The structural representation of this carrier is as shown in Figure 7, can find out that the SacB in light chain zone is inserted between Bg1II, the Xba I; The SacB in heavy chain zone is inserted between Nco I, the Nhe I.
The acquisition of embodiment 2, phage antibody
1, construction recombination plasmid pDF-SacB-HBsL and pDF-SacB-HBsH
(Wang Chen, Hou Lihua, Du Guixin, Li Jianming, Chen Puyan, Tong Yigang make up preset CDR2 gene phage antibody library and screen anti-people's integrin alpha ν β with plasmid B4-HFLF
3The humanization Fab of mAb, cell and molecular immunology magazine, 2006,22 (1), 64-67; The public can obtain from the microorganism of military medical sciences academy epidemic research.) be template, with the two group primer PCRs of table among the 1-2 increase respectively κ chain (primer is with VK3-Bgl, IGK-Xba), the Fd fragment gene (primer is with IGG-Nhe, VH4-r-2Nco1) of anti-hepatitis b surface antigen antibody, reaction conditions is: 95 ℃ of preparatory sex change 2min; 95 ℃ of sex change 20s, 65 ℃ of annealing 20s, 72 ℃ are extended 15s, and 35 circulations are extended 5min for back 72 ℃.The result is: obtaining heavy chain Fd gene amplification product is the band about 680bp, and κ chain amplified production is the band about 680bp.
Reclaim above-mentioned PCR product, the light chain κ chain PCR product after reclaiming is cut with Bgl II, Xba I enzyme (the enzyme system of cutting is 50 μ l, and two enzymes (NEB company) respectively add 1 μ l; 37 ℃ of enzymes are cut 5h), be connected with the pDF-D-SacB carrier of cutting through same enzyme, transformed competence colibacillus intestinal bacteria Trans1-Blue (the full formula in Beijing King Company) is bed board then; Extract recombinant plasmid and cut evaluation with Dra I enzyme, the result is as shown in Figure 4, and 1-10 is a recombinant plasmid; Have 5 bands to be positive colony, but 6 bands are negative clone, from figure, find out; The positive plasmid of 1-10, called after pDF-SacB-HBsL.
Heavy chain Fd gene PCR product after reclaiming is cut (the enzyme system of cutting is 50 μ l, and two enzymes (NEB company) respectively add 1 μ l, and 37 ℃ of enzymes are cut 5h) with Nco I, Nhe I enzyme; Be connected with the pDF-D-SacB carrier of cutting through same enzyme, transformed competence colibacillus intestinal bacteria Trans1-Blue is bed board then, extracts recombinant plasmid and cuts evaluation with Dra I enzyme; The result is as shown in Figure 5, and 1-10 is a recombinant plasmid, has 5 bands to be positive colony; Article 6, band is negative clone; From figure, find out the positive plasmid of 1-10, called after pDF-SacB-HBsH.
Picking contains the single colony Trans1-Blue bacterium and the single colony Trans1-Blue bacterium that contains pDF-SacB-HBsH of pDF-SacB-HBsL respectively; Be inoculated into respectively in the LB substratum that contains 100ug/ml penbritin (Amp) and 10g/L glucose; 37 ℃ of concussion overnight cultures are got the 30ul bacterium that spends the night and are inoculated into SB nutrient solution (30g peptone, the 20g yeast extract that 3ml contains 100ug/ml Amp; 10g MOPS; Zero(ppm) water 1000ml, pH7.0.), 37 ℃ of concussions are cultured to OD600 and are about 0.5; (Wang Chen, Hou Lihua, Du Guixin, Li Jianming, Chen Puyan, Tong Yigang make up preset CDR2 gene phage antibody library and screen anti-people's integrin alpha ν β to add 20ul helper phage VCSM13 respectively
3The humanization Fab of mAb, cell and molecular immunology magazine, 2006,22 (1), 64-67; The public can obtain from the microorganism of military medical sciences academy epidemic research.) (titre is 10
12) recombinate, 30 ℃ of concussion incubated overnight, next day is centrifugal collection supernatant respectively, obtains containing the phage and the phage that contains pDF-SacB-HBsH of pDF-SacB-HBsL.
Get the suitably supernatant of dilution (contain the phage of pDF-SacB-HBsL or contain the phage of pDF-SacB-HBsH) of 10ul respectively; Mix with the Trans1-Blue bacterium of 100ul increased logarithmic phase; Hatch 30min for 25 ℃, the shop contains the LB plate of Amp, 37 ℃ of incubated overnight; Next day, counting was estimated titre respectively.The experiment triplicate, results averaged.
The result is: the titre that contains the phage supernatant of pDF-SacB-HBsL is 9 * 10
11
The titre that contains the phage supernatant of pDF-SacB-HBsH is 1 * 10
12
2, reorganization in the cell of antibody gene
Picking list colony BS1365 (Wang Yan, Wang Yuxiao, Chen Xiaosui, change ice, Liu Xiaolin; Be used for the structure and the evaluation of the expression vector of large vol phage antibody library; 2003 the 19th volumes of China's Journal of Immunology, the 93-96 page or leaf, the public can obtain from the microorganism of military medical sciences academy epidemic research.) (can express Cre proteolytic enzyme) bacterial cell; At the 2YT substratum that contains 50ug/ml kantlex and 10g/L glucose (prescription: peptone 16g, yeast extract 10g, NaCl 5g, agar powder 15g; Zero(ppm) water 1000ml, pH 7.0) in grow to logarithmic phase (OD600=0.5).Pass through titer determination; The phage supernatant of the above-mentioned pDF-SacB-HBsL of containing is mixed with identical titre with the phage supernatant that contains pDF-SacB-HBsH; Phage particle and mycetocyte infect the BS1365 bacterium with 20: 1 mixed; 37 ℃ of incubation 60min add Amp and mend to 100ug/ml 30 ℃ of joltings of spending the night.
Get the incubated overnight bacterium next day and join 2TY nutrient solution (the 2TY nutrient solution prescription: 2 * YT substratum: 17g peptone, 10g yeast powder, 5g NaCI that 3ml contains Amp; Add deionized water dissolving; NaOH with 10mol/L is adjusted to pH7.0, is settled to 1L, the high pressure autosterilization.) in, 37 ℃ are cultured to the OD value and are about at 0.5 o'clock, and (titre is that titre is 10 to add 20ulVCSM13
12) recombinate 37 ℃ of concussion incubated overnight.Next day, centrifugal recovery supernatant obtained the phage antibody sample, measured phage titre, and the result is 1 * 10 for phage antibody sample pnagus medius titre
8
With the intestinal bacteria Trans1-Blue of the above-mentioned phage antibody sample infection logarithmic phase that obtains, the shop contains the culture plate of Amp, 37 ℃ of overnight cultures, and the single colony of picking next day, amplification cultivation is extracted plasmid, carries out Analysis and Identification through restriction endonuclease EcoRI spectrum.
The result is: pDF-SacB-HBsL and pDF-SacB-HBsH carrier are in can secreting the BS1365 bacterium of Cre proteolytic enzyme, and after the reorganization of Cre-Loxp mediation, recombinate in the heavy chain zone of each plasmid, therefore obtain 4 kinds of different plasmids.Except that 2 kinds of parent vector, contain the pDF-HBsL-H of weight chain and the pDF-D-SacB of no antibody gene in addition simultaneously.Carry out reconstruction experiment altogether 9 times, identified 180 clones, obtained four kinds of carriers.
Above-mentioned four kinds of carriers are carried out enzyme through restriction endonuclease EcoR I cut evaluation, the result is: enzyme slitting band is 4583bp, and 2639bp is pDF-SacB-HBsL; Enzyme slitting band is 3800bp, and 3444bp is pDF-SacB-HBsH; Enzyme slitting band is 3804bp, and 2654bp, 2026bp are PDF-D-SacB; Enzyme slitting band is that 5966bp is PDF-HBsL-H.
3、ELISA
Is that the 200nmol/L carbonate buffer solution is diluted to 7.62ug/ml with HBsAg (Huamei Bio-Engrg Co.) with the concentration of pH9.8; Encapsulate elisa plate with the 50ul/ hole, 4 ℃ are spent the night, and discard liquid in the hole next day; Behind 37 ℃ of sealings of skimmed milk 100ul with 12% 2h; PBST (lavation buffer solution, prescription: tween 20, sodium-chlor 5g, Repone K 0.125g, potassium primary phosphate 0.125g, Sodium phosphate, dibasic 0.18g, zero(ppm) water adds to 1000ml) wash 5 times; (the antagonist sample carries out 10 times of dilutions of successively decreasing, and is diluted to 10 to add the above-mentioned 2 phage antibody samples that obtain
-6, dilute with 10% skimmed milk.), 37 ℃ hatch 2h after, again with PBST washing 5 times, adds the HRP-goat-anti M13 (Pharmacia) of dilution in 3: 1000, hatch 2h for 37 ℃, add the OPD substrate after the last PBST washing 5 times and develop the color, read the A490 value.With the negative contrast of carrier pDF-D-SacB.The experiment triplicate, results averaged.
The result is specific as follows: pDF-D-SacB and antibody sample (pDF-HBsL-H) be respectively 0.167 and 0.610 at 0 times of dilution OD490 (No. 1 dilute the result);
PDF-D-SacB and antibody sample (pDF-HBsL-H) be respectively 0.133 and 0.387 at 10 times of dilution OD490 (No. 2 dilute the result);
PDF-D-SacB and antibody sample (pDF-HBsL-H) be respectively 0.061 and 0.293 at 100 times of dilution OD490 (No. 3 dilute the result);
PDF-D-SacB and antibody sample (pDF-HBsL-H) be respectively 0.071 and 0.273 at 1000 times of dilution OD490 (No. 4 dilute the result);
PDF-D-SacB and antibody sample (pDF-HBsL-H) be respectively 0.068 and 0.212 at 10000 times of dilution OD490 (No. 5 dilute the result);
PDF-D-SacB and antibody sample (pDF-HBsL-H) be respectively 0.056 and 0.154 at 100000 times of dilution OD490 (No. 6 dilute the result);
PDF-D-SacB and antibody sample (pDF-HBsL-H) be respectively 0.083 and 0.127 at 1000000 times of dilution OD490 (No. 7 dilute the result);
It is as shown in Figure 6 to map, and wherein, the 1-7 difference is 10 times of doubling dilutions successively, from figure, finds out, the phage antibody sample is the activity that combines of HBsAg with hepatitis B surface antigen, apparently higher than the combine activity of carrier pDF-D-SacB with HBsAg.The phasmid carrier that utilizes present method to make up is described, is can be used in and make up large vol, high-quality phage antibody library.
Claims (9)
1. carrier, according to following method preparation:
1) encoding sox 1 with SacB is inserted between the MCS of pDF, obtains intermediate carrier PSBX;
2) the BssHII restriction enzyme site of the intermediate carrier PSBX that step 1) is obtained changes the BglII restriction enzyme site into, obtains intermediate carrier PSGX;
3) encoding sox 2 with SacB is inserted into step 2) between the Nhe I and Nco I restriction enzyme site of the intermediate carrier PSGX that obtains, obtain carrier pDF-D-SacB;
The encoding sox 1 of said SacB for the sequence 1 of sequence table from 5 ' terminal 30-1908 position Nucleotide;
The encoding sox 2 of said SacB for the sequence 2 in the sequence table from 5 ' terminal 7-1857 position Nucleotide.
2. carrier according to claim 1 is characterized in that:
In the step 1), the MCS that said encoding sox 1 with SacB is inserted into pDF is inserted between the BssHII and Xba I restriction enzyme site of pDF for the encoding sox 1 with SacB.
3. carrier, according to following method preparation:
1) with puc19-SacB is template, carries out pcr amplification to 1, obtain PCR product 1, PCR product 1 is inserted between the BssHII and Xba I restriction enzyme site of pDF, obtain intermediate carrier PSBX with primer; Said PCR product 1 is the encoding sox 1 of SacB, the encoding sox of said SacB 1 for the sequence 1 of sequence table from 5 ' terminal 30-1908 position Nucleotide;
Said primer is classified the sequence 5 in the sequence table as to the nucleotides sequence of a primer in 1, and said primer is classified the sequence 6 in the sequence table as to the nucleotides sequence of another primer in 1;
2) the BssHII restriction enzyme site on the intermediate carrier PSBX that step 1) is obtained is changed into the BglII restriction enzyme site, obtains intermediate carrier PSGX;
3) with pUC19-SacB be template, carry out pcr amplification to 2, obtain PCR product 2, the PCR product 2 that obtains is inserted into step 2 with primer) between the Nhe I and Nco I site of the intermediate carrier PSGX that obtains, obtain recombinant vectors pDF-D-SacB; Said PCR product 2 is the encoding sox 2 of SacB, the encoding sox of said SacB 2 for the sequence 2 in the sequence table from 5 ' terminal 7-1857 position Nucleotide;
Said primer is classified the sequence 7 in the sequence table as to the nucleotides sequence of a primer in 2, and said primer is classified the sequence 8 in the sequence table as to the nucleotides sequence of another primer in 2.
4. the transgenic cell line or reorganization bacterium or the recombinant virus that contain arbitrary described carrier among the claim 1-3.
5. the application of arbitrary described carrier in the preparation phage antibody library among the claim 1-3.
6. the said transgenic cell of claim 4 ties up to the application in the preparation phage antibody library.
7. the application of the said reorganization of claim 4 bacterium in the preparation phage antibody library.
8. the application of the said recombinant virus of claim 4 in the preparation phage antibody library.
9. according to arbitrary described application among the claim 5-8, it is characterized in that: in the said application, said antibody is the anti-hepatitis b surface antigen antibody.
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| Zhang GM, et al."Modification and identification of a vector for making a large phage antibody library.".《Chin Med J (Engl).》.2007,第120卷(第22期), |
| 乔媛媛 等."大容量噬菌体抗体库的构建及鉴定".《中华微生物学和免疫学杂志》.2004,第24卷(第3期), |
| 吴菁 等."枯草芽孢杆菌sacB基因的功能验证及应用".《核农学报》.2008,第22卷(第5期), |
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