CN105821480A - Anti-orf virus two-humped camel VHH heavy-chain single-domain antibody cDNA library and preparation method thereof - Google Patents
Anti-orf virus two-humped camel VHH heavy-chain single-domain antibody cDNA library and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an anti-orf virus two-humped camel VHH heavy-chain single-domain antibody cDNA library and a preparation method thereof. The library contains an antigen dosage of a camel subjected to orf vaccine immunity, an immune procedure and a serum antibody valence. The preparation method of the library comprises the following steps that camel peripheral blood lymphocyte separation, extraction of total RNA and separation and purification of mRNA are performed; reverse transcription is conducted on CDS III and SMART primers to synthesize total-length cDNA, two sets of amplification primers are designed, and a homologous recombination method is adopted to conduct yeast cell Y187cotransfection on amplification products and pGADT7; the anti-orf virus camel VHH gene cDNA library is established; the capacity of the library is calculated; cDNA library amplification is performed for quality evaluation. The anti-orf virus two-humped camel VHH heavy-chain single-domain antibody cDNA library is an antibody library having good virus specificity and biological activities, strong in antigen combining ability and a potential neutralizing function, especially is a novel single-domain type antibody and has the extremely important effect on establishment of a clinical orf virus diagnosis method and research of virus infection mechanism.
Description
Technical field
Two-humped camel VHH single domain heavy chain antibody cDNA library that the present invention relates to a kind of anti-sheep of virus and preparation method thereof, belongs to biological technical field.
Background technology
ORFV can cause the contact of sheep and the mankind to infect, and this disease is widely current in China in recent years, constitutes a threat to sheep husbandry and human health.As one of line-up of delegates that Poxviridae parapoxvirus belongs to, ORFV has phylogenetic feature and the multiformity of gene function of distinctness, its genome belongs to the distrand DNA of two ends Guan Bi, it is about 138kb, central authorities are core space (88kb), two ends are inverted repeat (Invertedterminalrepeat, ITR), titled with pili annulati clamping structure.Central core district gene is the most conservative, major regulatory virus duplication, pack and export.Research finds that ORFV carries some special genes by the carrier advantage of spontaneous stable heredity, by the immune clearance effect limiting host that various expression products are collaborative, development and a whole set of the immune modulatory strategies of having evolved, establish virus and replicated smoothly and design immunologic escape.As can be seen here, when ORFV and host's interaction, each one does his duty for the correlation function gene of virus, finally makes virus be replicated smoothly and immunologic escape.Therefore, ORFV Rapid&Early diagnosis technology to be set up, specifies the pathogenesis of ORFV, need to start with from viral function antibody, screening viral function antibody by virus novel nano VHH antibody library is the key solving this problem, and building VHHcDNA library is the basis solving this problem.
Nano antibody, from finding so far, has developed into extensive biologic applications and is worth and the highly versatile molecule of clinical value.Nano antibody has and derives from the minimum functional antigen binding fragment of HCAbs in adult camel body, has high stability and the high affinity being combined with antigen, can be with albumen crack and the interaction of enzyme active sites, and the effect of being allowed to is similar to inhibitor.Therefore, nano antibody can be to design, from peptide aids drug, the thinking that little molecule enzyme inhibitor provides new.Due to only heavy chain, the manufacture of nano antibody is easy compared with mAb.The peculiar property of nano antibody, as being in the stability in extreme temperature and pH environment, has the incomparable water solublity of conventional single domain antibody and conformational stability.This monomer domain energy high specific, be combined with antigen high-affinity, thus neutralize or close the most hidden epitope.Have great advantage compared with common antibody by above characteristic single domain antibody.Single domain antibody be currently available have complete function, stable, can the minimum unit of conjugated antigen, owing to relative molecular mass is little, stability is strong, solubility is good, antigen-binding can expression good, easy and immunogenicity is low etc. that feature becomes the important target molecule in current molecular imaging study, this provides a good source for engineered antibody, has broad application prospects.Therefore, nano antibody has the highest value in the treatment and diagnosis of disease.
Therefore, comprehensive at present at research and the utilization power of antibody art, it is seen that conventional antibodies class medicine still Challenge, it is necessary to design and produce a new generation's more effectively antibody molecule with the needs of satisfied clinic.In particular how realize antibody miniaturization so that it is efficiently act on specific part.To this end, realize antibody miniaturization target cancer cell internal effect molecule or other target cells are always important goal and the study hotspot of antibody engineering and anticancer research.
The capacity in library and multiformity largely affect therefrom elutriation and go out the probability of specific antibody and the affinity of antibody.Since first cDNA library built for 1976, its method is constantly improved, the cDNA library that wherein SMART (switchingmechanismat5 ' endoftheRNAtranscript) method builds can represent the abundance of mRNA in original sample, saves the integrity of biological heredity information.And the TotalRNA of the method mRNA or 50ng of having only to 25ng can be obtained by the cDNA storehouse of high-quality, high yield, the more important thing is that the cDNA obtained can represent the abundance of the mRNA in original sample, can apply to direct amplification gene, construction cDNA library.Yeast-two hybrid technique can filter out, from the host cell cDNA library built, the albumen interacted with known destination protein simultaneously.The present invention successfully constructs Yeast two-hybrid cDNA library, and the storage capacity in this library is 6.1 × 106Cfu/ml, library recombination fraction is 100%, can be sore mouth virus and other sheep diseases viral disease gene functions and pathogenesis lays the foundation.
Summary of the invention
Given this, two-humped camel VHH single domain heavy chain antibody cDNA library that the present invention relates to a kind of anti-sheep of virus and preparation method thereof, because of VHH nano antibody have that relative molecular weight is little, tissue penetration is strong, internal removing is fast, be prone to Escherichia coli fermentation produces and carry out the advantage of genetic engineering improvement, therefore has bigger advantage and development prospect than the monoclonal antibody of complete length.The present invention is directed to the VHHcDNA library that sheep of virus builds is a kind of to possess the antibody library that type specificity, biologic activity are good, conjugated antigen ability strong, have potential neutralization function, the especially novel antibodies of single domain antibody etc, for setting up the methods for clinical diagnosis of sheep of virus, the infection mechanism of research virus has extremely important effect.
The present invention is by the techniques below means above-mentioned technical problem of solution:
The two-humped camel VHH single domain heavy chain antibody cDNA library of a kind of anti-sheep of virus, it comprises the antigen dose of sore mouth virus vaccine immunity camel, immune programme for children and serum antibody titer.
A kind of anti-sheep of virus two-humped camel VHH single domain heavy chain antibody cDNA library, two-humped camel immune programme for children and antigen inoculation dosage are, head exempts from 15 parts, two exempts from 30 parts, three exempts from 30 parts, within 21st day, carry out after exempting from headed by described immune programme for children two exempting from, within the 35th day, carry out three and exempt from, venous blood is gathered before first immunisation and after each immunity, serum antibody titer, after antibody reaches requirement of experiment, venous blood collection is detected with ORFV antibody ELISA detection kit.
A kind of preparation method of anti-sheep of virus two-humped camel VHH single domain heavy chain antibody cDNA library, it comprises the following steps:
(1) camel separation of lymphocytes, Total RNAs extraction and mRNA separation, purification；
(2) CDS III and SMART primer reverse transcription synthesis full-length cDNA, design two set amplimer, use the method for homologous recombination by amplified production and pGADT7 cotransfection yeast cells Y187；
(3) the VHH gene cDNA library of the anti-sheep of virus of camel is built；
(4) library storage capacity is calculated；
(5) amplification cDNA library carries out quality evaluation.
A kind of preparation method of the two-humped camel VHH single domain heavy chain antibody cDNA library of anti-sheep of virus, step (1) camel separation of lymphocytes step is as follows, jugular vein gathers camel anticoagulation 100ml, equivalent adds whole blood diluent, mixing, lymphocyte separation medium is first added with the ratio of 5:9, add, after elder generation is slow afterwards, the anticoagulation diluted soon, horizontal centrifuge 1800rpm, 18-22 DEG C, 20min, careful collection buffy coat is in new collecting pipe, it is diluted with 5 times amount cell washing solution, horizontal centrifuge 1000rpm, 18-22 DEG C, 10min, abandon supernatant, precipitation dilutes with cell washing solution again, horizontal centrifuge 1000rpm, 18-22 DEG C, 10min is centrifuged；Abandoning supernatant, precipitation is the lymphocyte being separated to, and after taking suspension cell supernatant and counting and be suspended in RNAlater, puts-70 DEG C and saves backup.
A kind of preparation method of the two-humped camel VHH single domain heavy chain antibody cDNA library of anti-sheep of virus, step (1) Total RNAs extraction step is for drawing above-mentioned lymphocyte liquid 400ul, add 1mlTRizol, mixing, 4 DEG C stand 5min, add 200ul chloroform, mixing, room temperature stands 3min, 12000r/min, 4 DEG C, centrifugal 15min, draw 750ul supernatant, add equivalent isopropanol, room temperature stands 10min, 10000r/min, 4 DEG C, centrifugal 10min, gently abandon supernatant, precipitation adds 75% ethanol, 8000r/min, 4 DEG C, centrifugal 5min, precipitation is put fume hood 10min and is allowed to dry, 20ul is the total serum IgE of results without the water-soluble precipitation of RNase.
A kind of preparation method of the two-humped camel VHH single domain heavy chain antibody cDNA library of anti-sheep of virus, in described step (2), two set amplimers are, first round amplimer:
F1:5-GTCCTGGCTGCTTCTACAAGG-3
R1:5-GGTACGTGGTTGAACTGTTCC-3
Second takes turns amplimer:
F2:5-TTCCACCCAAGCAGTGGTATCAAGTGGGAGTCTGGGGGAGG-3
R2:5-GTATCGATGCCCACCCTCTAGCCGAGGCGGCCGACATGGAGACGGTGACCTGGGT-3
A kind of preparation method of anti-sheep of virus two-humped camel VHH single domain heavy chain antibody cDNA library, step (3) builds the step in VHH gene cDNA library of the anti-sheep of virus of camel for using PEG/LiAc method to prepare yeast Y187 competent cell, every 600uL competent cell converts 20ul double-strand cDNA and 6ulpGADT7-Rec, the bacterium solution of conversion is spread evenly across on the SD/-Leu flat board with 150mm of diameter 100mm, it is inverted for 30 DEG C and cultivates 3-5d until clone occurs, again flat board is placed in 4 DEG C of refrigerators, place 3-4h, 5mL frozen stock solution (the YPDA fluid medium of 25% glycerol) is added to every piece of flat board, and add sterile glass beads, horizontal direction is shaken repeatedly, collect bacterium solution afterwards.
A kind of preparation method of anti-sheep of virus two-humped camel VHH single domain heavy chain antibody cDNA library, in step (5), amplification cDNA library carries out quality evaluation for taking cotransformation product bacterium solution, by 1/10, 1/100 and 1/1000 dilution, it is coated with 100ul diluent respectively to 100mmSD/-Leu flat board, it is inverted for 30 DEG C and cultivates 3-5d until clone occurs, the now single colony counting to growth, and calculate library capacity, random 16 single bacterium colonies of picking, bacterium colony PCR is carried out with pGADT-Rec universal sequencing primer thing, analyze size and the recombination fraction of library inserts, PCR response parameter: 94 DEG C of denaturations 5min；94 DEG C of degeneration 1min, 56 DEG C of annealing 1min, 72 DEG C extend 2min, totally 30 circulations, and last 72 DEG C extend 10min.Take 7ulPCR product and carry out 1.2% agarose gel electrophoresis analysis.
nullThe invention have the advantage that the cDNA library that the present invention provides forgives whole VHH antibody information of Orf virus protein in theory，Research material is provided for screening the whole VHH genes for sheep of virus afterwards，Because of VHH nano antibody, to have relative molecular weight little、Tissue penetration is strong、Internal removing is fast、It is prone to Escherichia coli fermentation produce and carry out the advantages such as genetic engineering improvement，Therefore than the monoclonal antibody of complete length, there is bigger advantage and development prospect，The cDNA library that the present invention is directed to sheep of virus structure is that one possesses type specificity、Biologic activity is good、Conjugated antigen ability is strong、There is the antibody library of potential neutralization function，The especially novel antibodies of single domain antibody etc，For setting up the methods for clinical diagnosis of sheep of virus，The infection mechanism etc. of research virus has extremely important effect.
Accompanying drawing explanation
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Wherein Fig. 1 is camel peripheral blood lymphocyte total serum IgE electrophoretogram；
Fig. 2 is the camel peripheral blood lymphocyte dscDNA first round to expand agarose gel electrophoresis figure；
Fig. 3 is that camel peripheral blood lymphocyte dscDNA second takes turns amplification agarose gel electrophoresis figure；
Fig. 4 is camel peripheral blood lymphocyte Yeast two-hybrid cDNA library monoclonal PCR amplification.
Detailed description of the invention
Below with reference to accompanying drawing, the present invention is described in detail.
1.1.1 experimental animal, antigen, adjuvant and sheep of virus antibody assay kit
The 1 male two-humped camel in peak (1 one full year of life), the 1 female two-humped camel in peak (6 months are big)；ORFV antibody ELISA detection kit is purchased from BIOPORTO company of the U.S..
1.1.2 bacterial strain and reagent
Yeast libraries relevant carriers is purchased from Clontech, restricted enzyme reclaim test kit purchased from NEB, lymphocyte separation medium (1.077) purchased from the raw work in Shanghai, Trizol, nickel affinity chromatography resin, DNA fragmentation and plasmid extraction kit be OMEGA product, PCR related reagent purchased from precious biotech firm, molecular biology reagents from Sigma company；Other biochemical reagents are domestic analytical pure.
1.2 method
1.2.1 the mensuration of antigen inoculation dosage, immune programme for children and serum antibody titer
Cervical region two-humped camel is subcutaneous, dorsal sc and back leg is subcutaneous carries out branch inoculation, many 3 vaccinations in each position.Gather venous blood before first immunisation and after each immunity, detect serum antibody titer, after antibody reaches requirement of experiment, venous blood collection with ORFV antibody ELISA detection kit.Concrete animal immune program and antigen inoculation dosage are as follows:
1.2.2 separation of lymphocytes
After mensuration antibody titer reaches requirement, jugular vein gathers camel anticoagulation 100ml, and equivalent adds whole blood diluent, mixing；First add lymphocyte separation medium with the ratio of 5:9, the slowest afterwards after add the anticoagulation diluted soon, horizontal centrifuge 1800rpm, 18-22 DEG C, 20min；Careful collection buffy coat, in new collecting pipe, dilutes it with 5 times amount cell washing solution, horizontal centrifuge 1000rpm, 18-22 DEG C, 10min；Abandoning supernatant, precipitation dilutes with cell washing solution again, horizontal centrifuge 1000rpm, 18-22 DEG C, and 10min is centrifuged；Abandoning supernatant, precipitation is the lymphocyte being separated to, and after taking suspension cell supernatant and counting and be suspended in RNAlater, puts-70 DEG C and saves backup.
1.2.3 the extraction of lymphocyte total serum IgE and RT-PCR
Drawing above-mentioned lymphocyte liquid 400ul, add 1mlTRizol, mixing, 4 DEG C stand 5min；Adding 200ul chloroform, mixing, room temperature stands 3min, 12000r/min, 4 DEG C, centrifugal 15min；Drawing 750ul supernatant, add equivalent isopropanol, room temperature stands 10min, 10000r/min, 4 DEG C, centrifugal 10min；Gently abandoning supernatant, precipitation adds 75% ethanol, 8000r/min, 4 DEG C, is centrifuged 5min；Precipitation is put fume hood 10min and is allowed to dry；20ul is the total serum IgE of results without the water-soluble precipitation of RNase, and it is 4029ng/ul that ND2000 records TotalRNA concentration, and purity OD260/OD280 is 1.92.Its electrophoresis detection is shown in Fig. 1
1.2.4 the design of primer is prepared in library
First round amplimer:
F1:5-GTCCTGGCTGCTTCTACAAGG-3
R1:5-GGTACGTGGTTGAACTGTTCC-3
Second takes turns amplimer:
F2:5-TTCCACCCAAGCAGTGGTATCAAGTGGGAGTCTGGGGGAGG-3
R2:5-GTATCGATGCCCACCCTCTAGCCGAGGCGGCCGACATGGAGACGGTGACCTGGGT-3
1.2.5cDNA synthesis double-strand and purification
1.2.5.1 whole gene cDNA synthesis
Expand at the PCR of 0.2ml and pipe be sequentially added into following component:
Mixing, is positioned over centrifuge tube 72 DEG C of metal baths, hatches 2min, put on ice immediately, cools down 2min, is sequentially added into following component:
Mixing, is positioned over 42 DEG C of metal baths, reverse transcription 1h by centrifuge tube, and room temperature cools down, adds 1 μ lRNA enzyme inhibitor (2u/ul), and the product obtained is full-length genome cDNA, puts-20 DEG C and saves backup.
1.2.5.2 first round amplification
Expand at the PCR of 0.2ml and pipe be sequentially added into following component:
Above-mentioned amplification program: 95 DEG C of 1min；95 DEG C of 15s, 68 DEG C of 5min, 20 circulations；68℃5min.By amplified production electrophoresis, purified pool 600bp product ,-20 DEG C save backup.
1.2.5.3 second takes turns amplification
Expand at the PCR of 0.2ml and pipe be sequentially added into following component:
Above-mentioned amplification program: 95 DEG C of 1min；95 DEG C of 15s, 68 DEG C of 5min, 20 circulations；68℃5min.Amplified production electrophoresis, gel reclaims test kit purified pool 400bp product from gel, puts-20 DEG C and save backup.
1.2.7 the structure in library and results
PEG/LiAc method is used to prepare yeast Y187 competent cell.Every 600uL competent cell converts 20ul double-strand cDNA and 6ulpGADT7-Rec, the bacterium solution of conversion is spread evenly across on the SD/-Leu flat board with 150mm of diameter 100mm, is inverted for 30 DEG C and cultivates 3-5d until clone occurs.Again flat board is placed in 4 DEG C of refrigerators, places 3-4h.Adding 5mL frozen stock solution (the YPDA fluid medium of 25% glycerol) to every piece of flat board, and add sterile glass beads, horizontal direction is shaken repeatedly, collects bacterium solution afterwards.
1.2.8 the quality evaluation in library
Take cotransformation product bacterium solution, by 1/10,1/100 and 1/1000 dilution, be coated with 100ul diluent respectively and cultivate 3-5d until clone occurs to 100mmSD/-Leu flat board, 30 DEG C of inversions, the now single colony counting to growth, and calculate library capacity.Random 16 single bacterium colonies of picking, carry out bacterium colony PCR with pGADT-Rec universal sequencing primer thing, analyze size and the recombination fraction of library inserts.PCR response parameter: 94 DEG C of denaturations 5min；94 DEG C of degeneration 1min, 56 DEG C of annealing 1min, 72 DEG C extend 2min, totally 30 circulations, and last 72 DEG C extend 10min.Take 7ulPCR product and carry out 1.2% agarose gel electrophoresis analysis.
2. result
The extraction of 2.1 camel peripheral blood lymphocyte total serum IgE and mRNA's is isolated and purified
Extract total serum IgE through ultraviolet spectrophotometer measure, concentration be 4029ng/ul, OD260/OD280 be 1.92.28S, 18S, 5S3 bar clear band seen from Denaturing Agarose Gel electrophoresis, 28S:18S is about 2:1.Illustrate that RNA and mRNA purity is higher, meet the needs of library construction, as shown in Figure 1.M.DL5000 relative molecular mass standard；1. camel peripheral blood lymphocyte total serum IgE.
The agarose gel electrophoresis of 2.2 double-stranded cDNA library
The dscDNA product taking first round amplification carries out electrophoresis, collects 600bp band.Take the second dscDNA product taking turns amplification and carry out electrophoresis, collect 400bp band.As shown in Figure 2 and Figure 3.M.DL2000 relative molecular mass standard；1,2. first round amplification dsDNA, 600bp, M.DL2000 relative molecular mass standard；1,2. second take turns amplification dsDNA, 400bp
2.3 library capacity and identification of polymorphisms
The flat board in dilution factor libraries different to coating carries out single colony counting, according to formula: clump count/tiling bacterium solution volume (ml) × dilution gfactor on library titre=flat board, is computed pGADT7-Rec library titre and is about 6.1 × 106cfu/ml.From SD/-Leu flat board, random 15 clones of picking carry out bacterium colony PCR, and electrophoresis result display library inserts is 400-2000bp, and average Insert Fragment is about 1000bp.The recombination fraction being appreciated that library by bacterium colony PCR result is 100%.As shown in Figure 4.M.DL5000 relative molecular mass standard；The PCR result of 1-15: yeast colony.
Finally illustrate is, above example is only in order to illustrate technical scheme and unrestricted, although the present invention being described in detail with reference to preferred embodiment, it will be understood by those within the art that, technical scheme can be modified or equivalent, without deviating from objective and the scope of technical solution of the present invention, it all should be contained in the middle of scope of the presently claimed invention.
Claims (8)
1. the two-humped camel VHH single domain heavy chain antibody cDNA library of an anti-sheep of virus, it is characterised in that: comprise the antigen dose of sore mouth virus vaccine immunity camel, immune programme for children and serum antibody titer.
The two-humped camel VHH single domain heavy chain antibody cDNA library of a kind of anti-sheep of virus the most according to claim 1, it is characterized in that: described two-humped camel immune programme for children and antigen inoculation dosage are, head exempts from 15 parts, two exempts from 30 parts, three exempts from 30 parts, within 21st day, carry out after exempting from headed by described immune programme for children two exempting from, within the 35th day, carry out three and exempt from, venous blood is gathered before first immunisation and after each immunity, serum antibody titer is detected with ORFV antibody ELISA detection kit, after antibody reaches requirement of experiment, venous blood collection.
The preparation method of a kind of anti-sheep of virus two-humped camel VHH single domain heavy chain antibody cDNA library the most according to claim 1, it is characterised in that comprise the following steps:
(1) camel separation of lymphocytes, Total RNAs extraction and mRNA separation, purification；
(2) CDS III and SMART primer reverse transcription synthesis full-length cDNA, design two set amplimer, use the method for homologous recombination by amplified production and pGADT7 cotransfection yeast cells Y187；
(3) the VHH gene cDNA library of the anti-sheep of virus of camel is built；
(4) library storage capacity is calculated；
(5) amplification cDNA library carries out quality evaluation.
The preparation method of a kind of anti-sheep of virus two-humped camel VHH single domain heavy chain antibody cDNA library the most according to claim 3, it is characterized in that: described step (1) camel separation of lymphocytes step is as follows, jugular vein gathers camel anticoagulation 100ml, equivalent adds whole blood diluent, mixing, lymphocyte separation medium is first added with the ratio of 5:9, add, after elder generation is slow afterwards, the anticoagulation diluted soon, horizontal centrifuge 1800rpm, 18-22 DEG C, 20min, careful collection buffy coat is in new collecting pipe, it is diluted with 5 times amount cell washing solution, horizontal centrifuge 1000rpm, 18-22 DEG C, 10min, abandon supernatant, precipitation dilutes with cell washing solution again, horizontal centrifuge 1000rpm, 18-22 DEG C, 10min is centrifuged；Abandoning supernatant, precipitation is the lymphocyte being separated to, and after taking suspension cell supernatant and counting and be suspended in RNAlater, puts-70 DEG C and saves backup.
nullThe preparation method of a kind of anti-sheep of virus two-humped camel VHH single domain heavy chain antibody cDNA library the most according to claim 3，It is characterized in that: described step (1) Total RNAs extraction step is for drawing above-mentioned lymphocyte liquid 400ul，Add 1mlTRizol，Mixing，4 DEG C stand 5min，Add 200ul chloroform，Mixing，Room temperature stands 3min，12000r/min，4℃，Centrifugal 15min，Draw 750ul supernatant，Add equivalent isopropanol，Room temperature stands 10min，10000r/min，4℃，Centrifugal 10min，Gently abandon supernatant，Precipitation adds 75% ethanol，8000r/min，4℃，Centrifugal 5min，Precipitation is put fume hood 10min and is allowed to dry，20ul is the total serum IgE of results without the water-soluble precipitation of RNase.
The preparation method of a kind of anti-sheep of virus two-humped camel VHH single domain heavy chain antibody cDNA library the most according to claim 3, it is characterised in that: in described step (2), two set amplimers are, first round amplimer:
F1:5-GTCCTGGCTGCTTCTACAAGG-3
R1:5-GGTACGTGGTTGAACTGTTCC-3
Second takes turns amplimer:
F2:5-TTCCACCCAAGCAGTGGTATCAAGTGGGAGTCTGGGGGAGG-3
R2:5-GTATCGATGCCCACCCTCTAGCCGAGGCGGCCGACATGGAGACGGTGACCTGGGT-3。
nullThe preparation method of a kind of anti-sheep of virus two-humped camel VHH single domain heavy chain antibody cDNA library the most according to claim 3，It is characterized in that: described step (3) builds the step in VHH gene cDNA library of the anti-sheep of virus of camel for using PEG/LiAc method to prepare yeast Y187 competent cell，Every 600uL competent cell converts 20ul double-strand cDNA and 6ulpGADT7-Rec，The bacterium solution of conversion is spread evenly across on the SD/-Leu flat board with 150mm of diameter 100mm，It is inverted for 30 DEG C and cultivates 3-5d until clone occurs，Again flat board is placed in 4 DEG C of refrigerators，Place 3-4h，5mL frozen stock solution (the YPDA fluid medium of 25% glycerol) is added to every piece of flat board，And add sterile glass beads，Horizontal direction is shaken repeatedly，Collect bacterium solution afterwards.
The preparation method of a kind of anti-sheep of virus two-humped camel VHH single domain heavy chain antibody cDNA library the most according to claim 3, it is characterized in that: in described step (5), amplification cDNA library carries out quality evaluation for taking cotransformation product bacterium solution, by 1/10, 1/100 and 1/1000 dilution, it is coated with 100ul diluent respectively to 100mmSD/-Leu flat board, it is inverted for 30 DEG C and cultivates 3-5d until clone occurs, the now single colony counting to growth, and calculate library capacity, random 16 single bacterium colonies of picking, bacterium colony PCR is carried out with pGADT-Rec universal sequencing primer thing, analyze size and the recombination fraction of library inserts, PCR response parameter: 94 DEG C of denaturations 5min；94 DEG C of degeneration 1min, 56 DEG C of annealing 1min, 72 DEG C extend 2min, totally 30 circulations, and last 72 DEG C extend 10min.Take 7ulPCR product and carry out 1.2% agarose gel electrophoresis analysis.
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| CN108504654A (en) * | 2018-04-13 | 2018-09-07 | 中国农业科学院兰州兽医研究所 | The yeast cDNA library and its construction method and purposes of a kind of anti-Sai Nika paddy virus VHH antibody |
| CN108676792A (en) * | 2018-05-23 | 2018-10-19 | 中国农业科学院兰州兽医研究所 | One kind is based on anti-PPRV cDNA libraries in hunchbacked source and its preparation method and application |
| CN110759999A (en) * | 2018-07-27 | 2020-02-07 | 深圳康体生命科技有限公司 | Preparation method and vector of GFP antibody |
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| CN108676792B (en) * | 2018-05-23 | 2020-12-25 | 中国农业科学院兰州兽医研究所 | Camel source-based PPRV-resistant cDNA library and preparation method and application thereof |
| CN110759999A (en) * | 2018-07-27 | 2020-02-07 | 深圳康体生命科技有限公司 | Preparation method and vector of GFP antibody |
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