CN104233474A - Synthetic phage display nano antibody library and application thereof - Google Patents
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Abstract
The invention discloses a synthetic phage display nano antibody library which is based on a nano antibody conservative frame cAbBCII10, has randomized amino acid sequence in area CDR3 but doesn't contain cysteine and termination codon and can obtain a prealbumin nano antibody with specificity and detecting functions through library screening. The invention discloses an amino acid sequence of a realbumin nano antibody VHH chain and a gene sequence encoding the nano antibody. Besides, the invention further discloses a host cell capable of expressing the nano antibody and introduces an application of the prealbumin nano antibody in the aspect of detection of prealbumin.
Description
Technical field
The present invention relates to biomedicine and biology field, the nano antibody for prealbumin PA relating to a kind of phage display nano antibody library and obtain based on this library screening.
Background technology
Phage display is current nano antibody library construction method the most extensively.The nano antibody library produced by the camel of immunity can keep complete diversity.Although the nano antibody of high-affinity can screen acquisition at short notice, but in some cases, camel immunity cannot be realized, such as immunogen has high toxicity or pathogenic, protein misfolding forms inclusion body and cannot obtain soluble antigen, and antigen is from immunity and when some antigens are not micromolecular compounds with immunity etc.For addressing these problems, naive libraries or synthetic library become the selection obtaining the nano antibody that specificity is good, avidity is strong.The present invention is based on a kind of nano antibody consensus framework cAbBCII10, pass through Overlap extension PCR, in the changeless situation in maintenance FR1 district to FR3 district, utilize three base codon for raw material, what make CDR3 district has 16 amino acid and the random independent assortment sequence of these amino acid whose orders, gets rid of halfcystine simultaneously and occurs.By this new technology, the nano antibody library storage capacity that we obtain is 3 × 10
9, and quality is fine, and diversity is enriched.
Prealbumin (Prealbumin, PA), also known as transthyretin, molecular weight 5.4 ten thousand, is synthesized by liver cell, and when electrophoretic separation, be often presented at albuminous front, its transformation period is very short, only about 1.9 days.Therefore, measure its concentration in blood plasma, for understanding the malnutrition of protein, hepatic insufficiency, the albumin of ratio and Transferrins,iron complexes, there is higher susceptibility.Principle of work in the market for detecting the test kit of prealbumin realizes mainly through a kind of antibody being directed to prealbumin, but this traditional Antibody stability is poor, sensitivity is low, production cost is high, and all factors all limit the detection for prealbumin.Belgium scientist finds that in camel blood nano antibody can be combined closely with the target such as antigen as normal antibody, has good structural stability and antigen-binding activity.
Summary of the invention
Technical problem solved by the invention is: build a kind of CDR3 region amino acid sequence based on nano antibody consensus framework cAbBCII10 and freely arrange at random but the phage display nano antibody library getting rid of halfcystine and terminator codon, utilizes this library screening to obtain for the nano antibody of prealbumin PA and the purposes in preparation detection reagent thereof simultaneously.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: provide a kind of phage display nano antibody library, this library comprises whole segment DNA sequence corresponding to FR1 district to the FR3 district of nano antibody consensus framework cAbBCII10, the DNA sequence dna that FR4 district is corresponding, and utilize three base codon to make CDR3 district containing the arrangement but get rid of the corresponding DNA sequence dna of halfcystine and terminator codon arbitrarily at random of 16 amino acid and amino acid, the DNA fragmentation comprising above-mentioned sequence is obtained by Overlap extension PCR, this DNA fragmentation and phagemid vector recombination to construct recombinant plasmid transformed intestinal bacteria are built into phage display nano antibody library.
Also provided is a kind of prealbumin nano antibody, its VHH chain comprises framework region FR and complementary determining region CDR, it is characterized in that, described framework region FR comprises the aminoacid sequence of FR1, FR2, FR3, FR4, its aminoacid sequence is respectively FR1 shown in SEQ ID NO:1, FR4 shown in the NO:4 of FR3 shown in FR2, SEQ ID NO:3, SEQ ID shown in SEQ ID NO:2; Described complementary determining region CDR comprises the aminoacid sequence of CDR1, CDR2, CDR3, its aminoacid sequence is respectively the CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or the CDR3 shown in SEQ ID NO:12.
Preferably, the VHH chain of described prealbumin nano antibody has following any one group of aminoacid sequence: aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:7; Or aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:8; Or aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:9; Or aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:10; Or aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:11; Or aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:12.
In addition, also adopt a kind of DNA molecular, it is in order to the VHH chain of prealbumin nano antibody of the present invention of encoding.
Preferably, described DNA molecular, has following any one group of nucleotide sequence: nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16; Or nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:17; Or nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:18; Or nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:19; Or nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:20; Or nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:21.
The present invention also provides a kind of expression vector, and it comprises the nucleotide sequence of prealbumin nano antibody DNA molecular of the present invention of encoding, and a kind of host cell, it can express the nano antibody of prealbumin.
Finally present invention also offers the purposes of prealbumin nano antibody of the present invention albumin aspect before detection.
The invention has the beneficial effects as follows: compared with prior art, advantage of the present invention is as follows: the present invention is based on a kind of nano antibody consensus framework cAbBCII10, pass through Overlap extension PCR, in the changeless situation in maintenance FR1 district to FR3 district, utilize three base codon for raw material, CDR3 district is made to have 16 amino acid and the random independent assortment sequence of these amino acid whose orders, get rid of halfcystine and terminator codon appearance simultaneously, the gene fragment utilizing Overlap extension PCR to obtain and phagemid vector construction recombination plasmid thus set up phage nano antibody gene pool, above-mentioned recombinant plasmid transformed intestinal bacteria are built into phage display nano antibody library, simultaneously, in test, prealbumin PA is coupled on enzyme plate, prealbumin PA in this format utilizes display technique of bacteriophage to screen and builds nano antibody gene pool, thus obtain for prealbumin PA specific nano antibody gene, this gene is gone in intestinal bacteria, thus establish can in the nano antibody strain of E. coli.The present invention adopts the novel phage display nano antibody library of structure can screen the nano antibody obtaining and have specificity and measuring ability, can solve and cannot cause building corresponding phage display nano antibody library and the problem that cannot obtain nano antibody by immune camel because antigen factor causes, such phage display nano antibody library is using the strong resource as biological study and medical diagnosis.
Accompanying drawing explanation
Fig. 1 is the mode chart of phage display nano antibody library construction and prealbumin nano antibody screening process;
Fig. 2 is the triphasic DNA electrophorogram of Overlap extension PCR;
Fig. 3 is the bacterium colony PCR electrophorogram constructed phage display nano antibody library being carried out to the detection of insertion rate, wherein swimming lane 1 is DNA molecular standard, and swimming lane 2-24 is that the clone PCR of random picking in constructed prealbumin nano antibody library detects electrophorogram;
Fig. 4 is the gradient dilution coated plate figure identified the storage capacity size in built library;
Fig. 5 is the mode chart that enzyme-linked immunoassay method (ELISA) with phage screens the single positive colony of specificity;
Fig. 6 is the nano antibody for prealbumin PA of expressing, the electrophorogram of the SDS-PAGE after affinity chromatography purifying; Wherein swimming lane 1 is containing the sample of 50 mmole imidazole elution institute wash-outs, swimming lane 2 is the samples containing 100 mmole imidazole elution institute wash-outs, swimming lane 3 is samples of 250 mmole imidazole elution institute wash-outs, and swimming lane 4 is samples of 500 mmole imidazole elution institute wash-outs;
Fig. 7 is that the nano antibody for prealbumin of expression is for detecting the ELISA experimental result histogram of prealbumin PA, X-coordinate is the concentration (mcg/ml) of prealbumin PA, and ordinate zou is the ratio that experimental group corresponding to different prealbumin PA concentration and control group wavelength 405 nm read value.
Embodiment
Below in conjunction with Figure of description, the present invention is further illustrated.
The present invention first please whole segment DNA sequence corresponding to FR1 district to the FR3 district of biotech firm synthesis of nano antibody consensus framework cAbBCII10 and utilize three base codon to make CDR3 district containing 16 amino acid and amino acid at random arbitrarily arrangement but get rid of the corresponding DNA sequence dna of halfcystine and terminator codon and PCR put up a bridge needed for corresponding primer, obtain DNA fragmentation by Overlap extension PCR, then with phagemid vector recombinate and transformation of E. coli build nano antibody gene library.In addition, prealbumin PA is coupled on NUNC enzyme plate, antigen in this format utilizes display technique of bacteriophage screening from the library built in the nano antibody strain for prealbumin PA of E. coli, and to utilize this nano antibody to carry out detection experiment to prealbumin PA.
Below in conjunction with specific embodiment, set forth the present invention further.
Embodiment 1: based on nano antibody consensus framework cAbBCII10 the random liberalization of CDR3 region amino acid sequence but get rid of the structure in phage display nano antibody library of halfcystine:
(1) the whole segment DNA sequence of asking FR1 district to the FR3 district of biotech firm synthesis of nano antibody consensus framework cAbBCII10 corresponding, i.e. SEQ ID NO:15, 5 '-CAA GTT CAA TTG GTT GAA TCT GGT GGT GGT TCT GTT CAA GCT GG TGG TTC TTT GAG ATT GTC TTG TAC TGC TTC TGG TGG TTC TGA ATAT TCT TAT TCT ACT TTT TCT TTG GGT TGG TTT AGA CAA GCT CCA GGT CAA GAA AGA GAA GCT GTT GCT GCT ATT GCT TCT ATG GGT GGT TTG ACT TAT TAT GCT GAT TCT GTT AAA GGT AGA TTT ACT ATT TCT AGA GAT AAT GCT AAA AAT ACT GTT ACT TTG CAA ATG AAT AAT TTG AAA CCA GAA GAT ACT GCT ATT TAT TAT TGT GCT GCT-3 ', use as PCR masterplate 1, synthesis utilizes three base codon to make CDR3 district containing 16 amino acid and amino acid is arranged arbitrarily at random but got rid of the corresponding DNA sequence dna of halfcystine and terminator codon, i.e. SEQ ID NO:22,5 '-ACT GCT ATT TAT TAT TGT GCT GCT [N]
16tGG GGT CAA GGT ACT CAA-3 ', uses as upstream primer 2 simultaneously, synthesize all the other primers: upstream primer 1 (SEQ ID NO: 23), 5 '-CAT ATG CAA GTT CAA TTG GTT GAA-3 ', downstream primer 1 (SEQ ID NO: 24), 5 '-AGC AGC ACA ATA ATA AAT-3 ', downstream primer 2 (SEQ ID NO: 25), 5 '-GAA TTC CTA AGA AGA AAC AGT AAC TTG AGT ACC TTG ACC CCA-3 '.(2) above-mentioned masterplate or primer are carried out over-lap PCR extend obtain FR1, CDR1, FR2, CDR2, FR3 district and FR4 district constant, CDR3 district contains 16 amino acid and amino acid is arranged arbitrarily at random but do not contained the DNA fragmentation of the correspondence of halfcystine and terminator codon, result such as Fig. 2 shows, and the size of this fragment is about 381 base numbers.Fig. 2 is from left to right respectively: the left side is with whole segment DNA sequence corresponding to the FR1 district of cAbBCII10 to FR3 district for masterplate, and upstream primer 1 and downstream primer 1 are extend the DNA fragmentation that primer obtains; Centre is that arrangement but the corresponding DNA sequence dna getting rid of halfcystine and terminator codon are that masterplate is simultaneously for primer and downstream primer 2 increase the DNA fragmentation produced arbitrarily at random with CDR3 district amino acid; The right is that the final DNA fragmentation (3) that Overlap extension PCR produces uses restrictive restriction endonuclease Pst I and Not I enzyme cut 96 microgram pMECS Vector for Phage Display (Biovector supply) and 32 microgram VHH and connect two fragments.(5) connection product electricity is converted into Divine Land, competent cell TG1(Beijing red autumnal leaves Science and Technology Ltd.) in, build the CDR3 region amino acid sequence randomization based on nano antibody consensus framework cAbBCII10 but get rid of the phage display nano antibody library of halfcystine and terminator codon and measure storage capacity, the size of storage capacity is 3 × 10
9, it is 10 that extension rate is worked as in Fig. 4 display
6time, clone's quantity is about 32; Meanwhile, random picking 24 clone, by bacterium colony PCR detect build library insertion rate detected result insertion rate be that 100%, Fig. 3 shows bacterium colony PCR result.After library construction completes, random picking 6 cloning and sequencings, detect CDR3 district exactness and diversity, result shows CDR3 district, this library really containing 16 amino acid and not containing halfcystine and terminator codon, simultaneously 6 clones have 5 kinds of different CDR3 district DNA sequence dnas, and coding 5 kinds of different aminoacids sequences, show that diversity is fine.
Embodiment 2: the screening process for the nano antibody of prealbumin PA:
(1) getting 200 u. l transformation TG1 cells joins in 100 milliliters of 2 × TY substratum, cultivates 3 hours for 37 DEG C.(2) add 40 microlitre VCSM13 helper phages, room temperature leaves standstill 30 minutes.(3) centrifugal 10 minutes, by the resuspended access of the cell 250 milliliters of 2 × TY substratum be centrifugation down, 37 DEG C of overnight incubation; (4) by centrifugal for nutrient solution 8000 rpm 30 minutes, get supernatant liquor, with the phage after the amplification of PEG/NaCl precipitation, and the phage after precipitation is dissolved in PBS solution.(5) by the prealbumin PA be dissolved in 100 mmole pH 8.2 NaHCO3 totally 10 micrograms be coupled on NUNC enzyme plate, 4 DEG C of placements are spent the night, and set up negative contrast simultaneously.Add 100 microlitre 0.1% caseins in (26) second days two holes respectively, room temperature closes 2 hours.After (7) 2 hours, add 100 microlitres and adopt the phage that described in above-mentioned steps 4, method is collected, at room temperature act on 1 hour.(8) 0.05% polysorbas20 is contained with in PBST(PBS) wash 5 times, wash away uncombined phage.(9) dissociated down by the phage with prealbumin PA specific binding with triethylamine (100 mmole), and infect the e. coli tg1 being in logarithmic phase growth, produce also purified phage and be used for the screening of next round, identical screening process repeats 6 and takes turns.In the process of constantly screening, positive clone will constantly by enrichment, thus reaches the object utilizing display technique of bacteriophage sieve to get positive colony.The principle modes figure of this experiment as shown in Figure 5.
Embodiment 3: screen the single positive colony of specificity with the enzyme-linked immunoassay method (ELISA) of phage:
(1) contain the Tissue Culture Dish of phage after taking turns screening from above-mentioned 6, select 96 single bacterium colonies and be inoculated in TB substratum containing 100 microgram penbritin every milliliter (containing 2.3 grams of potassium primary phosphates in 1 liter of TB substratum, 12.52 gram dipotassium hydrogen phosphate, 12 grams of peptones, 24 grams of yeast extracts, 4 milliliters of glycerine) in, after growing to logarithmic phase, add the IPTG that final concentration is 1 mmole, 28 DEG C of overnight incubation.(2) utilize osmose process to obtain and slightly carry antibody, and antibody is transferred in antigen coated elisa plate, at room temperature place 1 hour.(3) wash away unconjugated antibody with PBST, add primary antibodie mouse anti-HA tag antibody(mouse-anti HA antibody, purchased from Beijing CoWin Bioscience Co., Ltd.), at room temperature place 1 hour.
(4) wash away unconjugated antibody with PBST, add anti-mouse alkaline phosphatase conjugate(goat-anti-mouse alkaline phosphatase enzyme mark antibody, purchased from the prompt Science and Technology Ltd. of Amy), at room temperature place 1 hour.(5) wash away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, on ELISA instrument, read absorption value at 405 nm wavelength.(6) when sample well OD value is greater than control wells OD value more than 2 times, positive colony hole is judged to.(7) positive colony is seeded to 5 milliliters to contain in the LB liquid of 100 microgram penbritin every milliliter to extract plasmid and to check order.
Analyze the gene order of each clone strain according to sequence alignment program Vector NTI, strain identical for CDR1, CDR2, CDR3 sequence is considered as same clone strain, and the different strain of its sequence is considered as different clone strain, finally has 1 strain antibody.The aminoacid sequence of its antibody is the FR1-CDR1-FR2-CDR2-FR3 district shown in SEQ ID NO:13,14 and 20, FR4 district and CDR3 district, forms whole VHH.
Embodiment 4: nano antibody is at Host Strains expression in escherichia coli, purifying:
(1) by sequencing analysis above obtain different clone strain plasmid electricity be transformed in intestinal bacteria WK6, and be coated on the plate containing 100 micrograms per millilitre penbritins and 2% glucose LB solid medium, 37 DEG C are spent the night, (2) single colony inoculation is selected in 15 milliliters of LB nutrient solutions containing penbritin, 37 DEG C of shaking table overnight incubation, (3) inoculate 1 milliliter spend the night in bacterial classification to 330 milliliter TB substratum, 37 DEG C of shaking tables are cultivated, when cultivation reaches 0.6-1.0 to OD value, add IPTG, 28 DEG C of shaking table overnight incubation, (4) second days, centrifugal receipts bacterium, (5) osmose process is utilized by thalline to make tropina discharge to obtain antibody crude extract, (6) through affinity chromatography antibody purification albumen, for obtaining highly purified antibody, adopt imidazole gradient elution method, lower concentration imidazole elution (50 mmoles, 100 mmoles) for washing away assorted band, high density imidazole elution (250 mmoles, 500 mmoles) finally can prepare the albumen that purity reaches more than 85%.Fig. 6 is the nano antibody for prealbumin PA of expressing, the electrophorogram of the SDS-PAGE after affinity chromatography purifying; Wherein swimming lane 1 is the sample containing 50 mmole imidazole elution institute wash-outs, swimming lane 2 is the samples containing 100 mmole imidazole elution institute wash-outs, swimming lane 3 is samples of 250 mmole imidazole elution institute wash-outs, and swimming lane 4 is samples of 500 mmole imidazole elution institute wash-outs.
Embodiment 5: the prealbumin PA nano antibody that screening obtains is to the detection of prealbumin PA:
(1) prealbumin PA is diluted to different concns (mcg/ml): 100,50,20,10,5,1,0.5,0.1,0.05,0,01,0.005,0.001, is coated on NUNC enzyme plate, 4 DEG C of placements are spent the night, and set up negative contrast simultaneously.(2) wash away unconjugated antibody with PBST, add prealbumin PA nano antibody (10 mcg/ml), incubated at room 1 hour.(3) wash away unconjugated antibody with PBST, add primary antibodie mouse anti-HA tag antibody(mouse-anti HA antibody, purchased from Beijing CoWin Bioscience Co., Ltd.), at room temperature place 1 hour.(4) wash away unconjugated antibody with PBST, add anti-mouse alkaline phosphatase conjugate(goat-anti-mouse alkaline phosphatase enzyme mark antibody, purchased from the prompt Science and Technology Ltd. of Amy), at room temperature place 1 hour.(5) wash away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, on ELISA instrument, read absorption value at 405 nm wavelength.(6) experimental group calculating each prealbumin PA concentration reads the ratio that value and control group read value, carries out 3 and independently tests repetition, statistical error, be depicted as histogram, as shown in Figure 7.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (8)
1. the phage display nano antibody library of a synthesis, it is characterized in that, this library comprises whole segment DNA sequence corresponding to FR1 district to the FR3 district of nano antibody consensus framework cAbBCII10, the DNA sequence dna that FR4 district is corresponding, and utilize three base codon to make CDR3 district containing the arrangement but get rid of the corresponding DNA sequence dna of halfcystine and terminator codon arbitrarily at random of 16 amino acid and amino acid, Overlap extension PCR is adopted to obtain the DNA fragmentation comprising above-mentioned sequence, and this DNA fragmentation and phagemid vector recombination to construct recombinant plasmid transformed intestinal bacteria are built into phage display nano antibody library.
2. the prealbumin nano antibody gone out based on phage display nano antibody library screening described in claim 1, it is characterized in that the VHH chain of this prealbumin nano antibody comprises framework region FR and complementary determining region CDR, described framework region FR comprises the aminoacid sequence of FR1, FR2, FR3, FR4, its aminoacid sequence is respectively FR1 shown in SEQ ID NO:1, FR2 shown in SEQ ID NO:2, FR4 shown in FR3 shown in SEQ ID NO:3, SEQ ID NO:4; Described complementary determining region CDR comprises the aminoacid sequence of CDR1, CDR2, CDR3, its aminoacid sequence is respectively the CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or the CDR3 shown in SEQ ID NO:12.
3. prealbumin nano antibody according to claim 2, is characterized in that the VHH chain of described prealbumin nano antibody has following any one group of aminoacid sequence: aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:7; Or aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:8; Or aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:9; Or aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:10; Or aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:11; Or aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:4 and SEQ ID NO:12.
4. a DNA molecular, is characterized in that, the VHH chain of its coding claim 2 or prealbumin nano antibody according to claim 3.
5. DNA molecular according to claim 4, is characterized in that, it has following any one group of nucleotide sequence: nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16; Or nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:17; Or nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:18; Or nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:19; Or nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:20; Or nucleotide sequence shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:21.
6. an expression vector, is characterized in that, it comprises the nucleotide sequence of DNA molecular described in claim 4.
7. a host cell, is characterized in that, it can express the nano antibody of above-mentioned prealbumin.
8. the purposes of the albumin aspect before detection of the prealbumin nano antibody described in Claims 2 or 3.
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| CN105985439A (en) * | 2015-02-04 | 2016-10-05 | 中国科学院上海生命科学研究院 | Nano antibody of specific identification heavy metal Cr and application of nano antibody |
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| CN114381472A (en) * | 2021-12-30 | 2022-04-22 | 华南农业大学 | Nano antibody total synthesis library and construction method thereof |
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| CN105985439A (en) * | 2015-02-04 | 2016-10-05 | 中国科学院上海生命科学研究院 | Nano antibody of specific identification heavy metal Cr and application of nano antibody |
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| CN111349159A (en) * | 2020-03-17 | 2020-06-30 | 暨南大学 | Anti-human serum albumin nano antibody and application thereof |
| CN112266926A (en) * | 2020-10-19 | 2021-01-26 | 上海亚联抗体医药有限公司 | Synthetic antibody library and construction method and application thereof |
| CN114381472A (en) * | 2021-12-30 | 2022-04-22 | 华南农业大学 | Nano antibody total synthesis library and construction method thereof |
| CN114381472B (en) * | 2021-12-30 | 2024-04-12 | 华南农业大学 | Nanobody total synthesis library and construction method thereof |
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Application publication date: 20141224 |