CN104479015A - Nano antibody aiming at NGAL epitope and application thereof - Google Patents
Nano antibody aiming at NGAL epitope and application thereof Download PDFInfo
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- CN104479015A CN104479015A CN201410734730.4A CN201410734730A CN104479015A CN 104479015 A CN104479015 A CN 104479015A CN 201410734730 A CN201410734730 A CN 201410734730A CN 104479015 A CN104479015 A CN 104479015A
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Abstract
The invention provides a nano antibody VHH chain aiming at neutrophil gelatinase-associated lipocalin. The nano antibody VHH chain comprises a frame region and a complementary determining region. The invention also provides a nano antibody aiming at NGAL. The nano antibody comprises the nano antibody VHH chain. The invention further provides an application of the nano antibody. The nano antibody provided by the invention is a nano antibody aiming at neutrophil gelatinase-associated lipocalin antigen epitope. The nano antibody can be efficiently expressed in Escherichia coli and can be applied to early detection and diagnostic study of acute kidney injury and malignant tumors.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of nano antibody for NGAL epi-position and application thereof.
Background technology
NGAL protein is the small molecules secreted protein of expressing in the neutrophil leucocyte and epithelial cell of some histoorgans comprising uriniferous tubules.Be one of Lipocalin protein family newcomer, be made up of a peptide chain containing 197 amino-acid residues, total length 5869bp that therefore its molecular weight little (25KDa) and not easily degrading is easy to detect from serum or urine.Nearest research shows, NGAL is one of the most effective Biomarkers of diagnosing acute injury of the kidney.Under normal circumstances, NGAL protein level maintains a lower level; When kidney is when being subject to toxicant infringement or generation ischemia injury stimulates, renal cells secretes the significantly rising rapidly in 2 hours of NGAL level, and is discharged in urine and blood plasma, therefore by the injury of the kidney biomarker as early stage sensitivity.In addition NGAL gene in Several Kinds of Malignancy as unconventionality expression in the Several Kinds of Malignancies such as mammary cancer, the esophageal carcinoma, liver cancer, carcinoma of the pancreas, also closely related with the transfer of cancer cells, potential using value may be there is in early diagnosis of malignant tumour etc.Research shows, NGAL may take part in the process of multiple vital movement, comprises the generation development etc. of fetal development, cytodifferentiation, inflammatory and immune response, apoptosis, signal transduction, lipid metabolism, tumour.The NGAL detection method that existing market is common, some is ELISA kit, board-like or tubular type is needed to fix envelope antigen or antibody, but there is certain limitation, sensitive not to the detection of NGAL albumen, and due to the requirement of monoclonal antibody to reaction environment itself higher, also limit the enforcement of the chemical detection method much more optimized.
Nano antibody technology is on basis prepared by conventional antibodies, uses the antibody engineering revolution that Protocols in Molecular Biology fusion nanoparticle concept is carried out, thus up-to-date, the minimum antibody molecule developed.Common antibody protein is made up of two heavy chains and two light chains, and 1993, Hamers-Casterman etc. report the novel antibody natural deletions light chain found from camel blood, only have two heavy chains, i.e. heavy chain antibody (HCAbs).Heavy chain antibody (heavy chainantibody, hcAb) also known as nano antibody (Nanobody) or single domain antibody (single domain antibody, sdAb), a kind of novel antibody formation, little with its molecular weight, the multiple advantage such as stability is strong, solubility good, easy expression, output is high, immunogenicity is low, high temperature resistant, acidproof potassium is well-known.It has unique variable region of heavy chain (VHH), a hinge area and two constant regions (CH2 and CH3), and nano antibody lacks light chain.Therefore, nano antibody has only just possessed special antigen binding capacity and high affinity by 3 complementary determining regions (complementarity-determining region, CDR), and common antibody then needs 6 CDRs.These " heavy chain antibodies " tightly can be combined with targets such as antigens as normal antibody, but are mutually adhered unlike single-chain antibody and are gathered into block.Its crystalline structure diameter 215nm, long 4nm, molecular weight only has 1/10 of common antibody, chemical property is very flexible, good stability, solubility is high, expresses easily, easily obtains and easy other molecules of coupling, and has unique identification structure epi-position, can be used as the instrument of medical diagnosis on disease and diagnosing tumor, have broad application prospects.
Summary of the invention
Goal of the invention: in order to overcome above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of nano antibody for NGAL epi-position and application thereof.
Technical scheme: one provided by the invention, for the nano antibody VHH chain of neutrophil gelatinase-associated lipocalin (neutrophilgelatinase-associated lipocalin NGAL), comprises framework region and complementary determining region:
Wherein, described framework region has 4, is respectively FR1 to FR4; Wherein, the aminoacid sequence of FR1 is selected from the one in SEQ IDNO.5 to SEQ ID NO.8, and the aminoacid sequence of FR2 is selected from the one in SEQ ID NO.9 to SEQ ID NO.12; The aminoacid sequence of FR3 is selected from the one in SEQ ID NO.13 to SEQ ID NO.16; The aminoacid sequence of FR4 is selected from the one in SEQ ID NO.17 to SEQ ID NO.20;
Wherein, described complementary determining region has 3, is respectively CDR1 to CDR3; Wherein, the aminoacid sequence of CDR1 is selected from the one in SEQ ID NO.21 to SEQ ID NO.24; The aminoacid sequence of CDR2 is selected from the one in SEQ ID NO.25 to SEQ ID NO.28; The aminoacid sequence of CDR3 is selected from the one in SEQ ID NO.29 to SEQ ID NO.32.
Present invention also offers a kind of nano antibody for NGAL, it comprises nano antibody VHH chain according to claim 1.
Preferably, the described nano antibody for NGAL, comprises NGAL#1, NGAL#2, NGAL#3 and NGAL#4; The aminoacid sequence of described NGAL#1 is as shown in SEQ ID NO.1; The aminoacid sequence of NGAL#2 is as shown in SEQ ID NO.2, and the aminoacid sequence of NGAL#3 is as shown in SEQ ID NO.3, and the aminoacid sequence of NGAL#4 is as shown in SEQ ID NO.4.
Present invention also offers a kind of DNA molecular, its nano antibody for NGAL described in any one of Claims 2 or 3 of encoding.
Present invention also offers a kind of nucleic acid molecule, it comprises framework region and variable region, and its nucleotide sequence is selected from the one in SEQ IDNO.33 to SEQ ID NO.36.
Present invention also offers a kind of host cell, it comprises the expression vector of the above-mentioned nano antibody for NGAL.
Present invention also offers the above-mentioned nano antibody for NGAL and prepare the application in albuminuria detection kit.
Beneficial effect: nano antibody provided by the invention is a kind of for neutrophil gelatinase-associated lipocalin (neutrophil gelatinase-associated lipocalin, NGAL) nano antibody of epitope (also claims single domain antibody fragment, single domain antibody fragment), this nano antibody can in intestinal bacteria high expression, can be applicable to acute injury of kidney and the early stage diagnosis research of malignant tumour.
First the present invention expresses people source NGAL antigen, then be coupled on enzyme plate, show the correct space structure of this albumen, antigen in this format utilizes display technique of bacteriophage to screen immune nano Antibody geometric mean titer (camel heavy chain antibody phage display gene pool), screening obtains the specific nano antibody gene of NGAL, this gene is gone in intestinal bacteria, thus establish can in the nano antibody strain of E. coli.
Emphasis of the present invention is for the special nano antibody of NGAL, and its specificity is mainly present in the sequence in complementary determining region i.e. CDR district; Due to the diversity of the nano antibody of this several NGAL, the difference namely between the different nano antibody of NGAL is also complementary determining region (CDR district).Meanwhile, the present invention obtains the sequence of framework region through lot of experiments.
Accompanying drawing explanation
Fig. 1 is NGAL antigen purification SDS-polyacrylate hydrogel electrophorogram, and from left to right each protein band respectively: Article 1 is protein molecular standard, and Article 2 is the NGAL protein after purifying.
Fig. 2 is the DNA electrophorogram of nano antibody, and from left to right the DNA band of gel pore respectively: first is the molecule marker of 200bp, and all the other ducts are PCR primer, and PCR primer band is about 500bp.
Fig. 3 is the nano antibody of the NGAL expressed, the electrophorogram of the SDS-PAGE after nickel post resin gel affinitive layer purification.
Embodiment
Embodiment 1
For the structure in neutrophil gelatinase-associated lipocalin (neutrophil gelatinase-associatedlipocalin NGAL) nano antibody library, comprise the following steps:
(1) mixed with freund's adjuvant equal-volume by 1mg NGAL, an immunity Xinjiang dromedary, once in a week, immunity 7 times, stimulates the specific nano antibody of B cell antigen expressed altogether;
After (2) 7 immunity, extract 100mL camel peripheral blood lymphocyte and extract total serum IgE;
(3) reverse transcription is synthesized cDNA and is utilized nest-type PRC to obtain VHH fragment through twice amplification, for the first time PCR primer: upstream primer: GTC CTG GCT GCT CTT CTA CAA GGC, downstream primer: GGT ACG TGC TGT TGAACT GTT CC; Second time PCR with first time PCR primer for template, upstream primer: GAT GTG CAG CTG CAG GAGTCT GGR GGA GG, downstream primer: GGA CTA GTG CGG CCG CTG GAG ACG GTG ACC TGG GT;
(4) restriction enzyme PstI and NotI enzyme is utilized to cut 20ug pMECS carrier and 10ug VHH and connect two fragments;
(5) turn in competent cell TG1 by connection product conversion to electricity, build NGAL nano antibody library and measure storage capacity, storage capacity size is 1.15 × 10
8.
Embodiment 2
NGAL nano antibody screening process:
(1) 100mM NaHCO will be dissolved in
3(pH=8.2) the 20ug NGAL in is coupled on NUNC enzyme plate, and 4 DEG C of placements are spent the night;
Within (2) second days, add 100uL 0.1% casein, room temperature closes 2h;
(3) 100uL phage (5 × 10 is added
11tfu immunity camel nano antibody phage display gene pool), room temperature effect 1h;
(4) 5 times are washed with 0.05%PBS+Tween-20, to wash uncombined phage off;
(5) dissociate and the phage of NGAL specific binding with 100mM TEA (triethylamine), infect the e. coli tg1 being in logarithmic phase, cultivate 1h for 37 DEG C, produce also purified phage and be used for the screening of next round, identical screening process repeats 3-4 wheel, progressively obtains enrichment.
Embodiment 3
The single positive colony of specificity is screened with the enzyme-linked immunoassay method (ELISA) of phage:
(1) through the screening of 3-4 wheel, from containing the cel culture plates of phage, select 96 single bacterium colonies and be inoculated in 24 porocyte culture plates, after growing to logarithmic phase, adding 1mM IPTG, 28 DEG C of overnight incubation;
(2) utilize osmose process to obtain and slightly carry antibody, and transferred to by antibody in antigen coated elisa plate, room temperature places 1h;
(3) wash away unconjugated antibody with PBST, add mouse anti-HA tag antibody, room temperature places 1h;
(4) wash away unconjugated antibody with PBST, add anti-mouse alkaline phosphataseconjugate, room temperature places 1h;
(5) wash away unconjugated antibody with PBST, add nitrite ion, on ELISA instrument, at 405nm wavelength, read light absorption value;
(6) when sample well OD value is greater than control wells OD value more than 2 times, positive colony is judged to;
(7) purifying positive colony plasmid and carry out sequence verification.
According to the gene order of each clone strain, strain identical for CDR1, CDR2, CDR3 sequence is considered as same clone strain, the strain that its sequence is different is considered as different clone strain.
Embodiment 4
Nano antibody is at 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 LA+GLU plate containing penbritin and glucose, 37 DEG C of overnight incubation;
(2) picking individual colonies is seeded in 5mL LA nutrient solution, 37 DEG C of shaking table overnight incubation;
(3) inoculate the bacterial classification that spends the night of 1mL in 330mL TB nutrient solution, 37 DEG C of shaking tables are cultivated, and when cultivation reaches 0.6-1 to OD value, add 1M IPTG, 28 DEG C of overnight induction;
(4) centrifugal, receive bacterium;
(5) utilize osmose process, obtain antibody crude extract;
(6) the higher albumen of purity can be prepared through nickel post ion affinity chromatography.
Embodiment 5
NGAL nano antibody temperature tolerance detects
(1) under the nano antibody of NGAL protein being put into condition of different temperatures: 25 DEG C of 24h, 30 DEG C of 24h, 37 DEG C of 24h, 45 DEG C of 24h, 75 DEG C of 10min, 75 DEG C of 1h, 95 DEG C of 10min, 95 DEG C of 1h place;
(2) be coated on enzyme plate by NGAL albumen after carbonate dialysis, do blank well contrast (only bag is by NaHCO simultaneously
3);
(3) the NGAL nano antibody of different treatment is transferred to respectively in antigen coated elisa plate, at room temperature place 1 hour;
(4) wash away unconjugated antibody with PBST, add primary antibodie mouse anti-HA tag antibody (the anti-HA antibody of against murine, purchased from Beijing CoWin Bioscience Co., Ltd.), at room temperature place 1 hour;
(5) unconjugated antibody is washed away with PBST, add two anti-anti-mouse alkaline phosphataseconjugate (goat-anti-mouse alkaline phosphatase enzyme mark antibody, purchased from the prompt Science and Technology Ltd. of Amy), at room temperature place 1 hour;
(6) wash away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, on ELISA instrument, at 405nm wavelength, read absorption value.The results are shown in Table 1, result shows: the nano antibody of NGAL has good temperature tolerance.
Table 1
And the resistant to elevated temperatures characteristic of NGAL nano antibody is also for we carry out high specific in conjunction with photoelectrochemistry immunosensor afterwards and highly sensitive detection provides possibility.The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (7)
1., for a nano antibody VHH chain for neutrophil gelatinase-associated lipocalin, comprise framework region and complementary determining region, it is characterized in that:
Described framework region has 4, is respectively FR1 to FR4; Wherein, the aminoacid sequence of FR1 is selected from the one in SEQ ID NO.5 to SEQ ID NO.8, and the aminoacid sequence of FR2 is selected from the one in SEQ ID NO.9 to SEQ ID NO.12; The aminoacid sequence of FR3 is selected from the one in SEQ ID NO.13 to SEQ ID NO.16; The aminoacid sequence of FR4 is selected from the one in SEQ ID NO.17 to SEQ ID NO.20;
Described complementary determining region has 3, is respectively CDR1 to CDR3; Wherein, the aminoacid sequence of CDR1 is selected from the one in SEQ IDNO.21 to SEQ ID NO.24; The aminoacid sequence of CDR2 is selected from the one in SEQ ID NO.25 to SEQ ID NO.28; The aminoacid sequence of CDR3 is selected from the one in SEQ ID NO.29 to SEQ ID NO.32.
2., for a nano antibody of NGAL, it comprises nano antibody VHH chain according to claim 1.
3. a kind of nano antibody for NGAL according to claim 2, is characterized in that: comprise NGAL#1, NGAL#2, NGAL#3 and NGAL#4; The aminoacid sequence of described NGAL#1 is as shown in SEQ ID NO.1; The aminoacid sequence of NGAL#2 is as shown in SEQ ID NO.2, and the aminoacid sequence of NGAL#3 is as shown in SEQ ID NO.3, and the aminoacid sequence of NGAL#4 is as shown in SEQ ID NO.4.
4. a DNA molecular, its nano antibody for NGAL described in any one of Claims 2 or 3 of encoding.
5. a nucleic acid molecule, it comprises framework region and variable region, and its nucleotide sequence is selected from the one in SEQ ID NO.33 to SEQ IDNO.36.
6. a host cell, it comprises the expression vector of the nano antibody for NGAL described in any one of Claims 2 or 3.
7. the nano antibody for NGAL described in any one of Claims 2 or 3 is preparing the application in albuminuria detection kit.
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| CN108864281A (en) * | 2018-07-13 | 2018-11-23 | 西北农林科技大学 | A kind of nano antibody of anti-Bacterium enteritidis and its application |
| CN109553682A (en) * | 2018-12-29 | 2019-04-02 | 江苏众红生物工程创药研究院有限公司 | Anti-human neutrophil gelatinase-associated lipocalin antibody and its application in Test paper card |
| JP2021516951A (en) * | 2018-03-30 | 2021-07-15 | ナンジン レジェンド バイオテック カンパニー，リミテッドＮａｎｊｉｎｇ Ｌｅｇｅｎｄ Ｂｉｏｔｅｃｈ Ｃｏ．，Ｌｔｄ． | Single domain antibody against LAG-3 and its use |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2021516951A (en) * | 2018-03-30 | 2021-07-15 | ナンジン レジェンド バイオテック カンパニー，リミテッドＮａｎｊｉｎｇ Ｌｅｇｅｎｄ Ｂｉｏｔｅｃｈ Ｃｏ．，Ｌｔｄ． | Single domain antibody against LAG-3 and its use |
| JP7346790B2 (en) | 2018-03-30 | 2023-09-20 | ナンジン レジェンド バイオテック カンパニー，リミテッド | Single domain antibodies against LAG-3 and their uses |
| US11958903B2 (en) | 2018-03-30 | 2024-04-16 | Nanjing Legend Biotech Co., Ltd. | Single-domain antibodies against LAG-3 and uses thereof |
| CN108864281A (en) * | 2018-07-13 | 2018-11-23 | 西北农林科技大学 | A kind of nano antibody of anti-Bacterium enteritidis and its application |
| CN109553682A (en) * | 2018-12-29 | 2019-04-02 | 江苏众红生物工程创药研究院有限公司 | Anti-human neutrophil gelatinase-associated lipocalin antibody and its application in Test paper card |
| CN109553682B (en) * | 2018-12-29 | 2022-04-05 | 江苏众红生物工程创药研究院有限公司 | Anti-human neutrophil gelatinase-associated lipocalin antibody and application thereof in detection of test paper card |
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Application publication date: 20150401 |