CN105524173A - VHH (variable domain of heavy chain of heavy-chain) antibody for humanized antibody Fc fragment and application of VHH antibody - Google Patents
VHH (variable domain of heavy chain of heavy-chain) antibody for humanized antibody Fc fragment and application of VHH antibody Download PDFInfo
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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Abstract
The invention discloses a VHH (variable domain of heavy chain of heavy-chain) antibody for a humanized antibody Fc fragment and an application. The VHH antibody has VHH chains of amino acid sequences shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, and encoded genetic sequences of the VHH antibody are shown in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO: 29 and SEQ ID NO:30. The VHH antibody can be efficiently expressed in Escherichia coli through the VHH antibody genetic sequences and host cells and can be applied to purification and detection of humanized antibodies and recombinant protein with a humanized antibody Fc fragment (label).
Description
Technical field
The present invention relates to a kind of nano antibody for human antibody Fc fragment and application thereof, belong to biotechnology or biomedical sector technical field.
Background technology
Antibody (Antibody) be a kind of by body immune system for allogenic material (albumen as bacterium, virus) a kind of globular proteins of producing, be therefore otherwise known as immunoglobulin (Ig) (Ig).Antibody is produced by B cell usually, in the immunity system of body, exercise multiple physiologic function.Antibody has broad variety, and the IgG type antibody secreted by the plasmocyte wherein differentiated by B cell is the most general, a most important type, is usually present in vertebrate body fluid, and in the blood of adult, content can reach about 10g/L.The said antibody of usual people, namely refers to IgG type antibody.Antibody is that formed by disulfide-bonded by the light chain that two identical heavy chains are identical with two, symmetrical y-type structure, and the two-arm end of y-type structure is the position that antigen combines, and be called as Fab fragment, the shank of Y type is called as Fc fragment.
Due to antibody can efficiently, specifically in body and external various antigen proteins be combined, make antibody can not only be applied to immunity moderation system function, various high-sensitive detection method can also be applied to.At present, antibody drug is the most important integral part of biotech drug, and antibody reagent is also one of the most frequently used reagent arrived in medical diagnosis and biological study.Therefore, the biological product that antibody is correlated with has high application prospect and commercial value.Antibody can be obtained by number of ways, and such as: the ascites etc. of the mouse of the blood of animal or human, cell cultures, injection hybridoma, but this all needs effective means to carry out purifying, to obtain the antibody product with using value.Antibody purification process the most frequently used at present utilizes the high-affinity between the Fc fragment of specific proteins and antibody to carry out affinity chromatography.In the industrial process of carrying out antibody product, affinity chromatography is a wherein the most key step, is also expend the highest part in whole production.
At present, antibody purification generally uses the Sepharose of ProteinA or ProteinG coupling to carry out affinity chromatography.But use ProteinA/G to carry out antibody purification, first will carry out the expression and purification of ProteinA/G, and the usual expression amount of ProteinA/G is lower, this just considerably increases the cost of antibody affinity chromatography.In scientific research, have a large amount of protein to adopt fusion people source Fc fragment to express, to identify and purifying, this all needs to obtain the antibody for human antibody Fc fragment.
Therefore, people are making great efforts to probe into new method to overcome these difficulties always, and the novel nano antibody for people Fc fragment will likely solve this difficult problem.Nano antibody is the distinct antibodies deriving from camellid or selachian.Research shows, there is a kind of natural deletions light chain only containing the antibody of heavy chain, be called heavy chain antibody in camel body.The variable region of cloned heavy chain antibody can obtain the single domain antibody be only made up of a variable region of heavy chain, is called VHH antibody.The crystal diameter of VHH antibody only has 2.5nm, long 4nm, and be therefore otherwise known as nano antibody.The size of nano antibody only has 1/10th of conventional I gG type antibody, is the naturally occurring minimal segment that can be combined with antigen.Nano antibody energy coverlet genes encoding, can utilize microorganism to produce easily, and have very high productive rate.
Summary of the invention
Goal of the invention: in order to solve the problems of the technologies described above, the present invention screens the nano antibody obtaining Anti-Fc, and the affinity chromatography method set up based on anti-human source Fc fragment nano antibody, for alternative ProteinA/G, to reduce laboratory and the industrialization cost when purifying human antibody and the fusion rotein containing human antibody Fc fragment.
Technical scheme: in order to realize foregoing invention object, the invention provides a kind of nano antibody for human antibody Fc fragment, described nano antibody has the VHH chain of the aminoacid sequence shown in SEQIDNO:21, SEQIDNO:22, SEQIDNO:23, SEQIDNO:24, SEQIDNO:25, and its coding nucleotide sequence is as shown in SEQIDNO:26, SEQIDNO:27, SEQIDNO:28, SEQIDNO:29, SEQIDNO:30.
As preferably, described VHH chain comprises framework region FR and complementary determining region CDR;
Described framework region FR comprises the aminoacid sequence of FR1, FR2, FR3, FR4, and its aminoacid sequence is respectively:
FR4 shown in FR3, SEQIDNO:4 shown in FR2, SEQIDNO:3 shown in FR1, SEQIDNO:2 shown in SEQIDNO:1;
Or FR4 shown in FR3, SEQIDNO:4 shown in FR2, SEQIDNO:3 shown in FR1, SEQIDNO:2 shown in SEQIDNO:8;
Or FR4 shown in FR3, SEQIDNO:4 shown in FR2, SEQIDNO:12 shown in FR1, SEQIDNO:11 shown in SEQIDNO:10;
Or FR4 shown in FR3, SEQIDNO:4 shown in FR2, SEQIDNO:3 shown in FR1, SEQIDNO:2 shown in SEQIDNO:16;
Or FR4 shown in FR3, SEQIDNO:18 shown in FR2, SEQIDNO:17 shown in FR1, SEQIDNO:2 shown in SEQIDNO:8;
Described complementary determining region CDR comprises the aminoacid sequence of CDR1, CDR2, CDR3, corresponding above-mentioned each FR, and the aminoacid sequence of described CDR is respectively:
CDR3 shown in CDR2, SEQIDNO:7 shown in CDR1, SEQIDNO:6 shown in SEQIDNO:5;
Or CDR3 shown in CDR2, SEQIDNO:7 shown in CDR1, SEQIDNO:6 shown in SEQIDNO:9;
Or CDR3 shown in CDR2, SEQIDNO:15 shown in CDR1, SEQIDNO:14 shown in SEQIDNO:13;
Or CDR3 shown in CDR2, SEQIDNO:7 shown in CDR1, SEQIDNO:6 shown in SEQIDNO:9;
Or CDR3 shown in CDR2, SEQIDNO:7 shown in CDR1, SEQIDNO:20 shown in SEQIDNO:19.
Present invention also offers a kind of DNA molecular, its above-mentioned nano antibody for human antibody Fc fragment of encoding, its nucleotide sequence is as shown in SEQIDNO:26, SEQIDNO:27, SEQIDNO:28, SEQIDNO:29, SEQIDNO:30.
Present invention also offers a kind of expression vector, it comprises the nucleotide sequence of above-mentioned DNA molecular.
Present invention also offers a kind of host cell, it expresses the above-mentioned nano antibody for human antibody Fc fragment.
Present invention also offers the described application of nano antibody in separation and purification human antibody for human antibody Fc fragment.
Present invention also offers the described application of nano antibody in the fusion rotein of separation and purification containing human antibody Fc label for human antibody Fc fragment.
The present invention finally additionally provides described human antibody Fc fragment nano antibody and is detecting the application in human antibody Fc fragment.
Technique effect: relative to prior art, the present invention has following technical superiority:
The present invention adopts human antibody Fc fragment immunity Xinjiang two-humped camel, utilize the foundation of this camel peripheral blood lymphocyte for the nano antibody gene library of human antibody Fc fragment subsequently, human antibody Fc fragment is coupled on enzyme plate, antigen in this format utilizes display technique of bacteriophage to screen the nano antibody gene pool of immunity, thus the gene obtained for the high-affinity of human antibody Fc and the nano antibody of high specific, this gene is proceeded in intestinal bacteria, establishing can in the nano antibody strain of E. coli, the gene order of each clone strain is analyzed according to sequence alignment program, obtain the aminoacid sequence for the nano antibody VHH chain of human antibody Fc fragment, and prove that this nano antibody can combine with human antibody Fc fragments specific, thus for the purifying of the recombinant protein to human antibody and coupling human antibody Fc fragment.
Accompanying drawing explanation
Fig. 1 is the electrophorogram constructed phage display nano antibody library being carried out to the bacterium colony PCR of insertion rate detection, wherein swimming lane 1 is DNA molecular marker, and swimming lane 2-25 constructed detects electrophorogram for picking DCRP PCR random in the nano antibody library of human antibody Fc fragment;
Fig. 2 is the nano antibody for human antibody Fc fragment of expressing, and the SDS-PAGE electrophorogram after nickel resin affinitive layer purification, swimming lane 1 is albumen marker, and swimming lane 1-4 is the nano antibody after nickel resin affinitive layer purification.
Fig. 3 is for core material is to the schematic diagram of the enrichment of human antibody and the purifying to coupling humanization Fc Fragment Protein with the nano antibody of human antibody Fc fragment.Reference numeral: 1. people source serum or the protein lysate containing human antibody Fc fragment and human antibody Fc fragment fusion protein; 2. the nano antibody of anti-human source antibody Fc fragment; 3. human antibody; 4. human antibody Fc fragment and human antibody Fc fragment fusion protein.
Embodiment
Specific embodiment of the invention scheme is further described below in conjunction with accompanying drawing.
The present invention adopts human antibody Fc fragment immunity Xinjiang two-humped camel, extracts this two-humped camel peripheral blood lymphocyte and set up the special nano antibody library of human antibody Fc fragment after 7 immunity.Human antibody Fc fragment is coupled on enzyme plate, show correct space structure, the epitope of human antibody Fc fragment is come out, antigen in this format utilizes display technique of bacteriophage to screen the nano antibody gene pool (camel heavy chain antibody phage display gene pool) of human antibody Fc fragment immunity, and obtain can in the nano antibody strain of E. coli.
Below in conjunction with specific embodiment, set forth the present invention further.
Embodiment 1: the structure for the nano antibody library of human antibody Fc fragment:
(1) 1mg human antibody Fc fragment (stemming from healthy and free from worry outstanding person auspicious) antigen is mixed with freund's adjuvant equal-volume, an immunity Xinjiang two-humped camel, once in a week, continuous immunity 7 times altogether, the nano antibody of immunologic process moderate stimulation B cell expression specificity; After (2) 7 immunity terminate, extract camel peripheral blood lymphocyte 100ml and extract total serum IgE; (3) synthesize cDNA and utilize sleeve type PCR amplification VHH; (4) restriction enzyme Pst I and Not I enzyme is utilized to cut 20ugpMECS Vector for Phage Display and 10ugVHH and connect two kinds of fragments; (5) turn in competent cell TG1 by connection product conversion to electricity, build human antibody Fc fragment nano antibody phage display library and measure storage capacity, the size of storage capacity is about 1.2 × 10
8; Simultaneously, detect institute by bacterium colony PCR and build the insertion rate in library, Fig. 1 shows bacterium colony PCR result, and random 24 clones that choose are cooked bacterium colony PCR, and result shows insertion rate and reaches more than 95%.
Embodiment 2: the nano antibody screening process for human antibody Fc fragment:
(1) get in 200uL restructuring TG1 cell to 2 × TY substratum and cultivate, period adds 40uL helper phage VCSM13 and infects TG1 cell, and overnight incubation is with phage of increasing, and utilizes PEG/NaCl precipitating phage next day, collected by centrifugation amplification phage; (2) be coupled on enzyme plate by the human antibody Fc fragment 200ug be dissolved in 100mMpH8.2NaHCO3,4 DEG C of placements are spent the night, and set up negative contrast simultaneously; Within (3) second days, add the 3%BSA of 100ul, room temperature closes 2h; (4), after 2h, 100ul amplification phage (2 × 10 is added
11tfu immunity camel nano antibody phage display gene pool), room temperature effect 1h; (5) 5 times are washed with PBS+0.05%Tween-20, to wash the phage of combination off; (6) be that the phage combined in human antibody Fc fragments specific is dissociated down by the trypsinase of 25mg/ml with final concentration, and infect the e. coli tg1 cell being in logarithmic phase, cultivate 1h for 37 DEG C, produce and collect the screening of phage for next round, identical screening process repeats 3 and takes turns, progressively to enrichment.
Embodiment 3: screen specific positive clone with the enzyme-linked immunoassay method (ELISA) of phage:
(1) take turns the rear Tissue Culture Plate of screening from above-mentioned 3, select in 96 deep-well plates that 175 single bacterium colonies are inoculated in respectively containing the TB substratum of 100ug/mL penbritin, and blank be set, 37 DEG C be cultured to logarithmic phase after, add the IPTG that final concentration is 1mM, 28 DEG C of overnight incubation; (2) utilize infiltration method of bursting to obtain and slightly carry antibody, and be transferred to by antibody on antigen coated elisa plate, room temperature places 1h; (3) wash away unconjugated antibody with PBST, add the Mouseanti-HAtagantibody (mouse-anti HA antibody, purchased from Ke Wensi) of 100ul after 1:2000 dilution, place 1h in room temperature; (4) unconjugated antibody is washed away with PBST, add Anti-mousealkalinephosphataseconjugate (the goat-anti-mouse alkaline phosphatase enzyme mark antibody of 100ul after 1:2000 dilution, purchased from Sigma), place 1h in room temperature; (5) wash away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, reaction 5-10min after in microplate reader 405 wavelength places, read absorption value; (6) when sample well OD value is greater than control wells more than 5 times, positive colony hole is judged to be; (7) bacterium in positive colony hole is turned shake in the LB substratum containing 100ug/ul penbritin to extract plasmid and to check order.
The gene order of each clone strain is analyzed according to sequence alignment program VectorNTI, strain identical for FR1, FR2, FR3, FR4, CDR1, CDR2, CDR3 sequence is considered as same clone strain, and the different strain of sequence is considered as different clone strain, finally obtain 1 strain human antibody Fc fragments specific nano antibody.The aminoacid sequence of its antibody is the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 district shown in SEQIDNO:21, SEQIDNO:22, SEQIDNO:23, SEQIDNO:24, SEQIDNO:25, forms whole VHH.
Embodiment 4: the Expression and purification of human antibody Fc fragments specific nano antibody in Host Strains intestinal bacteria
(1) by above-mentioned sequencing analysis obtain different clone strain plasmid point be transformed in intestinal bacteria WK6, and be coated on LB+amp+glucose namely containing on the culture plate of penbritin and glucose, 37 DEG C of overnight incubation; (2) selecting single colony inoculation contains in the LB nutrient solution of penbritin at 5ml, 37 DEG C of shaking table overnight incubation; (3) inoculate the incubated overnight bacterial classification of 1mL in 330mLTB nutrient solution, 37 DEG C of shaking tables are cultivated, and cultivate OD
600nmwhen value reaches 0.6-0.9, add 1MIPTG, 28 DEG C of shaking table overnight incubation; (4) centrifugal, collect intestinal bacteria, utilize infiltration to burst method, obtain antibody crude extract; (5) be purified into antibody by affinity chromatography method, obtain highly purified nano antibody, as shown in Figure 2.
Embodiment 5: for human antibody Fc fragment nano antibody separation and purification human antibody and containing human antibody Fc fragment fusion rotein in application
As shown in Figure 3, nano antibody is fixed on solid-state dielectric surface, add people source serum or the protein lysate containing human antibody Fc fragment and human antibody Fc fragment fusion protein wherein, room temperature places 2h.Wash away unconjugated albumen with PBS, the albumen of energy specific binding is the albumen containing people source Fc fragment, then uses the combining albumen wash-out of elutriant, separation and purification can go out human antibody or the nano antibody containing human antibody Fc fragment.
Embodiment 6: detecting the application in human antibody Fc fragment for human antibody Fc fragment nano antibody
First human antibody Fc fragment nano antibody is coated on elisa plate, add antigen people source Fc fragment standard substance (as 1ng/ml to 1mg/ml) of different gradient concentration, parallel running adds the sample of human antibody Fc fragment to be detected, places 1h in room temperature.Wash away unconjugated antigen with PBST, then add biotin labeled another kind of human antibody Fc fragment nano antibody, room temperature places 1h.Wash away unconjugated antibody with PBST, then add the avidin of coupling horseradish peroxidase, room temperature places 1h.Wash away unconjugated avidin with PBST, add horseradish peroxidase nitrite ion, on ELISA instrument, at 405nm wavelength, read light absorption value.First the absorption value of the people source Fc fragment standard substance of each concentration gradient is made concentration standard curve, then bring the absorption value reading of detected sample into concentration standard curve, the content of human antibody Fc fragment in detected sample can be judged.
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; be not restricted to the described embodiments in the present invention; description 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 nano antibody for human antibody Fc fragment, it is characterized in that, described nano antibody has the VHH chain of the aminoacid sequence shown in SEQIDNO:21, SEQIDNO:22, SEQIDNO:23, SEQIDNO:24 or SEQIDNO:25, and its coding nucleotide sequence is respectively as shown in SEQIDNO:26, SEQIDNO:27, SEQIDNO:28, SEQIDNO:29, SEQIDNO:30.
2. according to claim 1 for the nano antibody of human antibody Fc fragment, it is characterized in that, described VHH chain comprises framework region FR and complementary determining region CDR;
Described framework region FR comprises the aminoacid sequence of FR1, FR2, FR3, FR4, and its aminoacid sequence is respectively:
FR4 shown in FR3, SEQIDNO:4 shown in FR2, SEQIDNO:3 shown in FR1, SEQIDNO:2 shown in SEQIDNO:1;
Or FR4 shown in FR3, SEQIDNO:4 shown in FR2, SEQIDNO:3 shown in FR1, SEQIDNO:2 shown in SEQIDNO:8;
Or FR4 shown in FR3, SEQIDNO:4 shown in FR2, SEQIDNO:12 shown in FR1, SEQIDNO:11 shown in SEQIDNO:10;
Or FR4 shown in FR3, SEQIDNO:4 shown in FR2, SEQIDNO:3 shown in FR1, SEQIDNO:2 shown in SEQIDNO:16;
Or FR4 shown in FR3, SEQIDNO:18 shown in FR2, SEQIDNO:17 shown in FR1, SEQIDNO:2 shown in SEQIDNO:8;
Described complementary determining region CDR comprises the aminoacid sequence of CDR1, CDR2, CDR3, corresponding above-mentioned each FR, and the aminoacid sequence of described CDR is respectively:
CDR3 shown in CDR2, SEQIDNO:7 shown in CDR1, SEQIDNO:6 shown in SEQIDNO:5;
Or CDR3 shown in CDR2, SEQIDNO:7 shown in CDR1, SEQIDNO:6 shown in SEQIDNO:9;
Or CDR3 shown in CDR2, SEQIDNO:15 shown in CDR1, SEQIDNO:14 shown in SEQIDNO:13;
Or CDR3 shown in CDR2, SEQIDNO:7 shown in CDR1, SEQIDNO:6 shown in SEQIDNO:9;
Or CDR3 shown in CDR2, SEQIDNO:7 shown in CDR1, SEQIDNO:20 shown in SEQIDNO:19.
3. a DNA molecular, is characterized in that, the nano antibody for human antibody Fc fragment of its coding described in claim 1 or 2, its nucleotide sequence is as shown in SEQIDNO:26, SEQIDNO:27, SEQIDNO:28, SEQIDNO:29 or SEQIDNO:30.
4. an expression vector, is characterized in that, it comprises the nucleotide sequence of DNA molecular described in claim 3.
5. a host cell, is characterized in that, it expresses the nano antibody for human antibody Fc fragment described in claim 1 or 2.
6. the application of nano antibody in separation and purification human antibody for human antibody Fc fragment described in claim 1 or 2.
7. the application of nano antibody in the fusion rotein of separation and purification containing human antibody Fc fragment for human antibody Fc fragment described in claim 1 or 2.
8. described in claim 1 or 2, detecting the application in human antibody Fc fragment for human antibody Fc fragment nano antibody.
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| CN112574309A (en) * | 2019-12-05 | 2021-03-30 | 屈向东 | anti-PD-L1 nano antibody and application thereof |
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| CN112574309B (en) * | 2019-12-05 | 2023-06-16 | 启愈生物技术(上海)有限公司 | anti-PD-L1 nano antibody and application thereof |
| CN116284424A (en) * | 2023-05-18 | 2023-06-23 | 广州明药科技有限公司 | Nanobody of anti-mouse antibody crystallizable section and application thereof |
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