CN103755804B - Nano antibody for type A H3N2 influenza virus and application thereof - Google Patents
Nano antibody for type A H3N2 influenza virus and application thereof Download PDFInfo
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- CN103755804B CN103755804B CN201410038476.4A CN201410038476A CN103755804B CN 103755804 B CN103755804 B CN 103755804B CN 201410038476 A CN201410038476 A CN 201410038476A CN 103755804 B CN103755804 B CN 103755804B
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Abstract
The invention discloses a nano antibody for type A H3N2 influenza virus epitope and a gene sequence for encoding the nano antibody, and further discloses a host cell for expressing the nano antibody and application of the host cell to diagnosis and treatment. By adopting the nano gene antibody gene sequence and the host cell disclosed by the invention, the nano antibody can be efficiently expressed in escherichia coli, and can be applied to development, targeted therapy and the like of a type A H3N2 influenza virus diagnosis kit.
Description
Technical field
The invention belongs to biomedicine or biological pharmacy technical field, relate to be directed to A type H3N2 influenza virus nano antibody, its encoding sequence and the application in Diagnosis and Treat.
Background technology
Influenza virus is different according to nucleoprotein antigen, can be divided into first, second, the third three types.Influenza A virus often occurs with popular form, can cause worldwide flu outbreak; Type B influenza virus usually causes Local primitive exponent, does not cause worldwide flu outbreak; C type influenza virus mainly occurs to be dispersed in form, and primary attack infant, does not generally cause popular.Influenza a virus infection host range is the widest, comprises the many animals such as people, pig, horse, fowl, dog.Influenza A virus can be divided into 16 hypotypes (H1-H16) according to the difference of hemagglutinin (HA) antigen on its surface, can be divided into 9 hypotypes (N1-N9) according to the difference of neuraminidase (NA) antigen.Influenza virus changes (antigenic drift) by the accumulation of surface protein antigen or various flows Influenza Virus producer is reset and produced the Immune discrimination that a kind of new influenza virus (antigenic shift) escapes infection host, thus is infecting propagation in colony, procreation.A type H3N2 influenza is a kind of respiratory system disease caused because of A type H3N2 influenza virus.Nineteen sixty-eight Hong Kong outburst H3N2 influenza lose one's life to making million people.At the beginning of 2013, the multiple state outburst in eastern united states H3N2 seasonal influenza, classic city government announced the public health emergency state January 9.Current influenza vaccines can not effectively for these strains for the mankind provide protection.Conventional antibodies belongs to macromolecular substance, and each molecule contains two cover heavy chain and light chains, and overlaps with glycan molecule.The macromole characteristic of antibody drug constrains it and widely uses the performance with curative effect.The digested system of general antibody capable is degraded very soon, thus prevents it to enter the periphery of brain or some tumours, and numerous disease cannot be treated with monoclonal antibody drug.And monoclonal antibody can be decomposed under the condition of high temperature and strong acid and strong base, must preserve, otherwise can lose activity within several weeks under zero absolute temperature.In addition, before synthesis monoclonal antibody, usually need to isolate antibody in mouse body, then carry out complicated humanization operation and utilize bio-reactor to be prepared, this process length consuming time, costly.
And nano antibody is antibody molecule minimum at present, its molecular weight is 1/10 of common antibody, is found at first by Belgian scientist Hamers, R in camel blood, and it is the class received much concern in engineered antibody product.The unique advantages such as it has that molecule is little, stability is strong, solubility is good, immunogenicity is low, easy expression, easily transformation, and can according to application need personalized designs is easily carried out to it, be the large focus that current antibody product is developed.Nano antibody has developed into the general molecular having extensive biologic applications value and clinical value, and its application relates to multiple field such as medical diagnosis on disease and treatment, basic medical research, biological study.Utilize the acquisition of nano antibody technology for the nano antibody of A type H3N2 influenza virus epitope, thus be applied to clinical diagnosis and targeted therapy.
Summary of the invention
Goal of the invention: technical problem to be solved by this invention is to provide a kind of nano antibody for A type H3N2 influenza virus epitope, provides encoding sequence and the application of this nano antibody in diagnostic detection and treatment of this nano antibody simultaneously.
Technical scheme: for achieving the above object, a first aspect of the present invention, provide a kind of VHH chain of H3N2 nano antibody, comprise framework region FR and complementary determining region CDR, described framework region FR is made up of four framework regions, be respectively FRl, FR2, FR3 and FR4, their aminoacid sequence is selected from following aminoacid sequence:
FR1 is the aminoacid sequence shown in SEQ ID NO.1, and FR2 is the aminoacid sequence shown in SEQ ID NO.2, and FR3 is the aminoacid sequence shown in SEQ ID NO.3, and FR4 is the aminoacid sequence shown in SEQ ID NO.4;
Or FR1 is the aminoacid sequence shown in SEQ ID NO.5, FR2 is the aminoacid sequence shown in SEQ ID NO.6, and FR3 is the aminoacid sequence shown in SEQ ID NO.7, and FR4 is the aminoacid sequence shown in SEQ ID NO.8;
Or FR1 is the aminoacid sequence shown in SEQ ID NO.9, FR2 is the aminoacid sequence shown in SEQ ID NO.10, and FR3 is the aminoacid sequence shown in SEQ ID NO.11, and FR4 is the aminoacid sequence shown in SEQ ID NO.4;
Or FR1 is the aminoacid sequence shown in SEQ ID NO.12, FR2 is the aminoacid sequence shown in SEQ ID NO.13, and FR3 is the aminoacid sequence shown in SEQ ID NO.14, and FR4 is the aminoacid sequence shown in SEQ ID NO.4;
Or FR1 is the aminoacid sequence shown in SEQ ID NO.1, FR2 is the aminoacid sequence shown in SEQ ID NO.15, and FR3 is the aminoacid sequence shown in SEQ ID NO.16, and FR4 is the aminoacid sequence shown in SEQ ID NO:4;
Or FR1 is the aminoacid sequence shown in SEQ ID NO.1, FR2 is the aminoacid sequence shown in SEQ ID NO.17, and FR3 is the aminoacid sequence shown in SEQ ID NO.18, and FR4 is the aminoacid sequence shown in SEQ ID NO.4;
Described complementary determining region CDR is made up of three complementary determining regions, respectively CDRl, CDR2 and CDR3, and their aminoacid sequence is selected from following aminoacid sequence:
CDR1 is the aminoacid sequence shown in SEQ ID NO.19, and CDR2 is the aminoacid sequence shown in SEQ ID NO.20, and CDR3 is the aminoacid sequence shown in SEQ ID NO.21;
Or CDR1 is the aminoacid sequence shown in SEQ ID NO.22, CDR2 is the aminoacid sequence shown in SEQ ID NO.23, and CDR3 is the aminoacid sequence shown in SEQ ID NO.24;
Or CDR1 is the aminoacid sequence shown in SEQ ID NO.25, CDR2 is the aminoacid sequence shown in SEQ ID NO.26, and CDR3 is the aminoacid sequence shown in SEQ ID NO.27;
Or CDR1 is the aminoacid sequence shown in SEQ ID NO.28, CDR2 is the aminoacid sequence shown in SEQ ID NO.29, and CDR3 is the aminoacid sequence shown in SEQ ID NO.30;
Or CDR1 is the aminoacid sequence shown in SEQ ID NO.31, CDR2 is the aminoacid sequence shown in SEQ ID NO.32, and CDR3 is the aminoacid sequence shown in SEQ ID NO.33;
Or CDR1 is the aminoacid sequence shown in SEQ ID NO.34, CDR2 is the aminoacid sequence shown in SEQ ID NO.35, and CDR3 is the aminoacid sequence shown in SEQ ID NO.36.
Further improvement of the present invention is: the VHH chain of H3N2 nano antibody have SEQ ID NO.37,38,39,40, the aminoacid sequence shown in 41 or 42.
Second aspect present invention, a kind of H3N2 nano antibody, it is the nano antibody for A type H3N2 influenza virus epitope, comprise two have SEQ ID NO.37,38,39,40, the VHH chain of the aminoacid sequence shown in 41 or 42.
Third aspect present invention, provides a kind of DNA molecular, and its coding is selected from the protein of lower group: the VHH chain of the H3N2 nano antibody shown in claim 1 or 2, or the H3N2 nano antibody shown in claim 3.
Preferably, described DNA molecular, it has the DNA sequence dna being selected from lower group: the aminoacid sequence as shown in SEQ ID NO.43,44,45,46,47 or 48.
A fourth aspect of the present invention, provides a kind of expression vector, it containing SEQ ID NO:43,44,45,46, the aminoacid sequence shown in 47 or 48.
A fifth aspect of the present invention, provides a kind of host cell, and it can express the nano antibody for A type H3N2 influenza virus.
A sixth aspect of the present invention, provides the application that H3N2 nano antibody of the present invention detects A type H3N2 influenza virus.
Beneficial effect: compared with prior art, advantage of the present invention is as follows: this utilizes the A type H3N2 influenza virus natural antigen immunity camel of deactivation, obtains high-quality immune nano Antibody geometric mean titer.Then the A type H3N2 influenza virus molecule of deactivation is 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), thus obtain the specific nano antibody gene of H3N2, again this gene is gone in intestinal bacteria, thus establish can in the nano antibody strain of E. coli.
Accompanying drawing explanation
Fig. 1 is the insertion rate detection figure in built library, it is the insertion rate detected result in the single domain antibody library built, from left to right the DNA band of gel pore respectively: first is DNA molecular marker, and all the other ducts are the PCR primer detecting Insert Fragment, and PCR primer band is about 500bp; After testing, the insertion rate in this library reaches more than 90%.
Fig. 2 is a kind of H3N2 nano antibody purifying figure, after nickel post resin gel affinitive layer purification, the electrophorogram of a kind of SDS-PAGE of H3N2 nano antibody, IDA solution (imidazoles containing concentration gradient) is used to carry out wash-out, result shows, H3N2 nano antibody is through this purge process, and its purity can reach more than 95%.
Fig. 3 is six kinds of H3N2 nano antibody purifying figure, is after ni-sepharose purification, the electrophorogram of the SDS-PAGE of the six kinds of H3N2 nano antibodies obtained, its albumen of expressing respectively corresponding SEQ ID NO:45,46,47,43, the nucleotide sequence shown in 44 or 48.Result shows, and H3N2 nano antibody is through this purge process, and its purity can reach more than 95%.
Embodiment
The present invention's application is coupled on enzyme plate for H3N2, the correct space structure of display protein matter, antigen selection immune nano Antibody geometric mean titer (camel heavy chain antibody phage display gene pool) in this format, and obtain can in the nano antibody strain of E. coli.
A first aspect of the present invention, provide a kind of VHH chain of H3N2 nano antibody, comprise framework region FR and complementary determining region CDR, described framework region FR is made up of four framework regions, be respectively FRl, FR2, FR3 and FR4, their aminoacid sequence is selected from following aminoacid sequence:
FR1 is the aminoacid sequence shown in SEQ ID NO.1, and FR2 is the aminoacid sequence shown in SEQ ID NO.2, and FR3 is the aminoacid sequence shown in SEQ ID NO.3, and FR4 is the aminoacid sequence shown in SEQ ID NO.4;
Or FR1 is the aminoacid sequence shown in SEQ ID NO.5, FR2 is the aminoacid sequence shown in SEQ ID NO.6, and FR3 is the aminoacid sequence shown in SEQ ID NO.7, and FR4 is the aminoacid sequence shown in SEQ ID NO.8;
Or FR1 is the aminoacid sequence shown in SEQ ID NO.9, FR2 is the aminoacid sequence shown in SEQ ID NO.10, and FR3 is the aminoacid sequence shown in SEQ ID NO.11, and FR4 is the aminoacid sequence shown in SEQ ID NO.4;
Or FR1 is the aminoacid sequence shown in SEQ ID NO.12, FR2 is the aminoacid sequence shown in SEQ ID NO.13, and FR3 is the aminoacid sequence shown in SEQ ID NO.14, and FR4 is the aminoacid sequence shown in SEQ ID NO.4;
Or FR1 is the aminoacid sequence shown in SEQ ID NO.1, FR2 is the aminoacid sequence shown in SEQ ID NO.15, and FR3 is the aminoacid sequence shown in SEQ ID NO.16, and FR4 is the aminoacid sequence shown in SEQ ID NO:4;
Or FR1 is the aminoacid sequence shown in SEQ ID NO.1, FR2 is the aminoacid sequence shown in SEQ ID NO.17, and FR3 is the aminoacid sequence shown in SEQ ID NO.18, and FR4 is the aminoacid sequence shown in SEQ ID NO.4;
Described complementary determining region CDR is made up of three complementary determining regions, respectively CDRl, CDR2 and CDR3, and their aminoacid sequence is selected from following aminoacid sequence:
CDR1 is the aminoacid sequence shown in SEQ ID NO.19, and CDR2 is the aminoacid sequence shown in SEQ ID NO.20, and CDR3 is the aminoacid sequence shown in SEQ ID NO.21;
Or CDR1 is the aminoacid sequence shown in SEQ ID NO.22, CDR2 is the aminoacid sequence shown in SEQ ID NO.23, and CDR3 is the aminoacid sequence shown in SEQ ID NO.24;
Or CDR1 is the aminoacid sequence shown in SEQ ID NO.25, CDR2 is the aminoacid sequence shown in SEQ ID NO.26, and CDR3 is the aminoacid sequence shown in SEQ ID NO.27;
Or CDR1 is the aminoacid sequence shown in SEQ ID NO.28, CDR2 is the aminoacid sequence shown in SEQ ID NO.29, and CDR3 is the aminoacid sequence shown in SEQ ID NO.30;
Or CDR1 is the aminoacid sequence shown in SEQ ID NO.31, CDR2 is the aminoacid sequence shown in SEQ ID NO.32, and CDR3 is the aminoacid sequence shown in SEQ ID NO.33;
Or CDR1 is the aminoacid sequence shown in SEQ ID NO.34, CDR2 is the aminoacid sequence shown in SEQ ID NO.35, and CDR3 is the aminoacid sequence shown in SEQ ID NO.36.
Further improvement of the present invention is: the VHH chain of H3N2 nano antibody have SEQ ID NO.37,38,39,40, the aminoacid sequence shown in 41 or 42.
Second aspect present invention, a kind of H3N2 nano antibody, it is the nano antibody for A type H3N2 influenza virus epitope, comprise two have SEQ ID NO.37,38,39,40, the VHH chain of the aminoacid sequence shown in 41 or 42.
Third aspect present invention, provides a kind of DNA molecular, and its coding is selected from the protein of lower group: the VHH chain of the H3N2 nano antibody shown in claim 1 or 2, or the H3N2 nano antibody shown in claim 3.
Preferably, described DNA molecular, it has the DNA sequence dna being selected from lower group: the aminoacid sequence as shown in SEQ ID NO.43,44,45,46,47 or 48.
A fourth aspect of the present invention, provides a kind of expression vector, it containing SEQ ID NO:43,44,45,46, the aminoacid sequence shown in 47 or 48.
A fifth aspect of the present invention, provides a kind of host cell, and it can express the nano antibody for A type H3N2 influenza virus.
A sixth aspect of the present invention, provides the application that H3N2 nano antibody of the present invention detects A type H3N2 influenza virus.
This utilizes the immune camel of the A type H3N2 influenza virus natural antigen of deactivation, obtains high-quality immune nano Antibody geometric mean titer.Then the A type H3N2 influenza virus molecule of deactivation is 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), thus obtain the specific nano antibody gene of H3N2, again this gene is gone in intestinal bacteria, thus establish can in the nano antibody strain of E. coli.
Below in conjunction with specific embodiment, set forth the present invention further.
The structure in embodiment 1:H3N2 nano antibody library:
(1) mixed with freund's adjuvant equal-volume by the A type H3N2 influenza natural antigen of 0.1mg deactivation, an immunity Xinjiang two-humped camel, once in a week, immunity 7 times, stimulates the specific nano antibody of B cell antigen expressed altogether; After (2) 7 immunity terminate, extract 100 mL camel peripheral blood lymphocytes and extract total serum IgE; (3) synthesize cDNA and utilize sleeve type PCR amplification VHH; (4) restriction enzyme PstI and NotI enzyme is utilized to cut 20 ug pComb3 Vector for Phage Display (Biovector supply) and 10 ug VHH and connect two fragments; (5) turn in competent cell TG1 by connection product conversion to electricity, build H3N2 nano antibody library and measure storage capacity, storage capacity size is 1.0 × 10
9.Meanwhile, our random pickings 24 clones carry out bacterium colony PCR detection, and result shows that the insertion rate in built library is more than 90%, and Fig. 1 shows bacterium colony PCR result.
Embodiment 2: the nano antibody screening process for H3N2:
(1) 100 mM NaHCO will be dissolved in
3, 20 ug deactivation H3N2 antigens in pH 8.2 are coupled on NUNC enzyme plate, 4 DEG C of placements are spent the night; Within (2) second days, add 100 uL 0.1% caseins, room temperature closes 2 h; After (3) 2 h, add 100 uL phages (5 × 10
11tfu immunity camel nano antibody phage display gene pool), room temperature effect 1 h; (4) 5 times are washed with 0.05% PBS+Tween-20, to wash non-specific phage off; (5) with 100 mM TEA(triethylamine) phage with H3N2 specific binding is dissociated down, and infect the e. coli tg1 cell being in logarithmic phase growth, cultivate 1 h 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: 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 the screening of above-mentioned 3-4 wheel, select 96 single bacterium colonies and the TB substratum being inoculated in the penbritin containing 100 micrograms per millilitre (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 of final concentration 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 the anti-HA antibody of a mouse anti-HA tag antibody(against murine, 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, at 405nm wavelength, read absorption value.(6) when sample well OD value is greater than control wells OD value more than 3 times, positive colony hole is judged to.(7) bacterium in positive colony hole is turned shake containing 100 micrograms per millilitre LB liquid in 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 the antibody that 6 strains are different.The aminoacid sequence of the VHH chain of its antibody is respectively as shown in SEQ ID NO:37,38,39,40,41 or 42.
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 LA+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 5 mL, 37 DEG C of shaking table overnight incubation; (3) that inoculates 1 mL spends the night in bacterial classification to 330 mL TB nutrient solution, and 37 DEG C of shaking tables are cultivated, and cultivates when reaching 0.6 ~ 1 to OD value, adds IPTG, 28 DEG C of shaking table overnight incubation; (4) centrifugal, receive bacterium; (5) utilize osmose process, obtain antibody crude extract; (6) can prepare through nickel post ion affinity chromatography the albumen that purity reaches more than 90%.
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.
SEQUENCE LISTING
<110> Nantong Egens Biotechnology Co., Ltd.
<120> is for the nano antibody of A type H3N2 influenza virus and application thereof
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<210> 29
<211> 9
<212> PRT
<213> Artificial
<222> (1)..(9)
<223> GSDGSTSYA
<400> 29
Gly Ser Asp Gly Ser Thr Ser Tyr Ala
1 5
<210> 30
<211> 21
<212> PRT
<213> Artificial
<222> (1)..(21)
<223> AAQYDAGGWYGLLDQRRFGYW
<400> 30
Ala Ala Gln Tyr Asp Ala Gly Gly Trp Tyr Gly Leu Leu Asp Gln Arg
1 5 10 15
Arg Phe Gly Tyr Trp
20
<210> 31
<211> 9
<212> PRT
<213> Artificial
<222> (1)..(9)
<223> LTISTYPMS
<400> 31
Leu Thr Ile Ser Thr Tyr Pro Met Ser
1 5
<210> 32
<211> 10
<212> PRT
<213> Artificial
<222> (1)..(10)
<223> SSGGGRTYYA
<400> 32
Ser Ser Gly Gly Gly Arg Thr Tyr Tyr Ala
1 5 10
<210> 33
<211> 14
<212> PRT
<213> Artificial
<222> (1)..(14)
<223> AKSRAHSTTYYSSR
<400> 33
Ala Lys Ser Arg Ala His Ser Thr Thr Tyr Tyr Ser Ser Arg
1 5 10
<210> 34
<211> 9
<212> PRT
<213> Artificial
<222> (1)..(9)
<223> FTFSSSTIS
<400> 34
Phe Thr Phe Ser Ser Ser Thr Ile Ser
1 5
<210> 35
<211> 10
<212> PRT
<213> Artificial
<222> (1)..(10)
<223> YNDGTASYNG
<400> 35
Tyr Asn Asp Gly Thr Ala Ser Tyr Asn Gly
1 5 10
<210> 36
<211> 10
<212> PRT
<213> Artificial
<222> (1)..(10)
<223> LANLRGDNHR
<400> 36
Leu Ala Asn Leu Arg Gly Asp Asn His Arg
1 5 10
<210> 37
<211> 111
<212> PRT
<213> Artificial
<400> 37
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Arg Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Ile Tyr Ser
35 40 45
Asn Gly Thr Pro Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu
65 70 75 80
Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Thr Asn Ser Asn Lys Pro
85 90 95
Lys Phe Asp Ala Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
100 105 110
<210> 38
<211> 111
<212> PRT
<213> Artificial
<400> 38
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Lys Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Arg Phe Ser Ala Cys Gly Met Asp Trp Tyr
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ser Leu Ile Asn Ser
35 40 45
Asp Gly Thr Thr Ser Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asp Ser Leu
65 70 75 80
Lys Thr Glu Asp Thr Ala Met Tyr Thr Cys Ala Ala Trp Leu Asn Gly
85 90 95
Gly Asn Arg Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser
100 105 110
<210> 39
<211> 120
<212> PRT
<213> Artificial
<400> 39
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Val Arg Ser Gly Tyr Thr Tyr Ser Tyr Asp Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Lys Arg Glu Gly Val Ala Val Ile Asn Ile
35 40 45
Gly Gly Thr Thr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala His Asp Gly Tyr
85 90 95
Leu Asp Cys Trp Arg Gly Ala Ser Ala Asp Phe Gly Asn Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 40
<211> 121
<212> PRT
<213> Artificial
<400> 40
Glu Ser Gly Gly Gly Ser Val His Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Phe Ala Ser Trp Tyr Tyr Lys Gly Trp Phe
20 25 30
Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Gly Ser
35 40 45
Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Thr Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala Ala Gln Tyr Asp Ala
85 90 95
Gly Gly Trp Tyr Gly Leu Leu Asp Gln Arg Arg Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 41
<211> 115
<212> PRT
<213> Artificial
<400> 41
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Leu Thr Ile Ser Thr Tyr Pro Met Ser Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Ser
35 40 45
Gly Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser
65 70 75 80
Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Ala Lys Ser Arg Ala
85 90 95
His Ser Thr Thr Tyr Tyr Ser Ser Arg Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 42
<211> 111
<212> PRT
<213> Artificial
<400> 42
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser Thr Ile Ser Trp Ala
20 25 30
Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val Ala Thr Ile Tyr Asn
35 40 45
Asp Gly Thr Ala Ser Tyr Asn Gly Asp Val Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Ser Asp Asp Thr Ala Leu Tyr Tyr Cys Leu Ala Asn Leu Arg
85 90 95
Gly Asp Asn His Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
100 105 110
<210> 43
<211> 333
<212> DNA
<213> Artificial
<221> CDS
<222> (1)..(333)
<400> 43
gag tct ggg gga ggc ttg gtg cag cct ggg ggg tct ttg aga ctc tcc 48
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
tgt gca gcc tct gga ttc acc ttc agt agt tat gcc atg aga tgg gtc 96
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Arg Trp Val
20 25 30
cgc cag gct ccg ggg aag gga ctc gag tgg gtc tca acg att tat agt 144
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Ile Tyr Ser
35 40 45
aat ggt aca cca gcc tat gca gac tcc gtg aag ggc cga ttc acc atc 192
Asn Gly Thr Pro Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
tcc aga gac aac gcc aag aac acg ctg tat ctg caa ttg aac agc ctg 240
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu
65 70 75 80
aaa tct gag gat acg gcc atg tat tac tgt aca aat tcc aac aag ccc 288
Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Thr Asn Ser Asn Lys Pro
85 90 95
aaa ttt gac gcc cgg ggc cag ggg acc cag gtc acc gtc tcc tca 333
Lys Phe Asp Ala Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
100 105 110
<210> 44
<211> 333
<212> DNA
<213> Artificial
<221> CDS
<222> (1)..(333)
<400> 44
gag tct ggg gga ggc tcg gtg cag gct gga ggg tct ctg aaa ctc tcc 48
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Lys Leu Ser
1 5 10 15
tgt gca gcc tct gga tac aga ttc agt gcc tgc gga atg gac tgg tac 96
Cys Ala Ala Ser Gly Tyr Arg Phe Ser Ala Cys Gly Met Asp Trp Tyr
20 25 30
cgc cag gct cca ggg aag gag cgc gag ttg gtc tca ctt att aat agt 144
Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ser Leu Ile Asn Ser
35 40 45
gat ggt act aca agc tat gta gac tcc gtg aag ggc cga ttc acc atc 192
Asp Gly Thr Thr Ser Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
tcc cga gac aat gcc aag aac acg ctg tat ctg caa ctg gac agc ctg 240
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asp Ser Leu
65 70 75 80
aaa acg gag gac acg gcc atg tat acc tgt gcg gct tgg cta aac ggt 288
Lys Thr Glu Asp Thr Ala Met Tyr Thr Cys Ala Ala Trp Leu Asn Gly
85 90 95
ggc aac cgt tac tgg ggc aaa gga acc cag gtc acc gtc tcc tca 333
Gly Asn Arg Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser
100 105 110
<210> 45
<211> 360
<212> DNA
<213> Artificial
<221> CDS
<222> (1)..(360)
<400> 45
gag tct ggg gga ggc tcg gtg cag gct gga ggg tct ctg aga ctc tcc 48
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
tgt gta cgt tct gga tac acc tac agt tac gac tgc atg ggc tgg ttc 96
Cys Val Arg Ser Gly Tyr Thr Tyr Ser Tyr Asp Cys Met Gly Trp Phe
20 25 30
cgc cag gct cca ggg aag aag cgc gag ggg gtc gca gtt att aat att 144
Arg Gln Ala Pro Gly Lys Lys Arg Glu Gly Val Ala Val Ile Asn Ile
35 40 45
ggt ggg acg act aca tac tat ccc gac tcc gtg aag ggc cgg ttc acc 192
Gly Gly Thr Thr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
atc tcc caa gac aac gcc aag aac acg gtg tat ctg caa atg aac agc 240
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
ctg aaa cct gag gac act gcc acg tac tac tgt gcg cat gac ggg tat 288
Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala His Asp Gly Tyr
85 90 95
tta gac tgc tgg cgc ggc gca tcg gct gac ttt ggt aac tgg ggc cag 336
Leu Asp Cys Trp Arg Gly Ala Ser Ala Asp Phe Gly Asn Trp Gly Gln
100 105 110
ggg acc cag gtc acc gtc tcc tca 360
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 46
<211> 363
<212> DNA
<213> Artificial
<221> CDS
<222> (1)..(363)
<400> 46
gag tct ggg gga ggc tcg gtg cac cct gga ggg tct ctg aga ctc tcc 48
Glu Ser Gly Gly Gly Ser Val His Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
tgt gca gcc tct gga tac ttc gcc agt tgg tat tat aag ggc tgg ttc 96
Cys Ala Ala Ser Gly Tyr Phe Ala Ser Trp Tyr Tyr Lys Gly Trp Phe
20 25 30
cgc cag gtt cca ggg aag gag cgc gag ggg gtc gca gct atc ggc agt 144
Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Gly Ser
35 40 45
gat ggt agc aca agc tac gca gac tcc gtg aag ggc cga ttc acc atc 192
Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
tcc aca gac aac gcc aag aac act ctg tat ctg caa atg aac agc ctg 240
Ser Thr Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
aaa cct gag gac act ggc atg tac tac tgt gcg gca caa tac gat gcg 288
Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala Ala Gln Tyr Asp Ala
85 90 95
ggt ggc tgg tat ggc cta cta gac caa agg cga ttt ggt tac tgg ggc 336
Gly Gly Trp Tyr Gly Leu Leu Asp Gln Arg Arg Phe Gly Tyr Trp Gly
100 105 110
cag ggg acc cag gtc acc gtc tcc tca 363
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 47
<211> 345
<212> DNA
<213> Artificial
<221> CDS
<222> (1)..(345)
<400> 47
gag tct ggg gga ggc ttg gtg cag cct ggg ggg tct ctg aga ctc tcc 48
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
tgt gca gcc tct gga ctc acc atc agt acc tat ccg atg agc tgg gtc 96
Cys Ala Ala Ser Gly Leu Thr Ile Ser Thr Tyr Pro Met Ser Trp Val
20 25 30
cgc cag gct cca ggg aag gga ctc gag tgg gtc tca ggt att agt agt 144
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Ser
35 40 45
ggt ggt ggt agg aca tac tat gca gac tcc gtg aag ggc cga ttc acc 192
Gly Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
atc tcc aga gac aac gcc aag aac acg ctg tat ctg caa ttg aac agc 240
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser
65 70 75 80
ctg aaa act gag gac acg gcc atg tat tac tgt gca aaa tcc cgc gcc 288
Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Ala Lys Ser Arg Ala
85 90 95
cat agt act act tac tac tct agc cgg ggc cag ggg acc cag gtc aca 336
His Ser Thr Thr Tyr Tyr Ser Ser Arg Gly Gln Gly Thr Gln Val Thr
100 105 110
gtc tcc tca 345
Val Ser Ser
115
<210> 48
<211> 333
<212> DNA
<213> Artificial
<221> CDS
<222> (1)..(333)
<400> 48
gag tct ggg gga ggc ttg gtg cag cct ggg ggg tct ctg aga ctc tcc 48
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
tgt gca gcc tct gga ttc acc ttc agt agc tcc acc att agc tgg gcc 96
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser Thr Ile Ser Trp Ala
20 25 30
cgc cag gct cca ggg aag agc cta gag tgg gtg gcc act atc tat aat 144
Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val Ala Thr Ile Tyr Asn
35 40 45
gat ggt acc gct tca tac aat gga gac gtc gtg aag ggc cga ttc acc 192
Asp Gly Thr Ala Ser Tyr Asn Gly Asp Val Val Lys Gly Arg Phe Thr
50 55 60
atc tcc aga gac aac gcc aag agc acg gtg tat ctg caa atg aac agc 240
Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
ctg aaa tct gac gac acg gcc ctg tat tac tgt ttg gcc aat ttg cgc 288
Leu Lys Ser Asp Asp Thr Ala Leu Tyr Tyr Cys Leu Ala Asn Leu Arg
85 90 95
ggc gat aac cac agg ggc cag ggg acc cag gtc acc gtc tcc tca 333
Gly Asp Asn His Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
100 105 110
Claims (6)
1. for a VHH chain for H3N2 nano antibody, comprise framework region FR and complementary determining region CDR, it is characterized in that, described framework region FR is made up of four framework regions, is respectively FRl, FR2, FR3 and FR4; Described complementary determining region CDR is made up of three complementary determining regions, is respectively CDRl, CDR2 and CDR3, and their aminoacid sequence is following aminoacid sequence:
First group: FR1 is the aminoacid sequence shown in SEQ ID NO.1, and FR2 is the aminoacid sequence shown in SEQ ID NO.2, and FR3 is the aminoacid sequence shown in SEQ ID NO.3, and FR4 is the aminoacid sequence shown in SEQ ID NO.4; CDR1 is the aminoacid sequence shown in SEQ ID NO.19, and CDR2 is the aminoacid sequence shown in SEQ ID NO.20, and CDR3 is the aminoacid sequence shown in SEQ ID NO.21;
Second group: FR1 is the aminoacid sequence shown in SEQ ID NO.5, and FR2 is the aminoacid sequence shown in SEQ ID NO.6, and FR3 is the aminoacid sequence shown in SEQ ID NO.7, and FR4 is the aminoacid sequence shown in SEQ ID NO.8; CDR1 is the aminoacid sequence shown in SEQ ID NO.22, and CDR2 is the aminoacid sequence shown in SEQ ID NO.23, and CDR3 is the aminoacid sequence shown in SEQ ID NO.24;
3rd group: FR1 is the aminoacid sequence shown in SEQ ID NO.9, and FR2 is the aminoacid sequence shown in SEQ ID NO.10, and FR3 is the aminoacid sequence shown in SEQ ID NO.11, and FR4 is the aminoacid sequence shown in SEQ ID NO.4; CDR1 is the aminoacid sequence shown in SEQ ID NO.25, and CDR2 is the aminoacid sequence shown in SEQ ID NO.26, and CDR3 is the aminoacid sequence shown in SEQ ID NO.27;
4th group: FR1 is the aminoacid sequence shown in SEQ ID NO.12, and FR2 is the aminoacid sequence shown in SEQ ID NO.13, and FR3 is the aminoacid sequence shown in SEQ ID NO.14, and FR4 is the aminoacid sequence shown in SEQ ID NO.4; CDR1 is the aminoacid sequence shown in SEQ ID NO.28, and CDR2 is the aminoacid sequence shown in SEQ ID NO.29, and CDR3 is the aminoacid sequence shown in SEQ ID NO.30;
5th group: FR1 is the aminoacid sequence shown in SEQ ID NO.1, and FR2 is the aminoacid sequence shown in SEQ ID NO.15, and FR3 is the aminoacid sequence shown in SEQ ID NO.16, and FR4 is the aminoacid sequence shown in SEQ ID NO:4; CDR1 is the aminoacid sequence shown in SEQ ID NO.31, and CDR2 is the aminoacid sequence shown in SEQ ID NO.32, and CDR3 is the aminoacid sequence shown in SEQ ID NO.33;
6th group: FR1 is the aminoacid sequence shown in SEQ ID NO.1, and FR2 is the aminoacid sequence shown in SEQ ID NO.17, and FR3 is the aminoacid sequence shown in SEQ ID NO.18, and FR4 is the aminoacid sequence shown in SEQ ID NO.4; CDR1 is the aminoacid sequence shown in SEQ ID NO.34, and CDR2 is the aminoacid sequence shown in SEQ ID NO.35, and CDR3 is the aminoacid sequence shown in SEQ ID NO.36.
2. the VHH chain of H3N2 nano antibody according to claim 1, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.37,38,39,40,41,42.
3. a H3N2 nano antibody, is characterized in that, it is the nano antibody for A type H3N2 influenza virus epitope, the VHH chain of the aminoacid sequence as shown in SEQ ID NO.37,38,39,40,41,42.
4. a DNA molecular, is characterized in that, its coding is selected from the protein of lower group: the VHH chain of the H3N2 nano antibody shown in claim 1 or 2, or the H3N2 nano antibody shown in claim 3.
5. DNA molecular according to claim 4, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.43,44,45,46,47,48.
6. an expression vector, is characterized in that, it containing SEQ ID NO:43,44,45,46, the nucleotide sequence shown in 47,48.
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| CN104650227B (en) * | 2014-12-22 | 2018-03-13 | 浙江省医学科学院 | It is a kind of for VSG Trypanosoma evansi nano antibody and its coded sequence and application |
| CN115028726B (en) * | 2022-03-31 | 2024-01-09 | 浙江特瑞思药业股份有限公司 | anti-PD-1 nano antibody and application thereof |
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