CN109535249A - A kind of monoclonal antibody ZKns3G2 and its application - Google Patents
A kind of monoclonal antibody ZKns3G2 and its application Download PDFInfo
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- CN109535249A CN109535249A CN201811519058.1A CN201811519058A CN109535249A CN 109535249 A CN109535249 A CN 109535249A CN 201811519058 A CN201811519058 A CN 201811519058A CN 109535249 A CN109535249 A CN 109535249A
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- antibody
- monoclonal antibody
- zkns3g2
- heavy chain
- light chain
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- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of monoclonal antibody ZKns3G2 and its application.Monoclonal antibody ZKns3G2 provided by the invention is a kind of full human monoclonal antibody for zika virus NS1 albumen, including with amino acid sequence shown in SEQ ID NO.1 heavy chain variable domain and SEQ ID NO.2 shown in amino acid sequence light chain variable region.Heavy chain and light chain in monoclonal antibody ZKns3G2 provided by the invention are nature pairings, therefore the affinity of antibody is high.In conjunction with zika virus NS1 protein-specific, do not intersect with dengue virus NS 1, can be used as diagnosis or therapeutic antibodies.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of monoclonal antibody ZKns3G2 and its application.
Background technique
2015 to 2016 zika virus (ZIKV) world wide especially America area rapidly propagate, because it can
Lead to serious birth defect and adult neural's complication, international public health emergency is declared as by WHO.
ZIKV is equally propagated through yellow-fever mosquito, often appears in the Endemic Area dengue virus (DENV).ZIKV and DENV are often appeared in together
One area, but Clinical symptoms, harm and disposition are different.Urgent need distinguishes both virus infections as early as possible, especially to the woman of pregnancy
For female.ZIKV and DENV belongs to flavivirus, and affiliation is close.Due to the phase on genome and protein sequence and conformation
Like caused cross reaction, difficulty is brought to distinguish both virus infections.
Serum polyclonal antibody and the monoclonal antibody separated from the infected are studies have shown that ZIKV induction host's generation is directed to disease
There are extensive cross reactions for malicious memebrane protein E antibody.And high special is presented for the antibody of virus nonstructural protein 1 (NS1)
Property reaction, provide possibility to identify both virus infections.Balmaseda etc. (2017) is separated with from ZIKV the infected
Only in conjunction with ZIKV NS1 monoclonal antibody, the Inhibition ELISA based on NS1 is established, is used for ZIKV Infect And Diagnose, and distinguish other Huangs
Virus such as 4 serotypes of Dengue, west nile virus and yellow fever virus.
It there is no effective vaccine for ZIKV at present, more lack early diagnosis and effective treatment method.NS1 albumen exists
It plays an important role in flavivirus life cycle, including virus replication, immunologic escape and the effect in pathogenic mechanism.?
NS1 is specified to play an important role in dengue fever pathogenesis, it is related to the seriousness of disease.Therefore it is directed to zika virus NS1
Full people's monoclonal antibody of albumen is likely to become a kind of strategy of zika virus disease treatment.
The technology of preparing of monoclonal antibody experienced the hybridoma technology of source of mouse antibody, mouse people's chimeric antibody, mouse antibody source of people skill
Then there is people's monoclonal antibody technology of preparing in art.It is presently available for the predominantly phage display skill of extensive specificity antibody screening
Art and single B cell antibody technology.Display technique of bacteriophage can be used for screening Separated pin to the antibody of any target antigen.However
The specific antibody filtered out from the heavy chain of random combine antibody and light chain library, heavy chain and light chain pairing are not that nature produces
Raw antibody is original, influences the affinity and function of antibody.Single B cell antibody technology directly expands antibody from single B cell
The variable region gene of heavy chain and light chain constructs antibody expressing plasmid, antibody is then expressed in cell culture system.This skill
Art is separable to be primarily present the functional antibodies that conformation is special in the infected's body.
Chinese patent application CN107188935A discloses a kind of zika virus NS1 antigenic mutant and its application, this is anti-
Former mutant is by carrying out knock out mutants to the high segment of homology, then through gene chemical synthesis, vector construction, protokaryon table
It reaches, antigen purification and immunologic function Analysis and Screening obtain, and have the function of reducing Dengue positive sample recall rate.But
This antigenic mutant mutation rate is low, is only reduction of zika virus and intersects with what dengue virus antibody detected, can not eradicate.
In summary, it is low that specificity exists in the prior art, can not thoroughly distinguish zika virus and dengue virus, screens
The disadvantages of process is cumbersome.
Summary of the invention
It is an object of the invention to overcome shortcoming and deficiency existing in the prior art, a kind of monoclonal antibody is provided
ZKns3G2 and its application.Monoclonal antibody ZKns3G2 provided by the invention has affinity high, can be used for distinguishing zika virus
And the advantages of dengue virus infection.
Currently, there is no effective vaccine for this sick new infections disease of zika virus, more lack early diagnosis and
Effective treatment method.The present invention screens by the culture to finite population memory B cell and obtains heavy chain of antibody and light chain
Variable region gene, construct antibody expression vector, antibody is then expressed in cell culture system.This method is suitable for building
Full human monoclonal antibody.
In order to achieve the above object, the present invention provides a kind of monoclonal antibody ZKns3G2, including heavy chain and light chain, institutes
The amino acid sequence information of the Variable Area of heavy chain is stated as shown in SEQ ID NO.1；The amino acid of the Variable Area of the light chain
Sequence information is as shown in SEQ ID NO.2；
EVQLLESGGGLIQPGGSLRLSCAASGLTVSNNYMNWVRQAPGKGLEWV SIIYSSGSTYYADSVKGRF
TISRDTRKNTLYLQMHSLRVEDTAVYYCARYRGW L(SEQ ID NO.1)；
VIWMTQSPSSLSASVGDRVTITCRASQSVSSHLNWYQQKPGKAPKLLIY AVSSLQSGVPSRFSGGDS
GTDFTLTIASLQPEDFATYYCQQTYTIPRTFGQGTK VEIK(SEQ ID NO.2)。
The present invention also provides a kind of Variable Area for encoding the heavy chain or the amino acid of the Variable Area of above-mentioned light chain
The polynucleotide sequence of sequence；The polynucleotide sequence of the heavy chain variable domain is encoded as shown in SEQ ID NO.3, is compiled
The polynucleotide sequence of the code light chain variable region is as shown in SEQ ID NO.4；
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGATCCAGCCTGGGGG GTCCCTGAGACTCTCCTGTG
CAGCCTCTGGGCTCACCGTCAGTAACAACTA CATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAGTGGG
TCTCAA TTATTTATAGTAGTGGTAGCACGTACTACGCAGACTCCGTGAAGGGCCGCTT CACCATCTCCAGAGAC
ACTCGCAAGAACACTCTGTATCTTCAAATGCACAG CCTGAGAGTCGAGGACACGGCCGTATATTACTGTGCGAGA
TACCGCGGCTG GCTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCAG(SEQ ID NO.3)；
GTCATCTGGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTT
GTCGGGCAAGTCAGAGCGTTAGCAGCCATTT AAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGA
TCTATG CTGTGTCCAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGTGGCGGTGAT TCTGGCACAGATTTCACT
CTCACCATCGCCAGTTTGCAACCTGAAGACTTT GCAACTTACTACTGTCAACAGACTTACACTATCCCTCGGACG
TTCGGCCAA GGGACCAAGGTGGAAATCAAAC(SEQ ID NO.4)。
The monoclonal antibody ZKns3G2's the preparation method comprises the following steps:
S1, it is screened from the infected's peripheral blood mononuclear cells using fluorescence antibody, obtains memory B cell；
S2, the resulting memory B cell of incubation step S1 are added cell factor and are converted using EBV, using capture
ELISA screens the presence of antibody in B cell culture supernatant, obtains the B cell containing positive antibody；
The RNA of S3, the resulting B cell of extraction step S2, reverse transcription is at cDNA, heavy chain and light chain through PCR amplification antibody
Variable region is cloned on antibody expression vector, obtains the expression vector of the ZKns2E11 containing monoclonal antibody；The PCR primer and
PCR amplification program is shown in document (J Immunol Methods.2008 Jan 1；329(1-2):112-24.)；
S4, the expression vector of the resulting ZKns2E11 containing monoclonal antibody of step S3 is transfected to 293T cell, 5-6 days
Afterwards, collect supernatant to get.
Preferably, the fluorescence antibody in the step S1 includes IgD-FITC, CD19-ECD, CD27-PC7, CD38-
APCA750, IgM-PB and CD45-KO.
Preferably, the Porcine HGF in the step S2 includes the health that CpG, IL21, IL2 and radiating irradiation are crossed
The cell (PBMC) of human peripheral monokaryon.
The present invention also provides a kind of monoclonal antibody ZKns3G2 in preparation clinical diagnosis or treatment zika virus disease
Application.
Compared with prior art, monoclonal antibody ZKns3G2 provided by the invention, has the advantage that
(1) monoclonal antibody ZKns3G2 provided by the invention is obtained through B cell culture binding antibody clone technology
Target full people's monoclonal antibody ZKns3G2 of zika virus NS1 albumen；
(2) monoclonal antibody ZKns3G2 provided by the invention, the heavy chain and light chain for being included are nature pairings, therefore anti-
The affinity of body is very high；
(3) monoclonal antibody ZKns3G2 provided by the invention zika virus specificity with higher, can be by stockaded village's card disease
Poison and dengue virus separate；
(4) monoclonal antibody ZKns3G2 provided by the invention is that one kind can separate and exist and be difficult in vitro in vivo
The functional antibodies of the conformational domains imitated.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the heavy chain expression vector of monoclonal antibody ZKns3G2；
Fig. 2 is the structural schematic diagram of the light chain expression vector of monoclonal antibody ZKns3G2；
Fig. 3 is the result figure of the association reaction of monoclonal antibody ZKns3G2 and virus N S1 albumen；
Fig. 4 is ZKns3G2 affinity measurement result figure；
Fig. 5 is the result figure that ZKns3G2 prepares that colloid gold test paper detects recombinant virus NS1 albumen.
Specific embodiment
The present invention is further explained combined with specific embodiments below, it is noted that following embodiment is only
To explain the present invention, and it cannot be used to limit the present invention, all technical solutions same or similar with the present invention are in this hair
Within bright protection scope.Unless otherwise specified, what technological means used in embodiment was well known to those skilled in the art
Conventional means, raw materials used is commercial goods.
Fluorescence antibody, antibody expression vector and flow cytometer are purchased from Beckman Coulter Inc., the U.S.
(Beckman Coulter,Inc.)；CDNA synthetic agent box (SuperScript III First Strand Synthesis
System, invitrogen, #18080051) it is purchased from Shanghai Yu Bo Biotechnology Co., Ltd；Antibody expression vector is shown in text
Offer (J Immunol Methods.2008 Jan 1；329(1-2):112-24.).
The preparation of 1 monoclonal antibody ZKns3G2 of embodiment
1. the identification and sorting of memory B cell
The separation of 1.1 peripheral blood mononuclear cells: stockaded village's card the infected's convalescence peripheral vein EDTA anticoagulation is taken, utilization is close
Spend gradient centrifugation procedure separating peripheral blood mononuclear cells, packing 5 × 106/ pipe, is placed in liquid nitrogen and freezes；
The dyeing of 1.2 fluorescent labeled antibodies: peripheral blood mononuclear cells is placed in water-bath in 37 DEG C and dissolves, and is buffered using PBS
Liquid washs 3 times, is subsequently added into antibody and is dyed, and room temperature, which is protected from light, is incubated for 15min, after washing using PBS buffer solution, is added 400
μ L PBS buffer solution suspension cell up flow type instrument, the streaming instrument are Beckman Kurt MoFlo Astrios EQ ultrahigh speed
Fluidic cell separation system；
If 9 analyzer tubes, such as table 1, the antibody of corresponding single fluorescent marker is added in 1-7 pipe, and 7 kinds of mixing are added in the 9th pipe
Fluorescent marker antibody, the 8th pipe for only cell blank tube, sample cell with the 9th pipe equally dyes；
The sorting of 1.3 memory B cells: machine is analyzed on sample cell, and the CD45 positive living is irised out according to 7-AAD and CD45
Leucocyte.B cell is irised out according to CD19.IgD-IgM-CD27+CD38low is defined in the B cell group of IgM and IgD jack to jack adapter
Group is memory B cell.Memory B cell is sorted into 96 porocyte culture plates containing cell culture fluid, 25-50 cell/every
Hole.
The classification for the fluorescent labeled antibody that 1 1-7 pipe of table is added
| Guan Hao | Dyeing pipe setting | The fluorescence of antibody and label | Volume (μ L) | Fluorescence channel |
| 1 | Single dye | IgD-FITC | 10 | FL1 |
| 2 | Single dye | CD19-ECD | 5 | FL3 |
| 3 | Single dye | 7-AAD | 10 | FL4 |
| 4 | Single dye | CD27-PC7 | 5 | FL5 |
| 5 | Single dye | CD38-APC A750 | 2 | FL8 |
| 6 | Single dye | IgM-PB | 5 | FL9 |
| 7 | Single dye | CD45-KO | 5 | FL10 |
| 8 | Blank tube | Antibody is not added | ||
| 9 | Sample cell | 7 kinds of antibody mixed liquors |
2. memory B cell culture and the screening of culture supernatant antibody.
The Healthy People that CpG, IL21, IL2, radiating irradiation are crossed is added into 96 porocyte culture plates containing memory B cell
PBMC and B95.8 cells and supernatant culture 7-10 days containing EBV.With needle in capture ELISA screening B cell culture supernatant
Presence to zika virus NS1 protein antibodies.
3. the clone of antibody
3.1cDNA synthesis: for having in the supernatant that filters out for B cell existing for zika virus NS1 protein antibodies,
It first proposes RNA another mistake and is transcribed into cDNA.CDNA synthesizes kit (SuperScript III First Strand Synthesis
System,invitrogen,#18080051)。
The heavy chain and light-chain variable region gene of 3.2 nested PCR amplification antibody: PCR primer sequence bibliography (J
Immunol Methods.2008 Jan 1；329 (1-2): 112-24.), reaction uses the super fidelity dna polymerase of Phusion
(Phusion High-Fidelity PCR Master Mix with GC Buffer,NEB,#M0532s)。
The heavy chains of 3.3 antibody and light chain variable region PCR product through digestion (VH, AgeI/SalI, VK, AgeI/Xhol, VL,
AgeI/BsiWI it) is cloned on the antibody expression vector containing human IgG1's constant region, heavy chain expression vector is shown in that Fig. 1, human IgG1 are permanent
The antibody expression vector for determining area is shown in Fig. 2.The heavy chain measured and light-chain variable region gene sequence are analyzed through IMGT/V-Quest.
The specificity analysis of 2 antibody of embodiment
1. the production of antibody: the heavy chain for the antibody that the sequence of building determines and light chain vector cotransfection are entered into 293T cell,
The culture supernatant containing antibody is harvested after 5-6 days；
2. the culture supernatant containing antibody is carried out the affine column purification of proteinA by the purifying of antibody, and measures protein content；
3. the reaction of antibody: antibody and zika virus (ZIKA) and dengue virus 1-4 type (DENV1, DENV2, DENV3,
DENV4) the association reaction of NS1 albumen, using capture NS1 antigen ELISA method, reaction result is shown in Fig. 3；
4. affinity of antibody measures (BLI): using the Octet RED384 system of Fortebio company, selecting Anti-
Human IgG Fc (AHC) sensor, affinity testing result are shown in Fig. 4, wherein affinity size constant KD is 4.62E-10,
Binding constant Ka is 5.57E+0.5, and dissociation constant Kd is 2.57E-0.4；Using antibody as Ligand, first curing antibody to biography
On sensor (20 μ g/mL of concentration)；Antigen detects the association and dissociation ability of antibody and antigen, detection recombination disease as analyte
Malicious NS1 albumen result is shown in Fig. 5.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although elaborating referring to preferred embodiment to the present invention, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Sequence table
<110>Guangzhou City No.8 People's Hospital
<120>a kind of monoclonal antibody ZKns3G2 and its application
<130> 2018.11.29
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 102
<212> PRT
<213>zika virus heavy chain of antibody amino acid sequence (Zika virus anti-weight chain amino acid
sequence)
<400> 1
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Val Ser Asn Asn
20 25 30
Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Thr Arg Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met His Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Tyr Arg Gly Trp Leu
100
<210> 2
<211> 107
<212> PRT
<213>zika virus antibody light chain amino acid sequence (Zika virus anti-weight chain amino acid
sequence)
<400> 2
Val Ile Trp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser His
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Val Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Gly Asp Ser Gly Thr Asp Phe Thr Leu Thr Ile Ala Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Ile Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 3
<211> 346
<212> DNA
<213>polynucleotide sequence (the Polynucleotide sequences of zika virus heavy chain of antibody amino acid is encoded
encoding Zika virus anti-weight chain amino acids)
<400> 3
gaggtgcagc tgttggagtc tgggggaggc ttgatccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctgggct caccgtcagt aacaactaca tgaactgggt ccgccaggct 120
ccaggaaagg ggctggagtg ggtctcaatt atttatagta gtggtagcac gtactacgca 180
gactccgtga agggccgctt caccatctcc agagacactc gcaagaacac tctgtatctt 240
caaatgcaca gcctgagagt cgaggacacg gccgtatatt actgtgcgag ataccgcggc 300
tggcttgact actggggcca gggaaccctg gtcactgtct cctcag 346
<210> 4
<211> 322
<212> DNA
<213>polynucleotide sequence (the Polynucleotide sequences of zika virus antibody light chain amino acid is encoded
encoding Zika virus anti-weight chain amino acids)
<400> 4
gtcatctgga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgtc gggcaagtca gagcgttagc agccatttaa attggtatca gcagaaacca 120
gggaaagccc ctaaactcct gatctatgct gtgtccagtt tacaaagtgg ggtcccatca 180
aggttcagtg gcggtgattc tggcacagat ttcactctca ccatcgccag tttgcaacct 240
gaagactttg caacttacta ctgtcaacag acttacacta tccctcggac gttcggccaa 300
gggaccaagg tggaaatcaa ac 322
Claims (4)
1. a kind of monoclonal antibody ZKns3G2, including heavy chain and light chain, which is characterized in that the amino of the heavy chain variable domain
Acid sequence is SEQ ID NO.1；The amino acid sequence of the light chain variable region is SEQ ID NO.2.
2. monoclonal antibody ZKns3G2 as described in claim 1, which is characterized in that encode the heavy chain Variable Area or
The amino acid sequence of the Variable Area of light chain is polynucleotide sequence.
3. monoclonal antibody ZKns3G2 as claimed in claim 2, which is characterized in that encode the Variable Area of the heavy chain
Polynucleotide sequence encodes the polynucleotide sequence such as SEQ of the Variable Area of the light chain as shown in SEQ ID NO.3
Shown in ID NO.4.
4. monoclonal antibody ZKns3G2 as described in claim 1, the application in preparation diagnosis or treatment zika virus disease.
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Cited By (1)
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| CN113122659A (en) * | 2021-04-27 | 2021-07-16 | 北京瀚梅生物科技有限公司 | Kit for detecting virus |
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