CN108727489A

CN108727489A - A kind of monoclonal antibody ZK2C2 and application

Info

Publication number: CN108727489A
Application number: CN201710257084.0A
Authority: CN
Inventors: 张复春; 庾蕾; 张林琦; 王若珂; 高菲
Original assignee: Guangzhou City No8 People's Hospital; Tsinghua University
Current assignee: Guangzhou City No8 People's Hospital; Tsinghua University
Priority date: 2017-04-19
Filing date: 2017-04-19
Publication date: 2018-11-02
Anticipated expiration: 2037-04-19
Also published as: CN108727489B

Abstract

The invention discloses a kind of monoclonal antibody ZK2C2 and applications.The present invention provides a kind of IgG antibodies, are named as ZK2C2, are made of light chain and heavy chain；CDR1, CDR2 and CDR3 in heavy chain variable region in the heavy chain is followed successively by the sequence 3 of sequence table from N-terminal 45-52 amino acids residue, 70-77 amino acids residue and 116-122 amino acids residues；CDR1, CDR2 and CDR3 in light chain variable region in the light chain is followed successively by the sequence 5 of sequence table from N-terminal 45-52 amino acids residue, 70-72 amino acids residue and 109-119 amino acids residues.The present invention also protects application of the IgG antibody in preparing the drug for inhibiting zika virus.The present invention has great application value for the prevention and control of zika virus disease, will generate far-reaching social effect.

Description

A kind of monoclonal antibody ZK2C2 and application

Technical field

The invention belongs to biotechnologies, and in particular to a kind of monoclonal antibody ZK2C2 and application.

Background technology

Zika virus, dengue fever virus, West Nile Virus and yellow fever virus belong to flavivirus.Zika virus disease It is a kind of mosquito matchmaker propagation infectious diseases caused by zika virus, clinical manifestation is slight or symptom does not occur.Main performance Slightly to generate heat, erythema (majority is maculopapule), headache, arthralgia, courbature, inability and apyetous conjunctivitis etc..Closely Zika virus is successively in French Polynesia and Brazilian eruption and prevalence over year, and continues to be especially Latin in worldwide America and Caribbean region are propagated rapidly, it has also become international public health emergency.

Zika virus infection only causes slight symptom.But there is serious nerve in the adult in the groove of Pohle Ni Xi Systematic complication-Guillain Barre syndrome (Guillain-Barr é syndrome, GBS), incidence is up to 1/4000.More draw People is concerned with, and as zika virus infection is in the appearance of Brazil, Severe birth defect microcephaly's deformity also obviously increases.According to estimates Mother infects probability of its fetus microcephaly's deformity incidence of zika virus higher than adult neural's complication.Also there is input in China Case report.Effective vaccine is there is no for this sick new infections disease of stockaded village's card at present, more lacks early diagnosis and effectively Therapy.

Occur leading to several RNA virus of mankind's severe infections in succession into 21 century, such as SARS, MERS coronavirus, And nearest Ebola virus and zika virus.The main problem shown especially in face of these new communicable diseases has been a lack of The medical counter-measure of effect includes antiviral therapy.Antiviral therapy is dependent on the research and development of broad-spectrum antiviral medicament or from existing Effective medicine is filtered out in drug quickly to cope with these new communicable diseases.

Monoclonal antibody can mass production, high-affinity and high specific with antigen binding greatly reduce clinic Using when adverse reaction.Antibody molecule can be transformed to increase its antiviral efficacy simultaneously.Antibody is with its specificity Become very promising means in infectious diseases treatment with the flexibility used.

Zika virus and the dengue virus for belonging to Flavivirus are propagated through same mosquito matchmaker, often in areal prevalence. Some researches show that the dengue virus of people elder generation postoperative infection different serotypes, there is the blood that the risk for the seriousness that aggravates disease infects in advance The antibody that clear type dengue virus generates, can be in conjunction with the different serotypes dengue virus of subinfection again, but cannot neutralize virus, instead And increase the entrance of virus, aggravate the state of an illness.Therefore, the treatment or prevention of zika virus disease with antibody development need to avoid antibody with The cross reaction of dengue virus.

Invention content

The object of the present invention is to provide a kind of monoclonal antibody ZK2C2 and applications.

The present invention provides a kind of IgG antibodies, are named as ZK2C2, are made of light chain and heavy chain；Heavy chain in the heavy chain CDR1, CDR2 and CDR3 in variable region are followed successively by the sequence 3 of sequence table from N-terminal 45-52 amino acids residue, 70- 77 amino acids residues and 116-122 amino acids residues；CDR1, CDR2 in light chain variable region in the light chain and CDR3 is followed successively by the sequence 5 of sequence table from N-terminal 45-52 amino acids residue, 70-72 amino acids residue and 109- 119 amino acids residues.

The heavy chain variable region is specifically made of the sequence of sequence table 3 from the 20th to 133 amino acids residue of N-terminal.

The light chain variable region is specifically made of the sequence of sequence table 5 from the 20th to 129 amino acids residue of N-terminal.

The heavy chain is concretely following (a) or (b)：(a) sequence 3 of sequence table is from N-terminal 20-463 amino acids The protein of residue composition；(b) protein shown in the sequence 3 of sequence table.

The light chain is concretely following (c) or (d)：(c) sequence 5 of sequence table is from N-terminal 20-235 amino acids The protein of residue composition；(d) protein shown in the sequence 5 of sequence table.

The present invention also protects the gene for encoding the IgG antibody.

The gene for encoding the heavy chain is specially following (1) or (2) or (3)：

(1) sequence 4 of sequence table DNA molecular shown in the nucleotide of 5 ' end 889-2280；

(2) sequence 4 of sequence table DNA molecular shown in the nucleotide of 5 ' end 946-2280；

(3) DNA molecular shown in the sequence 4 of sequence table.

The gene for encoding the light chain is specially following (4) or (5) or (6)：

(4) sequence 6 of sequence table DNA molecular shown in the nucleotide of 5 ' end 889-1596；

(5) sequence 6 of sequence table DNA molecular shown in the nucleotide of 5 ' end 946-1596；

(6) DNA molecular shown in the sequence 6 of sequence table.

The present invention also protects application of the IgG antibody in preparing the drug for inhibiting zika virus.

It is the IgG antibody that the present invention, which also protects a kind of drug for inhibiting zika virus, active constituent,.

The present invention also protects application of the IgG antibody in preparation is used for and in the drug of zika virus.

It is the IgG antibody that the present invention, which also protects a kind of drug for neutralizing zika virus, active constituent,.

The present invention also protects the IgG antibody answering in preparing the drug for preventing and/or treating zika virus disease With.

It is the IgG that the present invention, which also protects a kind of drug for preventing and/or treating zika virus disease, active constituent, Antibody.

Concretely zika virus GZ01 plants of any description above zika virus.

The present invention is screened anti-using the E protein of zika virus as bait from the peripheral blood mononuclear cells of the infected The memory B cell that body generates, obtaining can be the same as the monoclonal antibody of E protein specific bond.It is reduced and is tested by zika virus plaque Model, screening have obtained with neutralization activity while having had the monoclonal antibody of good specificity.

The present invention has great application value for the prevention and control of zika virus disease, will generate far-reaching social effect.

Description of the drawings

Fig. 1 is neutralization activity result of the ZK2C2 antibody to zika virus and dengue fever virus.

Fig. 2 is protection activity result of the ZK2C2 antibody to animal.

Specific implementation mode

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.

Plasmid pcDNA3.1 (+)：Invitrogen companies, catalog number V790-20.293T cells：Gaede, CRL- 11268.PMD18T carriers：Takara, catalog number D101A.Vero cells：ATCC companies, catalog number CCL-81. C6/36 cells：ATCC companies, catalog number CRL-1660.

Zika virus used in embodiment is GZ01 plants of zika virus (Zika virus isolate GZ01)：With reference to Document：GenBankACCESSION NO.KU820898.

Dengue 1-type virus used in embodiment is Hawaii plants.Bibliography：Complete Genome Sequences of Dengue Virus Type 1 to 4 StrainsUsed for the Development of CBER/FDA RNA Reference Reagentsand WHO International Standard Candidates for Nucleic Acid Testing；Genome Announcements；January/February 2016 Volume 4 Issue 1 e01583-15。

Dengue 2-type virus used in embodiment is Guinea plants of New.Bibliography：Complete Genome Sequences of Dengue Virus Type 1 to 4 StrainsUsed for the Development of CBER/FDA RNA Reference Reagentsand WHO International Standard Candidates for Nucleic Acid Testing；Genome Announcements；January/February 2016 Volume 4 Issue 1 e01583-15。

AG6 mouse (AG6mice)：AG6 mouse are the small of the receptor deficient of interferon-' alpha ', interferon beta and interferon gamma Mouse is attacked dead in several days after the zika virus of malicious doses.Bibliography：Flavivirus NS1 protein in infected host sera enhances viral acquisition by mosquitoes；Jianying Liu,etc； Nature microbiology.2016；1(9):16087.

The discovery of embodiment 1, antibody

One, the preparation of albumen

1, construction recombination plasmid

(1) double chain DNA molecule shown in the sequence 2 of composition sequence table.

Protein shown in the sequence 1 of double chain DNA molecule polynucleotide shown in the sequence 2 of sequence table, wherein open Reading frame is sequence 2 from the nucleotide of 5 ' end 13-1296.

In the sequence 1 of sequence table, signal peptide, 20-421 amino acids are formed from the 1st to 19 amino acids residue of N-terminal Residue forms the E protein of zika virus, and the 422nd to 427 amino acids residue forms His₆Label.By 1 institute of sequence of sequence table The protein shown is named as E-His₆Fusion protein, it is contemplated that molecular weight 50kDa.

(2) double chain DNA molecule obtained with restriction enzyme BamHI and NotI double digestion step 1, recycling digestion production Object.

(3) restriction enzyme BamHI and NotI double digestion plasmid pcDNA3.1 (+) is used, the carrier of about 5400bp is recycled Skeleton.

(4) digestion products that step (2) recycles are connected with the carrier framework that step (3) recycles, obtains recombinant plasmid pcDNA3.1-ZIKV-E.According to sequencing result, structure is carried out to recombinant plasmid pcDNA3.1-ZIKV-E and is described as follows：In plasmid The sequence 2 of sequence table is inserted between BamHI the and NotI restriction enzyme sites of pcDNA3.1 (+) from the core of 5 ' end the 7th to 1299 Double chain DNA molecule shown in thuja acid.

2, albumen is prepared

(1) recombinant plasmid pcDNA3.1-ZIKV-E is transfected into 293T cells, is then trained in the DMEM containing 2% fetal calf serum Base culture 72h is supported, then 4000rpm centrifuges 30min, collects supernatant.

(2) affinity chromatography

The chromatographic column specification of affinity chromatography：Length 3cm, internal diameter 1cm；

The column packing of affinity chromatography：Nickel column beads (is purchased from Qiagen companies, catalog number 30230)；

Step is proceeded as follows successively：1. the supernatant that 300mL steps (1) obtain is splined on affinity column, 4 DEG C It is incubated 3 hours；2. washing pillar with the sample-loading buffer of 100mL imidazoles containing 20mM；3. the elution with 30mL imidazoles containing 500mM is slow Fliud flushing elutes destination protein, collects solution after column.

Sample-loading buffer, that is, HEPEs buffer (pH7.2,1M).

(3) solution after crossing column that step (2) obtains is taken, (Merck companies are purchased from, catalog number is with 30kD concentration tubes UFC800396 it) is concentrated, obtains the concentrate that volume is 1mL.

(4) gel permeation chromatography

The chromatographic column specification of gel permeation chromatography：Length 24cm, internal diameter 2cm；

The column packing of gel permeation chromatography：Superdex200increase 10/300GL are (public purchased from GEHealthcare Department, catalog number 28-9909-44)；

Operating procedure：The concentrate that loading 0.5mL steps (3) obtain, the PBS buffer solution for being 0.5mL/min with flow velocity (pH7.2,10mM) is eluted, and collects peak corresponding solution, as E-His after crossing column that retention volume is 11mL₆Fusion protein is molten Liquid.

Two, the identification, sorting and culture of the infected's memory B cell, the screening of culture supernatant antibody

1, peripheral blood mononuclear cells detaches：Stockaded village's card the infected's convalescence peripheral vein EDTA anticoagulations are taken, density is utilized Gradient centrifugation procedure separating peripheral blood mononuclear cells, packing 5 × 10⁶/ pipe, is placed in liquid nitrogen and freezes.

2, fluorescent labeled antibody dyes：Peripheral blood mononuclear cells is positioned over water-bath in 37 DEG C and dissolves, and uses PBS buffer solution Washing 3 times, the antibody for being subsequently added into fluorescent marker, which is dyed, (sets 9 analyzer tubes, corresponding single fluorescence mark is added in 1-7 pipes The antibody of the fluorescent marker of 7 kinds of mixing is added in the antibody of note, the 9th pipe, and the 8th pipe is the blank tube of only cell), room temperature, which is protected from light, incubates 15min is educated, is washed using PBS buffer solution, 400 μ l PBS buffer solution suspension cell loading streaming instrument (Beckman libraries are then added That spy's MoFlo Astrios EQ ultrahigh speed streamings cell sorting system).

3, the sorting of memory B cell：Machine is analyzed on sample cell, and the white of the CD45 positives living is irised out according to 7-AAD and CD45 Cell.B cell is irised out according to CD19.IgD-IgM-CD27+CD38low groups are defined in the B cell group of IgM and IgD jack to jack adapters Body is memory B cell.Memory B cell is sorted into 96 porocyte culture plates containing cell culture fluid, 25-50 cell/every Hole.

4, memory B cell culture and the screening of culture supernatant antibody：Add into 96 porocyte culture plates containing memory B cell Enter Healthy People PBMC that CpG, IL21, IL2, radiating irradiation are crossed and B95.8 cells and supernatants culture 7-10 days containing EBV. With the E-His obtained in step 1₆Fusion protein is screened in B cell culture supernatant with ELISA method and is directed to as coating protein The presence of zika virus E protein antibody.

Three, the acquisition of antibody sequence

1, cDNA is synthesized：Have for B cell existing for zika virus E protein antibody for filtering out in supernatant, first carries RNA Another mistake is transcribed into cDNA.CDNA synthesizes kit (SuperScript III First Strand Synthesis System,invitrogen,#18080051)。

2, the heavy chain variable region gene and chain variable region gene of nested PCR amplification antibody：PCR primer sequence reference document (J Immunol Methods.2008 Jan 1；329(1-2):112-24.), reaction is polymerize using the super fidelity dnas of Phusion Enzyme (Phusion High-Fidelity PCR Master Mix with GC Buffer, NEB, #M0532s).

3, the heavy chain variable region of antibody and light chain variable region PCR product are 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.

Based on above step, a variety of IgG have been obtained, one of which is named as ZK2C2.

The amino acid sequence of the total length heavy chain of ZK2C2 is as shown in the sequence 3 of sequence table, the amino of the full-length light chains of ZK2C2 Acid sequence is as shown in the sequence 5 of sequence table.

In the sequence 3 of sequence table, the 1st to 19 amino acids residue forms signal peptide (pilot protein is secreted into extracellularly), 20th to 133 amino acids residue forms heavy chain variable region, and (CDR1, CDR2 and CDR3 are followed successively by 3 45-52 amino acids of sequence Residue, 70-77 amino acids residue and 116-122 amino acids residue), the 134th to 463 amino acids residue composition weight Chain constant region.

In the sequence 5 of sequence table, the 1st to 19 amino acids residue forms signal peptide (pilot protein is secreted into extracellularly), 20th to 129 amino acids residue forms light chain variable region, and (CDR1, CDR2 and CDR3 are followed successively by 5 45-52 amino acids of sequence Residue, 70-72 amino acids residue and 109-119 amino acids residue), the 130th to 235 amino acids residue composition is light Chain constant region.

The preparation of embodiment 2, ZK2C2 antibody

One, the structure of recombinant plasmid

Double chain DNA molecule shown in sequence 4 by sequence table is inserted into PMD18T carriers, obtains heavy chain expression vector.Sequence In the sequence 4 of table, the 1st to 888 nucleotide forms CMV promoter, the sequence of the 889th to 2280 nucleotide coding sequence table Total length heavy chain shown in 3, the 2333rd to 2478 nucleotide are ployA segments.

Double chain DNA molecule shown in sequence 6 by sequence table is inserted into PMD18T carriers, obtains light chain expression vector.Sequence In the sequence 6 of table, the 1st to 888 nucleotide forms CMV promoter, the sequence of the 889th to 1596 nucleotide coding sequence table Full-length light chains shown in 5, the 1597th to 1742 nucleotide are ployA segments.

Two, the preparation of antibody

1, by heavy chain expression vector and light chain expression vector cotransfection 293T cells, then containing 2% fetal calf serum DMEM medium culture 72h, then 4 DEG C, 4000rpm centrifugation 30min, collect supernatant.

2, affinity chromatography

The column packing of affinity chromatography：Protein A beads (Thermo, catalog number 10006D)；

Step is proceeded as follows successively：1. the supernatant that 300mL steps 1 obtain is splined on affinity column, 4 DEG C incubate It educates 16 hours；2. washing pillar with 60mL combination buffers；3. destination protein is eluted with 30mL elution buffers, after collecting column Solution.

Combination buffer：Glycine 112.6g, sodium chloride 175.2g are taken, water is dissolved in and is settled to 1L with water, uses hydroxide Sodium tune pH to 8.0.

Elution buffer：Glycine 7.5g is taken, water is dissolved in and is settled to 500mL with water, with hydrochloric acid tune pH to 3.0.

3, the solution after crossing column that step 2 obtains is taken, is concentrated with ultrafiltration concentration pipe and system is replaced into PBS buffer solution (pH7.2,10mM) obtains the antibody-solutions (antibody concentration is with albumen densimeter) that 1mL antibody concentrations are 2mg/mL, is ordered Entitled ZK2C2 solution.

The neutralization activity of embodiment 3, ZK2C2 antibody on viral

One, viral preparation

By zika virus inoculation C6/36 cells (using the DMEM culture mediums containing 10%FBS), it is placed in 28 DEG C, 5%CO₂Item Stationary culture 4 days under part, then 3000rpm centrifugations 5min, collects supernatant, as zika virus liquid.

Two, the neutralization activity detection of monoclonal antibody

1, ZK2C2 solution prepared by Example 2, is diluted with PBS buffer solution (pH7.2,10mM), it is dilute to obtain antibody Release liquid.

2, by Vero cell inoculations to six orifice plates (per hole 4 × 10⁵A cell), stationary culture is overnight, cell density grow to 90% and uniformly it is paved with orifice plate.

3, zika virus liquid prepared by step 1 is mixed with antibody diluent prepared by step 1, obtains each mixed liquor (in every milliliter of mixed liquor, viral level 100pfu, antibody concentration is 40 μ g/mL, 8 μ g/mL, 1.6 μ g/mL, 0.32 μ g/mL Or 0.064 μ g/mL)；Zika virus liquid prepared by step 1 is mixed with the PBS buffer solution of pH7.2,10mM, it is dense to obtain virus Degree is the mixed liquor (blank control) of 100pfu/mL；37 DEG C of stationary incubations 1 hour.

4, six orifice plates of step 2 are taken into, supernatant is abandoned in suction, and mixed liquor (each mixing that 1mL completes step 3 is added per hole 2 multiple holes are arranged in liquid), 37 DEG C of stationary incubations 1 hour.

5, six orifice plates of step 4 are taken into, supernatant is abandoned in suction, is washed twice with PBS buffer solution (pH7.2,10mM), is then added Enter the DMEM culture mediums containing 1% low melting-point agarose and 2%FBS, waits for that culture medium solidification is placed on 37 DEG C of stationary cultures 6 days.

6, six orifice plates of step 5 are taken into, paraformaldehyde is added and terminates reaction, 1% violet staining is then added, counts Virus plaques number.

Percent neutralization=(the virus plaques number of virus plaques number-test group of blank control group)/blank control Virus plaques number × 100% of group.

It the results are shown in Table 1 and Fig. 1.

Table 1

Antibody concentration when percent neutralization is 50%, the i.e. IC of antibody₅₀Value, the IC of ZK2C2₅₀Value is 3.8 μ g/mL.

The specificity of embodiment 4, ZK2C2 antibody

One, viral preparation

Dengue 1-type virus is inoculated with C6/36 cells, is placed in 28 DEG C, 5%CO₂Under the conditions of stationary culture 6 days, then 3000rpm centrifuges 5min, collects supernatant, as dengue 1-type virus liquid.

Dengue 2-type virus is inoculated with C6/36 cells, is placed in 28 DEG C, 5%CO₂Under the conditions of stationary culture 6 days, then 3000rpm centrifuges 5min, collects supernatant, as dengue 2-type virus liquid.

Two, neutralization activity detects

Use dengue 1-type virus liquid and dengue 2-type virus liquid as virus liquid to be measured respectively.

3, virus liquid to be measured is mixed with antibody diluent prepared by step 1, obtains each mixed liquor (every milliliter of mixed liquor In, viral level 100pfu, antibody concentration is 0.064 μ g/mL, 0.32 μ g/mL, 1.6 μ g/mL, 8 μ g/mL or 40 μ g/mL)； Virus liquid to be measured is mixed with the PBS buffer solution of pH7.2,10mM, obtains the mixed liquor (blank that virus concentration is 100pfu/mL Control)；37 DEG C of stationary incubations 1 hour.

The result is shown in Figure 1.The result shows that ZK2C2 is 30% to the neutralization activity of dengue 1-type virus and dengue 2-type virus Hereinafter, illustrating that ZK2C2 does not have cross-neutralization activity to 1 type of the dengue virus of Flavivirus and dengue type 2 virus.

Embodiment 5, ZK2C2 antibody are to the protection activity of animal

AG6 mouse are divided into two groups, every group 4, handle respectively as follows：

First group：(every mouse is attacked for zika virus liquid prepared by the step of testing the 1st day, embodiment 3 are injected intraperitoneally one Toxic dose is 1.5 × 10⁴pfu)；It tests the 2nd day, the ZK2C2 solution (administration of every mouse prepared by intraperitoneal injection embodiment 2 Dosage is 300 μ g antibody)；The weight and survival condition of record mouse daily.

Second group：ZK2C2 solution is replaced with isometric PBS buffer solution (pH7.2,10mM), other are the same as first group.

Since the timing testing initial time, the 1st 24 hours is the 1st day, the 2nd 24 hours be the 2nd day, class successively It pushes away.The measurement result of 2nd day survival results and relative body weight result i.e. the 48th hour end time, and so on.

Weight × 100% of the weight of relative body weight=the n-th day/experiment initial time.

Survival results are shown in the A of Fig. 2.Relative body weight result is shown in the B of Fig. 2.It tests the 6th day, second group has 2 mouse dead It dies, tests the 7th day, second group of remaining 2 dead mouse.First group of experiment was at the 9th day and had 2 and 1 respectively on the 10th day Dead mouse, until the 12nd day, first group of mouse was just all dead.The result shows that ZK2C2 can be to AG6 mouse to zika virus Infected with certain inhibiting effect, the dead number of days for the mouse that can delay.

SEQUENCE LISTING

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Tsinghua University

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Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

245 250 255

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

260 265 270

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

275 280 285

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

290 295 300

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

305 310 315 320

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

325 330 335

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

340 345 350

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

355 360 365

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

370 375 380

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

385 390 395 400

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

405 410 415

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

420 425 430

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

435 440 445

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455 460

<210> 4

<211> 2478

<212> DNA

<213>Artificial sequence

<400> 4

agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 60

ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 120

tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 180

atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 240

ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 300

gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 360

ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 420

tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 480

aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 540

tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct 600

gttttgacct ccatagaaga caccgggacc gatccagcct ccgcggccgg gaacggtgca 660

ttggaacgcg gattccccgt gccaagagtg acgtaagtac cgcctataga gtctataggc 720

ccaccccctt ggcttcgtta gaacgcggct acaattaata cataacctta tgtatcatac 780

acatacgatt taggtgacac tatagaataa catccacttt gcctttctct ccacaggtgt 840

ccactcccag gtccaactgc acctcggttc tatcgattga attccaccat gggatggtca 900

tgtatcatcc tttttctagt agcaactgca accggtgtac attccgaggt gcagctgttg 960

gagtctgggg gaggcgtggt ccaacctggg aggtccctga gactctcctg tgcaggctct 1020

ggattcacct tcagtagcta tggcatgcac tgggtccgcc aggctccagg gaagggactg 1080

gagtgggtgg cagttatatc atatgatgga agtaataaat actatgtaga ctccgtgaag 1140

ggccgattca ccatctccag agacaattcc aggaacacac tgtatctgca aatgaacaac 1200

ctgaaagctg aagacacggc tgtgtattac tgtgcgaaag ggcatcttat agactggggc 1260

cagggaaccc tggtcactgt ctcctcagcg tcgaccaagg gcccatcggt cttcccgcta 1320

gcaccctcct ccaagagcac ctctgggggc acagcggccc tgggctgcct ggtcaaggac 1380

tacttccccg aacctgtgac ggtctcgtgg aactcaggcg ccctgaccag cggcgtgcac 1440

accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 1500

ccctccagca gcttgggcac ccagacctac atctgcaacg tgaatcacaa gcccagcaac 1560

accaaggtgg acaagagagt tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 1620

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 1680

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 1740

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1800

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1860

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1920

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1980

tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct gacctgcctg 2040

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 2100

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctatagc 2160

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 2220

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtcccc gggtaaatga 2280

gtgcgacggc cggcaagccc ccgctccccg ggctctcgcg gtcgtacgag gaaagcttgg 2340

ccgccatggc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 2400

acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 2460

atcaatgtat cttatcat 2478

<210> 5

<211> 235

<212> PRT

<213>Artificial sequence

<400> 5

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val Leu Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr

20 25 30

Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile

35 40 45

Gly Arg Asn Ile Val Asn Trp Tyr Gln Gln Phe Pro Gly Thr Thr Pro

50 55 60

Lys Leu Leu Ile Tyr Gly Thr Asn Gln Arg Pro Ser Gly Val Pro Asp

65 70 75 80

Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser

85 90 95

Gly Leu Gln Ser Glu Asp Glu Thr Asn Tyr Tyr Cys Ala Ala Trp Asp

100 105 110

Asp Ser Leu Asn Gly Trp Leu Phe Gly Gly Gly Thr Lys Leu Thr Val

115 120 125

Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser

130 135 140

Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser

145 150 155 160

Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser

165 170 175

Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn

180 185 190

Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp

195 200 205

Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr

210 215 220

Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

225 230 235

<210> 6

<211> 1744

<212> DNA

<213>Artificial sequence

<400> 6

agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 60

ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 120

tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 180

atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 240

ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 300

gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 360

ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 420

tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 480

aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 540

tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct 600

gttttgacct ccatagaaga caccgggacc gatccagcct ccgcggccgg gaacggtgca 660

ttggaacgcg gattccccgt gccaagagtg acgtaagtac cgcctataga gtctataggc 720

ccaccccctt ggcttcgtta gaacgcggct acaattaata cataacctta tgtatcatac 780

acatacgatt taggtgacac tatagaataa catccacttt gcctttctct ccacaggtgt 840

ccactcccag gtccaactgc acctcggttc tatcgattga attccaccat gggatggtca 900

tgtatcatcc tttttctagt agcaactgca accggtgtcc tctcccagct tgtgctgact 960

cagccaccct cagcgtctgg gacccccggg cagagggtca ccatctcttg ttctggaagc 1020

agctccaaca tcggaagaaa tattgtaaac tggtaccagc agttcccagg aacgaccccc 1080

aaactcctca tctatggtac taatcagcgg ccctcagggg tccctgaccg attctctggc 1140

tccaagtctg gcacctcagc ctccctggcc atcagtgggc tccagtctga ggatgagact 1200

aattattact gtgcagcatg ggatgacagc ctgaatggtt ggctgttcgg cggagggacc 1260

aagctgaccg tcctaggtca gcccaaggct gccccctcgg tcactctgtt cccaccctcg 1320

agtgaggagc ttcaagccaa caaggccaca ctggtgtgtc tcataagtga cttctacccg 1380

ggagccgtga cagtggcctg gaaggcagat agcagccccg tcaaggcggg agtggagacc 1440

accacaccct ccaaacaaag caacaacaag tacgcggcca gcagctacct gagcctgacg 1500

cctgagcagt ggaagtccca cagaagctac agctgccagg tcacgcatga agggagcacc 1560

gtggagaaga cagtggcccc tacagaatgt tcatagaagc ttggccgcca tggcccaact 1620

tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 1680

aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 1740

atgt 1744

Claims

1. a kind of IgG antibody, is made of light chain and heavy chain；CDR1, CDR2 and CDR3 in heavy chain variable region in the heavy chain The sequence 3 of sequence table is followed successively by from N-terminal 45-52 amino acids residue, 70-77 amino acids residue and 116-122 Amino acids residue；CDR1, CDR2 and CDR3 in light chain variable region in the light chain is followed successively by the sequence 5 of sequence table from N End 45-52 amino acids residue, 70-72 amino acids residue and 109-119 amino acids residues.

2. IgG antibody as described in claim 1, it is characterised in that：

The heavy chain variable region is made of the sequence 3 of sequence table from the 20th to 133 amino acids residue of N-terminal；

The light chain variable region is made of the sequence 5 of sequence table from the 20th to 129 amino acids residue of N-terminal.

3. IgG antibody as claimed in claim 2, it is characterised in that：

The heavy chain is for following (a) or (b)：(a) sequence 3 of sequence table is formed from N-terminal 20-463 amino acids residues Protein；(b) protein shown in the sequence 3 of sequence table；

The light chain is for following (c) or (d)：(c) sequence 5 of sequence table is formed from N-terminal 20-235 amino acids residues Protein；(d) protein shown in the sequence 5 of sequence table.

4. encoding the gene of IgG antibody described in claim 3, it is characterised in that：

The gene for encoding the heavy chain is following (1) or (2) or (3)：

(3) DNA molecular shown in the sequence 4 of sequence table；

The gene for encoding the light chain is following (4) or (5) or (6)：

(6) DNA molecular shown in the sequence 6 of sequence table.

5. the application of claims 1 or 2 or 3 IgG antibodies in preparing the drug for inhibiting zika virus.

6. a kind of drug for inhibiting zika virus, active constituent is claims 1 or 2 or 3 IgG antibodies.

7. the application of claims 1 or 2 or 3 IgG antibodies in preparation is used for and in the drug of zika virus.

8. a kind of drug for neutralizing zika virus, active constituent is claims 1 or 2 or 3 IgG antibodies.

9. claims 1 or 2 or 3 IgG antibodies answering in preparing the drug for preventing and/or treating zika virus disease With.

10. a kind of drug for preventing and/or treating zika virus disease, active constituent is described in claims 1 or 2 or 3 IgG antibody.