CN106478815A - Quickly prepare the zika virus specifically method of full human monoclonal antibody and application - Google Patents
Quickly prepare the zika virus specifically method of full human monoclonal antibody and application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to field of pharmaceutical biology is and in particular to a kind of quickly prepare the zika virus specifically method of full human monoclonal antibody and application.The method of what the present invention provided quickly prepare zika virus specifically full human monoclonal antibody includes that PERIPHERAL BLOOD MONONUCLEAR CELL separates, plasma cell separates, the PCR amplification of antibody variable gene, the clone of antibody and the step such as cotransfection and identification.The method simple and fast of what the present invention provided quickly prepare zika virus specifically full human monoclonal antibody, do not need the antigen of labelling, separable presence in vivo and be difficult to the functional antibodies of conformational domains imitating in vitro, the specific antibody obtaining has important directive significance to the development of stockaded village's card vaccine, and some antibody are likely to be of the application prospect of clinical treatment.
Description
Technical field
The invention belongs to field of pharmaceutical biology is and in particular to one kind quickly prepares zika virus, and specifically full people's resource monoclonal resists
The method of body and application.
Background technology
Monoclonal antibody (monoclonal antibodies, mAbs) be undoubtedly a most potential class be used for treatment and
The biological preparation of diagnosis, current monoclonal antibody has had and has been applied to examining of tumor, autoimmune disease and infectious disease
Break and treat.Application with monoclonal antibody is more and more extensive, and what the technology of preparing of monoclonal antibody also obtained develops rapidly.
Chen Hui has delivered the paper of entitled " producing Measles viruss antibody with mouse hybridoma technology ", and this paper is little by BALB/e
The splenocyte of the rat bone marrow tumour p3x63Ags cell and Syngenic mice Measles viruss LEC strain immunity through vero passage is in PEG
Effect is lower to be hybridized.Hybrid cell divides the in the hole of kind of microplate, is incubated, and with HAT liquid medium-selection monoclonal cell, uses
RIA detects, in monoclonal cell hole, 60 handkerchief measles antibodies are positive.But, the antibody in the Mus source of above-mentioned employing is used for mankind's disease
The treatment of disease can occur the risk of the adverse immune reaction for foreign protein.
For overcoming the shortcomings of Mus source antibody, people's immortalised B-cell technology of people's hybridoma technology and EBV transfection meet the tendency and
Raw.Gu Ruolan has delivered the paper of entitled " setting up immortalized hepatocyte studying medicament metabolism with hybridoma technology ", should
Primary hepatocyte is merged from different parent's tumor cell strains by paper using hybridoma technology, and foundation meets drug metabolism study will
Seek, possess the liver hybridoma cell strain of good drug metabolism enzymatic activity, and its biological characteristics and function are identified；Evaluate
The liver hybridoma cell strain using value in drug metabolism study in vitro.But there is high selection, low fusion in these technical methods
And the defect that transfection efficiency is low, be not suitable for the screening of big antibody library and resist as separated specificity from thousands of memory cell
Body.
Zika virus can cause the Guillain-Barre syndrome of neonate microcephaly and adult to be closed by the world
Note.There is no effective vaccine and Therapeutic Method currently for stockaded village's card disease.Single B cell antibody technology (single B cell
Antibody technologies), separate and Cloning of Immunoglobulin encoding gene from single B cell, maintain original B
The heavy chain of antibody and light chain pairing in cell.The separation of the full people's monoclonal antibody for virus infection commonly used at present mainly adopts fluorescence
The memory cell of antigen selection the infected IgG+ of labelling, carries out single cell clone.As in HIV person with fluorescently-labeled
Gp140 is successfully separated the memory cell with the restructuring antigen reactive IgG+ of trimer gp140.This method usually selects extensive
Memory cell in the PERIPHERAL BLOOD MONONUCLEAR CELL of multiple phase patient, can only screen known antigens, but can lose some not with restructuring
Antigen protein combines, but has the important function antibody of the neutralization effect such as virus, be unfavorable for the method large-scale promotion and
Application.
Therefore, study and send out output can prevent and treat zika virus method be current urgent need to resolve a difficult problem.
Content of the invention
In order to overcome deficiency of the prior art, it is an object of the invention to provide one kind quickly to prepare zika virus special
The method of full human monoclonal antibody and application, above-mentioned to solve the problems, such as.
The invention provides a kind of method quickly preparing zika virus specifically full human monoclonal antibody, include following step
Suddenly：
S1 PERIPHERAL BLOOD MONONUCLEAR CELL separates：Take stockaded village's card the infected's acute stage peripheral vein EDTA anticoagulation, using density
Gradient centrifugation procedure separating peripheral blood mononuclear cells, subpackage 2-4 × 106/ pipe, is placed in frozen in liquid nitrogen；
S2 plasma cell separates：PERIPHERAL BLOOD MONONUCLEAR CELL frozen for step S1 is positioned in 37 DEG C of water-baths and dissolves, use PBS
Buffer solution 3-4 time, is subsequently added into fluorescent-labeled antibody and is dyeed, and room temperature lucifuge is incubated 12-16min, is buffered using PBS
After liquid washing, add 300-500 μ l PBS suspension cell up flow type instrument, sub-elect single plasma cell in 96 hole PCR plate
In frozen；Described antibody is IgD, CD19, CD27, CD38, IgM and CD45, the fluorescence of described labelling is FITC, ECD, PC7,
APC A750, PB and KO；
The PCR amplification of S3 antibody variable gene and the clone of antibody：The single slurry being obtained from step S2 using PCR method
Expand antibody variable gene in cell, comprise the following steps that：
Reverse transcription synthesizes first chain of cDNA：96 orifice plates containing single B cell are added random primer and reverse transcription
Deng, vortex mixing, it is placed on ice after reaction heating, reverse transcription obtains cDNA；
PCR expands antibody gene：Prepare PCR system, through denaturation, degeneration, annealing, extend circulation, PCR expands antibody
Variable region gene .PCR product is identified through gel electrophoresiss, carries out sequencing analysis to positive genetic fragment. will be from same sample hole
A pair of heavy chain that middle screening obtains, chain variable region gene are connected respectively to structure antibody expressing plasmid on carrier；
S4 cotransfection and identification：The antibody expressing plasmid that step S3 is built adopts 96 orifice plate transient transfection 293T cells,
Cell culture is collected culture supernatant and is identified for ELISA after 2-3 days.
Further, being paired into of antibody and mark fluorescent in described step S2：IgD-FITC、CD19-ECD、CD27-
PC7, CD38-APC A750, IgM-PB and CD45-KO.
Further, define CD27++- in the flow cytometer showed of described step S2 in the B cell colony of IgM and IgD jack to jack adapter
++ CD138++ colony is plasma cell.
Further, the polymerase in PCR amplification in described step S3 is Phusion super fidelity dna polymerase.
Further, in described step S3, pcr amplification reaction condition includes：Stage 1：98℃ 30s；Stage 2：Circulation 17
Secondary, 98 DEG C of 10s, 60-68 DEG C of 30s, 68 DEG C of 30s；Stage 3：Circulation 13 times, 98 DEG C of 10s, 68 DEG C of 30s, 72 DEG C of 30s；Rank
Section 4：72℃ 5min；It is incubated at 4 DEG C.
Further, transfection reagent during described step S4 transfection is transfection reagent Lipofectamine 2000.
Further, during described step S4 transfection, the addition of transfection reagent and the volume ratio of antibody expressing plasmid are 3:1.
In addition, present invention also offers the described method system quickly preparing zika virus specifically full human monoclonal antibody
Standby zika virus specifically full human monoclonal antibody answering in the medicine preventing or treating zika virus disease or diagnostic reagent
With.
The method of what the present invention provided quickly prepare zika virus specifically full human monoclonal antibody is using single B cell
Antibody technique obtains antibody from viral acute stage of infection plasma cell, further antagonist is estimated, it is to avoid can only screen
The antibody being combined with known antigens, is lost the antibody that some are not combined with recombinant antigen protein and function is important, greatly improves
The efficiency of screening and saved the time.
What the present invention provided quickly prepare zika virus specifically full human monoclonal antibody method simple and fast it is not necessary to
The antigen of labelling it is only necessary to relatively small number of B cell, and separable presence in vivo and be difficult to the conformation knot imitating in vitro
The functional antibodies in structure domain, the specific antibody of acquisition has important directive significance to the development of stockaded village's card vaccine, and some are anti-
Body is likely to be of the application prospect of clinical treatment.
In a word, the method for what the present invention provided quickly prepare zika virus specifically full human monoclonal antibody, with existing skill
Art is compared has following advantage：
(1) method of what the present invention provided quickly prepare zika virus specifically full human monoclonal antibody does not need labelling
Antigen, simple to operate；
(2) specifically the method for human monoclonal antibody can be with Separated pin pair entirely for the zika virus of quickly preparing of present invention offer
The functional antibodies of the conformational domains existing in vivo and being difficult in vitro imitate, greatly improve the efficiency of screening.
Brief description
Fig. 1 is the gating programme diagram of machine analysis on sample cell；
Fig. 2 be the heavy chain of antibody and light chain variable district PCR primer through enzyme action (VH, AgeI/SalI, VK, AgeI/Xhol,
VL, AgeI/BsiWI) gel electrophoresis figure, wherein：Mark is the horizontal swimming lane of the gel electrophoresis figure of VH, VK and VL respectively：
1-1,1-2,2-1,2-2,3-1,3-2,4-1,4-2, M2K, M5K, 5-1,5-2,6-1,6-2,7-1,7-2,8-1,8-2；Fig. 3 is
The heavy chain of antibody and light chain variable district PCR primer are through enzyme action (VH, AgeI/SalI, VK, AgeI/Xhol, VL, AgeI/BsiWI)
Connect with corresponding carrier and build antibody expressing plasmid figure；
Fig. 4 is antibody cloning flow chart.
Specific embodiment
Further describe the present invention below by way of specific embodiment, the present invention is not limited only to following examples.At this
In bright scope or without departing from the change in present disclosure, spirit and scope, the present invention being carried out, combination or replace
Change, will be apparent to the person skilled in the art, and be included within the scope of the present invention.
Embodiment 1, a kind of method quickly preparing zika virus specifically full human monoclonal antibody
S1 PERIPHERAL BLOOD MONONUCLEAR CELL separates：Take stockaded village's card the infected's acute stage peripheral vein EDTA anticoagulation, using density
Gradient centrifugation procedure separating peripheral blood mononuclear cells, subpackage 5 × 106/ pipe, is placed in frozen in liquid nitrogen；
S2 plasma cell separates：PERIPHERAL BLOOD MONONUCLEAR CELL frozen for step S1 is positioned in 37 DEG C of water-baths and dissolves, use PBS
Buffer solution 3 times, is subsequently added into fluorescent-labeled antibody and is dyeed, and room temperature lucifuge is incubated 15min, is washed using PBS
After washing, add 400 μ l PBS suspension cell up flow type instrument, sub-elect single plasma cell frozen in 96 hole PCR plate；Institute
Stating antibody is IgD, CD19, CD27, CD38, IgM and CD45, and the fluorescence of described labelling is FITC, ECD, PC7, APC A750, PB
And KO；
The PCR amplification of S3 antibody variable gene and the clone of antibody：The single slurry being obtained from step S2 using PCR method
Expand antibody variable gene in cell, comprise the following steps that：
Reverse transcription synthesizes first chain of cDNA：96 orifice plates containing single B cell are added random primer and reverse transcription
Deng, vortex mixing, it is placed on ice after reaction heating, reverse transcription obtains cDNA；
PCR expands antibody gene：Prepare PCR system, through denaturation, degeneration, annealing, extend circulation, PCR expands antibody
Variable region gene .PCR product is identified through gel electrophoresiss, carries out sequencing analysis to positive genetic fragment. will be from same sample hole
A pair of heavy chain that middle screening obtains, chain variable region gene are connected respectively to structure antibody expressing plasmid on carrier；
S4 cotransfection and identification：The antibody expressing plasmid that step S3 is built adopts 96 orifice plate transient transfection 293T cells,
Cell culture is collected culture supernatant and is identified for ELISA after 2-3 days.
Embodiment 2, a kind of concrete operation step quickly preparing zika virus specifically full human monoclonal antibody
S1 PERIPHERAL BLOOD MONONUCLEAR CELL separates：Take stockaded village's card the infected's acute stage peripheral vein EDTA anticoagulation, using density
Gradient centrifugation procedure separating peripheral blood mononuclear cells, subpackage 5 × 106/ pipe, is placed in frozen in liquid nitrogen.
S2 plasma cell separates：PERIPHERAL BLOOD MONONUCLEAR CELL frozen for step S1 is positioned over water-bath in 37 DEG C dissolve, uses
PBS washs 3 times, is subsequently added into antibody and is dyeed, and room temperature lucifuge is incubated 15min, after PBS washing,
Add 400 μ l PBS suspension cell up flow type instrument, described streaming instrument is Beckman Kurt MoFlo Astrios EQ
Ultrahigh speed streaming cell sorting system, fluorescently-labeled antibody staining：If 9 analyzer tubes, such as table 1,1-7 pipe adds single accordingly
Individual fluorescently-labeled antibody, the 9th pipe adds the fluorescently-labeled antibody of 7 kinds of mixing, and the 8th manages the blank tube for only cell,
The fluorescently-labeled antibody staining of table 1
| Guan Hao | Dyeing pipe is arranged | Antibody and the fluorescence of labelling | 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-APCA750 | 2 | FL8 |
| 6 | Single dye | IgM-PB | 5 | FL9 |
| 7 | Single dye | CD45-KO | 5 | FL10 |
| 8 | Blank tube | It is not added with antibody | - | - |
| 9 | Sample cell | 7 kinds of antibody mixed liquors | - | - |
Single plasma cell sorting：Machine analysis on sample cell, according to Fig. 1 gating.Iris out CD45 alive according to 7-AAD and CD45
Positive leukocyte.Iris out B cell according to CD19.CD27++-++CD138+ is defined in the B cell colony of IgM and IgD jack to jack adapter
+ colony is plasma cell.Single plasma cell is sorted in 96 hole PCR plate.
The PCR amplification of S3 antibody variable gene and the clone of antibody：The single slurry being obtained from step S2 using PCR method
Expand variable region gene in cell, comprise the following steps that：
Reverse transcription synthesizes first chain of cDNA：96 orifice plates containing single B cell are added random primer and reverse transcription
Deng, vortex mixing, it is placed on ice after reaction heating, reverse transcription obtains cDNA, and described cDNA synthesizes test kit
(SuperScript III First Strand Synthesis System,invitrogen,#18080051).
PCR expands antibody gene：Prepare PCR system, through denaturation, degeneration, annealing, extend circulation, PCR expands antibody
Variable region gene, PCR primer is identified through gel electrophoresiss, carries out sequencing analysis to positive genetic fragment. will be from same sample hole
A pair of heavy chain that middle screening obtains, chain variable region gene are connected respectively to structure antibody expressing plasmid on carrier；
PCR primer sequence reference document (J Immunol Methods.2008Jan 1；329(1-2):112-24.), through 2
Wheel PCR expands heavy chain (VH) and the light chain variable district (VK/VL) of antibody.Reaction uses Phusion super fidelity dna polymerase
(Phusion High-Fidelity PCR Master Mix with GC Buffer,NEB,#M0532s).Reaction system and
Cycling condition such as table 2 below：
Table 2 reaction system and cycling condition
| Composition | Volume (μ l) |
| 2X Phusion master GC Buffer | 12.5 |
| 5 ' primer mixtures (10um) | 1 |
| 3 ' primer mixtures (10um) | 1 |
| H2O | To 25 |
| Template | 1-2 |
Described pcr amplification reaction condition includes：Stage 1：98℃ 30s；Stage 2：Circulation 17 times, 98 DEG C of 10s, 60-68
DEG C 30s, 68 DEG C of 30s；Stage 3：Circulation 13 times, 98 DEG C of 10s, 68 DEG C of 30s, 72 DEG C of 30s；Stage 4：72℃ 5min；4
DEG C insulation.
Described PCR primer utilizes gel electrophoresis figure as shown in Figure 2.The heavy chain of described antibody and light chain variable district PCR primer
Connect with corresponding carrier through enzyme action (VH, AgeI/SalI, VK, AgeI/Xhol, VL, AgeI/BsiWI) and build antibody expression matter
Grain is as shown in Figure 3.Confirm through sequence analysis.
It is separated to 4 monoclonal antibodies being combined with virus E protein from stockaded village card patient, its allele uses and is shown in Table 3.
Table 3 heavy chain of antibody and the allele of light chain
| Name | V gene | D gene | J gene | |
| LJF-8-4 | VH | IGHV4-39*02F | HD4-11*01 | HJ5*01F |
| VK | KV1-9*01F | KJ3*01F | ||
| LJF-5-2 | VH | IGHV3-23*01F | IGHD3-22*01F | IGHJ4*02F |
| VK | IGKV1-12*01F | IGKJ1*01F | ||
| ZHJ-6-6 | VH | IGHV4-59*01F | IGHD1-26*01F | IGHJ1*01F |
| VK | IGKV1-39*01F | IGKJ2*01F | ||
| ZHJ-8-5 | VH | IGHV5-51*01F | IGHD2-15*01F | IGHJ6*02F |
| VK | IGKV3-20*01F | IGKJ1*01F |
The heavy chain of antibody and light-chain variable sequence are as shown in table 4 below：
The heavy chain of table 4 antibody and light-chain variable sequence
S4 cotransfection and identification：Step S3 is built antibody expressing plasmid and adopts 96 orifice plate transient transfection 293T cells, carefully
Born of the same parents collect culture supernatant and identify for ELISA after cultivating 2-3 days.
The expression of antibody：Using 96 orifice plate transient transfection 293T cells.The every hole VH and each 0.125 μ g of VK plasmid, transfection examination
Agent Lipofectamine 2000 (Invitrogen) presses 1:3 ratios.Cell culture is collected culture supernatant after 2-3 days and is used for
ELISA.
ELISA identifies：Detect IgG simultaneously and be combined with virus E protein.Elisa plate is coated kappa and lamda first
Antibody or be coated zika virus E protein.Add 10 times or 1：The culture supernatant of 1 dilution.The anti-human igg of 2 anti-employing HRP labellings.
After substrate colour developing terminates, read 450nm OD.Testing result is as shown in table 5 and table 6.
The OD value of table 5 LJF-8-4 and LJF-5-2
| Name | IgG OD value | E protein OD value |
| LJF-8-4 | 3.5867 | 3.0564 |
| 3.4064 | 3.0674 | |
| LJF-5-2 | 3.382 | 3.5785 |
| 3.2394 | 3.4914 | |
| Positive control | 3.0836 | 2.3292 |
| 3.3328 | 2.3275 | |
| Negative control | 0.0558 | 0.2586 |
| 0.0537 | 0.2278 |
The OD value of table 6 ZHJ-6-6 and ZHJ-8-5
| Name | IgG OD value | E protein OD value |
| ZHJ-6-6 | 1.8558 | 0.2139 |
| 1.8974 | 0.2078 | |
| ZHJ-8-5 | 2.1006 | 0.2127 |
| 2.2153 | 0.2051 | |
| Positive control | 1.8634 | 1.0207 |
| 1.8347 | 1.0104 | |
| Negative control | 0.0454 | 0.1251 |
| 0.438 | 0.1154 |
Described antibody cloning flow process such as Fig. 4.
Claims (8)
1. a kind of method quickly preparing zika virus specifically full human monoclonal antibody is it is characterised in that comprise the following steps：
S1 PERIPHERAL BLOOD MONONUCLEAR CELL separates：Take stockaded village's card the infected's acute stage peripheral vein EDTA anticoagulation, using density gradient
Centrifugal method separating peripheral blood mononuclear cells, subpackage 2-4 × 106/ pipe, is placed in frozen in liquid nitrogen；
S2 plasma cell separates：PERIPHERAL BLOOD MONONUCLEAR CELL frozen for step S1 is positioned in 37 DEG C of water-baths and dissolves, buffered with PBS
Liquid washs 3-4 time, is subsequently added into fluorescent-labeled antibody and is dyeed, and room temperature lucifuge is incubated 12-16min, is washed using PBS
After washing, add 300-500 μ lPBS buffer suspension cell up flow type instrument, sub-elect single plasma cell and freeze in 96 hole PCR plate
Deposit；Described antibody is IgD, CD19, CD27, CD38, IgM and CD45, and the fluorescence of described labelling is FITC, ECD, PC7, APC
A750, PB and KO；
The PCR amplification of S3 antibody variable gene and the clone of antibody：The single plasma cell being obtained from step S2 using PCR method
Middle amplification antibody variable gene, comprises the following steps that：
Reverse transcription synthesizes first chain of cDNA：96 orifice plates containing single B cell are added random primer and reverse transcription, is vortexed
Mix, be placed on ice after reaction heating, reverse transcription obtains cDNA；
PCR expands antibody gene：Prepare PCR system, through denaturation, degeneration, annealing, extend circulation, PCR expands antibody variable
Area's gene .PCR product is identified through gel electrophoresiss, carries out sequencing analysis to positive genetic fragment. will sieve from same sample hole
A pair of the heavy chain obtaining, chain variable region gene is selected to be connected respectively to structure antibody expressing plasmid on carrier；
S4 cotransfection and identification：The antibody expressing plasmid that step S3 is built adopts 96 orifice plate transient transfection 293T cells, cell
Culture is collected culture supernatant and is identified for ELISA after 2-3 days.
2. quickly prepare as claimed in claim 1 zika virus specifically full human monoclonal antibody method it is characterised in that
In described step S2, antibody and mark fluorescent is paired into：IgD-FITC、CD19-ECD、CD27-PC7、CD38-APCA750、
IgM-PB and CD45-KO.
3. quickly prepare as claimed in claim 1 zika virus specifically full human monoclonal antibody method it is characterised in that
Defining CD27++-++CD138++ colony in the flow cytometer showed of described step S2 in the B cell colony of IgM and IgD jack to jack adapter is slurry
Cell.
4. quickly prepare as claimed in claim 1 zika virus specifically full human monoclonal antibody method it is characterised in that
Polymerase in PCR amplification in described step S3 is Phusion super fidelity dna polymerase.
5. quickly prepare as claimed in claim 1 zika virus specifically full human monoclonal antibody method it is characterised in that
In described step S3, pcr amplification reaction condition includes：Stage 1：98℃30s；Stage 2：Circulation 17 times, 98 DEG C of 10s, 60-68 DEG C
30s,68℃30s；Stage 3：Circulation 13 times, 98 DEG C of 10s, 68 DEG C of 30s, 72 DEG C of 30s；Stage 4：72℃5min；It is incubated at 4 DEG C.
6. quickly prepare as claimed in claim 1 zika virus specifically full human monoclonal antibody method it is characterised in that
Transfection reagent during described step S4 transfection is transfection reagent Lipofectamine 2000.
7. quickly prepare as claimed in claim 6 zika virus specifically full human monoclonal antibody method it is characterised in that
During described step S4 transfection, the addition of transfection reagent and the volume ratio of antibody expressing plasmid are 3:1.
8. as arbitrary in claim 1-7 described quickly prepare the zika virus specifically method preparation of full human monoclonal antibody
Zika virus specifically full human monoclonal antibody in the medicine or diagnostic reagent of preparation prevention or treatment zika virus disease should
With.
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