CN109593758A - Multi-primers group and the method that human B cell's immune group library is constructed based on high-flux sequence using the primer sets - Google Patents

Multi-primers group and the method that human B cell's immune group library is constructed based on high-flux sequence using the primer sets Download PDF

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CN109593758A
CN109593758A CN201811597255.5A CN201811597255A CN109593758A CN 109593758 A CN109593758 A CN 109593758A CN 201811597255 A CN201811597255 A CN 201811597255A CN 109593758 A CN109593758 A CN 109593758A
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primer
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CN109593758B (en
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李艳艳
邱盟轩
靖相密
汪朝晖
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Shandong Acv Biotechnologies Co Ltd
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Abstract

A kind of method the invention discloses multi-primers group and using the primer sets based on high-flux sequence building human B cell's immune group library, belongs to molecular Biological Detection field.Multi-primers group of the invention is made of one group of upstream primer and one group of downstream primer, and upstream primer is connected in series by connector 1, primer bar code 1, sequencing primer 1, the specific primer V1-V18 designed for the variable region area V；Downstream primer by connector 2, primer bar code 2, sequencing primer 2, for IgA, lgD, IgE, IgG, IgM gene constant region C area's conserved sequence design specific primer Va, Vd, Ve, Vg, Vm be connected in series；Primer bar code 1 is different with 2 sequence of primer bar code；Sequencing primer 1 is identical as 2 sequence of sequencing primer.BCR immune group library can be constructed based on DNA sample or RNA sample using the method for the present invention, the diversity information of BCR can be covered comprehensively.And building is high-efficient, it is at low cost.

Description

Multi-primers group and the utilization primer sets are based on high-flux sequence building human B cell The method in immune group library
Technical field
The present invention relates to molecular Biological Detection field, in particular to a kind of multi-primers group and it is based on using the primer sets The method in high-flux sequence building human B cell's immune group library.
Background technique
Immune group library refers in any specified time, all functional diversity B cells and T in the circulatory system of some individual The summation of cell.
Human lymphocyte mainly includes T cell, B cell.B cell antigen receptor ((B cell receptor, BCR) It is a kind of surface membrane immunoglobulin (SmIg) of B cell identification antigen, there is antigen-binding specificity.BCR is by two heavy chains It is formed by connecting with two light chains, wherein heavy chain is divided into variable region (area V), constant region (area C), transmembrane region and cytoplasmic region；Light chain is then The only area V and the area C.The area V is made of two structural domains of VH and VL, they respectively by three complementary determining regions (CDR1, CDR2 and CDR3) form, the amino acid of CDR composition and put in order great diversity is presented, it is same in vivo, up to 109~ 1012, the huge library BCR of capacity is constituted, the tremendous potential for assigning the various antigens of individual identification, generating specific antibody, these three CDR both participates in the identification to antigen, codetermines the antigentic specificity of BCR.Exactly this species diversity plays to Guan Chong health The hypotype of the effect wanted, immune protein is more, is more highly resistant to pathogen, the more fewer easier infectious disease of hypotype.Except this it Outside, the factors such as other many ages, environment, disease induction and medication also affect the diversity in immune group library.
The immune groups base construction method coverage areas such as SSCP technology, GeneScan technology, fluorescent quantitation solubility curve technology It is narrow, the diversity in immune group library cannot comprehensively, be balancedly reacted, high-flux sequence can satisfy huge, various sequence and survey Sequence requirement.
In the experimental procedure of high-flux sequence, the preparation of sequencing library is a very crucial step.It is more due to BCR Sample, the factors such as difference for expanding Preference and template abundance of primer, design of primers and PCR amplification method are aobvious when building library It obtains particularly important.In addition, the preparation of traditional sequencing library needs sample nucleic acid extraction, enzyme pre-treatment or mechanical shearing, adjunction head It is attached, PCR amplification etc. process, it is complicated for operation.
Therefore it provides a kind of BCR immune group base construction method based on high-flux sequence, simplifies laboratory operating procedures, drop The Preference of low PCR primer amplification, improves amplification efficiency, particularly necessary.
Summary of the invention
In order to make up for the deficiencies of the prior art, the present invention provides one kind is exempted from based on high-flux sequence building mankind B cell The method in epidemic disease group library.
The technical solution of the present invention is as follows:
A kind of multi-primers group is made of one group of upstream primer and one group of downstream primer, the upstream primer by connector 1, Primer bar code 1, sequencing primer 1, the specific primer V1-V18 designed for the variable region area V are connected in series；The downstream primer By connector 2, primer bar code 2, sequencing primer 2, set for the area the gene constant region C conserved sequence of IgA, lgD, IgE, IgG, IgM Specific primer Va, Vd, Ve, Vg, Vm of meter are connected in series；Primer bar code 1 is different with the sequence of primer bar code 2；Sequencing primer 1 is identical as 2 sequence of sequencing primer.
Preferably, 1 sequence of connector is AAT GAT ACG GCG ACC ACC GAG ATC TAC AC；Connector 2 Sequence is CAA GCA GAA GAC GGC ATA CGA GAT.
Preferably, the sequence of sequencing primer 1 and sequencing primer 2 is GCT CTT CCG ATC T.
Preferably, primer bar code 1, primer bar code 2 are made of 6 or 8 nucleotide, the primer of different samples The difference of at least one nucleotide between bar code 1, primer bar code 2.
Primer bar code 1, primer bar code 2 can for TACGTA, TGAGCG, TACAGT, CATGTC etc. or TCCGTTCG, TGTGCGCT, TACCCTCA or GATTCATC.
Preferably, specific primer V1-V18 is respectively as follows: in upstream primer
V1:5 '-GCTGGGTGCGCCAGATGCCC
V2:5 '-TGGATCCGTCAGCCCCCAGG
V3:5 '-TGGATCCGTCAGCCCCCGGG
V4:5 '-GTGCGACAGGCCCCTGGACAA
V5:5 '-GTYCCGCAGGCCCCCGSACAA
V6:5 '-GGGTGMGACTCGCTCGRGGACAA
V7:5 '-GTGGCACAYGCCCCCGGACAA
V8:5 '-GTGCGACAGGCWCGGAGACAA
V9:5 '-TCCGCCAGCKCGGAGGGAAGG
V10:5 '-TCCGRCTGCCCGCYCCGAA
V11:5 '-TCCGGCAGMCGCCCCGGAA
V12:5 '-TCCGGCAGCCCGCTCYGAAGG
V13:5 '-GMAGCGCCAGSCTCCAGGGAA
V14:5 '-GGAGKGCCAGGCTTCSGGGAA
V15:5 '-GAAGCGCCWGGCTCCAGGGAA
V16:5 '-GGAGCGCCAGGCTCCAMGGAA
V17:5 '-GGAGCGCRAGGCTCCGGGCAA
V18:5 '-GGAGCGCCAGCCTCYAGGGAA；
Specific primer Va, Vd, Ve, Vg, Vm are respectively as follows: in downstream primer
Vg:5-ATGAYCGATGGGCCCTTGT
Va:5-GCAGACCTTRGGGCTGGTCA
Vm:5-GCGAATTCTCACWGGAGACG
Vd:5-GCGTGSCTGCAGCCTGATA
Ve:5-GTAGAYGGATGGGCTCSGT.
Method using the multi-primers group based on high-flux sequence building human B cell's immune group library, comprising steps of
1) sample nucleic acid DNA or RNA are extracted；
2) sample nucleic acid DNA or RNA first round PCR amplification, obtain pcr amplification product 1；
3) pcr amplification product 1 recycles；
4) the second wheel PCR amplification is carried out by template of the recovery product of pcr amplification product 1, obtains PCR amplified production 2；
5) pcr amplification product 2 recycles, and recovery product constitutes BCR sequencing library；
6) library detection:
Gained BCR sequencing library detects the purity and size in library using Aglilent 2100Bioanalyser；It utilizes Nanodrop one measures BCR library concentration；
7) high-flux sequence:
The library BCR after purification is sequenced using the Hiseq2500 of Illumina company, sequencing mode is PE150；
8) data are analyzed:
After sequencing data filters out sequencing background, by lower machine data and IMGT immunocyte receptoire data carry out V D J Gene compares analysis, establishes B cell immune group library spectrum.
Preferably, when the use of sample being DNA, in step 2), the PCR amplification body of the first round PCR amplification System prepares according to following: 5 × PCR Buffer, 5.0 μ L, dNTP Mix final concentration 0.5-1 mM, multiple PCR primer group upstream is drawn 3-5 μM of object group, 3-5 μM of downstream primer group, Mg2+Final concentration 3-4 mM, Taq enzyme 1.5-3unit, DNA sample 200-800ng add Enter DEPC water and supplies volume to 25 μ L.
Further, the amplification program of first round PCR amplification is as follows:
95 DEG C of thermal starting enzyme activation 10-20min；94-95 DEG C of denaturation 15sec, 55-60 DEG C of annealing 60-120sec, 72 DEG C Extend 30-40sec, carries out 6-10 circulation；94-95 DEG C of denaturation 15sec, 65-70 DEG C of annealing 40-60 sec, 72 DEG C of extension 30- 40sec carries out 10-20 circulation；16 DEG C of preservations.
Preferably, when the use of sample being RNA, in step 2), the PCR amplification body of the first round PCR amplification System prepares according to following: 5 × RT-PCR Buffer, 5.0 μ L, dNTP Mix final concentration 0.5-1mM, in multiple PCR primer group 3-5 μM of primer sets of trip, 3-5 μM of downstream primer group, RNase inhibitor 0.25 μ L, Mg2+Final concentration 3-4mM, reverse transcriptase, Taq enzyme Mixture 2.0-3.5unit, RNA sample 200-800ng is added DEPC water and supplies volume to 25 μ L.
Further, the amplification program of first round PCR amplification is as follows:
45-50 DEG C of reverse transcription 35-40min；95 DEG C of thermal starting enzyme activation 10-20min；94-95 DEG C of denaturation 15sec, 55- 60 DEG C of annealing 60-120sec, 72 DEG C of extension 30-40sec carry out 6-10 circulation；94-95 DEG C of denaturation 15sec, 65-70 DEG C is moved back Fiery 40-60sec, 72 DEG C of extension 30-40sec carry out 10-20 circulation；16 DEG C of preservations.
Preferably, in step 3) and step 5), the recycling of pcr amplification product 1 and the recycling of pcr amplification product 2 are used Agarose gel electrophoresis, purifying column purification or magnetic beads for purifying mode carry out purification and recovery.
Preferably, in step 4), the PCR amplification system of the second wheel PCR amplification is prepared according to following:
5 × PCR Buffer, 5.0 μ L, dNTP Mix final concentration 0.2-0.4mM, 0.2-0.5 μM of 1 final concentration of connector connect First 0.1-0.2 μM of 2 final concentration, Mg2+Final concentration 2-2.5mM, Taq enzyme 1.5-2unit, 1 recovery product of pcr amplification product, 5 μ L, DEPC water is added and supplies volume to 50 μ L.
Further, the amplification program of the second wheel PCR amplification is as follows:
95 DEG C of 10~20min of thermal starting enzyme activation；94~95 DEG C of 10~20sec of denaturation, 50~55 DEG C of annealing 30~40 Sec, 72 DEG C of extension 30-40s carry out 30-40 circulation, 16 DEG C of preservations.
Preferably, multiple PCR primer group upstream primer group is mixed by following 18 upstream primers:
1:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGCTGGGTGCGCCAGATGCCC
2:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TTGGATCCGTCAGCCCCCAGG
3:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TTGGATCCGTCAGCCCCCGGG
4:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGTGCGACAGGCCCCTGGACAA
5:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGTYCCGCAGGCCCCCGSACAA
6:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGGGTGMGACTCGCTCGRGGACAA
7:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGTGGCACAYGCCCCCGGACAA
8:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGTGCGACAGGCWCGGAGACAA
9:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TTCCGCCAGCKCGGAGGGAAGG
10:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TTCCGRCTGCCCGCYCCGAA
11:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TTCCGGCAGMCGCCCCGGAA
12:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TTCCGGCAGCCCGCTCYGAAGG
13:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGMAGCGCCAGSCTCCAGGGAA
14:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGGAGKGCCAGGCTTCSGGGAA
15:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGAAGCGCCWGGCTCCAGGGAA
16:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGGAGCGCCAGGCTCCAMGGAA
17:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGGAGCGCRAGGCTCCGGGCAA
18:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN (NN) GCT CTT CCG ATC TGGAGCGCCAGCCTCYAGGGAA；
Wherein, NNNNNN (NN) indicates primer bar code 1 in upstream primer group；
Downstream primer group is mixed by following 5 downstream primers:
1:5 '-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN (NN) GCT CTT CCG ATC TATGAYCGATGGGCCCTTGT
2:5 '-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN (NN) GCT CTT CCG ATC TGCAGACCTTRGGGCTGGTCA
3:5 '-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN (NN) GCT CTT CCG ATC TGCGAATTCTCACWGGAGACG
4:5 '-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN (NN) GCT CTT CCG ATC TGCGTGSCTGCAGCCTGATA
5:5 '-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN (NN) GCT CTT CCG ATC TGTAGAYGGATGGGCTCSGT；
Wherein, NNNNNN (NN) indicates primer bar code 2 in downstream primer.
Further, upstream primer group is mixed by 18 upstream primer equimolars；Downstream primer group is by 5 downstreams Primer equimolar mixes.
Preferably, upstream primer group total mole number is identical as downstream primer group total mole number.
The invention has the benefit that
BCR immune group library can be constructed based on DNA sample or RNA sample using method of the invention, after high-flux sequence Available millions of BCR sequences, can cover the diversity information of BCR comprehensively.From operating method, the present invention is direct Primer bar code and sequencing primer are introduced into the library of building by way of PCR amplification, reduce the numerous of library construction step It is trivial, building efficiency is improved, the time has been saved, reduces the cost consumptions such as artificial, reagent.Pass through the PCR amplification of two-step method The deflection of multiplexed PCR amplification is reduced, the multifarious time of day of BCR has preferably been reacted.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art To obtain other drawings based on these drawings.
Fig. 1 is that the present invention is based on the schematic diagrams that high-flux sequence constructs human B cell's immune group library；
Fig. 2 be embodiment 2 obtained in the library BCR using Aglilent 2100Bioanalyser detect library purity and The testing result of size；
Fig. 3 is CDR3 staple diagram in embodiment 2；Wherein, horizontal axis indicates the length of nucleotides of CDR3, longitudinal axis expression pair Answer ratio shared by the CDR3 of length；
Fig. 4 is high abundance CDR3 sequence analysis chart in embodiment 2；
Fig. 5 be embodiment 3 obtained in the library BCR using Aglilent 2100Bioanalyser detect library purity and The testing result of size；
Fig. 6 is CDR3 staple diagram in embodiment 3；Wherein, horizontal axis indicates the length of nucleotides of CDR3, longitudinal axis expression pair Answer ratio shared by the CDR3 of length；
Fig. 7 is high abundance CDR3 sequence analysis chart in embodiment 3.
Specific embodiment
Material and reagent explanation
Healthy volunteer's informed consent.No special explanation, the reagent that the present invention uses are commercial goods, the present invention The database that embodiment uses is disclosed online database.
Embodiment 1
A kind of multi-primers group is made of one group of upstream primer and one group of downstream primer, and upstream primer is by connector 1, primer Bar code 1, sequencing primer 1, the specific primer V1-V18 designed for the variable region area V are connected in series；Downstream primer by connector 2, Primer bar code 2, sequencing primer 2, for IgA, lgD, IgE, IgG, IgM gene constant region C area's conserved sequence design it is special Property primer Va, Vd, Ve, Vg, Vm are connected in series.Primer bar code 1 is different with the sequence of primer bar code 2；
1 sequence of connector is AAT GAT ACG GCG ACC ACC GAG ATC TAC AC；
2 sequence of connector is CAA GCA GAA GAC GGC ATA CGA GAT.
The sequence of sequencing primer 1 and sequencing primer 2 is GCT CTT CCG ATC T.
Primer bar code 1, primer bar code 2 are indicated by NNNNNN (NN), are made of 6 or 8 nucleotide, convenient for not same The upper machine sequencing together of this library, the difference of at least one nucleotide of the primer sequence of barcodes of different samples and are drawn at primer bar code 1 The sequence of object bar code 2 is different.
Primer bar code 1, primer bar code 2 be TACGTA, TGAGCG, TACAGT, CATGTC etc. or TCCGTTCG, TGTGCGCT, TACCCTCA or GATTCATC.
Specific primer is designed according to following method: being with people BCR CDR3 sequence disclosed in IMGT database Reference sequences, for the gene constant region in all variable region areas V (variable) and IgA, lgD, IgE, IgG, IgM (constant) area C gene is compared, using Primer Premier 5.0 carry out design of primers, using oligo7 into Row primer dimer and stem ring mispairing are analyzed, and design 18 upstream primer V1- for the variable region area V conserved sequence V18 is respectively designed under 1 for the area gene constant region (constant) the C gene conserved sequence of IgA, lgD, IgE, IgG, IgM Swim primer Va, Vd, Ve, Vg, Vm.
1 specific primer group of table
Note: R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, W=A/T
Therefore it is as shown in table 2 for realizing multi-primers group sequence used in library construction.
2 multiplex amplification primer sets of table
The present invention provides a kind of library constructing method in human B cell's immune group library, main includes the use ratio of primer Example and PCR amplification program.Sample used in the present invention can be DNA sample and be also possible to RNA sample.
When using DNA sample, aim sequence is gone out by multiplexed PCR amplification and carries out library construction, carries out two-wheeled PCR altogether.
When using RNA sample, two-step method RT-PCR amplification is can be used in library construction, i.e., is first carried out using reverse transcriptase Reverse transcription synthesizes cDNA, then carries out the synthesis of the second chain DNA；Also One step RT-PCR amplification can be used and carry out library construction. Embodiment below selects One step RT-PCR to carry out multiplex PCR amplification.But those skilled in the art can be according to one-step method RT-PCR amplification method is inferred to two-step method RT-PCR amplification not paying method in the case where substantive labour.
Embodiment 2
Using DNA sample, aim sequence is gone out by multiplexed PCR amplification and carries out library construction:
1, the quick separating of peripheral blood lymphocytes
1) it checks: taking out the anticoagulant separating pipe of lymphocyte, whether observe on separation gel has free separating liquid, if Have, 2000g, room temperature is centrifuged 1min.
2) it samples: peripheral blood 5ml being added into anticoagulant separating pipe.
3) be centrifuged: 800g, soft room temperature are centrifuged 15min.
4) it is inhaled with pasteur pipet or pipettor and abandons plasma layer, until closing on PBMC cellular layer, then careful suction PBMC (tunica albuginea Layer: between separation gel and blood plasma), it is transferred in a new 15ml centrifuge tube.
5) physiological saline or 1*PBS to 15ml is added, washs 1 time, 300g, soft room temperature is centrifuged 10min.
6) it discards supernatant, 2ml physiological saline or 1*PBS is added, be resuspended, add to 5ml, counted after mixing.
7) 300G is centrifuged 10min, discards supernatant, and adjusts cell concentration to 20*10 by counting6/ml。
2, sample nucleic acid DNA is extracted
Extract DNA using commercial kit, such as using QIAGEN blood, tissue DNA extracts kit (CAT: 65904) DNA is extracted, or genomic DNA is extracted using the method for phenol chloroform.The present embodiment uses QIAGEN blood, group It knits DNA extraction kit (CAT:65904) and extracts DNA, the method is known to those skilled in the art, does not add to repeat herein.
DNA extraction finishes, using purity, the concentration of Nanodrop one spectrophotometric determination DNA, 1% agarose The quality of electrophoresis detection DNA.
After measured, the DNA concentration of extraction is 198ng/ul, OD260/280 1.93.
3, first round PCR amplification
It is added following substance in PCR pipe: 5 × RT-PCR Buffer 5.0 μ L, dNTP Mix final concentration 0.7mM is more 2.7 μM of group of weight PCR primer group upstream primer, 2.7 μM of downstream primer group, Mg2+Final concentration 3mM, Taq enzyme 1.5unit, DNA sample 2.5 μ L are added DEPC water and supply volume to 25 μ L.
It is expanded according to following amplification condition, obtains pcr amplification product 1:
95 DEG C of thermal starting enzyme activation 10min；95 DEG C of denaturation 15sec, 58 DEG C of annealing 120sec, 72 DEG C of extension 40sec, into Row 10 circulations；95 DEG C of denaturation 15sec, 70 DEG C of annealing 60sec, 72 DEG C of extension 30sec carry out 20 circulations；16 DEG C of preservations.
4, pcr amplification product 1 recycles
Pcr amplification product 1 carries out purification and recovery, mould of the recovery product as the second wheel PCR amplification with magnetic beads for purifying mode Plate.Product recovery purifying eliminates the primer, dNTP, enzyme etc. for not participating in reaction, also eliminates these factors to the second wheel PCR The influence of amplification.
Magnetic beads for purifying specific steps are as follows:
1) it takes out magnetic bead oscillation and mixes 5min, the amount for taking out 45 μ l/ samples is put in room temperature 10min；
2) above-mentioned pcr amplification product 1 is taken out, 30 μ l H is added thereto2O is mixed, the magnetic bead finished to equilibrium at room temperature The 45 diluted pcr amplification products of μ l of middle addition blow 10 mixings with rifle suction, are put in room temperature 2min；
3) uncap and be put in 1min on magnetic frame, liquid become clarification can sucking liquid, be added 125 μ l 85% ethyl alcohol, It washes out and discards after 1min, magnetic bead is put in room temperature 8min and dries；
4) 30 μ l dd H2O back dissolvings are added.
5, the second wheel PCR amplification
Following substance: 5 × PCR Buffer, 5.0 μ L, dNTP Mix final concentration, 0.3 mM, connector 1 is added in PCR pipe 0.35 μM of final concentration, 0.1 μM of 2 final concentration of connector, Mg2+ final concentration 2mM, Taq enzyme 1.8unit, the recycling of pcr amplification product 1 produce 5 μ L of object is added DEPC water and supplies volume to 50 μ L.
It is expanded according to following amplification condition, obtains pcr amplification product 2.
95 DEG C of thermal starting enzyme activation 10min；95 DEG C of denaturation 15sec, 55 DEG C of annealing 40sec, 72 DEG C of extension 30s carry out 35 A circulation, 16 DEG C of preservations.
6, pcr amplification product 2 recycles
Obtained pcr amplification product 2 carries out purification and recovery with magnetic beads for purifying mode, and purifying specific method is referring to step 4.Institute Obtained recovery product constitutes BCR sequencing library.Recovery product eliminates responseless primer, dNTP etc., can be into one Step improves the sequencing efficiency and accuracy of constructed sequencing library.
7, library detection
Purity and size of the obtained library BCR using Aglilent 2100Bioanalyser detection library, detection As a result as shown in Fig. 2, library size is in 355bp, and library purity is higher, and has no other non-specific amplification sequences.It utilizes Nanodrop one measures BCR library concentration, and the library concentration of recycling is 21ng/ul.
8, high-flux sequence
Sequencing company is delivered in the library BCR after purification to be sequenced using the Hiseq2500 of Illumina company, is sequenced Mode is PE150.
9, data are analyzed
To being sequenced after resulting initial data depolluted, removes connector and gone low quality to filter, by lower machine data with Reference sequences on IMGT are compared, and by searching for corresponding genetic fragment, acquisition VDJ gene frequency clones frequency distribution, The information such as peculiar BCR sequence, item number establish B cell immune group library spectrum.Shown in partial data result table 3.
3. library sequencing result of table counts
Note:
1) Sample: sample names；
2) Total reads: all reads (sequence) number of sample sequencing；
3) Used reads: reads (sequence) number completely cloned again in sample；
4) Percent (%): Used reads accounts for the percentage of Total reads；
5) Clones: clone's number of sample.
CDR3 distribution of lengths is as shown in Figure 3, wherein horizontal axis indicates the length of nucleotides of CDR3, and the longitudinal axis indicates corresponding length CDR3 shared by ratio.The analysis of high abundance CDR3 sequence is as shown in Figure 4.
The preceding 10 CDR3 sequences for listing abundance highest (reads number is most) side by side are found in immune group library.Its In, horizontal axis indicates the length of CDR3, and the longitudinal axis indicates the frequency that the sequence of corresponding length occurs, lists frequency respectively most in legend 10 high sequences (frequency is successively successively decreased from top to bottom).
Embodiment 3
When using RNA sample, aim sequence is gone out by multiplex RT-PCR amplification and carries out library construction:
1, peripheral blood lymphocytes quick separating
1) it checks: taking out the anticoagulant separating pipe of lymphocyte, whether observe on separation gel has free separating liquid, if Have, 2000g, room temperature is centrifuged 1min.
2) it samples: peripheral blood 5ml being added into anticoagulant separating pipe.
3) be centrifuged: 800g, soft room temperature are centrifuged 15min.
4) it is inhaled with pasteur pipet or pipettor and abandons plasma layer, until closing on PBMC cellular layer, then careful suction PBMC (tunica albuginea Layer: between separation gel and blood plasma), it is transferred in a new 15ml centrifuge tube.
5) physiological saline or 1*PBS to 15ml is added, washs 1 time, 300g, soft room temperature is centrifuged 10min.
6) it discards supernatant, 2ml physiological saline or 1*PBS is added, be resuspended, add to 5ml, counted after mixing.
7) 300G is centrifuged 10min, discards supernatant, and adjusts cell concentration to 20*10 by counting6/ml。
2, sample nucleic acid RNA is extracted
RNA is extracted using commercial kit, is such as mentioned using QIAGEN RNeasy midi Kit (CAT:75142) RNA is taken, or total serum IgE is extracted using Trizol method.The present embodiment extracts total serum IgE using Trizol method, and extraction step is As it is known to those skilled in the art that not adding to repeat herein.
RNA extraction finishes, using the concentration of Nanodrop one spectrophotometric determination RNA, 1% agarose electrophoresis inspection Survey the quality of RNA.
The RNA concentration of extraction is 156ng/ul, OD260/280 2.01.
3, first round PCR amplification
It is added following substance in PCR pipe: 5 × RT-PCR Buffer, 5.0 μ L, dNTP Mix final concentration 0.8 mM is more 1.8 μM of group of weight PCR primer group upstream primer, 1.8 μM of downstream primer group, RNase inhibitor 0.25 μ L, Mg2+Final concentration 3.5mM, Reverse transcriptase, 3.5 μ L of Taq enzyme mixture 2.5unit, RNA sample are added DEPC water and supply volume to 25 μ L.
It is expanded according to following amplification condition, obtains pcr amplification product 1:
50 DEG C of reverse transcription 35min；95 DEG C of thermal starting enzyme activation 10min；95 DEG C of denaturation 15sec, 58 DEG C of annealing 120sec, 72 DEG C extend 40sec, carry out 8 circulation；95 DEG C of denaturation 15sec, 69 DEG C of annealing 60sec, 72 DEG C of extension 30sec carry out 15 and follow Ring；16 DEG C of preservations.
4, pcr amplification product 1 recycles
Pcr amplification product 1 carries out purification and recovery, mould of the recovery product as the second wheel PCR amplification with magnetic beads for purifying mode Plate.Product recovery purifying eliminates the primer, dNTP, enzyme etc. for not participating in reaction, also eliminates these factors to the second wheel PCR The influence of amplification.
Magnetic beads for purifying specific steps are as follows:
1) it takes out magnetic bead oscillation and mixes 5min, the amount for taking out 45 μ l/ samples is put in room temperature 10min；
2) above-mentioned pcr amplification product 1 is taken out, 30 μ l H is added thereto2O is mixed, the magnetic bead finished to equilibrium at room temperature The 45 diluted pcr amplification products of μ l of middle addition blow 10 mixings with rifle suction, are put in room temperature 2min；
3) uncap and be put in 1min on magnetic frame, liquid become clarification can sucking liquid, be added 125 μ l 85% ethyl alcohol, It washes out and discards after 1min, magnetic bead is put in room temperature 8min and dries；
4) 30 μ l dd H2O back dissolvings are added.
5, the second wheel PCR amplification
Following substance: 5 × PCR Buffer, 5.0 μ L, dNTP Mix final concentration, 0.3 mM, connector 1 is added in PCR pipe 0.35 μM of final concentration, 0.2 μM of 2 final concentration of connector, Mg2+Final concentration 2mM, Taq enzyme 1.8unit, 1 recovery product of pcr amplification product 5 μ L are added DEPC water and supply volume to 50 μ L.
It is expanded according to following amplification condition, obtains pcr amplification product 2.
95 DEG C of thermal starting enzyme activation 10min；95 DEG C of denaturation 15sec, 53 DEG C of annealing 35sec, 72 DEG C of extension 30s carry out 35 A circulation, 16 DEG C of preservations.
6, pcr amplification product 2 recycles
Obtained pcr amplification product 2 carries out purification and recovery with magnetic beads for purifying mode, and purifying specific method is referring to step 4.Institute Obtained recovery product constitutes BCR sequencing library.Recovery product eliminates responseless primer, dNTP etc., can be into one Step improves the sequencing efficiency and accuracy of constructed sequencing library.
Note: pcr amplification product 2 is 50 μ L, needs to be recycled in two times.
7, library detection
Purity and size of the obtained library BCR using Aglilent 2100Bioanalyser detection library, detection As a result as shown in figure 5, library size is in 356bp, and library purity is higher, and has no other non-specific amplification sequences.It utilizes Nanodrop one measures BCR library concentration, and the library concentration of recycling is 19ng/ul.
8, high-flux sequence
Sequencing company is delivered in the library BCR after purification to be sequenced using the Hiseq2500 of Illumina company, is sequenced Mode is PE150.
9, data are analyzed
After sequencing data filters out sequencing background, by lower machine data and IMGT immunocyte receptoire data carry out V D J Gene compares, and by searching for corresponding genetic fragment, obtains VDJ gene frequency, clones frequency distribution, peculiar BCR sequence, item number Etc. information, establish T cell immune group library spectrum.The results are shown in Table 4 for partial data.
4 library sequencing result of table counts
Note:
1) Sample: sample names；
2) Total reads: all reads (sequence) number of sample sequencing；
3) Used reads: reads (sequence) number completely cloned again in sample；
4) Percent (%): Used reads accounts for the percentage of Total reads；
5) Clones: clone's number of sample.
CDR3 distribution of lengths is as shown in Figure 6, wherein horizontal axis indicates the length of nucleotides of CDR3, and the longitudinal axis indicates corresponding length CDR3 shared by ratio.The analysis of high abundance CDR3 sequence is as shown in Figure 7.
The preceding 10 CDR3 sequences for listing abundance highest (reads number is most) side by side are found in immune group library.Its In, horizontal axis indicates the length of CDR3, and the longitudinal axis indicates the frequency that the sequence of corresponding length occurs, lists frequency respectively most in legend 10 high sequences (frequency is successively successively decreased from top to bottom).
B cell immune group library possesses wide application field, thin by the huge flux measurement T of high-flux sequence platform Born of the same parents' immune group library diversity, it is possible to provide a certain specific period complete diversity information of individual probes into antibody and BCR in adaptability Variation in immunologic process.B cell immune group library sequencing can be applied to vaccine and medicine research and development, biomarker discovery, The fields such as monitoring after minimal residual disease detection, the research and transplanting of autoimmune disease.
SEQUENCE LISTING
<110>Shandong Acv Biotechnologies Co., Ltd.
<120>multi-primers group and the method using the primer sets based on high-flux sequence building human B cell's immune group library
<130> 2018
<160> 49
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> DNA
<213>artificial sequence
<400> 1
aatgatacgg cgaccaccga gatctacac 29
<210> 2
<211> 24
<212> DNA
<213>artificial sequence
<400> 2
caagcagaag acggcatacg agat 24
<210> 3
<211> 13
<212> DNA
<213>artificial sequence
<400> 3
gctcttccga tct 13
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<211> 20
<212> DNA
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gctgggtgcg ccagatgccc 20
<210> 5
<211> 20
<212> DNA
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<400> 5
tggatccgtc agcccccagg 20
<210> 6
<211> 20
<212> DNA
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<400> 6
tggatccgtc agcccccggg 20
<210> 7
<211> 21
<212> DNA
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gtgcgacagg cccctggaca a 21
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<211> 21
<212> DNA
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gtyccgcagg cccccgsaca a 21
<210> 9
<211> 23
<212> DNA
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gggtgmgact cgctcgrgga caa 23
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gtggcacayg cccccggaca a 21
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<212> DNA
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gtgcgacagg cwcggagaca a 21
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<211> 21
<212> DNA
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tccgccagck cggagggaag g 21
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tccgrctgcc cgcyccgaa 19
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<212> DNA
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tccggcagmc gccccggaa 19
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tccggcagcc cgctcygaag g 21
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<212> DNA
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gmagcgccag sctccaggga a 21
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<212> DNA
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ggagkgccag gcttcsggga a 21
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gaagcgccwg gctccaggga a 21
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ggagcgccag gctccamgga a 21
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<212> DNA
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ggagcgcrag gctccgggca a 21
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ggagcgccag cctcyaggga a 21
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<211> 19
<212> DNA
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atgaycgatg ggcccttgt 19
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gcagaccttr gggctggtca 20
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gcgaattctc acwggagacg 20
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gcgtgsctgc agcctgata 19
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<212> DNA
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gtagayggat gggctcsgt 19
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<212> DNA
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<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gctgggtgcg 60
ccagatgccc 70
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<211> 70
<212> DNA
<213>artificial sequence
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<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tggatccgtc 60
agcccccagg 70
<210> 29
<211> 70
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tggatccgtc 60
agcccccggg 70
<210> 30
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 30
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gtgcgacagg 60
cccctggaca a 71
<210> 31
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 31
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gtyccgcagg 60
cccccgsaca a 71
<210> 32
<211> 73
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 32
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gggtgmgact 60
cgctcgrgga caa 73
<210> 33
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 33
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gtggcacayg 60
cccccggaca a 71
<210> 34
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 34
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gtgcgacagg 60
cwcggagaca a 71
<210> 35
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 35
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tccgccagck 60
cggagggaag g 71
<210> 36
<211> 69
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 36
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tccgrctgcc 60
cgcyccgaa 69
<210> 37
<211> 69
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 37
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tccggcagmc 60
gccccggaa 69
<210> 38
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 38
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct tccggcagcc 60
cgctcygaag g 71
<210> 39
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 39
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gmagcgccag 60
sctccaggga a 71
<210> 40
<211> 71
<212> DNA
<213>artificial sequence
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<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 40
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct ggagkgccag 60
gcttcsggga a 71
<210> 41
<211> 71
<212> DNA
<213>artificial sequence
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<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
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aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct gaagcgccwg 60
gctccaggga a 71
<210> 42
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 42
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct ggagcgccag 60
gctccamgga a 71
<210> 43
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 43
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct ggagcgcrag 60
gctccgggca a 71
<210> 44
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 44
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct ggagcgccag 60
cctcyaggga a 71
<210> 45
<211> 71
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (30)..(37)
<223> n is a, c, g, or t
<400> 45
aatgatacgg cgaccaccga gatctacacn nnnnnnngct cttccgatct ggagcgccag 60
cctcyaggga a 71
<210> 46
<211> 65
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (25)..(32)
<223> n is a, c, g, or t
<400> 46
caagcagaag acggcatacg agatnnnnnn nngctcttcc gatctgcaga ccttrgggct 60
ggtca 65
<210> 47
<211> 65
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (25)..(32)
<223> n is a, c, g, or t
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caagcagaag acggcatacg agatnnnnnn nngctcttcc gatctgcgaa ttctcacwgg 60
agacg 65
<210> 48
<211> 64
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (25)..(32)
<223> n is a, c, g, or t
<400> 48
caagcagaag acggcatacg agatnnnnnn nngctcttcc gatctgcgtg sctgcagcct 60
gata 64
<210> 49
<211> 64
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (25)..(32)
<223> n is a, c, g, or t
<400> 49
caagcagaag acggcatacg agatnnnnnn nngctcttcc gatctgtaga yggatgggct 60
csgt 64

Claims (16)

1. a kind of multi-primers group, it is characterised in that: be made of one group of upstream primer and one group of downstream primer, the upstream primer It is connected in series by connector 1, primer bar code 1, sequencing primer 1, the specific primer V1-V18 designed for the variable region area V；It is described Downstream primer by connector 2, primer bar code 2, sequencing primer 2, for IgA, lgD, IgE, IgG, IgM the gene area constant region C protect Specific primer Va, Vd, Ve, Vg, the Vm for keeping sequence design are connected in series；Primer bar code 1 is different with the sequence of primer bar code 2； Sequencing primer 1 is identical as 2 sequence of sequencing primer.
2. multi-primers group as described in claim 1, it is characterised in that: 1 sequence of connector is AAT GAT ACG GCG ACC ACC GAG ATC TAC AC；2 sequence of connector is CAA GCA GAA GAC GGC ATA CGA GAT.
3. multi-primers group as described in claim 1, it is characterised in that: the sequence of sequencing primer 1 and sequencing primer 2 is GCT CTT CCG ATC T。
4. multi-primers group as described in claim 1, it is characterised in that: primer bar code 1, primer bar code 2 are by 6 or 8 Nucleotide composition, the difference of at least one nucleotide between the primer bar codes 1 of different samples, primer bar code 2.
5. multi-primers group as described in claim 1, which is characterized in that
Specific primer V1-V18 is respectively as follows: in upstream primer
V1:5 '-GCTGGGTGCGCCAGATGCCC
V2:5 '-TGGATCCGTCAGCCCCCAGG
V3:5 '-TGGATCCGTCAGCCCCCGGG
V4:5 '-GTGCGACAGGCCCCTGGACAA
V5:5 '-GTYCCGCAGGCCCCCGSACAA
V6:5 '-GGGTGMGACTCGCTCGRGGACAA
V7:5 '-GTGGCACAYGCCCCCGGACAA
V8:5 '-GTGCGACAGGCWCGGAGACAA
V9:5 '-TCCGCCAGCKCGGAGGGAAGG
V10:5 '-TCCGRCTGCCCGCYCCGAA
V11:5 '-TCCGGCAGMCGCCCCGGAA
V12:5 '-TCCGGCAGCCCGCTCYGAAGG
V13:5 '-GMAGCGCCAGSCTCCAGGGAA
V14:5 '-GGAGKGCCAGGCTTCSGGGAA
V15:5 '-GAAGCGCCWGGCTCCAGGGAA
V16:5 '-GGAGCGCCAGGCTCCAMGGAA
V17:5 '-GGAGCGCRAGGCTCCGGGCAA
V18:5 '-GGAGCGCCAGCCTCYAGGGAA；
Specific primer Va, Vd, Ve, Vg, Vm are respectively as follows: in downstream primer
Vg:5-ATGAYCGATGGGCCCTTGT
Va:5-GCAGACCTTRGGGCTGGTCA
Vm:5-GCGAATTCTCACWGGAGACG
Vd:5-GCGTGSCTGCAGCCTGATA
Ve:5-GTAGAYGGATGGGCTCSGT.
6. the method using multi-primers group as described in claim 1 based on high-flux sequence building human B cell's immune group library, Characterized in that it comprises the following steps:
1) sample nucleic acid DNA or RNA are extracted；
2) sample nucleic acid DNA or RNA first round PCR amplification, obtain pcr amplification product 1；
3) pcr amplification product 1 recycles；
4) the second wheel PCR amplification is carried out by template of the recovery product of pcr amplification product 1, obtains pcr amplification product 2；
5) pcr amplification product 2 recycles, and recovery product constitutes BCR sequencing library；
6) library detection:
Gained BCR sequencing library detects the purity and size in library using 2100 Bioanalyser of Aglilent；It utilizes Nanodrop one measures BCR library concentration；
7) high-flux sequence:
The library BCR after purification is sequenced using the Hiseq2500 of Illumina company, sequencing mode is PE150；
8) data are analyzed:
After sequencing data filters out sequencing background, by lower machine data and IMGT immunocyte receptoire data carry out V D J gene Analysis is compared, B cell immune group library spectrum is established.
7. the method as claimed in claim 6 based on high-flux sequence building human B cell's immune group library, which is characterized in that when When the use of sample being DNA, in step 2, the PCR amplification system of the first round PCR amplification is prepared according to following: 5 × PCR 5.0 μ L, dNTP Mix final concentration 0.5-1 mM of Buffer, 3-5 μM of group of multiple PCR primer group upstream primer, downstream primer group 3-5 μM, Mg2+Final concentration 3-4 mM, Taq enzyme 1.5-3 unit, DNA sample 200-800ng are added DEPC water and supply volume extremely 25 µL。
8. the method as claimed in claim 7 based on high-flux sequence building human B cell's immune group library, which is characterized in that the The amplification program of one wheel PCR amplification is as follows:
95 DEG C of thermal starting enzyme activation 10-20 min；94-95 DEG C of 15 sec of denaturation, 55-60 DEG C of annealing 60-120 sec, 72 DEG C are prolonged 30-40 sec is stretched, 6-10 circulation is carried out；94-95 DEG C of denaturation 15 sec, 65-70 DEG C of annealing 40-60 sec, 72 DEG C of extension 30- 40 sec carry out 10-20 circulation；16 DEG C of preservations.
9. the method as claimed in claim 6 based on high-flux sequence building human B cell's immune group library, which is characterized in that when When the use of sample being RNA, in step 2, the PCR amplification system of the first round PCR amplification is prepared according to following: 5 × RT-PCR 5.0 μ L, dNTP Mix final concentration 0.5-1 mM of Buffer, 3-5 μM of group of multiple PCR primer group upstream primer, downstream primer group 3-5 μM, RNase inhibitor 0.25 μ L, Mg2+Final concentration 3-4 mM, reverse transcriptase, Taq enzyme mixture 2.0-3.5 unit, RNA Sample 200-800ng is added DEPC water and supplies volume to 25 μ L.
10. the method as claimed in claim 9 based on high-flux sequence building human B cell's immune group library, which is characterized in that the The amplification program of one wheel PCR amplification is as follows:
45-50 DEG C of reverse transcription 35-40 min；95 DEG C of thermal starting enzyme activation 10-20 min；94-95 DEG C of denaturation 15 sec, 55-60 DEG C annealing 60-120 sec, 72 DEG C of extensions 30-40 sec, carry out 6-10 recycle；94-95 DEG C of 15 sec of denaturation, 65-70 DEG C is moved back Fiery 40-60 sec, 72 DEG C of extension 30-40 sec carry out 10-20 circulation；16 DEG C of preservations.
11. the method as claimed in claim 6 based on high-flux sequence building human B cell's immune group library, which is characterized in that step Rapid 3) and in step 5), the recycling of pcr amplification product 1 and the recycling of pcr amplification product 2 are pure using agarose gel electrophoresis, purification column Change or magnetic beads for purifying mode carries out purification and recovery.
12. the method as claimed in claim 6 based on high-flux sequence building human B cell's immune group library, which is characterized in that step It is rapid 4) in, the PCR amplification system of the second wheel PCR amplification is prepared according to following:
5 × PCR Buffer, 5.0 μ L, dNTP Mix final concentration 0.2-0.4 mM, 0.2-0.5 μM of 1 final concentration of connector, connector 2 0.1-0.2 μM of final concentration, Mg2+Final concentration 2-2.5mM, Taq enzyme 1.5-2 unit, 1 recovery product of pcr amplification product, 5 μ L, add Enter DEPC water and supplies volume to 50 μ L.
13. the method as claimed in claim 12 based on high-flux sequence building human B cell's immune group library, which is characterized in that The amplification program of second wheel PCR amplification is as follows:
95 DEG C of 10 ~ 20 min of thermal starting enzyme activation；94 ~ 95 DEG C of denaturation 10 ~ 20 sec, 50 ~ 55 DEG C of annealing 30 ~ 40 sec, 72 DEG C Extend 30-40s, carries out 30-40 circulation, 16 DEG C of preservations.
14. the method based on high-flux sequence building human B cell's immune group library as described in claim 7 or 9, feature exist In:
Multiple PCR primer group upstream primer group is mixed by following 18 upstream primers:
1:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGCTGGGTGCGCCAGATGCCC
2:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTGGATCCGTCAGCCCCCAGG
3:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTGGATCCGTCAGCCCCCGGG
4:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGTGCGACAGGCCCCTGGACAA
5:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGTYCCGCAGGCCCCCGSACAA
6:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGGGTGMGACTCGCTCGRGGACAA
7:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGTGGCACAYGCCCCCGGACAA
8:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGTGCGACAGGCWCGGAGACAA
9:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTCCGCCAGCKCGGAGGGAAGG
10:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTCCGRCTGCCCGCYCCGAA
11:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTCCGGCAGMCGCCCCGGAA
12:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TTCCGGCAGCCCGCTCYGAAGG
13:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGMAGCGCCAGSCTCCAGGGAA
14:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGGAGKGCCAGGCTTCSGGGAA
15:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGAAGCGCCWGGCTCCAGGGAA
16:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGGAGCGCCAGGCTCCAMGGAA
17:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGGAGCGCRAGGCTCCGGGCAA
18:5 '-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACNNNNNN(NN) GCT CTT CCG ATC TGGAGCGCCAGCCTCYAGGGAA；
Wherein, NNNNNN(NN in upstream primer group) indicate primer bar code 1；
Downstream primer group is mixed by following 5 downstream primers:
1:5 '-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN(NN) GCT CTT CCG ATC TATGAYCGATGGGCCCTTGT
2:5 '-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN(NN) GCT CTT CCG ATC TGCAGACCTTRGGGCTGGTCA
3:5 '-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN(NN) GCT CTT CCG ATC TGCGAATTCTCACWGGAGACG
4:5 '-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN(NN) GCT CTT CCG ATC TGCGTGSCTGCAGCCTGATA
5:5 '-CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN(NN) GCT CTT CCG ATC TGTAGAYGGATGGGCTCSGT；
Wherein, NNNNNN(NN in downstream primer) indicate primer bar code 2.
15. the method as claimed in claim 14 based on high-flux sequence building human B cell's immune group library, which is characterized in that Upstream primer group is mixed by 18 upstream primer equimolars；Downstream primer group is mixed by 5 downstream primer equimolars.
16. the method as claimed in claim 14 based on high-flux sequence building human B cell's immune group library, which is characterized in that Upstream primer group total mole number is identical as downstream primer group total mole number.
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