CN104560978B - The multiple PCR primer and method of people's BCR heavy chain libraries are built based on high-flux sequence - Google Patents
The multiple PCR primer and method of people's BCR heavy chain libraries are built based on high-flux sequence Download PDFInfo
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- CN104560978B CN104560978B CN201510027844.XA CN201510027844A CN104560978B CN 104560978 B CN104560978 B CN 104560978B CN 201510027844 A CN201510027844 A CN 201510027844A CN 104560978 B CN104560978 B CN 104560978B
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Abstract
The invention discloses the multiple PCR primer and method of people's BCR heavy chain libraries is built based on high-flux sequence, its primer sequence is as shown in SEQ ID NO.7～17, the primer is the rule design according to b lymphocyte receptor heavy chain (IGH genes) Somatic Rearrangement, non-masterplate radom insertion disappearance, somatic hypermutation and type conversion restructuring, and in the upstream addition sequence measuring joints of forward primer and reverse primer, the primer energy efficient amplification template, can be used in building immune group storehouse；The invention also discloses the method for building people's BCR heavy chain libraries, the method is simple, can rapid build people's BCR heavy chain libraries, to further appreciating that immune group storehouse Changing Pattern, to disclose disease incidence mechanism significant.
Description
Technical field
The invention belongs to biological technical field, and in particular to build the multiple of people's BCR heavy chain libraries based on high-flux sequence
PCR primer and the method for building people's BCR heavy chain libraries using the primer.
Background technology
Have benefited from the fast development and extensively application of high throughput sequencing technologies of future generation, DNA and RNA microarray datasets are in gene
The various aspects that group is learned play prominent impetus.Meanwhile, this emerging technical field be applied to T cell and
The immune group storehouse sequencing of B-cell receptor.In acquired immunity, T cell and B cell φt cell receptor and B cell by surface
The identification antigenic peptides of receptor-specific-MHC (pMHC) and then startup immune system activation.By surveying
The CDR3 immune group storehouse of sequence lymphocyte receptor, determines the composition of acceptor molecule in T, bone-marrow-derived lymphocyte, can be to evaluate immunity
The characteristic parameters such as the multiformity in group storehouse, and further study the response generating process and its mechanism of acquired immune system.B
Cell receptor (BCR) by two heavy chains and two light chains, totally four peptide chains compositions, heavy chain by IGH gene codes, light chain respectively by
IGL (Lamda chains) and IGK (Kappa chains) codings, simultaneously because heavy chain there is more complicated inside composition thus can be preferably anti-
Reflect the composition situation of BCR.The germline gene of IGH genes is arranged in order by multiple open reading frame and is formed, and can be divided into IGHV,
Tetra- pack section of IGHD, IGHJ, IGHC, is realized by Somatic Rearrangement in bone-marrow-derived lymphocyte growth course random between different fragments
Combination produces ripe IGH molecules, and the multiformity for BCR provides Molecular and genetic basis.In IGHD-IGHJ, IGHV-IGHD's
Junction, due to radom insertion and the disappearance of non-masterplate nucleotide, considerably increases the diversity level of BCR molecules.B cell is lived
During change, due to the effect that somatic hypermutation (i.e. affinity maturation) and recombinant type are changed, the multiformity of BCR is further
Increase.And the complementary determining region 3 (CDR3) of IGH genes just covers IGHV-Junction-IGHD-Junction-IGHJ areas
Domain, includes the most diversity information of IGH genes, therefore, for the sequence in bone-marrow-derived lymphocyte IGH gene Cs DR3 region
Composition is carried out being sequenced and can be very good the composition and response situation that reflect BCR immune group storehouse.
The content of the invention
In view of this, an object of the present invention is to provide to build many of people's BCR heavy chain libraries based on high-flux sequence
Weight PCR primer；The second object of the present invention is to provide the method for building people's BCR heavy chain libraries using the multiple PCR primer.
For achieving the above object, the present invention provides following technical scheme：
1st, the multiple PCR primer of people's BCR heavy chain libraries, the multiple PCR primer such as SEQ are built based on high-flux sequence
Shown in ID NO.7～17.
2nd, the method for building people's BCR heavy chain libraries using multiple PCR primer described in claim 1, first extracts normal gastric and sticks
Membrane tissue, stomach organization or peripheral blood total serum IgE, then synthesize by reverse transcription primer reversion of sequence shown in SEQ ID NO.1～6
CDNA, then with the cDNA of synthesis, as template, shown in SEQ ID NO.7～17, sequence carries out multiplex PCR for primer, reclaims PCR and produces
PCR primer is carried out microemulsion drop PCR, then carries out PGM sequencings, obtain people's BCR heavy chain libraries by thing.
Preferably, the amplification condition of the multiplex PCR is：95 DEG C of denaturations 10min；95 DEG C of degeneration 30s, 59 DEG C of annealing
90s, 72 DEG C of extension 90s, circulates 35 times；Extend 10min after last 72 DEG C.
The beneficial effects of the present invention is：The invention discloses building the multiple PCR primer of people's BCR heavy chain libraries, this draws
Thing can expand IGH gene Cs DR3 area diverse sequence, be built into BCR heavy chain libraries, establish structure people's IGH gene Cs DR3 area
The operating process of sequencing library, realizes the stable detection in B-cell receptor immune group storehouse, to further appreciating that immune group storehouse changes
Rule, announcement disease incidence mechanism are significant.
Description of the drawings
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below：
Fig. 1 is gel imaging testing goal histogram (1：DL2000；2、3：People's BCR heavy chain library bands).
Fig. 2 is BCR heavy chain library statistical results.
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.It is unreceipted concrete in embodiment
The experimental technique of condition, generally according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brookers etc. write)
Described in condition, or according to the condition proposed by manufacturer.
Total serum IgE in embodiment 1, extraction people's tissue
In people's tissue, the extracting method of total serum IgE, comprises the steps：
A. take after people's normal mucosa tissues, stomach organization and peripheral blood add Trizol and blow and beat, be stored at room temperature 5min, directly
Meet extraction RNA or to be put into -80 DEG C of refrigerators frozen；
B. 200 μ l chloroforms are added by every microlitre of Trizol, overturn and mix (30s), room temperature places 3min, then in 4 DEG C,
12000g is centrifuged 15min；
C. fetch water phase, add by every microlitre of Trizol that 200 μ l chloroforms are reverse to mix (30s), room temperature places 3min, Ran Houyu
12000g centrifugations 15min under the conditions of 4 DEG C；
D. upper strata aqueous phase being taken again, 0.5ml isopropanols is added by every microlitre of Trizol, being overturned and is mixed, room temperature places 10min,
Then the 12000g centrifugations 10min under the conditions of 4 DEG C, abandons supernatant；
E. precipitating and the ethanol that 1ml volume fractions are 75% being added by every microlitre of Trizol, gentle to shake, suspend precipitation, so
After 4 DEG C, 8000g centrifugation 5min, supernatant is sucked, room temperature (18～25 DEG C) is deposited in and is dried；
D. with without RNase water dissolution after drying, obtain RNA.
Gained RNA epoch will be extracted and determine RNA concentration and quality.Testing result shows, OD260/OD280 1.8～
2.0 left and right.
Recycle degeneration gel electrophoresis detection 28s, 18s and 5s.Testing result shows that substantially, 28s/18s is left 2.0 for band
It is right.
Above-mentioned testing result shows that the RNA mass satisfaction that the present embodiment is extracted builds storehouse demand, and storehouse is continued after can be used in.
Embodiment 2, reverse transcription and multiplex PCR
The total serum IgE sample that embodiment 1 is obtained carries out reverse transcription respectively, and reverse transcription uses RevertAid First
Strand cDNA Systhesis kit, concrete operations are carried out by kit specification, and its reaction system is：Total serum IgE 0.1～
5 μ g, reverse transcription primer 1 μ L of the concentration for 20pmol, add DEPC to process water and are settled to 12 μ L, then by mixed liquor in PCR instrument
In 65 DEG C incubation 5min, be then put into rapidly ice bath 5min in ice, be subsequently adding 5 × reaction buffer, 4 μ L,
RibolockTMRNase inhibitor (20 μ/μ L) 1 μ L, 2 μ L of 1mM dNTP Mix, revertAid TM M-MuLV reverse transcription
200 μ/μ L, 1 μ L, after mixing, then centrifugation under the conditions of 42 DEG C is incubated 1h, obtains the first chain cDNA.Wherein reverse transcription primer
Particular sequence is：
hsIGG RT UID vlmr：5’-tgactggagttcagacgtgtgnnnnnnnnaagaccgatgggcccttg-3’(SEQ
ID NO.1)；hsIGA RT UID vlmr：5’-tgactggagttcagacgtgtgnnnnnnnngaagaccttggggctggt-3’
(SEQ ID NO.2)；hsIGM RT UID vlmr：5’-tgactggagttcagacgtgtgnnnnnnnngggaattctcacaggaga
cg-3’(SEQ ID NO.3)；hsIGD RT UID vlmr：5’-tgactggagttcagacgtgtgnnnnnnnngggtgtct
gcaccctgata-3’(SEQ ID NO.4)；hsIGE.1RT UID vlmr：5’-tgactggagttcagacgtgtgnnnnnn
nngaagacggatgggctctgt-3’(SEQ ID NO.5)；hsIGE.2RT UID vlmr：5’-tgactggagttcagacg
Tgtgnnnnnnnnttgcagcagcgggtcaaggg-3 ' (SEQ ID NO.6), n=a/c/g/t.
Surpassed according to b lymphocyte receptor heavy chain (IGH genes) Somatic Rearrangement, non-masterplate radom insertion disappearance, somatic cell
Mutation and type change the rule of restructuring, design multiple PCR primer, for convenience of sequencing in forward primer and the upstream of reverse primer
The sequence measuring joints of addition, concrete primer are as shown in table 1：
Table 1, people BCR libraries multiple PCR primer
| Primer | 5’→3’ | Sequence number |
| trP1-hsIGHV1-vlmr | cctctctatgggcagtcggtgatagcctacatggagctgagc | SEQ ID NO.7 |
| trP1-hsIGHV2-vlmr | cctctctatgggcagtcggtgataggtggtccttacaatgaccaac | SEQ ID NO.8 |
| trP1-hsIGHV3.1-vlmr | cctctctatgggcagtcggtgattctgcaaatgaacagcctga | SEQ ID NO.9 |
| trP1-hsIGHV3.2-vlmr | cctctctatgggcagtcggtgattgttcaaatgagcagtctgagag | SEQ ID NO.10 |
| trP1-hsIGHV3.3-vlmr | cctctctatgggcagtcggtgattctgcaaatgggcagcctga | SEQ ID NO.11 |
| trP1-hsIGHV4/6-vlmr | cctctctatgggcagtcggtgatttctccctgaagctgaactctg | SEQ ID NO.12 |
| trP1-hsIGHV5-vlmr | cctctctatgggcagtcggtgatgcctacctgcagtggagcag | SEQ ID NO.13 |
| trP1-hsIGHV6-vlmr | cctctctatgggcagtcggtgatttctccctgcagctgaactctg | SEQ ID NO.14 |
| trP1-hsIGHV7.1-vlmr | cctctctatgggcagtcggtgatgcatatctgcagatcagcagc | SEQ ID NO.15 |
| trP1-hsIGHV7.2-vlmr | cctctctatgggcagtcggtgatcagatcagcagcctaaaggc | SEQ ID NO.16 |
| Acbcd01-adaptor21m | ccatctcatccctgcgtgtctccgactcagttacctctgactggagttcagacgtgtg | SEQ ID NO.17 |
Then with the first chain cDNA for obtaining as template, with SEQ ID NO.7～sequence shown in SEQ ID NO.17 to draw
Thing, enters performing PCR amplification, and the reaction system of PCR amplifications is as follows：25 μ L of multiplex-PCR premixed liquids, 5 μ L of forward primer, reversely
5 μ L of 5 μ L of primer, cDNA, 10 μ L of water, altogether 50 μ L；PCR amplification conditions are：95 DEG C of denaturations 10min；95 DEG C of degeneration 30s, 59
DEG C annealing 90s, 72 DEG C extension 90s, circulate 35 times；Extend 10min after last 72 DEG C.The product mass fraction that amplification is obtained
TAE agarose gel (low melting-point agarose gel) for 3%, the electrophoresis 3h under 50V voltages will contain mesh under ultraviolet after electrophoresis
The gel of band cut, be put in 1.5mL EP pipes, be subsequently adding 1mL QG Solubilization buffer, at 45 DEG C
Under the conditions of 5～10min of water-bath until blob of viscose is completely dissolved, then ice bath 1-2min；The sol solutionses for having dissolved are added by ice prognosis
To on adsorption column, 500 μ L are added every time, 1min is centrifuged in 18000g then, the waste liquid in collecting pipe is outwelled after centrifugation, will absorption
Post is put back in collecting pipe, adds 300 μ L QG Solubilization buffer, 18000g centrifugation 1min, in adsorption column
Fall the waste liquid in collecting pipe, adsorption column is put back in collecting pipe, adsorption column is being placed on new 1.5ml by 18000g centrifugation 2min
In EP pipes, the ethanol remained on adsorption column is thoroughly dried up with hair-dryer and add in backward adsorption column 25 DEG C of preheatings (95 DEG C of preheatings)
Ultra-pure water, stand 2min, under last 18000g, be centrifuged 2min, collect eluent, the eluent is to build storehouse sample.
In order to determine that building storehouse sample quality meets the requirements, to build storehouse sample mass fraction be 1.5% agarose gel,
Electrophoresis 30min under 100V voltages, then by gel imaging testing goal band, as a result as shown in Figure 1.Above-mentioned testing result table
The storehouse sample satisfaction of building of bright acquisition builds storehouse demand, can be used in next step sequencing.
Embodiment 3, em-PCR (microemulsion drips PCR) and PGM sequencings
The method of diluted sample is：Assume that the concentration that Qubit is measured is N ng/ μ L, the sub- degree of amplified library is M kb, then
The extension rate in library is N*1 .515*1000/26*M.
Then with the library after dilution as template, emPCR is carried out, emPCR uses Ion OneTouchTMSequencing system is provided
Reagent, operating procedure are carried out by kit specification, then carry out PGM sequencings, and sequencing result is people's BCR heavy chain libraries.So
Afterwards sequencing result is counted, as a result as shown in Figure 2.As a result show, the people BCR heavy chains text built using the method for the present invention
Storehouse can cover the diversity information of people's BCR heavy chains.
Finally illustrate, preferred embodiment above is only unrestricted to illustrate technical scheme, although logical
Cross above preferred embodiment to be described in detail the present invention, it is to be understood by those skilled in the art that can be
Various changes are made to which in form and in details, without departing from claims of the present invention limited range.
Claims (3)
1. the multiple PCR primer of people's BCR heavy chain libraries is built based on high-flux sequence, it is characterised in that：The multiple PCR primer
As shown in SEQ ID NO.7～17.
2. the method for building people's BCR heavy chain libraries using multiple PCR primer described in claim 1, it is characterised in that：Just first extract
Often gastric mucosa tissue, stomach organization or peripheral blood total serum IgE, then anti-as reverse transcription primer with sequence shown in SEQ ID NO.1～6
Turn synthesis cDNA, then sequence carries out multiplex PCR for primer as template, shown in SEQ ID NO.7～17 with the cDNA of synthesis, reclaims
PCR primer is carried out microemulsion drop PCR, then carries out PGM sequencings, obtain people's BCR heavy chain libraries by PCR primer.
3. method according to claim 2, it is characterised in that：The amplification condition of the multiplex PCR is：95 DEG C of denaturations
10min；95 DEG C of degeneration 30s, 59 DEG C of annealing 90s, 72 DEG C of extension 90s, circulate 35 times；Extend 10min after last 72 DEG C.
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| CN105087560B (en) * | 2015-08-07 | 2016-06-01 | 深圳市瀚海基因生物科技有限公司 | A kind of multiple PCR primer and method building pig BCR heavy chain library based on high-flux sequence |
| CN107345241A (en) * | 2016-05-12 | 2017-11-14 | 眭维国 | B cell antigen receptor H chains CDR3 processing method |
| CN106319064A (en) * | 2016-09-13 | 2017-01-11 | 北京天科雅生物科技有限公司 | Multi-PCR-primer and method for constructing human TCRA library on basis of high-throughput sequencing |
| CN106319066B (en) * | 2016-09-14 | 2020-01-17 | 中国医学科学院北京协和医院 | Primer and kit for detecting Systemic Lupus Erythematosus (SLE) B cell receptor library |
| CN108070586A (en) * | 2016-11-18 | 2018-05-25 | 杭州拓宏生物科技有限公司 | Pcr amplification primer and its application |
| CN107513560B (en) * | 2017-05-19 | 2018-09-18 | 武汉康录生物技术股份有限公司 | A kind of quick detection probe of IGH gene breaks of low cost and its preparation method and application |
| CN108004304B (en) * | 2017-12-20 | 2021-04-23 | 北京旌准医疗科技有限公司 | Method for detecting clonality of lymphocyte related gene rearrangement |
| CN108893464A (en) * | 2018-07-13 | 2018-11-27 | 广州华银医学检验中心有限公司 | A kind of construction method of immune group library high-throughput sequencing library |
| CN109593758B (en) * | 2018-12-26 | 2021-11-26 | 山东艾克韦生物技术有限公司 | Multiplex primer set and method for constructing human B cell immune repertoire based on high-throughput sequencing by using same |
| CN109536491A (en) * | 2018-12-27 | 2019-03-29 | 北京迈基诺基因科技股份有限公司 | A kind of method and its primer special group based on high-flux sequence detection people DNA BCR IGH immune group library |
| CN113234808A (en) * | 2021-04-09 | 2021-08-10 | 深圳荻硕贝肯精准医学有限公司 | Primer group and method for detecting IGH immune repertoire of human DNA by high-throughput sequencing |
| CN114107287A (en) * | 2021-12-13 | 2022-03-01 | 云测智能科技有限公司 | Preparation method for comprehensively amplifying humann TCR beta chain library by adopting a small amount of degenerate primers |
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| CN103184216B (en) * | 2011-12-27 | 2015-03-18 | 深圳华大基因科技有限公司 | Primer composition for amplifying coding sequence of immunoglobulin heavy chain CDR3 and use thereof |
| CN103710454B (en) * | 2013-12-31 | 2016-02-17 | 南方科技大学 | Method for TCR or BCR high-throughput sequencing and method for correcting multiple PCR primer deviation by using tag sequence |
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