CN108949743A - A kind of full-length genome hybridizing clones method - Google Patents
A kind of full-length genome hybridizing clones method Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
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- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
Abstract
A kind of full-length genome hybridizing clones method for biological field, it is characterized in that, genophore and competent cell used in traditional gene cloning are replaced with mouse sp2/0 cell, it is intended to make the sp2/0 mixing with cells of the cell of genome cloning and energy Immortalization, it is digested with 0.25% trypsase and II Collagenase Type mixed liquor, then it is acted on through PEG, after birth successively occurs for two parental cells, endochylema, karyon, the fusion of chromosome and genome hybridizes, it is screened through HAT and obtains the hybrid cell for inheriting two parental generation cellular genetic materials both having to colone genome energy Immortalization, competent cell in i.e. traditional gene cloning, clone the full-length genome in hybrid cell with the Immortalization of hybrid cell, short segment DNA can only be cloned and cannot synchronize clone cell by solving The problem of traditional gene clone technology of interior full-length genome cann't be solved.
Description
Technical field
The invention discloses a kind of full-length genome hybridizing clones method for biological field, it is mainly used in intracellular complete
The synchronization clonal expansion of genome.
Background technique
Gene cloning is the revolutionary technology that has of the invention seventies, and it is by purpose that gene cloning, which is also known as DNA recombination,
Gene is connect in vitro with the carrier DNA with autonomous replication capacity, constructs recombinant DNA, is then introduced into recipient cell, carries out mesh
Gene masculine recipient cell screening and vegetative propagation.
Gene cloning is created by inventions such as P.Berg, the DNA and bacteriophage a kind of simian virus such as P.Berg in 1972
After DNA is cut with same restriction enzyme, then with DNA ligase both DNA moleculars are connected, is constituted a kind of new
Recombinant DNA molecules.S.Cohen in 1973 etc. connects one section of exogenous dna fragment with Plasmid DNA, constitutes a weight
Group plasmid, and the recombinant plasmid is transferred in Escherichia coli and is replicated, complete gene cloning system is established for the first time.Gene gram
Grand to experienced three developing stage till now from invention, first stage is the early 1970s being still widely used so far
Need the creation of the classical gene clone technology of restriction enzyme and ligase.Second stage be early 1990s not
The appearance of the cloning process of ligase is relied on, this method does not need the participation of ligase, the limitation of unrestricted restriction endonuclease,
So that gene cloning is more flexible.Recombinase at the beginning of three phases are 21 century is applied in gene cloning, makes gene cloning
Targeting is stronger, operates easier.
Gene cloning can be summarized as " divide, cut, connecting, turning, selecting " step, and final purpose is that target gene importing host is thin
Born of the same parents, and the massive duplication in host cell.Wherein " divide " DNA for referring to that separation preparation is qualified, including as carrier DNA and
The target DNA to be cloned, these DNA moleculars or from purpose biological genome DNA or from aim cell mRNA reverse transcription close
At doublestranded cDNA molecule；" cutting ", which refers to, cuts carrier DNA with the restriction enzyme of sequence specific or cuts out target gene,
Since genomic DNA is larger, it is unfavorable for cloning, it is therefore necessary to be processed into being suitble to the DNA small fragment of clone, common side
Method has machine cuts and nucleic acid restriction endonuclease digestion, if gene order it is known and also it is smaller can manually chemistry it is straight
It is bonded into.If the both ends partial sequence of fruit gene is it is known that according to known array design primer, lead to from genomic DNA or cDNA
Target gene can be obtained by crossing round pcr；" company ", which refers to, is connected target DNA and carrier DNA with DNA ligase, is formed
The DNA molecular of recombination；" turning ", which refers to that the DNA molecular of recombination is sent into host cell by special method, is replicated and is expanded
Increase；" choosing " is then that the individual for carrying recombinant DNA molecules is picked out from host group.By the operating procedure of gene cloning, need
1. DNA or RNA is carried out to extract；2. the PCR amplification of DNA or the cDNA reverse transcription PCR amplification of RNA；3. pcr amplification product passes through
1% agarose gel electrophoresis and Ago-Gel DNA QIAquick Gel Extraction Kit purification and recovery；4. the DNA recycled is long with restriction fragment
Spend endonuclease digestion；5. T4 DNA ligase connects DNA and carrier through digestion, recombinant DNA is constructed；6. recombinant DNA conversion sense
By state Escherichia coli, and carries out positive bacteria and fall screening；7. Testing and appraisals such as the culture of the positive colony screened and PCR.
In summary, traditional gene clone technology has the following problems: 1. it is related to a variety of high end equipments such as PCR instrument, it is right
Equipment requirement is high；2. being related to kinds of experiments reagent, reagent cost is high；3. trivial operations, technical requirements are high；4. especially can only gram
The DNA of grand short-movie section can not clone the DNA molecular compared with long segment, can not synchronize the full-length genome of clone cell level.
Summary of the invention
In order to solve the problems, such as that traditional gene clone technology cann't be solved, present inventors have proposed the present invention.
The invention aims to provide a kind of full-length genome hybridizing clones method.
The object of the present invention is achieved like this: being replaced used in traditional gene cloning with rat bone marrow tumour sp2/0 cell
Genophore and competent cell are intended to make the sp2/0 cell of the cell of genome cloning and energy Immortalization with 2~5: 1
Mixing is digested 30 minutes with 0.25% trypsase and II Collagenase Type mixed liquor, is then acted on through 30~50%PEG, amphiphilic
Successively occur for cell after birth, endochylema, karyon, chromosome and genome fusion hybridization, screened through HAT, obtain both have to
The hybrid cell for inheriting two parental generation cellular genetic materials of colone genome and energy Immortalization, i.e., in tradition gene cloning
Competent cell clones the full-length genome in hybrid cell with the Immortalization of hybrid cell.
Further, according to the surface marker of cell to be fused, based on the above technical solution, one is drawing
Use immunoglobulin (Ig) as fusion agent is promoted, make to have the cell (B cell) of the genomic clone to be made of IgG Fc receptor with
Sp2/0 cell with IgG Fab receptor is connected as immune complex by IgG, then is carried out carefully with the effect of PEG or electro fusion method
Born of the same parents merge hybridization；The second is quoting immunoglobulin (Ig) and staphylococcal protein A (SPA) simultaneously as rush fusion
Agent makes to all have being combined by IgG Fab to genomic clone cell and sp2/0 cell for IgG Fab receptor, then is had
The SPA of IgG Fc receptor is connected as immune complex, and then carries out cell fusion and hybridization with the effect of PEG or electro fusion method.
The genome cloning method that the present invention creates solves the problems, such as that traditional gene clone technology cann't be solved,
The shortcomings that can only cloning short segment DNA and traditional gene cloning of full-length genome in clone cell cannot be synchronized is overcome, and
The more traditional gene clone method of the present invention is easy to operate, at low cost, because the present invention is will be to be cloned complete by cell hydridization
Genome introgression hybrid cell is cloned, so thin DNA is imported host without recombinating in traditional gene cloning for DNA
The carrier of born of the same parents, thus without the building of carrier, the expense of carrier, carrier and DNA attended operation and its restriction endonuclease used, T4
The experimental procedures such as DNA ligase, PCR amplification, agarose gel electrophoresis and required equipment, but because the present invention is to pass through hybridization
The Immortalization of cell and clone its full-length genome intracellular, so experiencing without importing recombinant DNA in traditional gene cloning
In state host cell the step of duplication, thus without traditional competence host cell, it is thin it is not necessary that recombinant DNA is imported host
Experimental procedures and the experiment equipment such as the operation of born of the same parents and its screening, the identification of positive host cell, and the present invention is with 0.25% pancreas
Protease and the digestion of II Collagenase Type mixed liquor are conducive to cell fusion, demonstrate,prove through experiment to gene cloning cell and sp2/0 cell
The real present invention can synchronize multiple mutated genes in clone cell, and the biological industry of the application clone and to(for) full-length genome have pole
Its important meaning.
Specific embodiment
Fig. 1 is that IgG of the invention promotees fusion schematic diagram.
Fig. 2 is that SPA collaboration double antibody of the invention promotees fusion schematic diagram.
Fig. 3 is real scene shooting cell fusion figure of the invention.
Fig. 4 is the DNA electrophoretogram of hybrid cell of the invention.
In Fig. 1,1 indicates the sp2/0 cell with IgG Fab receptor, and 2 indicate monoclonal IgG, and 3 indicate there is IgG
Fc receptor to genomic clone cell, cell to be hybridized is linked together by monoclonal antibody, is conducive to cell and melts
It closes.
In Fig. 2,1 indicates the staphylococcal protein A (SPA) with IgG Fc receptor, and 2 indicate that number is 6 and 7
The monoclonal antibody to genomic clone cell, 3 indicate number be 4 and 5 the monoclonal antibody to genomic clone cell,
Monoclonal antibody [2] and number be 6 and 7 to genomic clone cell combination, monoclonal antibody [3] and number be 4 and 5
After genomic clone cell combination, then the connection through SPA, the cell of number 4,5,6 and 7 are conducive to merge.
In Fig. 3, under monoclonal antibody and/or the fusion of the rush of SPA, screens 2 weeks through HAT, clapped under inverted microscope
(40X) is taken the photograph, hybridoma cell clone is obtained.
In Fig. 4,1 is Mark, represents the DNA of different molecular weight different location locating in electrophoretogram；2,3 and 4 points
DNA band of E47, E49 and E50 gene of normal control in electrophoretogram is not represented；5,6 and 7 respectively represent DMD patient's
The DNA band that E47, E49 and E50 gene are likely to occur in electrophoretogram, wherein DNA occurs in electrophoretogram in only E47 gene
Band and E49 and E50 gene are without DNA band；8,9 and 10 E47, E49 and E50 gene of sp2/0 cell are respectively represented in electricity
The DNA band being likely to occur in swimming figure, wherein only E49 gene occur in electrophoretogram DNA shallowly band and E47 and E50 gene is equal
Without DNA band；11,12 and 13 DMD cell is respectively represented and E47, E49 and ES0 gene of the fused hybrid cell of sp2/0 exist
There is the DNA band from DMD, E49 base in electrophoretogram in the DNA band being likely to occur in electrophoretogram, E47 gene therein
Because occurring the shallow band of DNA from sp2/0 in electrophoretogram.Illustrate that hybrid cell (competent cell) has both DMD's and sp2/0
Gene, the breeding for having passed through competent cell have achieved the purpose that target gene group is cloned.
Below with reference to Fig. 1, Fig. 2, Fig. 3 and Fig. 4, the technical side of genome cloning of the present invention is described with specific embodiment
Case.
1, the acquisition of genome cloning cell
The present invention includes the gene mutations such as monogenic disease, polygenic disease, chromosomal disorder, mitochondriopathy lymphocyte, monokaryon
Cell, amniocyte or villus cell, but using the lymphocyte of DMD patient and amniocyte as test cell.
2, experiment reagent
(1) myeloma cell: mouse SP2/0 cell or people H929 myeloma cell, the present invention use mouse SP2/0 cell.
(2) promote fusion agent: being 1. incorporated the CD138 with SP2/0 cell equivalence in the DMEM culture medium of 10% fetal calf serum
Monoclonal antibody, i.e. SP2/0 cell: monoclonal antibody=1 CD138: 1；2. it is thin to be incorporated corresponding hybridization in the DMEM culture medium of 10% fetal calf serum
The IgG antibody of equal value and SPA of born of the same parents.
(2) other reagents: DMEM culture medium, HAT Selective agar medium, fetal calf serum (FBS), DMSO (dimethyl sulfoxide).
3, the preparation (cell fusion method) of competent cell
(1) preparation of SP2/0 cell: fusion the last week takes out the SP2/0 cell frozen out of liquid nitrogen container, sets 37 DEG C immediately
Appropriate complete culture solution is added in water-bath recovery after recovery, 1000r/m is centrifuged 3min, is repeated 1 times, and sediment is moved into cell training
Bottle to be supported, adds 10%DMEM culture solution, sets CO2 incubator, passed within 3~4 days, fusion adjusts cell state in first 24 hours,
Guaranteeing that cellular morphology is good before merging, growth is vigorous, when fusion, blows down SP2/0 from culture bottle, it is transferred in centrifuge tube,
1000r/m is centrifuged 5~10min, washes repeatedly cell 2 times, gently beats mixing, takes a small amount of suspension to count, adjusts density, make
Density when fusion is 80% or so.
(2) preparation to gene cloning cell: lymphocyte or amniocyte are with DMEM culture solution adjustment total cell number to 1
×108~2 × 108, with Trypan Blue, phase-contrast microscopy, viable count should be higher than that 80% is qualified.
(3) preparation (cell fusion method) of competent cell: 1. direct cell fusion method prepares competent cell: with sp2/
0 cell replaces genophore and competent cell used in traditional gene cloning, by the DMD patient of genome cloning to be made
Lymphocyte is mixed with the sp2/0 cell of energy Immortalization with 2~5: 1, is mixed with 0.25% trypsase and II Collagenase Type
Liquid digests 30 minutes, and then through the effect of 30~50%PEG, after birth, endochylema, karyon, chromosome successively occur for two parental cells
And the fusion hybridization of genome, it is screened through HAT, obtaining not only has that energy Immortalization inherits two parental generations again to colone genome
The hybrid cell of cellular genetic material, i.e. competent cell in tradition gene cloning, can make the full-length genome in hybrid cell
It is cloned with the Immortalization of hybrid cell.2. CD138 monoclonal antibody collaboration cell fusion method prepares competent cell: first will be wait melt
It closes cell to digest 30 minutes with 0.25% trypsase and II Collagenase Type mixed liquor, then by CD138 monoclonal antibody and sp2/0
Cell combines the CD138 receptor of sp2/0 cell just by CD138 monoclonal antibody with 1: 1 allotment, then mono- by CD138
The bone-marrow-derived lymphocyte of the DMD patient of the IgG Fc targeted capture genome cloning to be made of clonal antibody, is allowed to be formed that " sp2/0 is thin
The immune complex of born of the same parents-CD138 monoclonal antibody-bone-marrow-derived lymphocyte ", then the effect through PEG, two parental generation cell fusions are that can clone carefully
The hybrid cell of full-length genome intracellular, i.e. competent cell in tradition gene cloning.3. SPA and IgG cooperates with cell fusion method
Prepare competent cell: to containing IgG Fab receptor DMD Patient cells and sp2/0 cell, through 0.25% trypsase and
After II Collagenase Type mixed liquor digests 30 minutes, it is allowed to respective with corresponding IgG Fab connection, then with SPA connection IgG Fc, shape
It is at the immune complex of " sp2/0 cell-IgG-SPA-IgG-DMD cell ", then the effect through PEG, two parental generation cell fusions
Can in clone cell full-length genome hybrid cell, i.e., the competent cell in traditional gene cloning.
(4) cell fusion method prepares the experimental implementation of competent cell: cell to be fused is after above-mentioned processing, in 37 DEG C of water
In bath gently rotation preheating centrifuge tube, after taking-up aseptically by the 50%PEG3000 of 1000 μ L of preheating in 60s edge
Tube wall is added drop-wise in fusion pipe, while gently rotating centrifugal pipe, later by the 25mL basal medium of preheating in 3~5min
It being added drop-wise in centrifuge tube along tube wall, lightly rotating centrifugal pipe during addition is then allowed to stand in 37 DEG C of water-bath 10min,
Transposition 56 DEG C of water-baths release antibody 5 minutes (need to remove binding antibody person), is centrifuged 5min immediately with 1500r/m, discards supernatant, add
Enter 50mL HAT culture medium, is inoculated into 96 well culture plates after appropriate mixing, is placed in 37 DEG C of 5%CO2 incubators and cultivates.
4, the screening of competent cell (fused cell) clone
96 orifice plate cell growth status are observed, is only that the cell of effective integration can be grown after 7~10 days, discards at this time
HAT culture medium replaces the complete medium containing HT, continues to cultivate, to obtain competence cell clone.
5, the screening of monoclonal competent cell (fusion or hybrid cell)
When the clonal growth area of fused cell reaches 1/10 cell hole, culture supernatant is removed, selects hybrid cell
Growth conditions good culture hole marks cell growth position and size under microscope, using sterile pipette tips in the position of mark
Cell clone is drawn in the new culture hole for having complete medium, then successively doubling dilution makes below to hole is counted below
Cell number in each hole is 0~1, and culture 1 week or so in 37 DEG C of 5%CO2 incubators, microscopically observation cell grows feelings
Condition, when the cell clone when below in (inverse) hole is covered with to 1/10 or more of hole floor space, the cell in hole is again below for selection
The screening of row monoclonal, preparation can clone the monoclonal competent cell of its full-length genome intracellular.
6, the passage amplification of monoclonal competent cell
The monoclonal competent cell screened to the present invention carries out secondary culture respectively, with amplifying cells quantity, and respectively
Monoclonal competent cell from each clone is dispensed and indicated, is frozen spare in -196 DEG C of liquid nitrogen.
7, the DMD genetic test of monoclonal competent cell
It freezes after the present invention that learns from else's experience passage amplification in the monoclonal competent cell of -196 DEG C of liquid nitrogen, using Standard PCR, base
The DMD genes such as E47, E49 and E50 because of the methods of chip or gene sequencing detection monoclonal competent cell, contain corresponding mesh
Mark gene person continues to freeze, and can take the circumstances into consideration to abandon without target gene person.
8, the STABILITY MONITORING of monoclonal competent cell
(1) cell growth characteristics monitor: periodically cultivating freeze-stored cell, 3 monoclonal competence frozen were taken every 1 month
Cell carries out recovery culture, observes the survival rate of cell and the growing state of secondary culture, and stabilization person continues to freeze.
(2) cellular genome STABILITY MONITORING: every cell for passing for 5~10 generations or freezing 1~3 month is taken to carry out target gene
The detection such as PCR, genetic chip or gene sequencing, verify the stability of target Disease-causing gene, stabilization person continues to freeze.
9, the verification result of monoclonal competent cell DMD gene
The monoclonal competent cell for being passaged to that the 15th generation was still in logarithmic growth is taken, using PCR amplification and Ago-Gel
As a result this 3 kinds of DMD genes of electrophoresis detection its E47, E49 and E50 obtain the DNA electrophoretogram for having both two parental genes, such as specification
Attached drawing 4.From attached drawing 4 it is found that monoclonal competent cell has the gene of DMD and sp2/0 simultaneously, illustrate to have passed through monoclonal sense
By state cell (hybrid cell) breeding and reached the clone of target DMD gene, its gene magnification 2 when theory uploaded for 15 generation15
Times.Therefore, the present invention can prepare monoclonal competent cell with other hereditary disease cells, the full-length genome gram for biological industry
It is grand.
10, the industry application of genome cloning
(1) industrialization prepares rare expressed genes: in the present inventive method, making rare base by preparing monoclonal competent cell
It is cloned due to the unlimited amplification with competent cell, so that industrialization prepares rare expressed genes, keeps rare expressed genes unrare, be phase
The research of disease is answered to provide scientific research material.
(2) the immortalization gene library of rare disease is constructed: can in the form of monoclonal competent cell freezes in -196 DEG C of liquid nitrogen
Reproducibility saves the full-length genome of corresponding disease, provides scientific research material for the research of corresponding disease.
(3) solve the problems, such as that traditional gene clone technology cann't be solved, overcome can only clone short segment DNA without
The shortcomings that traditional gene cloning of full-length genome in clone cell can be synchronized.
(4) quality as Quality Control Reference Strains for genetic test controls
1. Quality Control Reference Strains are made: by DNA extraction kit specification, extracting monoclonal competent cell DNA, measure mesh
The DNA concentration of mark gene is scaled comparable cell quantity, according to target gene by DNA dosage as defined in gene detecting kit
The type of the relationship and target gene of DNA concentration and cell content is prepared respectively, is dispensed, and the calibration as genetic test refers to
Strain.
2. being used for the Internal Quality Control of genetic test: 1. judge the accuracy of testing result: 2 plants of calibration Reference Strains of selection, with
The tested sample of the Disease-causing gene containing same target detects under the same conditions, if the known target Disease-causing gene of Quality Control Reference Strains
All detected, and the target Disease-causing gene not contained is not detected, and illustrates that detection system is in Quality Control in control state, same to batch
Similar target Disease-causing gene testing result it is reliable, capable of emitting report.
3. calibrating cdna detection platform: according to the Internal Quality Control of Quality Control Reference Strains as a result, if it find that detection system is not
In Quality Control in control state, then reason out of control should be searched, including calibrate or replace detecting instrument and detection reagent, use data point instead
Analyse software and mutated gene comparison data library.
4. verify data analyze software: detector and detection reagent all in Quality Control under the premise of control, available reference
Whether the detection data verify data analysis software of strain is accurate and reliable, analyzes its standard in the processing such as screening and splicing of data
True property.
5. verifying gene database: existing in detector, detection reagent and analysis software and its analysis method all in Quality Control
Under the premise of control, the reliability in the detection data verifying gene mutation comparison data library of Quality Control Reference Strains can be used, analyze it and joining
It examines in the comparison and screening of plant targeted mutagenesis gene and whether is consistent with contained known mutations gene.
6. proving the testing result of mutated gene: obtained by Quality Control Reference Strains and tested sample are detected under the same conditions
Detection data be compared simultaneously with comparison data library, if the mutated gene that is detected of Reference Strains and contained known prominent
Become gene to be consistent, then reliable result should be judged as by being tested the mutated gene identical with Reference Strains that sample is detected.
7. the room interstitial for carrying out genetic test is commented: Quality Control Reference Strains periodically can be issued to the list that respectively participates in evaluation and electing by quality management mechanism
Position, carries out the quality evaluation of genetic test, is respectively participated in evaluation and electing the accuracy of laboratory genetic test and each according to the evaluation of result of return
The difference of room testing result, to find quality problems in time and to rectify and improve in time.
8. recognizing each other testing result between room: commented according to room interstitial as a result, if it find that identical Quality Control Reference Strains are because detecting unit
Difference and provide different as a result, quality is problematic between then illustrating room, reason should be searched, uniformly use reliable genetic test instead
System, it is ensured that the accuracy and consistency of constituent parts testing result promote recognizing each other for each room testing result, in case clients are every
One medical institutions goes to a doctor and each need to sample reinspection.
9. creating with Quality Control Reference Strains is the gene tester compared: traditional gene tester is by gene core
The comparison of piece and sequencing data and gene database obtains gene diagnosis or screening results, such as by genetic test data and normally
Mutation database compares, and removes burst-normal gene, then compare with pathogenic mutation database, obtains similar pathogenic mutation gene.
It is this to compare because tested ethnic group and testing conditions etc. are different, lack comparativity, creation Quality Control Reference Strains substitute existing gene data
Library is compared, the same detection data based on tested sample with Reference Strains with same detection condition and both inferring has phase
Same mutated gene makes gene diagnosis or screening report according to this.
11, the comparison of the present invention and traditional gene cloning
1 present invention of table is compared with traditional gene cloning
As shown in Table 1, the present invention has different purposes compared with traditional gene cloning, overcomes traditional gene cloning only
The shortcomings that short segment DNA capable of being cloned and cannot synchronizing full-length genome in clone cell, solving traditional gene cloning cannot solve
Certainly the problem of.And reagent according to the present invention, instrument are few, it is easy to operation.
12, the design sense of cell to be fused is digested
The present invention is devised with 0.25% trypsase and II Collagenase Type mixture slaking 30 minutes steps of cell to be fused
Suddenly, i.e., will first disappear to the cell of gene cloning and sp2/0 mixing with cells, then with 0.25% trypsase and the mixing of II Collagenase Type
Change 30 minutes.In theory, it is digested using 0.25% trypsase and II Collagenase Type, wherein the concentration of " 0.25% " is most
Common digestion concentration；Clostridiopetidase A is only capable of the albumen of the collagen without vitellophag wall on vitellophag surface, thus to cell without
Injury, it is evenly dispersed to be conducive to cell, avoid because cell surface is there are collagen make cytoadherence to easily formed invalid fusion,
Reduce the fusion rate of cell；Trypsase increases the permeability of cell wall because of the slight vitellophag wall-held protein of energy, is conducive to thin
Born of the same parents' fusion.And digestion time is determined as 30 minutes, is the best digestion time through testing, and can increase the clone of fused cell
Number improves the fusion rate of cell, and as a result see Table 2 for details.
The time (min) of 2 0.25% trypsase of table and II Collagenase Type mixture slaking cell to be fused and clone's number
Relationship
As shown in Table 2,5 cells to be fused are divided respectively with 0.25% trypsase and II Collagenase Type mixture slaking 15
Clock, 30 minutes and 60 minutes, being formed by corresponding cell clone total number is respectively 26,55 and 29, illustrate digestion to
It is most that clone's total number is formed by fused cell 30 minutes.Its corresponding fusion rate highest.
Claims (5)
1. a kind of full-length genome hybridizing clones method, which is characterized in that with can in vitro Immortalization mouse sp2/0 cell replacement
The genophore and competent cell of traditional gene cloning will import sp2/0 cell by cell fusion to clone gene, be allowed to
Hybridize with the DNA of sp2/0 cell and/or RNA and cloned with the Immortalization of sp2/0 cell, short-movie can only be cloned by solving
The problem of segment DNA and the traditional gene clone technology that cannot synchronize full-length genome in clone cell cann't be solved.
2. a kind of full-length genome hybridizing clones method according to claim 1, which is characterized in that the cell fusion will contain
Cell and sp2/0 cell to clone gene are divided before fusion with 0.25% trypsase and II Collagenase Type mixture slaking 30
Clock.
3. a kind of full-length genome hybridizing clones method according to claim 1, which is characterized in that the cell fusion uses
PEG effect or the regular growth fusion of electro' asion.
4. a kind of full-length genome hybridizing clones method according to claim 1, which is characterized in that the cell fusion uses
IgG as fusion agent is promoted, make to have IgG Fc receptor to gene cloning cell with the sp2/0 cell of IgG Fab receptor
Immune complex is connected as by IgG, and then carries out regular growth fusion.
5. a kind of full-length genome hybridizing clones method according to claim 1, which is characterized in that the cell fusion uses
IgG and SPA as fusion agent is promoted, make to all have IgG Fab receptor to gene cloning cell and sp2/0 cell by IgG
Fab is combined, then is connected as immune complex by the SPA with IgG Fc receptor, and then carry out regular growth fusion.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109810949A (en) * | 2019-02-05 | 2019-05-28 | 翁炳焕 | A kind of construction method in hybridoma technology and rare sick full-length genome immortalization gene library |
| CN109913500A (en) * | 2019-02-05 | 2019-06-21 | 翁炳焕 | A kind of hybridoma technology based on the improvement of sp2/0 cell immortalityization |
| CN109943534A (en) * | 2019-02-05 | 2019-06-28 | 翁炳焕 | A kind of building in monogenic disease full-length genome immortalization gene library |
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