CN106497976A

CN106497976A - A kind of HBB gene kit for correcting severe beta Thalassemia autologous patient candidate stem cell

Info

Publication number: CN106497976A
Application number: CN201610930594.5A
Authority: CN
Inventors: 邢晓; 王雯倩; 宋珂慧; 郭栋; 麦玉蝉; 陈莉; 郭伟
Original assignee: Guangdong Science And Technology Co Ltd
Current assignee: Guangdong Science And Technology Co Ltd
Priority date: 2016-10-31
Filing date: 2016-10-31
Publication date: 2017-03-15

Abstract

The invention discloses a kind of HBB gene kit for correcting severe beta Thalassemia autologous patient candidate stem cell, the reagent for infecting candidate stem cell by a set of reagent for preparing 101 HIV slow virus of specific HBB and a set of HIV slow virus is constituted.The invention also discloses the kit makes severe beta Thalassemia autologous patient candidate stem cell be changed into the application that can have in the candidate stem cell for normally synthesizing beta globin function.Experiment confirms that the kit of the present invention is simple to operate, virus titer is higher, viral efficiency of infection is up to 56.99%, candidate stem cell after to correction enter performing PCR identification and DNA sequencing identification the positive after can carry out venous re-transfusion so as to realize healing severe beta Thalassemia patient purpose, in clinical research and treatment use, development prospect is wide.

Description

A kind of HBB for correcting severe β-patients with thalassemia autologous stem cell Kit gene

Technical field

The present invention relates to a kind of gene therapy agents box, suffers from for correcting severe β-thalassemia more particularly, to a kind of The HBB gene kit of person's autologous stem cell.

Background technology

β-thalassemia is a kind of heredity hemoglobinopathy, belongs to autosomal dominant inheritance, is by beta-globin base Because of beta-globin dyssynthesis caused by (HBB) mutation, it is global modal single gene inheritance disease.

HBB gene is located at No. 11 1st area of the short arm of a chromosome, 2 bands, and this disease is in addition to a few is several nucleotide deletions, big absolutely Caused by part is all point mutation (single nucleotide acid is replaced, and is increased or is lacked), mutation cause the suppressed person of β chain composite parts claim " β+ Thalassemia "；The completely suppressed person of β chains is caused to claim " β o thalassemias ".β o/ β o homozygotes show as heavy type, β+/ β o heterozygosis Much more sub show as osculant, β+/ β shows as light-duty, as beta globin genes mutation deletion type is various, there is multiple heterozygosis State, and the compensatory factor of γ, δ peptide chain, clinical manifestation is from completely asymptomatic carrier to not having the heavy type of severe anemia There is substantially boundary.The whole world has been found that 100 kinds of gene mutation types, China have 20 kinds, is mainly distributed on province on the south the Changjiang river, especially Which is with provinces such as Guangdong, Guangxi, Hainan, Sichuan, Yunnan, Taiwan as multiple.The ratio that ground lean patient in each department accounts for：Yunnan： 4.8%th, Sichuan：2.37%th, Guizhou：2.21%, Fujian：1.83%th, Guangxi：1.52%th, Guangdong：1.08%.At present in β-ground Extra large anaemia relies primarily on periodic transfusions and carries out maintaining treatment, brings white elephant to patient family and society.Allogene is made Hemocytoblast transplanting (allo-HSCT) is the only clinical means for effecting a radical cure β thalassemia major disease at present.It is well known that Rejection behind the source of candidate stem cell and transplanting strongly limit its clinical practice.

With developing rapidly for molecular biology and molecular genetics theory and technology, people have been carried out extensively to gene therapy General and deep basic research, and take at aspects such as implementation, transfer efficiency, gene expression, animal experiment, safety evaluatios Very big progress was obtained, gene therapy gradually starts to be received by people as a kind of clinical treatment.β-Mediterranean is lean Blood is that gene therapy β-thalassemia is used as new therapeutic modality due to the genetic disease of the point mutation of HBB gene Defective for patient gene is substituted or compensating defective function is become new study hotspot.But retrieve and find：At present not There is self inactivation type slow virus (HIV) using the normal HBB gene immunodeficiency type of carrying function to infect β-thalassemia trouble Person's autologous stem cell, is then fed back to the candidate stem cell that has corrected in patient's body and realizes treatment β-thalassemic Related product.

Content of the invention

For in prior art, also without Suitable therapeutic β-thalassemic gene reagent problem, the purpose of the present invention is A kind of HBB gene kit for correcting severe β-patients with thalassemia autologous stem cell is provided.

HBB gene kit for correcting severe β-patients with thalassemia autologous stem cell of the present invention, The reagent that candidate stem cell is infected by a set of reagent for preparing specific HBB-101HIV slow virus and a set of HIV slow virus Composition；

It is characterized in that：

The reagent for preparing specific HBB-101HIV slow virus is：Concentration is 500～1000ng/ μ l specificity 500 μ l of pSINHBB-101HIV slow virus expression plasmid, the concentration for negative control is 500～1000ng/ μ l green fluorescences The 200 μ l of pSIN GFP HIV slow virus expression plasmid that GFP (GFP) is marked, concentration are 500～1000ng/ μ l 200 μ l of psPAX2 packaging plasmids, concentration are 500～1000ng/ μ l pMD2G packaging plasmids, 200 μ l, and concentration is 0.1～5M's CaCl₂10ml, 2 × HBS aqueous solution 100ml；Wherein, 280mM NaCl, 1.5mM are contained in 2 × HBS aqueous solution Na₂HPO₄With 50mM 4- HEPESs (HEPES)；

The reagent that the HIV slow virus infects candidate stem cell is：Concentration is 5 μ of 0.5mg/ml polybrenes (polybrene) L, sterilize distilled water 5ml.

In the above-mentioned HBB gene kit for correcting severe β-patients with thalassemia autologous stem cell：Described It is preferably for preparing the reagent of specific HBB-101HIV slow virus：Concentration is 800ng/ μ l specificity pSIN HBB- 500 μ l of 101HIV slow virus expression plasmid, the concentration for negative control are marked for 800ng/ μ l green fluorescence protein genes (GFP) The 200 μ l of pSIN GFP HIV slow virus expression plasmid of note, concentration are 200 μ l of 800ng/ μ l psPAX2 packaging plasmids, and concentration is 200 μ l of 800ng/ μ l pMD2G packaging plasmids, CaCl of the concentration for 2M₂10ml, 2 × HBS aqueous solution 100ml；Wherein, described 2 Contain 280mM NaCl, 1.5mM Na in the × HBS aqueous solution₂HPO₄With 50mM 4- HEPESs (HEPES).

The construction method of above-mentioned specific pSIN HBB-101HIV slow virus expression plasmids：Design HBB gene (Gene Bank NO.NC_000011) specific primer, annealing forms double-stranded DNA of the two ends containing restriction enzyme site cohesive end, with through Not1 and Connection product is converted bacillus coli DH 5 alpha competent cell by the pSIN carriers connection that Spe1 double digestions are reclaimed, and culture, to clone Product enters performing PCR identification, DNA sequencing, and it is slow that the positive colony for identifying is the specific pSIN HBB-101HIV for successfully constructing Viral expression plasmids.

The construction method of above-mentioned pSIN GFP HIV slow virus expression plasmids：Design GFP genes (Gene Bank NO.NC_ 011521.1) specific primer, annealing form double-stranded DNA of the two ends containing restriction enzyme site cohesive end, and through Not1 and Spe1 double digestions Connection product is converted bacillus coli DH 5 alpha competent cell by the pSIN carriers connection of recovery, and culture is carried out to clone products PCR identifications, DNA sequencing, the positive colony for identifying are the specific pSIN GFP HIV slow virus expression matter for successfully constructing Grain.

CaCl of the above-mentioned concentration for 2M₂Solution is by adding anhydrous CaCl in 40ml ultra-pure waters₂Solid 4.4g, is settled to 50ml, filters during (0.22 μm of filter membrane) is dispensed afterwards to 10ml reagent bottles and is obtained.

Above-mentioned 2 × HBS aqueous solution is by addition NaCl 16.38g, Na in 900ml ultra-pure waters₂HPO₄0.309g, 4- hydroxyl second Base piperazine ethanesulfonic acid (HEPES) 12g, adjusts pH to 7.05, is settled to 1L, filters (0.22 μm of filter membrane) and is dispensed to 100ml reagents afterwards It is obtained in bottle.

Above-mentioned concentration is by addition 0.5mg in 0.9% physiological saline 1ml for 0.5mg/ml polybrenes (polybrene) Polybrene powder is obtained in dispensing to 5 μ l test tubes after carrying out fully dissolving.

Of the present invention for correcting the HBB gene kit of severe β-patients with thalassemia autologous stem cell Reagent content is people's deal, and special messenger is special.

HBB gene kit for correcting severe β-patients with thalassemia autologous stem cell of the present invention exists It is changed into severe β-patients with thalassemia autologous stem cell and there can be the Hematopoietic Stem for normally synthesizing beta-globin function Application in cell.

Specifically, the method for above-mentioned application is：

(1) by specific pSIN HBB-101HIV slow virus expression plasmid and psPAX2 packaging plasmids and pMD2G packaging matter Grain is according to volume ratio 2～8:3:Then 1 ratio mixing, cotransfection 293T cells is collected on the cell rich in lentiviral particle Clear liquid, after concentration, is obtained the slow virus concentrate of high titre；

(2) obtained slow virus concentrate is added the severe β-Mediterranean that with the addition of polybrene (polybrene) lean In blood autologous patient candidate stem cell, slow-virus infection candidate stem cell, culture is made to obtain having normal synthesis beta-globin The candidate stem cell of function.

In above-mentioned application：The specific pSIN HBB-101HIV slow virus expression plasmid and psPAX2 packaging plasmids and PMD2G packaging plasmids are preferably according to volume ratio 4:3:1 ratio mixing, cotransfection 293T cells, obtained slow virus titre is most High.

The invention discloses a kind of HBB gene for correcting severe β-patients with thalassemia autologous stem cell is tried Agent box, retrieval show that the kit of the present invention has filled up the blank for the treatment of severe β-thalassemia gene prod.Solve biography Rule and treat the various problems occurred in severe β-thalassemia.Gene therapy β-new treatment side of thalassemia is hewed out Method.

Provided by the present invention for correcting the HBB gene kit of severe β-patients with thalassemia autologous stem cell There is complete operating process, application operating is simple, and virus titer is higher, and viral efficiency of infection is up to 56.99%.Indicate this examination Agent box can have normally synthesis beta-globin function being changed into severe β-patients with thalassemia autologous stem cell In candidate stem cell, tool is widely used.Candidate stem cell after to correction enter performing PCR identification and DNA sequencing identification the positive after i.e. Can carry out venous re-transfusion so as to realize cure severe β-patients with thalassemia purpose.Send out in clinical research and treatment use Exhibition has a extensive future.

Description of the drawings

Fig. 1 is beta-globin gene (HBB) total length PCR primer.

Fig. 2 is pSIN HBB-101 plasmid order-checking result figures.

Fig. 3 is slow virus expression plasmid and packaging plasmid collection of illustrative plates.

Wherein：

A.pSIN HBB-101HIV slow virus expression plasmid collection of illustrative plates；

B.pSIN GFP HIV slow virus expression plasmid collection of illustrative plates；

C.psPAX2 packaging plasmid collection of illustrative plates；

D.pMD2G packaging plasmid collection of illustrative plates.

Fig. 4 is the percentage variation diagram of CD34+ cells before and after magnetic bead sorting.

Wherein：Left figure is the content accounting of candidate stem cell in peripheral blood before sorting, and right figure is for making in peripheral blood after sorting The content accounting of hemocytoblast.

Fig. 5 is the dye efficiencies schematic diagram that different proportion packs that restrovirus infects candidate stem cell sense.

Fig. 6 is restructuring candidate stem cell Colony forming datagram.

Fig. 7 is HBB gene expression in RT-PCR detection restructuring candidate stem cells.

Fig. 8 is extraction HBB gene DNA sequencing and normal HBB gene pair after 2 lentiviral particle of ratio infection candidate stem cell The situation of ratio.

Wherein：A is that HBB gene code area SNP is sequenced peak value comparison diagram, and B is compared for HBB gene code area sequencing sequence Figure.

Specific embodiment

General explanation：Enzyme involved by following examples is purchased from Thermo companies, blood DNA extracts kit, matter Grain extracts kit, Ago-Gel reclaim DNA fragmentation kit, RNA extracts kits and reverse transcription reagent box and are purchased from Tiangeng Biochemical technology (Beijing) Co., Ltd, operation are carried out fully according to corresponding instructions.In plasmid construction, gene sequencing and primer are closed Into being completed by Huada gene company.GFP egfp expression plasmids pEGFP-N3 from ATCC, (protect by American Type Culture Tibetan center)；Escherichia coli (E.coli) DH5 α competent cells are purchased from Quan Shijin Bioisystech Co., Ltd.Its in embodiment His experimental technique and reagent if no special instructions, are this area conventional method and commercial reagent.

The LB fluid nutrient mediums are：Peptone 10g/L, dusty yeast 5g/L, NaCl 5g/L.

Embodiment 1：The acquisition of specific HBB gene sequence

According to the human HBB gene sequences of NCBI retrievals, Serial No. NC_000011 is retrieved, design specific primer Obtain HBB gene total length.

1. normal human peripheral blood is gathered, and STb gene is extracted using Chelex-100 methods and (is said referring to blood DNA extracts kit Bright book).

2. using following (the SEQ ID No.1＆SEQ ID of 5 Software for Design specific primers of Primer Premier No.2)：5 '-GGATCTTCCA GAGATTGTAC GGCTGTCATC ACTTAG-3 ' HBB of HBB gene full-length clone upstream primer 5 '-CTGCCGTTCG ACGATTAAGG AACACTTCAGGGGAAAG-3 ' of full length gene cloned downstream primer

3. the reaction system according to table 1 enters performing PCR amplification.

Table 1

4.Bio-Rad C1000PCR instrument (Bio-Rad, the U.S.) enters performing PCR reaction, and reaction system is 98 DEG C of denaturations 3min；98 DEG C of denaturation 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 2min, totally 30 circulations；Last 72 DEG C of extensions 2min, 4 DEG C of end Reaction.

5. detect pcr amplification product and reclaim using 1% agarose gel electrophoresis (DNA pieces are reclaimed referring to Ago-Gel Section kit specification), as a result see Fig. 1, and be named as the human hemoglobin beta-globin gene HBB-101.

6. couple HBB-101 carries out sequencing and detects its accuracy,

The nucleotide sequence of HBB-101 is as shown in SEQ ID No.3.

Embodiment 2：The structure of pSIN HBB-101HIV slow virus expression plasmids

1. digestion carried out to HBB-101 genetic fragments using SpeI and NotI, 37 DEG C of digestion 2h, endonuclease reaction system is such as Under：

2. pSIN HIV slow virus expression plasmids are reclaimed through SpeI and NotI double digestions and make which linear, be carried out according to the following table 37 DEG C of endonuclease reaction 2h, endonuclease reaction system are as follows：

3. prepared by clone

(1) connect

Reaction system is：

(2) convert

1. the aseptic pipette tips with cooling take 200 μ l and are transferred in sterile centrifugation tube, plus 2 μ l connection liquids, mix, place on ice 30min.

2. 90s is heated in 42 DEG C of waters bath with thermostatic control, is then immediately placed in cold shock 2min on ice.

3. the mixture of connection conversion is added in 500 μ l antibiotic-free LB fluid nutrient mediums, 180rpm, 37 DEG C of incubations 50min, makes bacterium rejuvenate.

4. 200 μ L bacterium solutions are taken equably to be applied on the LB solid medium flat boards containing Amp, was cultivated under the conditions of 37 DEG C Night.

4. the identification of positive colony and DNA sequencing

(1) white monoclonal bacterium colony of the positive colony screening on pipette tips picking flat board, is inoculated into the LB liquid containing Amp respectively In body culture medium, 37 DEG C of 200rpm cultivate 5-10 hours.

(2) bacterium solution PCR is identified with bacterium solution as template, and HBB gene full-length clone primer carries out 10 μ l systems and carries out regular-PCR Identification, 10 μ l reaction systems：1 μ l of bacterium solution, 0.5 μ l of upstream primer, 0.5 μ l of downstream primer, 10 × PCR Buffer (Mg²⁺Plus) 0.5 μ l of 1 μ l, dNTP Mixture (each 2.5mM), 0.1 μ l of TaKaRa LA Taq, ddH₂O 6.4μl.

(3) PCR reaction responses condition is：94 DEG C of denaturations 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extensions 40s, totally 33 circulations；Last 72 DEG C of extensions 10min.

After PCR primer is through 1% agarose gel electrophoresis detection, positive colony is selected.

(4) positive colony bacterium colony is selected, and plasmid is extracted using plasmid extraction kit and (is illustrated referring to plasmid extraction kit Book), carry out DNA sequencing.(such as Fig. 2)

Obtain pSIN HBB-101HIV slow virus expression plasmids.

Embodiment 3：The structure of pSIN GFP HIV slow virus expression plasmids

1. the GFP gene orders according to NCBI retrievals are obtained, Serial No. NC_011521.1 is retrieved, design specificity is drawn Thing obtains GFP full length genes.

2. using following (the SEQ ID No.4＆SEQ of 5 Software for Design GFP gene-specific primers of Primer Premier ID No.5)：

GFP upstream region of gene primers：5’-GGACTAGTTGAGTAAAGG-3’

GFP downstream of gene primers：5’-TTGCGGCCGCTTATTTGTA-3’

3. with plasmid pEGFP-N3 as template, GFP full length genes are cloned, GFP genes are reclaimed by agarose gel electrophoresis Fragment.

4. digestion is carried out to GFP genetic fragments using SpeI and NotI, 37 DEG C of digestion 2h, endonuclease reaction system are as follows：

5. pSIN HIV slow virus expression plasmids are reclaimed through SpeI and NotI double digestions and make which linear, be carried out according to the following table 37 DEG C of endonuclease reaction 2h, endonuclease reaction system are as follows：

6. prepared by clone

(1) connect

Reaction system is：

(2) convert

7. the identification of positive colony and DNA sequencing

(2) bacterium solution PCR is identified with bacterium solution as template, and GFP full length gene cloning primers carry out 10 μ l systems and carry out regular-PCR Identification, 10 μ l reaction systems：1 μ l of bacterium solution, 0.5 μ l of upstream primer, 0.5 μ l of downstream primer, 10 × PCR Buffer (Mg²⁺Plus) 0.5 μ l of 1 μ l, dNTP Mixture (each 2.5mM), 0.1 μ l of TaKaRa LA Taq, ddH₂O 6.4μl.

(4) positive colony bacterium colony is selected, and plasmid is extracted using plasmid extraction kit and (is illustrated referring to plasmid extraction kit Book).

Obtain pSIN GFP HIV slow virus expression plasmids.

Embodiment 3：The packaging of HIV slow virus.

HIV slow virus be made up of three kinds of plasmids (such as Fig. 3) include psPAX2 packaging plasmids, pMD2G packaging plasmids and PSIN HBB-101HIV slow virus expression plasmids or pSIN GFP HIV slow virus expression plasmid packaging, four kinds of plasmids are passed through The extracting of endotoxin-free high-purity is obtained.

1. the preparation of Viral packaging cell：Concentration is taken for 1 × 10⁶Individual/ml 293T cells (purchased from ATCC companies of the U.S.) 8ml is inoculated in 10cm cell cultures, 1640 culture mediums (purchased from Hyclone companies of the U.S.) for adding 10ml fresh, is put into 37 DEG C, 5%CO₂After cultivating 24 hours in cell culture incubator, all culture mediums are suctioned out, 1640 culture mediums of 5ml are added, stand-by.

2. it is respectively added in 50ml centrifuge tubes according to the order and volume of following reagent.

Wherein pSIN HBB-101HIV slow virus expression plasmid and pSIN GFP HIV slow virus expression plasmid respectively with PsPAX2 packaging plasmids, pMD2G packaging plasmids are with volume ratio respectively according to 2:3:1、4:3:1 and 8:3:1 ratio is reacted. Each reaction is respectively designated as ratio 1, ratio 2, ratio 3 successively.

3. CaCl is separately added into₂156.25 μ l of solution, quickly shake up, about 10s；

4. 1250 μ l of 2xHBS are separately added into, 30s is quickly shaken up, 2min 30s are stood；

5. draw 2.5ml fresh cultures to add in said mixture, fully piping and druming is mixed, and obtains compound；

6. complexes drop-wise is equably separately added in 3 parts of 293T cells for having finished changing fresh culture, is mixed, in 37 DEG C, 5%CO₂Cultivate in cell culture incubator；

7. after culture 12-14h, cell changes liquid entirely, mixes, in 37 DEG C, 5%CO₂Cultivate in cell culture incubator；

8., after continuing cultured cells 36h, observation of cell form and transfected condition under fluorescence microscope will along culture dish side wall During vial supernatant is suctioned out to 50ml centrifuge tubes；

9. the filtration syringe filter virus liquid for the viral supernatants that collects being used 0.45 μm is into collection virus pipe；

10. fill it up with into 293T fresh mediums to the viral mouth of pipe in collection virus pipe；

11.50000rpm/min, 4 DEG C, it is centrifuged 2.5h；

12. remove supernatant, using the resuspended viruses of 100 μ l ice PBS (volume), dispense, -80 DEG C of preservations.

Embodiment 4：The measure of slow virus titre

The lentiviral particle of three groups of different proportion packagings that embodiment 3 is obtained is respectively while carry out following 1～8 step behaviour Make.

1. 7 clean EP pipes are taken out, 1～7 is numbered successively.990 μ lDMEM+10%FBS are added to cultivate in each EP pipe Base.Take out 10 μ l concentrating virus to add in No. 1 EP pipe, mix, now concentration is 1/10 to virus.10 μs are taken out from No. 1 EP pipe again L is added in No. 2 centrifuge tubes, is mixed.3～No. 7 pipes repeat No. 2 pipe operations.Now virus is diluted into 1/10～1/10⁷Times. 10 μ l, 1640 culture mediums are filled in each centrifuge tube, are now 1ml viral suspensions in each centrifuge tube.

2. prepare 7 six orifice plates, add the viral suspension of 10 μ l, stand-by.

3. take the logarithm the NIH3T3 cells (purchased from ATCC companies of the U.S.) in growth period, 0.25% pancreas enzyme -EDTA digests 2min, It is put into basis of microscopic observation cell state.When cell shrinkage is rounded, you can add complete medium to terminate digestion, cell is blown and beaten Get off.

4. cell is collected in centrifuge tube, and trim, 300 × g are centrifuged 3min.

5. supernatant is discarded, 1640 culture medium re-suspended cells of 1ml is added, is taken 10 μ l cell counts.

6. cell suspension is diluted to 2 × 10⁶/ ml, adds 1ml cell suspensions to six orifice plates per hole, while adding 2 per hole μ l concentration is 0.5mg/ml polybrenes (polybrene).

After 7.48h, clear, collection cell, flow cytometer detection GFP% is sucted.

8. calculated closest to 1 extension rate with flow cytometer detection GFP%, virus infection drop is calculated with following equation Degree：

Virus infection titer (PFU/ml)=[(2 × 10⁶Cells/well) × GFP%]/(virus liquid volume × dilution times Number).

9. the slow virus titre of three groups of different proportion packagings is shown in Table 2.

Table 2

Embodiment 5：The sorting of candidate stem cell and culture (U.S.'s day Ni immunological magnetic bead sorting kits)

1. coated cell culture plate：The use of density is 5 μ g/cm²Fibronectin (fibronectin) be coated 12 hole cells Culture plate, 37 DEG C of standing 6h.

2. recover frozen severe β-patients with thalassemia PMNC, 37 DEG C of water-baths are melted, by cell Be transferred to from cryopreservation tube in 15ml centrifuge tubes, be added dropwise with Pasteur's pipe and F12 culture mediums be settled to 8ml, during when being added dropwise Rock.Centrifugation 300g, 3min.

3. supernatant is outwelled, and using 1mlFACS flushing liquors (1 × PBS containing 2%FBS), re-suspended cell, after piping and druming is mixed It is filled in new 15ml centrifuge tubes with 70 μm of cell screen clothes.

4. cell count.It is centrifuged again, 500g, 10min.Suct clearly, according to the cell number for counting, per 1 × 10⁸Individual cell With 300 μ l re-suspended cells.

5. antibody is incubated：Lucifuge is operated, according to per 1 × 10⁸Individual cell adds 50 μ l FCR confining liquids micro- with 50 μ l CD34 Pearl, 4 DEG C, 30min.

6. 2ml FACS flushing liquors are added, be centrifuged 300g, 3min.Supernatant is removed, 1ml FACS flushing liquor re-suspended cells are added, With 70um cell screen clothes filtration cells.

7. absorption magnet and absorption pillar are prepared.3ml FACS flushing liquor rinse adsorption columns are used first, after the completion of rinse Cell suspension is added in adsorption column, after the completion for the treatment of which drips naturally, addition 3ml FACS flushing liquors are simultaneously repeated 5 times, altogether plus Enter 15ml FACS flushing liquors.

8. remove adsorption column to be placed in new 15ml centrifuge tubes, add 3ml FACS flushing liquors, liquid is extruded using piston Wash-out CD34 cells.

9. inhale 100 μ l cells, lucifuge add 1 μ l CD34 antibody, 4 DEG C, 30min.The percentage of flow cytometer detection CD34+ cells Than (such as Fig. 4).

10. sampling is counted.500g, 5min is centrifuged after the completion of counting.

11. remove supernatant, add 20 culture mediums of X-VIVO (purchased from Lonza companies of Switzerland) re-suspended cell, be proportionally added into The pre-stimulation factor of lower concentration, is dispensed in 12 well culture plates after fully mixing per hole 2ml, cultivates stand-by.

Embodiment 6：The checking of lentiviral particle validity

1.3 groups of virus infection peripheral blood hematopoietic stem cells and flow cytomery slow virus infect efficiency

(1) observation of cell form and upgrowth situation, count, and control CD34 viable counts 1 × 10⁵Individual.

(2) inhale and abandon the old culture mediums of 1.5ml, and add fresh 20 culture mediums of X-VIVO of 1.5ml.

(3) add polybrene (polybrene) into culture medium, volume is 32 μ l, after mixing, stand 1～3min.

(4) virus after calculate the slow virus titre according to three groups of different proportion packagings is separately added in above-mentioned cell, Viral addition such as table 3, is well mixed.

Table 3

Observation of cell form fresh 20 culture mediums of X-VIVO are changed after (5) 37 DEG C of culture 24h.

(6) 37 DEG C of cultures after 48 hours are collected cell in centrifuge tube, are centrifuged, and are cultivated with fresh X-VIVO 20 The resuspended results of base.

(7) using the percentage (such as Fig. 5) of flow cytomery cell expressing green fluorescent protein.

2. the expression sequencing of candidate stem cell colony-forming test and external source HBB gene

(1) viral metainfective candidate stem cell is collected, is counted.

(2) resuspended 3000 candidate stem cells of 200 μ l IMDM+2%FBS are used.

(3) 1ml methylcellulose is added in cell suspension, mixed, add 2.5mm culture plates.

(4) culture plate is put into 37 DEG C, 5%CO₂Cultivate 12～14 days in cell culture incubator, forbid moving in incubation Dynamic.

Later observe colony form within (5) 12～14 days, count erythroid colonies quantity (such as Fig. 6).

(6) draw erythroid colonies using the long glass tube of tip to be put in the EP pipes for having 500 μ l PBS.

(7) 500 μ l PBS are added after colony picking is complete, is mixed, centrifugation, 500g, 5min.

(8) supernatant is removed, 200 μ l TRIZOL cell lysis are added.

(9) extract RNA and reverse transcription is cDNA (referring to RNA extracts kits and reverse transcription reagent box specification).

(10) RT-PCR detects that the expression of HBB gene in erythroid colonies cDNA, the specific primer being directed to are shown in SEQ ID No.6-9.

1. reaction system see the table below

2. using the PCR instrument of Bio-Rad CFX96 systems, reaction condition is：95 DEG C of denaturations 3min；94 DEG C of denaturation 10s, 58 DEG C of annealing 20s, 72 DEG C extend 20s (plus Plate Read), totally 40 circulations；Last 65 DEG C of Melt Curve (melting curve) To 95 DEG C, 0.5 DEG C is raised per 30s.

3. 2 are adopted according to a peak period^-ΔΔCtMethod calculates the relative expression quantity of each gene, carries out data analysis (as schemed 7).

Embodiment 7：The checking of 2 lentiviral particle validity of ratio

1. observation of cell form and upgrowth situation, count, and control CD34 viable counts 1 × 10⁵Individual.

2. inhale and abandon the old culture mediums of 1.5ml, and add fresh 20 culture mediums of X-VIVO of 1.5ml.

3. add polybrene (polybrene) into culture medium, volume is 32 μ l, after mixing, stand 1～3min.

4. take 470 μ l ratios, 2 slow virus and add cell, be well mixed.

Observation of cell form fresh 20 culture mediums of X-VIVO are changed after 5.37 DEG C of culture 24h.

Cell is collected in centrifuge tube after 6.37 DEG C of culture 48h, be centrifuged, harvest.

7. resuspended 3000 candidate stem cells of 200 μ l IMDM+2%FBS are used.

8. 1ml methylcellulose is added in cell suspension, mixed, add 2.5mm culture plates.

9. culture plate is put into 37 DEG C, 5%CO₂Cultivate 12～14 days in cell culture incubator, forbidden moves in incubation.

Later observe colony form within 10.12～14 days, count erythroid colonies quantity.

11. draw erythroid colonies using the long glass tube of tip is put in the EP pipes for having 500 μ l PBS.

500 μ l PBS are added after 12. colony pickings are complete, is mixed, centrifugation, 500g, 5min.

13. remove supernatant, add 200 μ l TRIZOL cell lysis.

14. extract RNA, and simultaneously reverse transcription is cDNA.

The product of 15.PCR send sequencing, can detect that specific SNP bimodal (such as Fig. 8).

Sequence table

<110>Guangdong Yi Ke gene biologicals Science and Technology Ltd.

<120>A kind of HBB gene kit for correcting severe β-patients with thalassemia autologous stem cell

<141> 2016-10-16

<160>9

<210> 1

<211> 36

<212> DNA

<213>Artificial sequence

<221>HBB gene full-length clone upstream primer

<222>（1）…（36）

<400> 1

ggatcttcca gagattgtac ggctgtcatc acttag 36

<210> 2

<211> 37

<212> DNA

<213>Artificial sequence

<221>HBB gene full-length clone downstream primer

<222>（1）…（37）

<400> 2

ctgccgttcg acgattaagg aacacttcag gggaaag 37

<210> 3

<211> 2088

<212> DNA

<213>Artificial sequence

<221>The human hemoglobin beta-globin gene HBB-101

<222>（1）…（2088）

<400>3

tttttagtag caatttgtac tgatggtatg gggccaagag atatatctta gagggagggc 60

tgagggtttg aagtccaact cctaagccag tgccagaaga gccaaggaca ggtacggctg 120

tcatcactta gacctcaccc tgtggagcca caccctaggg ttggccaatc tactcccagg 180

agcagggagg gcaggagcca gggctgggca taaaagtcag ggcagagcca tctattgctt 240

acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgcatc 300

tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac gtggatgaag 360

ttggtggtga ggccctgggc aggttggtat caaggttaca agacaggttt aaggagacca 420

atagaaactg ggcatgtgga gacagagaag actcttgggt ttctgatagg cactgactct 480

ctctgcctat tggtctattt tcccaccctt aggctgctgg tggtctaccc ttggacccag 540

aggttctttg agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag 600

gtgaaggctc atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac 660

aacctcaagg gcacctttgc cacactgagt gagctgcact gtgacaagct gcacgtggat 720

cctgagaact tcagggtgag tctatgggac gcttgatgtt ttctttcccc ttcttttcta 780

tggttaagtt catgtcatag gaaggggata agtaacaggg tacagtttag aatgggaaac 840

agacgaatga ttgcatcagt gtggaagtct caggatcgtt ttagtttctt ttatttgctg 900

ttcataacaa ttgttttctt ttgtttaatt cttgctttct ttttttttct tctccgcaat 960

ttttactatt atacttaatg ccttaacatt gtgtataaca aaaggaaata tctctgagat 1020

acattaagta acttaaaaaa aaactttaca cagtctgcct agtacattac tatttggaat 1080

atatgtgtgc ttatttgcat attcataatc tccctacttt attttctttt atttttaatt 1140

gatacataat cattatacat atttatgggt taaagtgtaa tgttttaata tgtgtacaca 1200

tattgaccaa atcagggtaa ttttgcattt gtaattttaa aaaatgcttt cttcttttaa 1260

tatacttttt tgtttatctt atttctaata ctttccctaa tctctttctt tcagggcaat 1320

aatgatacaa tgtatcatgc ctctttgcac cattctaaag aataacagtg ataatttctg 1380

ggttaaggca atagcaatat ctctgcatat aaatatttct gcatataaat tgtaactgat 1440

gtaagaggtt tcatattgct aatagcagct acaatccagc taccattctg cttttatttt 1500

atggttggga taaggctgga ttattctgag tccaagctag gcccttttgc taatcatgtt 1560

catacctctt atcttcctcc cacagctcct gggcaacgtg ctggtctgtg tgctggccca 1620

tcactttggc aaagaattca ccccaccagt gcaggctgcc tatcagaaag tggtggctgg 1680

tgtggctaat gccctggccc acaagtatca ctaagctcgc tttcttgctg tccaatttct 1740

attaaaggtt cctttgttcc ctaagtccaa ctactaaact gggggatatt atgaagggcc 1800

ttgagcatct ggattctgcc taataaaaaa catttatttt cattgcaatg atgtatttaa 1860

attatttctg aatattttac taaaaaggga atgtgggagg tcagtgcatt taaaacataa 1920

agaaatgaag agctagttca aaccttggga aaatacacta tatcttaaac tccatgaaag 1980

aaggtgaggc tgcaaacagc taatgcacat tggcaacagc ccctgatgca tatgccttat 2040

tcatccctca gaaaaggatt caagtagagg cttgatttgg aggttaaa 2088

<210> 4

<211> 18

<212> DNA

<213>Artificial sequence

<221>GFP upstream region of gene primers

<222>（1）…（18）

<400> 4

ggactagttg agtaaagg 18

<210> 5

<211> 19

<212> DNA

<213>Artificial sequence

<221>GFP downstream of gene primers

<222>（1）…（19）

<400> 5

ttgcggccgc ttatttgta 19

<210> 6

<211> DNA

<212>

<213>Artificial sequence

<221>Reference gene GAPDH upstream primers

<222>（1）…（21）

<400> 6

tgacttcaac agcgacaccc a 21

<210> 7

<211> DNA

<212>

<213>Artificial sequence

<221>Reference gene GAPDH downstream primers

<222>（1）…（21）

<400> 7

caccctgttg ctgtagccaa a 21

<210> 8

<211> DNA

<212> 18

<213>Artificial sequence

<221>HBB gene specific forward primer

<222>（1）…（18）

<400> 8

atgaagttgg tggtgagg 18

<210>9

<211> DNA

<212> 18

<213>Artificial sequence

<221>HBB gene specific Down Stream primer

<222>（1）…（18）

<400> 9

tgaggttgtc caggtgag 18

Claims

1. a kind of HBB gene kit for correcting severe β-patients with thalassemia autologous stem cell, is applied mechanically by one In the reagent composition that the reagent and a set of HIV slow virus for preparing specificity HBB-101HIV slow virus infects candidate stem cell；

It is characterized in that：

The reagent for preparing specific HBB-101HIV slow virus is：Concentration is 500～1000ng/ μ l specificity pSIN 500 μ l of HBB-101HIV slow virus expression plasmid, the concentration for negative control is 500～1000ng/ μ l green fluorescent proteins The 200 μ l of pSIN GFP HIV slow virus expression plasmid that gene (GFP) is marked, concentration are 500～1000ng/ μ l psPAX2 bags Dress 200 μ l of plasmid, concentration is 500～1000ng/ μ l pMD2G packaging plasmids, 200 μ l, and concentration is the CaCl of 0.1～5M₂ 10ml, 2 × HBS aqueous solution 100ml；Wherein, 280mM NaCl, 1.5mM Na are contained in 2 × HBS aqueous solution₂HPO₄With 50mM 4- HEPESs (HEPES)；

The reagent that the HIV slow virus infects candidate stem cell is：Concentration is 5 μ l of 0.5mg/ml polybrenes (polybrene), goes out Bacterium distilled water 5ml.

2. the HBB gene examination for correcting severe β-patients with thalassemia autologous stem cell is used for according to claim 1 Agent box, it is characterised in that：The reagent for preparing specific HBB-101HIV slow virus is：Concentration is that 800ng/ μ l are special Property 500 μ l of pSIN HBB-101HIV slow virus expression plasmid, the concentration for negative control is 800ng/ μ l green fluorescent proteins The 200 μ l of PSIN GFP HIV slow virus expression plasmid that gene (GFP) is marked, concentration are 800ng/ μ l psPAX2 packaging plasmids 200 μ l, concentration are 200 μ l of 800ng/ μ l pMD2G packaging plasmids, CaCl of the concentration for 2M₂10ml, the 2 × HBS aqueous solution 100ml；Wherein, 280mM NaCl, 1.5mM Na are contained in 2 × HBS aqueous solution₂HPO₄With 50mM 4- hydroxyethyl piperazine second Sulfonic acid (HEPES).

3. the HBB gene reagent for correcting severe β-patients with thalassemia autologous stem cell is used for described in claim 1 or 2 Box can have normally synthesis beta-globin function making being changed into severe β-patients with thalassemia autologous stem cell Application in hemocytoblast.

4. application according to claim 3, it is characterised in that application process is：

(1) specific pSIN HBB-101HIV slow virus expression plasmid and psPAX2 packaging plasmids and pMD2G packaging plasmids are pressed According to volume ratio 2～8:3:Then 1 ratio mixing, cotransfection 293T cells collect the cell supernatant rich in lentiviral particle, After concentration, the slow virus concentrate of high titre is obtained；

(2) obtained slow virus concentrate is added the severe β-thalassemia that with the addition of polybrene (polybrene) suffer from In person's autologous stem cell, slow-virus infection candidate stem cell, culture is made to obtain having normal synthesis beta-globin function Candidate stem cell.

5. application according to claim 4, it is characterised in that：The specific pSIN HBB-101HIV slow virus expression Plasmid is with psPAX2 packaging plasmids and pMD2G packaging plasmids according to volume ratio 4:3:1 ratio mixing, cotransfection 293T cells, Obtained slow virus titre highest.