CN106497976A - A HBB gene kit for correcting autologous hematopoietic stem cells of patients with severe β-thalassemia - Google Patents

Abstract

The present invention discloses a HBB gene kit for correcting autologous hematopoietic stem cells of patients with severe β-thalassemia, which is composed of a set of reagents for preparing specific HBB-101 HIV lentivirus and a set of reagents for HIV lentivirus to infect hematopoietic stem cells. The present invention also discloses the use of the kit in converting autologous hematopoietic stem cells of patients with severe β-thalassemia into hematopoietic stem cells that can have normal β-globin synthesis function. Experiments have confirmed that the kit of the present invention is simple to operate, has a high virus titer, and a virus infection efficiency of up to 56.99%. After PCR identification and DNA sequencing identification of the corrected hematopoietic stem cells are positive, intravenous reinfusion can be performed to achieve the purpose of curing patients with severe β-thalassemia, and the development prospects in clinical research and therapeutic applications are broad.

Description

A HBB for correcting autologous hematopoietic stem cells in patients with severe β-thalassemia Gene Kit

technical field

The invention relates to a gene therapy kit, in particular to an HBB gene kit for correcting autologous hematopoietic stem cells of severe beta-thalassemia patients.

Background technique

β-thalassemia is a hereditary hemoglobinopathy, which belongs to autosomal dominant inheritance. It is a disorder of β-globin synthesis caused by mutation of β-globin gene (HBB). It is the most common monogenic genetic disease in the world.

The HBB gene is located in zone 1, zone 2, short arm of chromosome 11. Except for a few types of the disease, most of them are caused by point mutations (single nucleotide substitutions, additions or deletions). Mutations Those who cause partial inhibition of β chain synthesis are called "β + thalassemia"; those who cause complete inhibition of β chain are called "βo thalassemia". βo/βo homozygotes are severe, β+/βo heterozygotes are mostly intermediate, and β+/β is light. Due to the variety of β globin gene mutations and deletions, there are multiple heterozygous states, and γ and δ peptides Chain compensatory factors, clinical manifestations from completely asymptomatic carriers to severe anemia between no clear boundaries. 100 types of gene mutations have been found in the world, 20 types in my country, mainly distributed in the provinces south of the Yangtze River, especially in Guangdong, Guangxi, Hainan, Sichuan, Yunnan, Taiwan and other provinces. The proportion of thalassemia patients in each region: Yunnan: 4.8%, Sichuan: 2.37%, Guizhou: 2.21%, Fujian: 1.83%, Guangxi: 1.52%, Guangdong: 1.08%. At present, β-thalassemia mainly relies on regular blood transfusion for maintenance treatment, which has brought a heavy burden to the patient's family and society. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only clinical means to cure β-thalassemia major. However, as we all know, the source of hematopoietic stem cells and the rejection after transplantation greatly limit its clinical application.

With the rapid development of molecular biology and molecular genetics theory and technology, people have conducted extensive and in-depth basic research on gene therapy, and have made great achievements in implementation methods, transfer efficiency, gene expression, animal experiments, and safety evaluation. Great progress, gene therapy as a clinical treatment has gradually begun to be accepted by people. β-thalassemia is a genetic disease caused by a point mutation of the HBB gene. Gene therapy for β-thalassemia as a new treatment method to replace the defective gene in patients or compensate for the defective function has become a new research hotspot. However, the search found that at present, there is no way to infect the autologous hematopoietic stem cells of patients with β-thalassemia with the immunodeficiency self-inactivating lentivirus (HIV) carrying the normal HBB gene, and then reinfuse the corrected hematopoietic stem cells into the patient's body. Related products for the treatment of β-thalassemia.

Contents of the invention

Aiming at the problem that there is no suitable gene reagent for treating β-thalassemia in the prior art, the purpose of the present invention is to provide a HBB gene kit for correcting autologous hematopoietic stem cells of patients with severe β-thalassemia.

The HBB gene kit for correcting autologous hematopoietic stem cells of patients with severe β-thalassemia according to the present invention consists of a set of reagents for preparing specific HBB-101 HIV lentivirus and a set of reagents for HIV lentivirus infecting hematopoietic stem cells;

It is characterized by:

The reagents for preparing specific HBB-101HIV lentivirus are: the concentration is 500-1000ng/μl specific pSINHBB-101HIV lentivirus expression plasmid 500μl, the concentration for negative control is 500-1000ng/μl green fluorescent protein gene (GFP) labeled pSIN GFP HIV lentiviral expression plasmid 200μl, the concentration is 500～1000ng/μlpsPAX2 packaging plasmid 200μl, the concentration is 500～1000ng/μl pMD2G packaging plasmid 200μl, the concentration is 0.1～5M CaCl ₂ 10ml, 2×HBS 100ml aqueous solution; wherein, the ₂ ×HBS aqueous solution contains 280mM NaCl, 1.5mMNa2HPO4 and 50mM ₄ -hydroxyethylpiperazineethanesulfonic acid (HEPES);

The reagents for infecting hematopoietic stem cells with the HIV lentivirus are: 5 μl of polybrene at a concentration of 0.5 mg/ml, and 5 ml of sterilized double distilled water.

In the above-mentioned HBB gene kit for correcting autologous hematopoietic stem cells of patients with severe β-thalassemia: the reagent for preparing specific HBB-101HIV lentivirus is preferably: a concentration of 800ng/μl specific pSIN HBB-101HIV lentivirus Expression plasmid 500μl, the concentration used for negative control is 800ng/μl green fluorescent protein gene (GFP) tagged pSIN GFP HIV lentiviral expression plasmid 200μl, the concentration is 800ng/μl psPAX2 packaging plasmid 200μl, the concentration is 800ng/μl pMD2G packaging plasmid 200 μl, 10 ml of 2M CaCl ₂ , 100 ml of 2×HBS aqueous solution; wherein, the 2×HBS aqueous solution contains 280 mM NaCl, 1.5 mM Na ₂ HPO ₄ and 50 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) .

The construction method of the specific pSIN HBB-101HIV lentiviral expression plasmid mentioned above: design HBB gene (GeneBank NO.NC_000011) specific primers, anneal to form double-stranded DNA with sticky ends containing restriction sites at both ends, and combine with Not1 and Spe1 double enzymes Cut and ligate the recovered pSIN vector, transform the ligated product into Escherichia coli DH5α competent cells, culture, and carry out PCR identification and DNA sequencing on the cloned product, and the identified positive clone is the successfully constructed specific pSIN HBB-101HIV lentiviral expression plasmid .

The construction method of the above-mentioned pSIN GFP HIV lentiviral expression plasmid: design GFP gene (Gene Bank NO.NC_011521.1) specific primers, anneal to form double-stranded DNA with sticky ends containing restriction sites at both ends, and combine with Not1 and Spe1 double enzymes The recovered pSIN vector was cut and ligated, the ligated product was transformed into Escherichia coli DH5α competent cells, cultured, and the cloned product was identified by PCR and DNA sequencing. The identified positive clone was the successfully constructed specific pSIN GFP HIV lentiviral expression plasmid.

The above-mentioned _CaCl2 solution with a concentration of 2M was prepared by adding 4.4 g of anhydrous _CaCl2 solids to 40 ml of ultrapure water, distilled to 50 ml, filtered (0.22 μm filter membrane) and then dispensed into 10 ml reagent bottles.

The above 2×HBS aqueous solution is prepared by adding 16.38g of NaCl, 0.309g of Na ₂ HPO ₄ , and 12g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) into 900ml of ultrapure water, adjust the pH to 7.05, and set the volume to 1L, filter (0.22μm filter membrane) and then dispensed into 100ml reagent bottles.

The above-mentioned concentration of 0.5 mg/ml polybrene (polybrene) is obtained by adding 0.5 mg of polybrene powder to 1 ml of 0.9% physiological saline to fully dissolve and then packing into 5 μl test tubes.

The reagent content of the HBB gene kit for correcting autologous hematopoietic stem cells of patients with severe beta-thalassemia according to the present invention is for one person, and it is specially designed for special use.

The application of the HBB gene kit for correcting the autologous hematopoietic stem cells of severe beta-thalassemia patients to transform the autologous hematopoietic stem cells of severe beta-thalassemia patients into hematopoietic stem cells capable of normally synthesizing beta-globin.

Specifically, the method of the above application is:

(1) Mix the specific pSIN HBB-101HIV lentiviral expression plasmid with psPAX2 packaging plasmid and pMD2G packaging plasmid in a volume ratio of 2-8:3:1, co-transfect 293T cells, and then collect lentiviral particles-rich After the cell supernatant is concentrated, a high-titer lentivirus concentrate is obtained;

(2) Add the prepared lentivirus concentrate to the autologous hematopoietic stem cells of patients with severe β-thalassemia to which polybrene has been added, so that the lentivirus can infect the hematopoietic stem cells, and culture them to obtain cells with normal synthesis of β-globin Functional hematopoietic stem cells.

In the above application: the specific pSIN HBB-101HIV lentivirus expression plasmid is preferably mixed with the psPAX2 packaging plasmid and pMD2G packaging plasmid in a volume ratio of 4:3:1, and co-transfected into 293T cells, and the obtained lentivirus titer is Highest.

The invention discloses a HBB gene kit for correcting autologous hematopoietic stem cells of patients with severe beta-thalassemia, and a search shows that the kit of the invention fills the blank of gene products for treating severe beta-thalassemia. Solve various problems in the traditional treatment of severe β-thalassemia. Opened up a new treatment method of gene therapy β-thalassemia.

The HBB gene kit for correcting autologous hematopoietic stem cells of patients with severe beta-thalassemia provided by the present invention has a complete operation process, simple application and operation, high virus titer, and virus infection efficiency as high as 56.99%. It is predicted that the kit will be widely used in transforming autologous hematopoietic stem cells of patients with severe β-thalassemia into hematopoietic stem cells capable of normally synthesizing β-globin. After the corrected hematopoietic stem cells are positively identified by PCR and DNA sequencing, they can be infused intravenously so as to achieve the goal of curing severe β-thalassemia patients. It has broad prospects for development in clinical research and therapeutic applications.

Description of drawings

Figure 1 is the full-length PCR product of β-globin gene (HBB).

Figure 2 is a diagram of the sequencing results of the pSIN HBB-101 plasmid.

Figure 3 is a map of lentiviral expression plasmids and packaging plasmids.

in:

A. pSIN HBB-101HIV lentiviral expression plasmid map;

B. pSIN GFP HIV lentiviral expression plasmid map;

C. psPAX2 packaging plasmid map;

D. Map of the pMD2G packaging plasmid.

Figure 4 is a graph showing the percentage change of CD34+ cells before and after magnetic bead sorting.

Among them: the left figure shows the proportion of hematopoietic stem cells in peripheral blood before sorting, and the right figure shows the proportion of hematopoietic stem cells in peripheral blood after sorting.

Figure 5 is a schematic diagram of the infection efficiency results of virus infection of hematopoietic stem cells after packaging in different proportions.

Fig. 6 is a graph showing colony formation data of recombinant hematopoietic stem cells.

Figure 7 shows the detection of HBB gene expression in recombinant hematopoietic stem cells by RT-PCR.

Figure 8 is a comparison of HBB gene DNA sequencing and normal HBB gene extracted after lentiviral particles infected hematopoietic stem cells in ratio 2.

Among them: A is the comparison map of SNP sequencing peaks in the coding region of HBB gene, and B is the comparison map of the sequencing sequences in the coding region of HBB gene.

detailed description

General description: the enzymes involved in the following examples were all purchased from Thermo Company, blood DNA extraction kit, plasmid extraction kit, agarose gel recovery DNA fragment kit, RNA extraction kit and reverse transcription kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., the operation was carried out in accordance with the corresponding instructions. Gene sequencing and primer synthesis in plasmid construction were completed by Huada Gene Company. GFP green fluorescent protein expression plasmid pEGFP-N3 was obtained from ATCC (American Type Culture Collection); Escherichia coli (E.coli) DH5α competent cells were purchased from Quanshijin Biotechnology Co., Ltd. Other experimental methods and reagents in the examples are conventional methods and commercially available reagents in the art unless otherwise specified.

The LB liquid medium is: peptone 10g/L, yeast powder 5g/L, NaCl 5g/L.

Embodiment 1: Acquisition of specific HBB gene sequence

According to the human HBB gene sequence retrieved by NCBI, the retrieval sequence number is NC_000011, and specific primers were designed to obtain the full length of the HBB gene.

1. Collect peripheral blood from normal people, and extract total DNA using the Chelex-100 method (see the instructions of the blood DNA extraction kit).

2. Use Primer Premier 5 software to design specific primers as follows (SEQ ID No.1&SEQ ID No.2): HBB gene full-length clone upstream primer 5'-GGATCTTCCA GAGATTGTAC GGCTGTCATC ACTTAG-3' HBB gene full-length clone downstream primer 5'- CTGCCGTTCG ACGATTAAGG AACACTTCAGGGGAAAG-3'

3. Carry out PCR amplification according to the reaction system in Table 1.

Table 1

4. Bio-Rad C1000 PCR instrument (Bio-Rad, USA) was used for PCR reaction. The reaction system was pre-denaturation at 98°C for 3min; Extend for 2 min, and end the reaction at 4°C.

5. Use 1% agarose gel electrophoresis to detect and recover the PCR amplification product (see the instructions of the agarose gel recovery DNA fragment kit), the results are shown in Figure 1, and named as human hemoglobin β-globin gene HBB-101.

6. Sequencing of HBB-101 to test its accuracy,

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

Example 2: Construction of pSIN HBB-101 HIV lentiviral expression plasmid

1. Use SpeI and NotI to digest the HBB-101 gene fragment, and digest at 37°C for 2 hours. The enzyme digestion reaction system is as follows:

2. Recover the pSIN HIV lentiviral expression plasmid by double digestion with SpeI and NotI and make it linear. Carry out the enzyme digestion reaction at 37°C for 2 hours according to the following table. The enzyme digestion reaction system is as follows:

3. Cloning Preparation

(1) connection

The reaction system is:

(2) Conversion

① Transfer 200 μl with a cooled sterile pipette tip to a sterile centrifuge tube, add 2 μl of connection solution, mix well, and place on ice for 30 minutes.

②Heat in a constant temperature water bath at 42°C for 90 seconds, and then quickly place it on ice for 2 minutes.

③ Add the ligated transformation mixture to 500 μl of antibiotic-free LB liquid medium, incubate at 180 rpm at 37°C for 50 min to revive the bacteria.

④ Take 200 μL of the bacterial liquid and evenly smear it on the LB solid medium plate containing Amp, and culture it overnight at 37°C.

4. Identification of positive clones and DNA sequencing

(1) Screening of positive clones Pick up the white monoclonal colonies on the plate with a pipette tip, inoculate them into LB liquid medium containing Amp respectively, and culture at 37° C. at 200 rpm for 5-10 hours.

(2) Bacterial liquid PCR identification: Bacterial liquid is used as a template, HBB gene full-length cloning primers are used for 10 μl system for ordinary PCR identification, 10 μl reaction system: bacterial liquid 1 μl, upstream primer 0.5 μl, downstream primer 0.5 μl, 10×PCR Buffer ( Mg ²⁺ Plus) 1 μl, dNTP Mixture (2.5 mM each) 0.5 μl, TaKaRa LA Taq 0.1 μl, ddH ₂ O 6.4 μl.

(3) The reaction conditions of PCR reaction are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, a total of 33 cycles; and finally extension at 72°C for 10 min.

After PCR products were detected by 1% agarose gel electrophoresis, positive clones were selected.

(4) Select positive clone colonies, use a plasmid extraction kit to extract the plasmid (see the instruction manual of the plasmid extraction kit), and perform DNA sequencing. (as picture 2)

Obtain pSIN HBB-101HIV lentiviral expression plasmid.

Example 3: Construction of pSIN GFP HIV lentiviral expression plasmid

1. Obtain the GFP gene sequence retrieved according to NCBI, the retrieval sequence number is NC_011521.1, and design specific primers to obtain the full length of the GFP gene.

2. Use Primer Premier 5 software to design GFP gene-specific primers as follows (SEQ ID No.4&SEQ ID No.5):

GFP gene upstream primer: 5'-GGACTAGTTGAGTAAAGG-3'

GFP gene downstream primer: 5'-TTGCGGCCGCTTATTTGTA-3'

3. Using the plasmid pEGFP-N3 as a template, the full-length GFP gene was cloned, and the GFP gene fragment was recovered by agarose gel electrophoresis.

4. Use SpeI and NotI to digest the GFP gene fragment, and digest at 37°C for 2 hours. The enzyme digestion reaction system is as follows:

5. Recover the pSIN HIV lentiviral expression plasmid by double digestion with SpeI and NotI and make it linear. Carry out the enzyme digestion reaction at 37°C for 2 hours according to the following table. The enzyme digestion reaction system is as follows:

6. Cloning Preparation

(1) connection

The reaction system is:

(2) conversion

① Transfer 200 μl with a cooled sterile pipette tip to a sterile centrifuge tube, add 2 μl of connection solution, mix well, and place on ice for 30 minutes.

②Heat in a constant temperature water bath at 42°C for 90 seconds, and then quickly place it on ice for 2 minutes.

③Add the ligated transformation mixture to 500 μL of antibiotic-free LB liquid medium, incubate at 180 rpm and 37°C for 50 min to revive the bacteria.

④ Take 200 μL of the bacterial liquid and evenly smear it on the LB solid medium plate containing Amp, and culture it overnight at 37°C.

7. Identification of positive clones and DNA sequencing

(1) Screening of positive clones Pick up the white monoclonal colonies on the plate with a pipette tip, inoculate them into LB liquid medium containing Amp respectively, and culture at 37° C. at 200 rpm for 5-10 hours.

(2) Bacterial solution PCR identification: Bacterial solution is used as a template, GFP gene full-length cloning primers are used for 10 μl system for common PCR identification, 10 μl reaction system: bacterial solution 1 μl, upstream primer 0.5 μl, downstream primer 0.5 μl, 10×PCR Buffer ( Mg ²⁺ Plus) 1 μl, dNTP Mixture (2.5 mM each) 0.5 μl, TaKaRa LA Taq 0.1 μl, ddH ₂ O 6.4 μl.

(3) The reaction conditions of PCR reaction are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, a total of 33 cycles; and finally extension at 72°C for 10 min.

After PCR products were detected by 1% agarose gel electrophoresis, positive clones were selected.

(4) Select positive clone colonies, and use a plasmid extraction kit to extract the plasmid (see the instruction manual of the plasmid extraction kit).

Obtain the pSIN GFP HIV lentiviral expression plasmid.

Example 3: Packaging of HIV lentivirus.

HIV lentivirus is composed of three plasmids (as shown in Figure 3), including psPAX2 packaging plasmid, pMD2G packaging plasmid and pSIN HBB-101HIV lentiviral expression plasmid or pSIN GFP HIV lentiviral expression plasmid packaging. The four plasmids are endotoxin-free and highly purified extracted.

1. Preparation of virus packaging cells: Take 8ml of 293T cells (purchased from ATCC, USA) at a concentration of ¹ ×106/ml and inoculate them into a 10cm cell culture dish, add 10ml of fresh 1640 medium (purchased from Hyclone, USA) , placed in a 37°C, 5% CO ₂ cell culture incubator for 24 hours, suck out all the medium, and then add 5ml of 1640 medium for use.

2. Add the following reagents into 50ml centrifuge tubes in order and volume.

Among them, the pSIN HBB-101HIV lentiviral expression plasmid and the pSIN GFP HIV lentiviral expression plasmid were respectively mixed with the psPAX2 packaging plasmid and the pMD2G packaging plasmid in a volume ratio of 2:3:1, 4:3:1 and 8:3:1. react. Each reaction is named as ratio 1, ratio 2 and ratio 3 in turn.

3. Add 156.25 μl of CaCl ₂ solution, and shake quickly for about 10 seconds;

4. Add 2xHBS 1250μl respectively, shake quickly for 30s, and let stand for 2min 30s;

5. Take 2.5ml of fresh medium and add it to the above mixture, blow and mix well to obtain the complex;

6. Add the compound dropwise and evenly to 3 parts of 293T cells that have been replaced with fresh medium, mix well, and culture at 37°C, 5% CO ₂ cell incubator;

7. After 12-14 hours of culture, the cells were completely replaced with medium, mixed well, and cultured in a 37°C, 5% CO ₂ cell incubator;

8. After continuing to culture the cells for 36 hours, observe the cell morphology and transfection status under a fluorescent microscope, and suck out the virus supernatant along the side wall of the culture dish into a 50ml centrifuge tube;

9. Use a 0.45 μm filter syringe to filter the collected virus supernatant into the virus collection tube;

10. Fill up the virus collection tube with 293T fresh culture medium to the virus tube mouth;

11.50000rpm/min, 4°C, centrifuge for 2.5h;

12. Remove the supernatant, use 100 μl ice-cold PBS (volume) to resuspend the virus, aliquot and store at -80°C.

Embodiment 4: the determination of lentivirus titer

The three groups of lentiviral particles packaged in different proportions obtained in Example 3 were subjected to the following steps 1 to 8 at the same time.

1. Take out 7 clean EP tubes, numbered 1-7 in sequence. Add 990 μl DMEM+10% FBS medium to each EP tube. Take out 10 μl of concentrated virus and add it to No. 1 EP tube, mix well, and the virus concentration at this time is 1/10. Then take 10 μl from No. 1 EP tube and add it to No. 2 centrifuge tube, and mix well. Repeat the operation for No. 2 tubes for No. 3-7 tubes. At this point the virus is diluted to 1/10 to 1/10 ⁷ times. Add 10 μl of 1640 medium into each centrifuge tube, and at this time, there is 1 ml of virus suspension in each centrifuge tube.

2. Prepare seven six-well plates, add 10 μl of virus suspension, and set aside.

3. Take NIH3T3 cells in the logarithmic growth phase (purchased from ATCC, USA), digest them with 0.25% trypsin-EDTA for 2 min, and observe the cell state under a microscope. When the cells become shrunken and round, complete medium can be added to stop the digestion, and the cells are blown off.

4. Collect the cells into a centrifuge tube, balance, and centrifuge at 300×g for 3 minutes.

5. Discard the supernatant, add 1ml 1640 medium to resuspend the cells, take 10μl of cells for counting.

6. Dilute the cell suspension to 2×10 ⁶ /ml, add 1 ml of the cell suspension to each well of the six-well plate, and simultaneously add 2 μl of polybrene (polybrene) at a concentration of 0.5 mg/ml to each well.

After 7.48 hours, the supernatant was aspirated, the cells were collected, and the GFP% was detected by flow cytometry.

8. Calculate the dilution factor of GFP% detected by flow cytometry closest to 1, and use the following formula to calculate the virus infection titer:

Virus infection titer (PFU/ml)=[(2×10 ⁶ cells/well)×GFP%]/(virus liquid volume×dilution factor).

9. See Table 2 for the lentivirus titers packaged in different proportions of the three groups.

Table 2

Example 5: Sorting and culturing of hematopoietic stem cells (Miltenyi Immunomagnetic Bead Sorting Kit)

1. Coating the cell culture plate: a 12-well cell culture plate was coated with Fibronectin (fibronectin) at a density of 5 μg/cm ² , and left standing at 37° C. for 6 hours.

2. Resuscitate the frozen peripheral blood mononuclear cells of patients with severe β-thalassemia, thaw in a 37°C water bath, transfer the cells from the frozen tube to a 15ml centrifuge tube, add F12 medium dropwise with a Pasteur tube and make up to 8ml, shake while adding dropwise. Centrifuge at 300g for 3min.

3. Pour off the supernatant, use 1ml FACS washing solution (1×PBS containing 2% FBS), resuspend the cells, mix well by pipetting, and filter them into a new 15ml centrifuge tube with a 70μm cell mesh.

4. Cell count. Centrifuge again, 500g, 10min. Aspirate the supernatant and resuspend the cells in 300 μl per 1×10 ⁸ cells according to the number of cells counted.

5. Incubate the antibody: operate in the dark, add 50 μl of FCR blocking solution and 50 μl of CD34 microbeads per 1×10 ⁸ cells, 4°C, 30 min.

6. Add 2ml of FACS washing solution and centrifuge at 300g for 3min. Remove the supernatant, add 1ml FACS washing solution to resuspend the cells, and filter the cells with a 70um cell mesh.

7. Prepare the magnet for adsorption and the column for adsorption. Rinse the adsorption column with 3ml FACS washing solution first, add the cell suspension to the adsorption column after the washing is completed, and add 3ml FACS washing solution after the natural dripping is completed, and repeat 5 times, adding a total of 15ml FACS washing solution.

8. Remove the adsorption column and place it in a new 15ml centrifuge tube, add 3ml FACS washing solution, and use the plunger to press the liquid out to elute CD34 cells.

9. Aspirate 100 μl of cells, and add 1 μl of CD34 antibody in the dark, at 4°C for 30 minutes. The percentage of CD34+ cells was detected by flow cytometry (as shown in Figure 4).

10. Sampling and counting. After counting, centrifuge at 500g for 5min.

11. Remove the supernatant, add X-VIVO 20 medium (purchased from Lonza, Switzerland) to resuspend the cells, add the following concentrations of pre-stimulation factors in proportion, mix well and distribute to 12-well culture plates, 2ml per well, Cultivate for use.

Example 6: Verification of the effectiveness of lentiviral particles

1.3 groups of viruses infected peripheral blood hematopoietic stem cells and flow cytometry detection of lentivirus infection efficiency

(1) Observe cell morphology and growth status, count, and control the number of CD34 living cells at 1×10 ⁵ .

(2) Discard 1.5ml of old medium and add 1.5ml of fresh X-VIVO 20 medium.

(3) Add polybrene (polybrene) to the culture medium with a volume of 32 μl, mix well and let stand for 1-3 minutes.

(4) The viruses whose titers were calculated according to the three groups of lentiviruses packaged in different proportions were added to the above-mentioned cells respectively, and the amount of viruses added was as shown in Table 3, and mixed evenly.

table 3

(5) After culturing at 37°C for 24 hours, observe the cell morphology and replace with fresh X-VIVO 20 medium.

(6) After culturing at 37°C for 48 hours, the cells were collected into a centrifuge tube, centrifuged, and resuspended with fresh X-VIVO 20 medium to harvest.

(7) Use a flow cytometer to detect the percentage of cells expressing green fluorescent protein (as shown in FIG. 5 ).

2. Hematopoietic stem cell colony formation test and exogenous HBB gene expression sequencing

(1) Hematopoietic stem cells after virus infection were collected and counted.

(2) Use 200 μl IMDM+2% FBS to resuspend 3000 hematopoietic stem cells.

(3) Add 1ml of methylcellulose to the cell suspension, mix well, and add to a 2.5mm culture dish.

(4) Put the culture plate into a 37° C., 5% CO ₂ cell culture incubator for 12 to 14 days, and prohibit movement during the culture process.

(5) After 12 to 14 days, observe the colony morphology and count the number of erythroid colonies (as shown in Figure 6).

(6) Use a pointed long glass tube to absorb the erythroid colony and place it in an EP tube containing 500 μl of PBS.

(7) After the colonies were picked, 500 μl of PBS was added, mixed evenly, and centrifuged at 500 g for 5 min.

(8) Remove the supernatant and add 200 μl TRIZOL to lyse the cells.

(9) RNA was extracted and reverse-transcribed into cDNA (see instructions for RNA extraction kit and reverse transcription kit).

(10) RT-PCR detection of the expression level of HBB gene in the cDNA of the erythroid colony, wherein the specific primers involved are shown in SEQ ID No. 6-9.

① See the table below for the reaction system

② Use the PCR instrument of Bio-Rad CFX96 system, and the reaction conditions are: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 10 s, annealing at 58°C for 20 s, extension at 72°C for 20 s (plus Plate Read), a total of 40 cycles; finally Melt Curve ( Melting curve) from 65°C to 95°C, increasing by 0.5°C every 30s.

③ The relative expression of each gene was calculated by using the 2- ^ΔΔCt method according to the number of peak cycles, and data analysis was performed (as shown in Figure 7).

Example 7: Verification of the effectiveness of lentiviral particles in ratio 2

1. Observe the cell morphology and growth status, count, and control the number of CD34 living cells at 1×10 ⁵ .

2. Discard 1.5ml of old medium and add 1.5ml of fresh X-VIVO 20 medium.

3. Add polybrene (polybrene) to the medium with a volume of 32 μl, mix well and let stand for 1-3 minutes.

4. Add 470 μl of the ratio 2 lentivirus to the cells and mix well.

5. After culturing at 37°C for 24 hours, observe the cell morphology and replace with fresh X-VIVO 20 medium.

6. After culturing at 37°C for 48 hours, the cells were collected into centrifuge tubes, centrifuged, and harvested.

7. Use 200 μl IMDM+2% FBS to resuspend 3000 hematopoietic stem cells.

8. Add 1ml of methylcellulose to the cell suspension, mix well, and add to a 2.5mm culture dish.

9. Put the culture plate into a 37°C, 5% CO ₂ cell culture incubator for 12 to 14 days, and it is forbidden to move during the culture.

10. After 12 to 14 days, observe the colony morphology and count the number of erythroid colonies.

11. Use a pointed long glass tube to absorb the erythroid colony and place it in an EP tube containing 500 μl of PBS.

12. After the colonies are picked, add 500 μl PBS, mix well, and centrifuge at 500 g for 5 min.

13. Remove the supernatant and add 200 μl TRIZOL to lyse the cells.

14. RNA was extracted and reverse transcribed into cDNA.

15. The PCR product is sent for sequencing, and specific SNP doublets can be detected (as shown in Figure 8).

sequence listing

<110>Guangdong Iridium Gene Biotechnology Co., Ltd.

<120> A HBB gene kit for correcting autologous hematopoietic stem cells in patients with severe β-thalassemia

<141> 2016-10-16

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acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgcatc 300

tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac gtggatgaag 360

ttggtggtga ggccctgggc aggttggtat caaggttaca aagacaggttt 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 catttattt 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 gene upstream primer

<222> (1)...(18)

<400> 4

ggactagttg agtaaagg 18

<210> 5

<211> 19

<212>DNA

<213> Artificial sequence

<221> GFP gene downstream primer

<222> (1)...(19)

<400> 5

ttgcggccgc ttatttgta 19

<210> 6

<211>DNA

<212>

<213> Artificial sequence

<221> Internal reference gene GAPDH upstream primer

<222> (1)...(21)

<400> 6

tgacttcaac agcgacaccc a 21

<210> 7

<211>DNA

<212>

<213> Artificial sequence

<221> Internal reference gene GAPDH downstream primer

<222> (1)...(21)

<400> 7

caccctgttg ctgtagccaa a 21

<210> 8

<211>DNA

<212> 18

<213> Artificial sequence

<221> HBB gene specific upstream primer

<222> (1)...(18)

<400> 8

atgaagttgg tggtgagg 18

<210>9

<211>DNA

<212> 18

<213> Artificial sequence

<221> HBB gene-specific downstream primers

<222> (1)...(18)

<400> 9

tgaggttgtc caggtgag 18

Claims (5)

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.