CN110467680B

CN110467680B - Fusion protein, medicine and coding gene prepared from fusion protein and MSC (mesenchymal stem cell) containing gene

Info

Publication number: CN110467680B
Application number: CN201910812884.3A
Authority: CN
Inventors: 贾永前; 吴鹏强; 王甫珏; 王永生
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-08-30
Filing date: 2019-08-30
Publication date: 2020-06-23
Anticipated expiration: 2039-08-30
Also published as: CN110467680A

Abstract

The invention discloses a fusion protein, a medicine and a coding gene prepared from the fusion protein, and MSC containing the gene. The amino acid sequence of the fusion protein is shown as SEQ ID NO. 1; the nucleotide sequence of the gene is shown in SEQ ID NO. 4. The mesenchymal stem cells prepared by the invention can over-express IDO and iNOS by hundreds of thousands of times, can obviously inhibit the proportion of Th17 cells in vivo and in vitro, improve the proportion of Treg cells, obviously adjust a Th17/Treg axis and obviously reduce the incidence rate of allogeneic hematopoietic stem cell transplantation mice aGVHD and the mortality rate related to aGVHD.

Description

Fusion protein, medicine and coding gene prepared from fusion protein and MSC (mesenchymal stem cell) containing gene

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a fusion protein, a medicine and a coding gene prepared from the fusion protein, and an MSC (mesenchymal stem cell) containing the gene.

Background

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is mainly characterized by the activation of donor T lymphocytes and dendritic cells, and the remaining leukemia cells of the recipient are killed to the maximum by the immune mechanism in order to achieve the graft-versus-leukemia (GVL) effect, while the activation of the donor T cells and dendritic cells in the recipient often causes acute graft-versus-host disease (aGVHD), which is the most serious complication and often increases the death rate of allo-HSCT. The mechanism is mainly that the proportion of T helper cells 17(Th17) is increased, and the proportion of T regulatory cells (Tregs) is reduced, so that the axial proportion of Th17/Treg is disordered. The current treatment recommendation aiming at aGVHD is mainly prevention after allo-HSCT, and the medicines comprise cyclosporine A, methotrexate, antithymocyte globulin and the like; while the first-line medication for aGVHD which has already occurred is recommended to be methylprednisolone, the second-line therapy comprises mycophenolate mofetil capsules, methotrexate, tacrolimus, anti-TNF antibodies, Mesenchymal Stem Cell (MSC) infusion and the like, and the treatment effect is still poor.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a fusion protein, a medicine prepared from the fusion protein, a coding gene and MSC containing the gene, which can reduce the pathological damage of aGVHD and reduce the fatality rate of aGVHD after allogeneic hematopoietic stem cell transplantation by regulating the Th17/Treg ratio of a xenogeneic hematopoietic stem cell transplantation organism.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a fusion protein has an amino acid sequence shown as SEQ ID NO. 1.

Furthermore, the fusion protein is formed by fusing the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO.3 through a molecular cloning method.

The nucleotide sequence of the gene for coding the fusion protein is shown as SEQ ID NO. 4.

Furthermore, the nucleotide sequence of the gene is the nucleotide sequence of the same functional protein which is obtained by substituting, deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 4.

Recombinant plasmids containing the above genes.

The mesenchymal stem cell containing the gene has the following specific preparation process:

(1) connecting the gene with a vector to prepare a recombinant plasmid pWPXld-IFNG-IFNGR 1;

(2) and (3) extracting the DNA of the recombinant plasmid pWPXld-IFNG-IFNGR1, packaging the DNA with lentivirus, and infecting mesenchymal stem cells.

A medicament for treating aGVHD, which comprises the fusion protein and a pharmaceutically acceptable carrier.

The invention has the beneficial effects that:

the mesenchymal stem cells prepared by the invention can over-express IDO by hundreds of thousands of times, can obviously inhibit the proportion of Th17 cells in vivo and in vitro, improve the proportion of Treg cells, obviously adjust the Th17/Treg axis, and obviously reduce the generation rate of allogeneic hematopoietic stem cell transplanted mice aGVHD and the mortality rate related to aGVHD.

Drawings

FIG. 1 is a diagram showing the results of electrophoresis;

fig. 2 is a detection result of detecting transgenic mesenchymal stem cell membrane expression IFNG by flow cytometry, wherein fig. 2a is a standard detection result graph, fig. 2b is a detection result of mesenchymal stem cells cultured in example 3, and fig. 2c is a detection result of a control group;

FIG. 3 is a graph showing the result of IDO secretion assay.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

EXAMPLE 1 ligation of Gene of interest to vector

1. Design of primers

An upstream primer: 5'-CGGAATTCGGATCCAGGCCTAAGCT-3', respectively; (SEQ ID NO.5)

A downstream primer: 5'-CGGGATCCGAATTCGAAGTTGAGCTC-3', respectively; (SEQ ID NO.6)

2. The target gene PCR was performed by using 1. mu.L of DNA template (IFNG-IFNGR1, SEQ ID NO. 4), 2. mu.L of Primer-F (10. mu.M), 2. mu.L of Primer-R (10. mu.M), 10. mu.L of 2 × MasterMix, and ddH₂O is complemented to 50 mu L;

the reaction procedure is as follows: preheating at 94 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 1min for 25 cycles, and extension at 72 deg.C for 10 min.

3. Respectively carrying out enzyme digestion on a target gene and a vector, wherein an enzyme digestion system of the target gene comprises: 25 μ L PCRThe product, 5. mu.L Buffer EcoRI, 2.5. mu.L BamHI, finally with ddH₂O is complemented to 50 mu L;

the carrier enzyme digestion system comprises: 20 μ L of pWPXLD, 5 μ L of Buffer EcoRI, 2.5 μ L of BamHI, finally with ddH₂O make up to 50. mu.L.

4. Construction of recombinant plasmid pWPXld-IFNG-IFNGR1

Respectively using an Axygen gel recovery kit to carry out gel recovery on a target gene enzyme digestion product and a carrier enzyme digestion product, and then connecting a target gene with a carrier, wherein the reaction system comprises: 2 mu L of Buffer, 1 mu L T4 ligase, and a target gene fragment and a vector with a total volume of 17 mu L, wherein the molar ratio of the target gene fragment to the vector is 1: 10.

5. After enzyme digestion, electrophoresis verification is carried out, and the result is shown in figure 1, wherein a band 1 is plasmid DNA; band 2 is Digetedwith BamH I/Spe I; the band M is DNA Marker.

EXAMPLE 2 Lentiviral packaging of recombinant plasmid pWPXld-IFNG-IFNGR1

Firstly, extracting recombinant plasmids by using a TIANGEN large endotoxin-free plasmid extraction kit, which comprises the following specific processes:

1. firstly, putting the adsorption column CP6 into a 50mL collecting tube, then adding 2.5mL balancing liquid BL, centrifuging for 2 minutes at room temperature of 8000r/min, pouring out waste liquid in the collecting tube, and putting the adsorption column into the collecting tube again;

2. adding 100mL of overnight-cultured bacterial liquid into a centrifuge tube, centrifuging at room temperature of 8000r/min for 3 minutes to collect bacteria, removing supernatant as much as possible, and removing water drops on the bottle wall by using clean absorbent paper;

3. adding 8mL of RNase A-containing solution P1 into the centrifuge tube with the bacterial precipitates, and vortexing to thoroughly suspend the bacterial precipitates;

4. adding 8mL of solution P2 into a centrifuge tube, immediately turning over for 6-8 times, fully cracking the thallus, and standing for 5 minutes at room temperature;

5. adding 8mL of solution P4 into a centrifuge tube, immediately turning over for 6-8 times, and fully mixing until white dispersed flocculent precipitate appears in the solution; standing at room temperature for 10 minutes, centrifuging at room temperature of 8000r/min for 5-10 minutes to separate white precipitate to the bottom of the tube, pouring all the solution into a filter CS1, and collecting the filtrate into another clean 50mL centrifuge tube;

6. adding isopropanol with the volume of 0.3 time into the filtrate, turning upside down, mixing uniformly, and transferring into an adsorption column CP 6;

7. centrifuging at room temperature of 8000r/min for 2 min, removing waste liquid in the collecting tube, and replacing adsorption column CP6 in the collecting tube;

8. adding 10mL of rinsing liquid PW containing absolute ethyl alcohol into the adsorption column CP6, centrifuging at room temperature of 8000r/min for 2 min, discarding the waste liquid in the collection tube, replacing the adsorption column into the collection tube, and repeating the steps once;

9. adding 3mL of absolute ethyl alcohol into the adsorption column CP6, centrifuging for 2 minutes at room temperature of 8000r/min, and pouring off waste liquid;

10. putting the adsorption column CP6 back into the collection tube again, centrifuging for 5 minutes at room temperature of 8000r/min, and removing residual rinsing liquid in the adsorption column;

11. placing the adsorption column CP6 in a clean 50mL collecting tube, hanging and dripping 1-2mL elution buffer TB into the middle part of the adsorption membrane, placing for 5 minutes at room temperature, and centrifuging for 2 minutes at room temperature 8000 r/min;

12. the eluate from the 50mL centrifuge tube was concentrated to 20. mu.L, and the remainder was transferred to a clean 1.5mL centrifuge tube and stored at-20 ℃.

II, preparation of virus

1. Placing DMEM medium containing 10% fetal calf serum and 100U/L streptomycin double antibody and pancreatin at 37 ℃ for preheating;

2. 2 hours, 25cm before transfection²Replacing the solution by using the preheating culture medium prepared in the step 1 when the fusion degree of the 293T cells in the culture dish reaches 70%;

3. transfection

(1) Adding 25cm into a 50mL centrifuge tube²Petri dish transfection System (including 20. mu.g of psPAX2, 9. mu.g of pMD2.0G, 21. mu.g of IFNGR1, 125. mu.L of 20 × CaCl₂1000. mu.L of 2 × BBS, finally ddH₂Supplementing O to 2.5mL), and standing for 15 minutes at room temperature;

(2) the transfection system was slowly instilled into 25cm containing 293T cells²In a culture dish, gently shaking and mixing, and adding 5% CO₂Culturing in an incubator at 37 ℃ for 12-16 hours, and changing the culture solution once;

4. and (3) virus collection:

(1) virus supernatants were collected 48 hours after transfection and replaced with DMEM medium containing 10% FBS, 100U/L double antibody preheated at 37 deg.C, and cells were plated in 5% CO₂Continuously culturing in an incubator at 37 ℃; filtering the virus supernatant with 0.22 μm filter, and storing at 4 deg.C;

(2) the viral supernatant was collected again 72 hours after transfection and filtered through a 0.22 μm filter and stored at 4 ℃.

5. Concentration of virus

Centrifuging the virus by an ultracentrifugation method, which comprises the following specific steps:

(1) an ultracentrifuge: beckmann coulter Optima Max; a rotary head and a rotor of SW32Ti, and an ultracentrifuge tube;

(2) adding the filtered virus supernatant into a centrifugal tube in a biological safety cabinet, strictly balancing by using a balance with the precision of 0.001g, wherein the mass difference between 6 rotating heads of the added virus supernatant is not more than 0.001 g;

(3) centrifuging: centrifuging at 20 deg.C and 70000g for 2 hr;

(4) after centrifugation, discarding the supernatant, and inversely placing the centrifuge tube on sterilized absorbent paper to suck dry the residual culture medium;

(5) resuspending the virus pellet in sterile PBS, sucking a little for virus titer detection, and subpackaging the rest into small EP tubes to be stored at-80 ℃ for later use.

6. Lentivirus titer detection (using the TIANGEN genomic DNA extraction kit, and the instructions for the steps specifically as follows):

(1) 293T cells according to 1 × 10⁵Well-inoculated in 6-well plate, 10% FBS, 100U/L double-antibody DMEM 2 mL/well in 5% CO₂Culturing in a cell culture box at 37 ℃ for 24 hours;

(2) adding 10 mu L of virus concentrated solution into DMEM culture medium containing 10% FBS and 100U/L double antibody, mixing uniformly, replacing the culture medium in a 6-well plate, establishing a blank control at the same time, and putting the blank control back to the incubator for continuous culture;

(3) after 24 hours, the virus-containing supernatant in the 6-well plate was aspirated, replaced with 2mL of fresh DMEM medium containing 10% FBS and 100U/L double antibody, and returned to 5% CO₂Continuing culturing in an incubator at 37 ℃;

(4) collecting 293T cells from 72 hours after infection, centrifuging the cell suspension at 10000r/min for 1 minute, removing supernatant, adding 200 mu L of buffer solution GA, and fully shaking;

(5) adding 20 mu L of ProteinaseK solution and mixing evenly;

(6) adding 200 μ L buffer solution GB, fully reversing and mixing until white precipitate is generated, placing in 70 deg.C water bath for 10min until the solution becomes clear, and centrifuging briefly to remove water drop on the inner wall of the tube cover;

(7) adding 200 μ L of anhydrous ethanol, shaking and mixing for 15 s to generate flocculent precipitate, and centrifuging briefly to remove water drops on the inner wall of the tube cover;

(8) putting the adsorption column C3 into a collecting pipe, adding the solution and the precipitate obtained in the previous step into the adsorption column C3, centrifuging at 12000r/min for 30 seconds, discarding the waste liquid, and putting the adsorption column back into the collecting pipe;

(9) adding absolute ethyl alcohol into 500 mu L of buffer GD according to the requirements of the specification, sucking 500 mu L of buffer GD, adding the buffer GD into an adsorption column CB3, centrifuging for 30 seconds at 12000 rpm, and placing an adsorption column C3 back into a collection tube after discarding waste liquid;

(10) adding the rinsing liquid into absolute ethyl alcohol according to the concentration of a specification, sucking 600 mu L of the rinsing liquid, adding the rinsing liquid into an adsorption column CB3, centrifuging at 12000r/min for 30 seconds, discarding waste liquid, and adding an adsorption column CB3 into a collecting pipe;

(11) repeating the step (10);

(12) putting the adsorption column C3 into a collecting pipe, centrifuging at 12000r/min for 2 minutes, discarding the waste liquid, standing at room temperature for 10 minutes, and completely airing the residual rinsing liquid;

(13) placing the adsorption column C3 into another new centrifuge tube, slowly suspending and dripping 50-200 μ L of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5 min, centrifuging at 12000r/min for 2 min, and collecting the solution into the centrifuge tube;

(14) virus titer was determined by RT-PCR using a defined concentration of plasmid as standard, in 12.5. mu.L of 2 × SYBR Premi Ex Taq II (Tli RNaseH)Plus), 1.0. mu.L of PCR Forward Primer (10. mu.M), 1.0. mu.L of PCR Reverse Primer (10. mu.M), 2. mu.L of reaction solution of LRT (cDNA solution), and finally ddH₂Make up to 25. mu.L of O.

(15) And after the reaction is finished, calculating the concentration of the target DNA in the gene through a linear relation constructed by a standard curve, thereby calculating the virus titer.

Example 3 infection of human mesenchymal Stem cells

1. The mesenchymal stem cells are processed into the product with the weight of 1.0 × 10⁵Well-plated with 6-well plate, 10% FBS, 100U/L double-antibody DMEM 2 mL/well in 5% CO₂Culturing in a 37 ℃ cell culture box;

2. when the inoculated mesenchymal stem cells are uniformly attached to the wall and the fusion degree reaches 30%, calculating the required virus volume according to the measured virus titer according to the Moi value 50;

3. adding the required virus volume into 2mL of DMEM medium containing 10% FBS and 100U/L double antibody;

4. absorbing original culture medium of mesenchymal stem cells in a 6-well plate, and adding 2mL of culture medium containing viruses into each well; at 5% CO₂Culturing at 37 ℃ in a cell culture box, wherein the cell state needs to be observed during the culture;

5. culturing for 12-16 h, and changing the culture medium;

6. after culturing for 72 hours, the cells were harvested, and the expression of membranes Ifn-gamma and Ifn-gamma R1 were detected by flow, and IDO expression was detected by RT-PCR, the results of which are shown in FIGS. 2 and 3, respectively.

Fig. 2 is a detection result of detecting transgenic mesenchymal stem cell membrane expression IFNG by flow cytometry, wherein fig. 2a is a standard detection result graph, fig. 2b is a detection result of mesenchymal stem cells cultured in example 3, and fig. 2c is a detection result of a control group.

Fig. 3 shows IDO secretion detection, wherein CON is the detection result of the control group, IFNR1-60 is the detection result of the cells cultured in example 3 of the present application, and it can be seen from fig. 3 that the IDO secretion value of the mesenchymal stem cells cultured in the present application is increased by hundreds of thousands of times compared with that of the common mesenchymal stem cells of the control group.

Sequence listing

<110> Sichuan university

<120> fusion protein, drug and coding gene prepared from same, and MSC containing gene

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Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser

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Gln Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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Ile Gln Tyr Lys Ile Leu Thr Gln Lys Glu Asp Asp Cys Asp Glu Ile

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Glu Lys Gln Ile Met Ile Asp Ile Phe His Pro Ser Val Phe Val Asn

100 105 110

Gly Asp Glu Gln Glu Val Asp Tyr Asp Pro Glu Thr Thr Cys Tyr Ile

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cggaaccctg ttcctcggaa tactgaagaa ctggaaggaa gagagcgaca gaaagataat 240

gcaaagccag atcgtcagct tctactttaa actgttcaag aacttcaagg acgaccaaag 300

catccaaaag tccgtggaga ccatcaaaga ggacatgaat gtgaagttct tcaacagcaa 360

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gcagagaaag gccattcacg agctgataca ggtgatggcc gagctgagcc cagccgcaaa 480

gacaggaaag agaaaaagaa gccagatgct gttcagaggc agaagagcca gccaaagtgg 540

cggcggaagt ggcggcggcg gatctggagg aggcggaagt ggaggaggag gaagcggagg 600

gggaagcctg caggagatgg gaacagcaga cctgggacca agcagcgtgc caaccccaac 660

aaacgtgaca attgagagct acaacatgaa ccccatcgtg tactgggagt atcagatcat 720

gccacaagtc cccgtgttca ccgtggaggt caagaactac ggagtgaaga acagcgaatg 780

gatagacgcc tgtatcaaca tctcccacca ctactgcaac atcagcgacc acgtcggcga 840

cccctccaat tccctgtggg tgagagtgaa agccagggtc ggacagaaag aaagcgccta 900

cgctaaaagc gaggaatttg ccgtctgcag ggacggaaag ataggaccac ccaagctgga 960

catcagaaag gaggagaagc agatcatgat tgacattttt cacccaagcg tgttcgtgaa 1020

tggagacgaa caggaggtgg actacgaccc cgagacaacc tgctacataa gagtttacaa 1080

cgtgtacgtg aggatgaacg gcagcgagat acagtacaag atcctgaccc agaaggagga 1140

tgactgcgat gagatccagt gccagctggc aatacccgtg agcagcctga atagccaata 1200

ttgcgtgagc gccgagggag tgctgcacgt gtggggcgtg actacagaaa agagcaaaga 1260

ggtgtgcata accatattca actcctctat caaagggagc ctgtggattc ctgtcgtggc 1320

cgccctgctg ctgtttctgg tgctgtctct ggtgtttatc tgcttctaca tcaagaagat 1380

caaccccctc aaggagaagt ctatcatcct gcctaagtcc ctgatttccg tggttcgttc 1440

cgccaccctc gaaaccaaac ccgagtccaa gtacgtgtcc ctgatcacct cctaccagcc 1500

cttctccctg gaaaaggagg tcgtgtgcga agaacccctg agccccgcca ccgtccccgg 1560

aatgcacacc gaagacaacc ccggcaaggt ggaacacacc gaggaactga gcagcatcac 1620

cgaggtggtg accaccgagg aaaacatacc cgatgtggtg cccggaagcc atctcacccc 1680

catcgaaaga gaaagcagca gccccctgag cagcaaccaa agcgaacccg gaagcatcgc 1740

cctgaacagc taccacagca gaaactgcag cgagagtgat cattccagaa acggcttcga 1800

caccgacagc agctgcctgg aatcccacag cagcctcagc gacagcgagt tcccccccaa 1860

caacaaaggc gaaatcaaaa ccgagggaca ggaactgatc accgtgatca aagccccaac 1920

cagcttcggc tacgacaaac cccacgtcct ggtcgatctg ctggtcgatg acagcggcaa 1980

agagagcctg atcggctaca gaccaaccga ggacagcaag gaatttagcg actacaaaga 2040

cgacgatgac aaggactaca aagatgacga tgacaaatga tccggactca gatctcgata 2100

gctagtagct agctagctag tcgagctcaa cttcgaatt 2139

<210>5

<211>25

<212>DNA

<213> Artificial sequence (Ichthyomyzon unicuspis)

<400>5

cggaattcgg atccaggcct aagct 25

<210>6

<211>26

<212>DNA

<213> Artificial sequence (Ichthyomyzon unicuspis)

<400>6

cgggatccga attcgaagtt gagctc 26

Claims

1. A fusion protein is characterized in that the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.

2. A gene encoding the fusion protein of claim 1, wherein the nucleotide sequence of the gene is represented by seq id No. 4.

3. A recombinant plasmid containing the gene of claim 2.

4. A mesenchymal stem cell containing the gene of claim 2.

5. A medicament for the treatment of aGVHD comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier.