WO2017214832A1

WO2017214832A1 - Lentiviral vector for specifically promoting high expression of foxp3 gene, and applications thereof

Info

Publication number: WO2017214832A1
Application number: PCT/CN2016/085637
Authority: WO
Inventors: 石庆学
Original assignee: 石庆学
Priority date: 2016-06-14
Filing date: 2016-06-14
Publication date: 2017-12-21

Abstract

Provided is a lentiviral expression vector for specifically promoting high expression of an Foxp3 gene, comprising a fundamental sequence, a resistance gene sequence, a multiple-cloning-sites sequence, a promoter sequence, and an Foxp3 gene cDNA sequence of a pLVX-IRES-puro expression vector. The multiple cloning sites comprise an EcoR I enzyme cutting site and an Spe I enzyme cutting site, the Foxp3 gene cDNA sequence comprises an EcoR I enzyme cutting site, an Foxp3 gene coding sequence and an Spe I enzyme cutting site, and the Foxp3 gene cDNA sequence is forwardly inserted into the multiple-cloning-sites sequence. The lentiviral expression vector can be used in the preparation of drugs related to Foxp3.

Description

Lentiviral vector specifically promoting the high expression of Foxp3 gene and its application

[0001] The present invention belongs to the field of genetic engineering technology, and particularly relates to a lentivirus expression which specifically promotes high expression of Foxp3 gene, and a construction method and application thereof.

Background technique

[0002] Foxp3 is a member of the transcription factor fork ead/winged-helix (fork-like/winged helix) family, FOX

(Fork eadbox) is a generic term for the vertebrate fork ead transcription factor. Different subclasses are represented by the following English letters and the last digits. Foxp3 comprises at least three distinct domains: the N-terminal C2H2 zinc finger structure, the leucine zipper motif, and the C-terminal fork ead DNA binding region (FKH). The FKH domain is a common feature of FOX family proteins and plays a key role in nuclear localization and DNA binding.

technical problem

[0003] Studies have shown that Foxp3 and human immune disorders - multiple endocrine adenopathy - enteropathy -X

It is related to a variety of autoimmune diseases such as chain syndrome (IPEX), multiple sclerosis (MS), and diabetes. Therefore, the study of Foxp3 can promote the progress of prevention and treatment of these autoimmune diseases, but there is a lack of specificity in the prior art. Lentiviral expression vectors that promote high expression of the Foxp3 gene greatly limit the conduct of the study.

Problem solution

Technical solution

[0004] In order to solve the problems existing in the prior art, the inventors conducted extensive research on the selection of vectors, the method of recombinant construction, and the like, and found that the EcoR I cleavage site and Spe are included.

The Foxp3 gene cDNA sequence of the I cleavage site was inserted into the multiple cloning site of the pLVX-IRES-Puro expression vector to construct a lentiviral expression vector which specifically promotes the high expression of the Foxp3 gene, thereby completing the present invention.

The present invention provides a lentiviral expression vector which specifically promotes high expression of the Foxp3 gene, including the basic sequence of the pLVX-IRE S-puro expression vector, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence, and Fo xp3 a gene cDNA sequence; the multiple cloning site comprises an EcoR I cleavage site and a Spe I cleavage site, and the Foxp3 gene cDNA sequence comprises an EcoR I cleavage site, a Foxp3 gene coding sequence, and a Spe I cleavage site At the point, the Foxp3 gene cDNA sequence is positively inserted into the multiple cloning site sequence.

[0006] According to the above technical solution, the lentiviral expression vector constructed by inserting the Foxp3 gene cDNA sequence into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase Foxp3. The advantages of gene expression can be used as a powerful tool in the preparation and treatment of drugs for the treatment of Foxp3 gene expression in diseases such as Alzheimer's disease.

[0007] As a further improvement of the present invention, the Foxp3 gene coding sequence is obtained by PCR amplification, and the PCR primer comprises an upstream primer and a downstream primer, and the sequence of the upstream primer is: 5'-GCGAATTCATGCCCAACCCCAGGCCTGG-3, ie, SEQ ID NO: 1, the sequence of the downstream primer is: 5'- GACTAGTTCAGGGGCCAGGTGTAGGGTTG -3', gPSEQ ID NO: 2. Using the above PCR primer sequence, the Foxp3 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the Foxp3 gene, which reduces the cost of sequence synthesis and lowers the cost.

Correspondingly, the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the Foxp3 gene, and comprises the following steps:

[0009] A) Foxp3 gene primer design: According to the Foxp3 gene coding sequence, using 01igo 7 analysis, select 5, -GCGAATTCATGCCCAACCCCAGGCCTGG-3, ie SEQ ID NO: 1 as the upstream primer, select 5,- GACTAGTTCAGGGGCCAGGTGTAGGGTTG -3', ie SEQ ID

NO: 2 is used as a downstream primer, and then the upstream primer and the downstream primer are synthesized; the upstream primer and the downstream primer have no primer dimer, and the annealing temperature difference is small;

[0010] B) obtaining the Foxp3 gene cDNA sequence: PCR amplification using the upstream primer and the downstream primer to obtain a large number of Foxp3 gene coding sequences, and then adding the A tail reaction to the sequence, using T4 DNA ligase The ligation product was obtained by ligating to the pGM-T vector, and the ligation product was transformed into competent E. coli DH50C, uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid was picked and subjected to PCR. Initial identification, the preliminary identification results indicated that the Foxp3 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification; the correct E. coli was identified by liquid LB medium culture sequencing, and the pGM-T vector containing the Foxp3 gene cDNA sequence was extracted and used. The restriction endonuclease EcoR I enzyme and Spe l enzyme are digested, and the fragment of about 1000 bp is recovered by electrophoresis and gel-cutting, and the fragment is the Foxp3 gene cDNA sequence; C)

Construction and identification of a lentiviral vector that specifically promotes high expression of Foxp3 gene: The plasmid pLVX-IRES-P uro was extracted, digested with restriction endonuclease EcoR I enzyme and Spe l enzyme, electrophoresis, gel-removed recovery vector, and then used. T4 DNA ligase ligated the Foxp3 gene cDNA sequence into the pLVX-IRES-Puro expression vector to obtain a ligation product, which was transformed into competent E. coli DH50C and uniformly coated onto an ampicillin-containing LB medium plate. The positive monoclonal colonies were picked and cultured to preserve the bacterial liquid, and the preliminary identification of PCR was carried out. The preliminary identification results indicated that the Foxp3 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification;

[0012] D)

Extraction of lentiviral vectors that specifically promote the high expression of Foxp3 gene: The sequencing results confirmed that the Foxp3 gene cD NA sequence was inserted into the successful bacterial cell expansion culture, and the recombinant plasmid was extracted to obtain the lentivirus expression which specifically promoted the high expression of Foxp3 gene. Carrier.

[0013] The present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of Foxp3 gene. After successful identification, it is packaged into a virus and introduced into Jurkat cells. After puromycin is used to screen cells, real-time quantitative PCR is used. Western Blot technology verified the change of Foxp3 gene expression from mRNA and protein levels respectively. The experimental results confirmed that the Foxp3 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-I RES-Puro expression vector, which can promote specifically, continuously, efficiently and stably. The Foxp3 gene is highly expressed.

The present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of the Foxp3 gene for the preparation of a medicament for treating a disease associated with abnormal expression of Foxp3 gene.

Advantageous effects of the invention

Beneficial effect

The lentiviral expression vector which specifically promotes the high expression of Foxp3 gene provided by the invention has the advantages of high transfection efficiency, low dosage, specific, sustained, high efficiency and stable promotion of high expression of Foxp3 gene, and can be used as a powerful tool. In drug research and development related to Foxp3; the present invention also provides specific promotion of Foxp

The method for constructing a lentiviral expression vector with high expression of 3 gene has good operation effect and reduced sequence synthesis cost

, the cost is lower.

Brief description of the drawing

DRAWINGS

1 is a plasmid map of the pLVX-IRES-Puro expression vector. 2 is a schematic diagram showing the results of fluorescent quantitative PCR detection after puromycin screening of cells.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0019] Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences. 293FT cells were purchased from Thermo Fisher, Premix PrimeSTAR HS enzyme, lentiviral expression vector pLVX-IRES-Puro, virus packaging auxiliary kit, and Lenti-X GoStix kit. Purchased from Takara, RNeasy Mini

Kit was purchased from QIAGEN, pGM-T was purchased from Tiangen, and Endo-Free Plasmid Mini Kit II was purchased from Omega bio-tek.

[0020] Example 1 Design of Foxp3 gene primers.

[0021] According to the Foxp3 gene coding sequence (GenBank NM_001114377.1), it was analyzed using 01igo7 to find upstream primers and downstream primers (requires as little primer-free dimer as possible and the annealing temperature difference is small), then upstream primers and The protective base and the restriction sites EcoR I and EcoR I were added to the 5' end of the downstream primer, respectively, and the designed primer sequences are shown in Table 1. The designed PCR primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

[0022] Table 1 PCRp3 gene PCR bow |

[] [Table 1]

[0023] Example 2 Construction of a lentiviral vector that specifically promotes high expression of Foxp3 gene

[0024] After diluting the synthetic antibody, the coding sequence of Foxp3 gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A tail reaction, and then ligated to pGM-T with T4 DNA ligase. The ligation product (Foxp3-T vector) was obtained on the vector, and the ligation product was transformed into competent E. coli DH 5α, uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h. Negative control group 1 (consistent cells were uniformly coated on ampicillin-free plates), negative control group 2 (peripheral cells were uniformly coated in 100)

g/ml ampicillin on the plate), positive control group 1 (the connection product of the double enzyme-cut empty vector was evenly coated in 100

g/ml ampicillin on the plate), positive control group 2 (the empty carrier was evenly spread on a plate containing 100 g/mL ampicillin). The experimental group grew a single colony, the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, the positive control group 2 did not grow colonies.

[0025] After picking up 8 single colonies from the experimental group, 0.5 culture solutions were taken and PCR-amplified with primers of Foxp3 gene for preliminary identification. The results showed that the cultures of 8 single colonies could be successfully amplified. The Foxp3 gene was exported, and the recombinant vector was sent to Shanghai Biotech Co., Ltd. for sequencing.

[0026] The bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the Foxp3 gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly used with EcoR I enzyme and Spe I, respectively. The enzyme was double-digested, electrophoresed and recovered, and the product was recovered by T4 DNA ligase, and then transformed into competent E. coli DH50C, uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h. The negative control group 1 was set up (the competent cells were uniformly coated on the ampicillin-free plate), and the negative control group 2 (the competent cells were uniformly coated on the plate containing 100 g/ml ampicillin), Positive control group 1 (the ligation product of the double enzyme-cut empty vector was uniformly coated on a plate containing 100 g/ml ampicillin), positive control group 2 (the empty vector was uniformly coated on 100 g/mL ampicillin) On the tablet). The experimental group grew a single colony, and the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, and the positive control group 2 did not grow colonies.

[0027] After picking up 6 single colonies from the experimental group, 0.5 culture solutions were taken and PCR-amplified with the primer of Foxp3 gene for preliminary identification. The results showed that all 6 cultures could successfully amplify the Foxp3 gene, and then the recombinant vector was sent to Shanghai Biotech Co., Ltd. for sequencing. The sequencing results were exactly as expected, and the pLVX-Foxp3 plasmid was obtained.

[0028] The previously collected recombinant plasmid bacterial solution was taken out, inoculated into 15 ml of LB medium (containing 100 g/ml ampicillin), cultured at 37 ° C, 300 rpm for 16 h, using Endo-Free Plasmid Mini Kit II The recombinant plasmid pLVX-Foxp3 was extracted and its purity and concentration were measured. The results are shown in Table 2.

Table 2 Purification and Concentration of Recombinant Plasmid [] [Table 2]

[0031] 293FT cells were cultured, and cells grown in good condition were inoculated into six wells, 1,000,000 cells per well, and the extracted recombinant plasmid pLVX-Foxp3 2μ _§ was transfected into 293FT cells using a lentiviral packaging auxiliary kit. After 48 h, the virus-containing supernatant medium was collected, and the virus solution was filtered through a sieve of 0.45 μm for infecting Jurka t cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 50,000,000 IFU.

Example 4 Lentiviral transduction Jurkat cells

[0033] Jurkat cells were inoculated into 6-well plates at 1000000 cells per well. After 12 hours, the cell density was about 50<3⁄4, and the virus solution was taken separately. The virus was diluted 10 times with DMEM complete medium, and polybrene was added. ) The final concentration is 8 g/mL. The medium in the 6-well plate was added to the virus-containing DMEM complete medium (containing 10% fetal bovine serum). After 24 hours, the virus-containing DMEM complete medium was discarded, and the fresh DMEM complete medium was replaced. After 24 hours, 0.5 was used. The cells were screened at a g/ml concentration of puromycin. After 10 days of screening, the medium was changed once every 3 days, and the concentration of puromycin was continuously increased to 1.00 g/ml.

Example 5 Fluorescence quantitative PCR was used to detect the expression level of Foxp3 gene.

[0035] According to the GAPDH and Foxp3 gene mRNA sequences, the primer design software Oligo 7.0 was used to design the bow.

[] [table 3]

Jurkat cells, pLVX empty vector control Jurkat cell group, and _P LVX-Foxp3 high expressing cells were inoculated into 6-well plates, respectively. The cell density reached 80<3⁄4-90<3⁄4吋, and the total RNA of each group was extracted with RNeasy Mini Kit, and PrimeScrip RT reagent was used for He ¹ J. Kit reverse transcribes mRNA into cDNA, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ∞. After the end of reverse transcription, add 9 (L of RNase Free dH ₂ 0 diluted cDNA, and store at -20 ° C for later detection. Take the cDNA of each group of cells.

Ιμΐ was used as a template, and GAPDH was used as an internal reference. Real-time quantitative PCR (QPCR) was used to detect the relative expression of Foxp3, and the reaction conditions were set: 95. C 30s, 1 cycle, 54°C 30s 40 cycles, 95. C 5s, 60. C

Lmin, 95 ° C for 15 s, the relative expression of Foxp3 gene in each group was detected by SYBR Primescript RT-PCR Kit. After the pLVX-Foxp3 cells were continuously cultured for 20 passages, the above experiment was repeated. The summarized results are shown in Figure 2. It can be seen that the expression of Foxp3 gene is more than 105-fold higher than that of Jurkat cells, whether it is just after screening or after 20 generations of pLVX-Foxp3 cells, and the expression of Foxp3 gene in pLVX empty vector cells. There is almost no change compared with Jurkat cells, indicating that the Foxp3 gene cDNA sequence provided by the present invention is successfully inserted into the pLVX-IRES-Puro expression vector, and can promote the high expression of Foxp3 gene specifically, continuously, efficiently and stably.

[0037] The above is a further detailed description of the present invention in conjunction with the specific preferred embodiments. It is not intended that the specific embodiments of the invention are limited to the description. It will be apparent to those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the invention.

Industrial applicability

The lentiviral expression vector which specifically promotes the high expression of Foxp3 gene provided by the invention has the advantages of high transfection efficiency, low dosage, specific, sustained, high efficiency and stable promotion of high expression of Foxp3 gene, and can be used as a powerful tool. In the pharmaceutical research and development related to Foxp3; the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of Foxp 3 gene, which has good operation effect, reduces sequence synthesis cost, and has low cost.

Claims

claims

[Claim 1] A lentiviral expression vector that specifically promotes high expression of the Foxp3 gene, characterized by: including the basic sequence of the pLVX-IRES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and Foxp3 gene cDNA sequence; the multiple cloning site includes EcoR I restriction site and Spe I restriction site, and the Foxp3 gene cDNA sequence includes EcoR I restriction site, Foxp3 gene coding sequence and Spe

I enzyme cutting site, the Foxp3 gene cDNA sequence is inserted into the multiple cloning site sequence in the forward direction.

[Claim 2] The lentiviral expression vector specifically promoting the high expression of Foxp3 gene according to claim 1, characterized in that: the Foxp3 gene coding sequence is obtained by PCR amplification, and the PCR primers include upstream primers and downstream primers, so The sequence of the upstream primer is: 5'- GCGAATTCATGCCCAACCCCAGGCCTGG -3, that is, SEQ ID NO: 1, and the sequence of the downstream primer is: 5'-

GACTAGTTCAGGGGCCAGGTGTAGGGTTG -3' , i.e. SEQ ID NO: 2

[Claim 3] The construction method of a lentiviral expression vector that specifically promotes the high expression of Foxp3 gene according to claim 2, characterized by: comprising the following steps:

A) Foxp3 gene primer design: According to the Foxp3 gene coding sequence, use 01igo 7 to analyze and select 5, - GCGAATTCATGCCCAACCCCAGGCCTGG -3', that is, SEQ ID NO: 1 as the upstream primer, select 5'-

GACTAGTTCAAGGGGCCAGGTGTAGGGTTG -3', i.e. SEQ ID NO: 2 as the downstream primer, and then the upstream primer and the downstream primer are synthesized;

B) Obtaining the Foxp3 gene cDNA sequence: Use the upstream primer and the downstream primer to perform PCR amplification to obtain a large number of Foxp3 gene coding sequences, then perform an A-tailing reaction on the sequence, and then connect it to pGM using T4 DNA ligase Obtain the ligation product on the -T vector, transform the ligation product into competent E. coli DH50C, spread it evenly on an LB medium plate containing ampicillin, pick out positive single clone colonies, culture and preserve the bacterial liquid, and conduct preliminary PCR identification. The preliminary identification results indicate that the Foxp3 gene cDNA sequence was successfully inserted into the bacteria. liquid for sequencing identification; use liquid LB medium to culture the correct E. coli for sequencing identification, extract the pGM-T vector containing the Foxp3 gene cDNA sequence, and double-digest it with the restriction endonuclease E coR I enzyme and Spel enzyme. , perform electrophoresis and gel cutting to recover a fragment of about 1000 bp, which is the Foxp3 gene cDNA sequence;

C) Construction and identification of lentiviral vectors that specifically promote the high expression of Foxp3 gene: Extract plasmid pLVX-IRES-Puro, double-digest with restriction endonucleases EcoR I and Spe I, electrophoresis, and gel cutting to recover the vector, and then Use T4 DNA ligase to connect the Foxp3 gene cDNA sequence into the pLVX-IRES-Puro expression vector to obtain a ligation product, transform the ligation product into competent E. coli DH50C, and spread it evenly onto an LB medium plate containing ampicillin On the top, select positive single clone colonies, culture and preserve the bacterial liquid and perform preliminary PCR identification. The preliminary identification results indicate that the Foxp3 gene cDNA sequence is successfully inserted into the bacterial liquid for sequencing identification;

D) Extraction of lentiviral vector that specifically promotes the high expression of the Foxp3 gene: The sequencing results confirm that the Foxp3 gene cDNA sequence has been successfully inserted into the bacterial liquid amplification culture, and the recombinant plasmid is extracted to obtain the lentivirus that specifically promotes the high expression of the Foxp3 gene. Expression vector.

[Claim 4] Use of the lentiviral expression vector specifically promoting the high expression of Foxp3 gene according to any one of claims 1 to 3 in the preparation of drugs for the treatment of diseases related to abnormal Foxp3 gene expression