WO2017214829A1

WO2017214829A1 - Lentiviral expression vector for specifically promoting high expression of pd-l1 gene, and applications thereof

Info

Publication number: WO2017214829A1
Application number: PCT/CN2016/085634
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

A lentiviral expression vector for specifically promoting high expression of a PD-L1 gene comprises a fundamental sequence, a resistance gene sequence, a multiple-cloning-sites sequence, a promoter sequence, and a PD-L1 gene cDNA sequence of a pLVX-IRES-puro expression vector. The multiple cloning sites comprise an EcoR I enzyme cutting site and an SpeI enzyme cutting site, the PD-L1 gene cDNA sequence comprises an EcoR I enzyme cutting site, a PD-L1 gene coding sequence and an SpeI enzyme cutting site, and the PD-L1 gene cDNA sequence is forwardly inserted into the multiple-cloning-sites sequence. The lentiviral expression vector has the advantages of high transfection efficiency and small usage and being capable of specifically, continuously, efficiently and stably expressing the PD-L1 gene, and can serve as a powerful tool applied to research and development of drugs related to PD-L1.

Description

Lentiviral expression vector for specifically promoting high expression of PD-L1 gene and application thereof

[0001] The present invention belongs to the field of genetic engineering technology, and particularly relates to a lentiviral expression vector which specifically promotes high expression of PD-L1 gene, and a construction method and application thereof.

Background technique

[0002] Non-small cell lung cancer (NSCLC) is a common malignant tumor in the world, and its incidence is increasing year by year. Studying NSCLC tumor recurrence factors and tumor immune escape mechanisms has important theoretical significance and clinical application value. T lymphocytes are important effector cells that mediate tumor immune responses, and T cell activation requires TCR-mediated antigen-specific signaling and costimulatory molecule-mediated costimulatory signals.

technical problem

[0003] PD-L1/B7-H1 is a negative T cell costimulatory molecule in the B7 family, PD-L1 passes its receptor PD

-1 binding, inhibits the proliferation and activation of CD4 and CD8 T cells, negatively regulates the immune response process of the body, thereby mediating tumor immune escape and promoting tumor growth. Therefore, the study on the role of PD-L1 in tumor escape has An important role, but the lack of lentiviral expression vectors that specifically promote the high expression of the PD-L1 gene in the prior art hinders the development of related research.

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 PD-L 1 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 PD-L1 gene, thereby completing the present invention.

The present invention provides a lentiviral expression vector which specifically promotes high expression of the PD-L1 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 P D-L1 gene cDNA sequence; the multiple cloning site includes an EcoR I cleavage site and a Spe I cleavage site, and the PD-L1 gene cDNA sequence includes an EcoR I cleavage site and a PD-L1 gene coding Sequence and Spe I cleavage At the point, the cDNA sequence of the PD-L1 gene is positively inserted into the multiple cloning site sequence.

[0006] According to the above technical solution, the lentiviral expression vector constructed by inserting the cDNA sequence of the PD-L1 gene into the pLVX-IRES-Puro expression vector has the advantages of high transfection efficiency, low dosage, sustainable, high efficiency and stability. The advantages of improving PD-L1 gene expression can be used as a powerful tool for the preparation and treatment of PD-L1 gene expression for diseases such as Alzheimer's disease.

[0007] As a further improvement of the present invention, the PD-L1 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'-GGAATTCATGAGGATATTTGCTGTC-3', ie SEQ ID NO: 1, the sequence of the downstream primer is: 5, - GACTAGTTTACGTCTCCTCCAAATGTG -3, ie SEQ ID NO: 2. Using the above PCR primer sequence, the PD-L1 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the PD-L1 gene, which reduces the cost of sequence synthesis and lowers the cost.

Accordingly, the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the PD-L1 gene, comprising the following steps:

[0009] A) PD-L1 gene primer design: According to the PD-L1 gene coding sequence, use Oligo

7 After analysis, select 5, - 00 11 ^ butyl 0 00 □ 1 ¹ 1 ¹ 00101: -3, ie SEQ ID NO: 1 as the upstream arch, select 5,- GACTAGTTTACGTCTCCTCCAAATGTG -3', ie SEQ ID NO : 2 as a downstream primer, then synthesizing the upstream primer and the downstream primer; the upstream primer and the downstream primer have no primer dimer, and the annealing temperature difference is small;

[0010] B) obtaining the PD-L1 gene cDNA sequence: PCR amplification is performed by using the upstream primer and the downstream primer to obtain a large number of PD-L1 gene coding sequences, and then the sequence is subjected to an A-tail reaction, and then The T4 DNA ligase was ligated to the pGM-T vector to obtain a ligation product, and the ligation product was transformed into competent E. coli DH50C, uniformly coated on an ampicillin-containing LB medium plate, and a positive monoclonal colony culture preservation strain was picked. The liquid was preliminarily identified by PCR, and the preliminary identification results indicated that the cDNA sequence of PD-L1 gene was inserted into the successful bacterial solution for sequencing and identification; the correct Escherichia coli was identified by liquid LB medium, and the cDNA with PD-L1 gene was extracted. Sequence pGM-T vector, digested with restriction endonuclease EcoR I enzyme and Spe l enzyme, electrophoresis, gelation recovery 500

a fragment of about bp, which is the cDNA sequence of the PD-L1 gene; [0011] C) Construction and identification of a lentiviral vector that specifically promotes high expression of the PD-L1 gene: The plasmid pLVX-I RES-Puro is extracted, digested with restriction endonuclease EcoR I enzyme and Spe l enzyme, electrophoresis, The collagen was recovered and the cDNA sequence of the PD-L1 gene was ligated into the ajpLVX-IRES-Puro expression vector by T4 DNA ligase.

The ligation product was obtained, and the ligation product was transformed into competent Escherichia coli DH50C, uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid was picked and subjected to preliminary identification by PCR, and preliminary identification was carried out. The results indicated that the cDNA sequence of the PD-L1 gene was successfully inserted into the bacterial cell for sequencing.

[0012] D) Extraction of a lentiviral vector that specifically promotes high expression of the PD-L1 gene: The sequencing result confirms that the cDNA sequence of the PD-L1 gene is inserted into a successful bacterial cell expansion culture, and the recombinant plasmid is extracted to obtain a specific promotion. A lentiviral expression vector with high expression of the PD-L1 gene.

[0013] The present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of the PD-L1 gene. After successful identification, it is packaged into a virus and introduced into Jurkat cells. After the cells are selected by puromycin, the real-time fluorescence is quantified. The PCR and Western Blot techniques were used to verify the expression of PD-L1 gene from mRNA and protein levels, respectively. The experimental results confirmed that the cDNA sequence of PD-L1 gene provided by the present invention was successfully inserted into the pLVX-I RES-Puro expression vector, which can be specific and sustained. Highly and stably promote high expression of PD-L1 gene.

The present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of the PD-L1 gene in the preparation of a medicament for treating a disease associated with abnormal PD-L1 gene expression.

Advantageous effects of the invention

Beneficial effect

The lentiviral expression vector which specifically promotes the high expression of the PD-L1 gene provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of the PD-L1 gene in a specific, sustained, efficient and stable manner. As a powerful tool for drug research and development related to PD-L1; the present invention also provides specific promotion of PD-L

A method for constructing a lentiviral expression vector with high expression of 1 gene, which 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

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.

Example 1 Design of PD-L1 gene primers.

[0021] According to the PD-L1 gene coding sequence (GenBank NM_001267706.1), it was analyzed using 01igo7 to find upstream primers and downstream primers (requiring as little primer-free dimer as possible and the annealing temperature difference is small), and then upstream The protective base and the restriction sites EcoR I and EcoR I were added to the 5' end of the primer and 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.

Table 1 PCR primer sequences of PD-L1 gene

[] [Table 1]

[0023] Example 2 Construction of a lentiviral vector that specifically promotes high expression of PD-L1 gene

[0024] After diluting the synthetic antibody, the coding sequence of the PD-L1 gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A-tail reaction, and then T4 was used.

The DNA ligase was ligated to the pGM-T vector to obtain the ligation product (PD-L1-T vector), and the ligation product was transformed into competent E. coli DH50C and uniformly coated onto an ampicillin-containing LB medium plate at 37 Cultured at °C for 12 h, the same control group was set to negative control group 1 (the competent cells were uniformly coated on the ampicillin-free plate), and the negative control group 2 (the competent cells were uniformly coated with 100 g/ml ampicillin). Penicillin On the plate), positive control group 1 (the connection product of the double enzyme-cut empty vector was uniformly coated on the plate containing 100 g/ml ampicillin), positive control group 2 (the empty carrier was evenly coated in 100 g) /mL ampicillin on the plate). 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.

[0025] After picking up 8 single colonies from the experimental group, 0.5 culture solutions were taken and PCR-amplified with primers of PD-L1 gene for preliminary identification. The results showed that the cultures of 8 single colonies were successful. The PD-L 1 gene was amplified, and then 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 PD-L1 gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly used with the EcoR I enzyme and The Spe I 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. h, the same negative control group 1 was set (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 carrier was uniformly coated in 100 g/mL ampicillin) Penicillin on the plate). 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 primers of PD-L1 gene for preliminary identification. The results showed that all the 6 culture broths could successfully amplify the PD-L1 gene, and then the recombinant vector broth was sent to Shanghai Biotech Co., Ltd. for sequencing, and the sequencing results were exactly as expected, and the pLVX-PD-Ll 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-PD-L1 was extracted and its purity and concentration were measured. The results are shown in Table 2.

[0029] Table 2 recombinant plasmid purity and concentration

[] [Table 2] Recombinant plasmid A260/A280 Concentration (mg/mL)

pLVX-PD-Ll 1.88 854.9 [0030] Example 3 slow virus packaging

[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-PD-Ll 2μ _§ was transfected into 293FT 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 Jur kat 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 PD-L1 gene.

[0035] According to the GAPDH and PD-L1 gene mRNA sequences, the primer design software Oligo 7.0 was used to design the bow.

[] [table 3]

[0036] Jurkat cells, pLVX empty vector control Jurkat cell group, and pLVX-PD-L1 high expressing cells were respectively inoculated into 6-well plates. 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 the reverse transcription, add 9 (L RNase Free dH20 diluted cDNA, and store at -20 °C for later detection. Take the cDNA of each group of cells.

Ιμΐ as a template, with GAPDH as an internal reference, real-time quantitative PCR (QPCR) detection of PD-L1 relative table Amount, set the reaction conditions: 95. C 30s, 1 cycle, 54. C 30s 40 cycles, 95. C 5s, 60 ° C lmin , 95 ° C 15s, SYBR Primescript RT-PCR Kit was used to detect the relative expression of PD-L1 gene in each group. After the pLVX-PD-L1 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 whether the pLVX-PD-L1 cells have been screened or have been cultured for 20 generations, the expression of PD-L1 gene is about 110 times higher than that of Jurkat cells, whereas the pLVX empty vector cells are The expression level of PD-L1 gene was almost unchanged from that of Jurkat cells, indicating that the cDNA sequence of PD-L1 gene provided by the present invention was successfully inserted into the pLVX-IRES-Puro expression vector, which can promote PD-specifically, continuously, efficiently and stably. The L1 gene is highly expressed.

[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 method for constructing a lentiviral expression vector with high expression of 1 gene has good operation effect, reduces the cost of sequence synthesis, and has low cost.

Claims

claims

[Claim 1] A lentiviral expression vector that specifically promotes high expression of the PD-L1 gene, characterized by: including the basic sequence of the pLVX-IRES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, and the promoter sequence and PD-L1 gene cDNA sequence; the multiple cloning site includes EcoR I restriction site and Spe I restriction site, and the PD-L1 gene cDNA sequence includes EcoR I restriction site, PD -L1 gene coding sequence and Spe

I enzyme cutting site, the PD-L1 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 PD-L1 gene according to claim 1

, which is characterized in that: the PD-L1 gene coding sequence is obtained by PCR amplification, the PCR primer includes an upstream primer and a downstream primer, the sequence of the upstream primer is: 5'-GGAATTCATGAGGATATTTGCTGTC-3, that is, SEQ ID NO : 1, the sequence of the downstream primer is: 5'-GACTAGTTTACGTCTCCTCCAAATGTG -3', that is, SEQ ID NO: 2.

[Claim 3] The construction method of a lentiviral expression vector that specifically promotes the high expression of PD-L1 gene according to claim 2, characterized in that: comprising the following steps:

A) PD-L1 gene primer design: According to the PD-L1 gene coding sequence, use Oligo 7 to analyze and select 5,-GGAATTCATGAGGATATTTCTGTC-3', that is, SEQ ID NO: 1 as the upstream primer, select 5'-

GACTAGTTTACGTCTCCTCCAAATGTG -3, that is, SEQ ID NO: 2 as the downstream primer, and then the upstream primer and the downstream primer are synthesized;

B) Obtaining the PD-L1 gene cDNA sequence: Use the upstream primer and the downstream primer to perform PCR amplification to obtain a large number of PD-L1 gene coding sequences, and then perform an A-tailing reaction on the sequence, and then connect it with T4 DNA The enzyme is connected to the pGM-T vector to obtain the ligation product. The ligation product is transformed into competent E. coli DH50C, evenly spread on an LB medium plate containing ampicillin, and positive single clone colonies are picked, cultured, and preserved. Preliminary identification by PCR, the preliminary identification results indicate that the PD-L1 gene cDNA sequence was successfully inserted into the bacterial liquid for sequencing identification; use liquid LB medium to culture and sequence the correct E. coli, And extract the pGM-T vector containing the PD-L1 gene cDNA sequence, double-digest it with the restriction endonuclease E coR I enzyme and Spel enzyme, electrophoresis, and gel cutting to recover a fragment of about 500 bp, which is PD-L1 gene cDNA sequence;

C) Construction and identification of lentiviral vectors that specifically promote the high expression of PD-L1 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 PD-L1 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 into a solution containing ampicillin On the LB medium plate, select positive single clone colonies, culture and preserve the bacterial liquid and perform preliminary PCR identification. The preliminary identification results indicate that the PD-L1 gene cDNA sequence is successfully inserted into the bacterial liquid for sequencing and identification;

D) Extraction of lentiviral vector that specifically promotes high expression of PD-L1 gene: Sequencing results confirm that the PD-L1 gene cDNA sequence has been successfully inserted into bacterial liquid amplification and culture, and the recombinant plasmid is extracted to obtain a vector that specifically promotes PD-L1 Lentiviral expression vector for high gene expression.

[Claim 4] Use of the lentiviral expression vector that specifically promotes high expression of the PD-L1 gene according to any one of claims 1 to 3 in the preparation of drugs for the treatment of diseases related to abnormal PD-L1 gene expression