WO2017214830A1

WO2017214830A1 - Lentiviral vector for specifically promoting high expression of pd-1 gene, and applications thereof

Info

Publication number: WO2017214830A1
Application number: PCT/CN2016/085635
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-1 gene comprises a fundamental sequence, a resistance gene sequence, a multiple-cloning-sites sequence, a promoter sequence, and a PD-1 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 PD-1 gene cDNA sequence comprises an EcoR I enzyme cutting site, a PD-1 gene coding sequence and an SpeI enzyme cutting site, and the PD-1 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-1 gene, and can serve as a powerful tool applied to research and development of drugs related to PD-1.

Description

Title: Lentiviral Vector Specifically Promoting High Expression of PD-1 Gene and Its Application

[0001] The present invention belongs to the field of genetic engineering technology, and particularly relates to a chronic virus vector which specifically promotes high expression of PD-1 gene, and a construction method and application thereof.

Background technique

[0002] Lung cancer is one of the most common malignancies in the world. In China, the incidence and mortality of lung cancer are the highest among malignant tumors, which are 53.57/100,000 and 45.57/100,000 respectively, which seriously threaten the health of the people. Non-small cell lung cancer (NSCLC) accounts for 84% of lung cancer. Most patients have advanced diagnosis, and their treatment is limited. The 5-year survival rate is only 2%. In recent years, research on tumor immunotherapy has advanced by leaps and bounds: in addition to immune surveillance to eliminate tumor cells, the immune system in the body also promotes tumor immune escape at certain stages, playing an extremely important role in tumorigenesis and development.

technical problem

[0003] Studies have shown that activation of the programmed death 1 (PD-1) / PD-1 ligand (PD-L1) signaling pathway can lead to the formation of immunosuppressive tumor microenvironment, Tumor cells evade immune surveillance and killing, while blocking PD-1/PD-L1 signaling pathway can reverse the tumor immune microenvironment and enhance endogenous anti-tumor immune effects. Therefore, the role of PD-1 in the treatment of lung cancer and its mechanism are very important, but the lack of lentiviral expression vectors that specifically promote the high expression of PD-1 gene in the prior art has caused the progress of related research. Certain obstacles.

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-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-1 gene, thereby completing the present invention.

The present invention provides a lentiviral expression vector which specifically promotes high expression of the PD-1 gene, including the basic sequence of the pLVX-IRES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and the PD - 1 gene cDNA sequence; the multiple cloning site includes EcoR I cleavage site and Spe

I cleavage site, the PD-1 gene cDNA sequence including EcoR

The I cleavage site, the PD-1 gene coding sequence and the Spe l cleavage site, and the PD-1 gene cDNA sequence is inserted into the cloning site sequence.

[0006] According to the above technical solution, the lentiviral expression vector constructed by inserting the cDNA sequence of the PD-1 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-1 gene expression can be used as a powerful tool for the preparation and treatment of PD-1 gene expression for diseases such as Alzheimer's disease.

[0007] As a further improvement of the present invention, the PD-1 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'-GGAATTCATGCAGATCCCACAGGCG-3, ie SEQ ID NO: 1, the sequence of the downstream primer is: 5, - GACTAGTTCAGAGGGGCCAAGAGCAGTG -3', ie SEQ ID NO: 2. Using the above PCR primer sequence, the PD-1 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-1 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 a PD-1 gene, comprising the following steps:

[0009] A) PD-1 gene primer design: According to the coding sequence of PD-1 gene, using Oligo 7 analysis, select 5 '- GGAATTCATGCAGATCCCACAGGCG -3', ie SEQ ID NO: 1 as the upstream primer, select 5, - GACTAGTTCAGAGGGGCCAAGAGCAGTG - 3, that is, SEQ ID NO: 2 as a downstream primer, and 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-1 gene cDNA sequence: using the upstream primer and the downstream primer for PCR amplification, obtaining a large number of PD-1 gene coding sequences, and then adding the A tail reaction to the sequence, 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 the PD-1 gene was inserted into the successful bacterial solution for sequencing and identification; the correct Escherichia coli was identified by liquid LB medium culture sequencing, and The pGM-T vector carrying the _cDNA sequence of PE gene was extracted and digested with the restriction enzyme EcoR I enzyme and Spe I enzyme. The fragment of about 1000 bp was recovered by electrophoresis and gel-cutting. This fragment is the PD-1 gene. cDNA sequence;

[0011] C)

Construction and identification of a lentiviral vector that specifically promotes high expression of PD-1 gene: The plasmid pLVX-IRES-Pur o is extracted, and the restriction endonuclease EcoR I enzyme and Spe I enzyme are double-digested, electrophoresis, and gel-removed to recover the vector. The cDNA sequence of the PD-1 gene was ligated into the ajpLVX-IRES-Puro expression vector by T4 DNA ligase to obtain a ligation product, which was transformed into competent E. coli DH50C and uniformly coated into ampicillin-containing LB culture. On the basal plate, the positive monoclonal colony culture supernatant was picked and preliminarily identified by PCR. The preliminary identification results indicated that the PD-1 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification;

[0012] D) Extraction of a lentiviral vector that specifically promotes high expression of the PD-1 gene: The sequencing result confirms that the cDNA sequence of the PD-1 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-1 gene.

[0013] The present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of PD-1 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 changes of PD-1 gene expression from mRNA and protein levels, respectively. The experimental results showed that the PD-1 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IRES-Puro expression vector, which was specific, sustained, and Highly and stably promote high expression of PD-1 gene.

The present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of the PD-1 gene for the preparation of a medicament for treating a disease associated with abnormal expression of PD-1 gene.

Advantageous effects of the invention

Beneficial effect

The lentiviral expression vector which specifically promotes the high expression of the PD-1 gene provided by the invention has the advantages of high transfection efficiency, low dosage, and can specifically, stably, efficiently and stably promote the high expression of the PD-1 gene. As a powerful tool for drug research and development related to PD-1; the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of PD-1 gene, which has a good operation effect and reduces the cost of sequence synthesis. 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.

Example 1 Design of PD-1 gene primers.

[0021] According to the PD-1 gene coding sequence (GenBank NM_005018.2), it was analyzed using 01igo7

, looking for upstream primers and downstream primers (requires no primer dimer as much as possible and the annealing temperature difference is small), then add the protective base and the restriction sites EcoR I and EcoR I at the 5' end of the upstream primer and the downstream primer, respectively. 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-1 gene

[] [Table 1]

[0023] Example 2 Construction of a lentiviral vector that specifically promotes high expression of PD-1 gene

[0024] After diluting the synthesized primer, the coding sequence of the PD-1 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 (PD-1-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.吋Set negative control group 1 (consistent cells were uniformly coated on ampicillin-free plates), negative control group 2 (consistent 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 PD-1 gene for preliminary identification. The results showed that the cultures of 8 single colonies were successful. The PD-1 gene was amplified, 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 PD-1 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-1 gene for preliminary identification. The results showed that all the 6 culture broths could successfully amplify the PD-1 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-Ι 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 get on The recombinant plasmid pLVX-PD-1 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]

[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-1 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 Jurka t cells, and the Lenti-X GoStix kit was used to detect the virus titer of 5000000 to 50000000 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-1 gene.

[0035] According to the GAPDH and PD-1 gene mRNA sequences, the primer design software Oligo 7.0 was used to design the bow.

[] [table 3]

Inoculated with Jurkat cells, pLVX empty vector control Jurkat cell group, and pLVX-PD-1 high expressing cells To 6-well plate. 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 PD-1, 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 PD-1 gene in each group was detected by SYBR Primescript RT-PCR Kit. After continuous culture of pLVX-PD-1 cells 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-Ι cells, which have just been screened or have been cultured for 20 generations, have a PD-1 gene expression level that is more than 105-fold higher than that of Jurkat cells, whereas pLVX empty vector cells The expression level of PD-1 gene was almost unchanged from that of Jurkat cells, indicating that the cDNA sequence of PD-1 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. 1 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

Claims

claims

[Claim 1] A lentiviral expression vector that specifically promotes high expression of the PD-1 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-1 gene cDNA sequence; the multiple cloning site includes EcoR I restriction site and Spe I restriction site, and the PD-1 gene cDNA sequence includes EcoR I restriction site, PD -1 gene coding sequence and Spel restriction site, and the PD-1 gene cDNA sequence is inserted into the multiple cloning site sequence in the forward direction.

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

[Claim 3] The construction method of a lentiviral expression vector that specifically promotes high expression of PD-1 gene according to claim 2, characterized in that: comprising the following steps:

A) PD-1 gene primer design: Based on the PD-1 gene coding sequence, use 01igo 7 to analyze and select 5, - GGAATTCATGCAGATCCCACAGGCG -3, that is, SEQ ID NO

: 1 as the upstream primer, select 5'-

GACTAGTTCAGAGGGGCCAAGAGCAGTG-3', that is, SEQ ID NO: 2 as the downstream primer, and then synthesize the upstream primer and the downstream primer;

B) Obtaining the PD-1 gene cDNA sequence: Use the upstream primer and the downstream primer to perform PCR amplification to obtain a large number of PD-1 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-1 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 gene cDNA sequence. , using restriction endonuclease EcoR I Double enzyme digestion with Spe l enzyme, electrophoresis, and gel cutting to recover a fragment of about 1000 bp. This fragment is the PD-1 gene cDNA sequence; construction and identification of a lentiviral vector that specifically promotes high expression of the PD-1 gene: Extract plasmid pLV

Ligase ligates the PD-1 gene cDNA sequence into the pLVX-IRES-Puro expression vector

, obtain the ligation product, 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 perform preliminary identification by PCR. The results showed that the PD-1 gene cDNA sequence was successfully inserted into the bacterial liquid for sequencing and identification; the lentiviral vector that specifically promoted the high expression of the PD-1 gene was extracted: the sequencing results confirmed that the PD-1 gene cDNA sequence was successfully inserted into the bacterial liquid and amplified Culture, extract the recombinant plasmid, and obtain a lentiviral expression vector that specifically promotes high expression of the PD-1 gene.

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