WO2017214939A1 - Lentiviral expression vector for improving expression level of ccr7 gene, and applications thereof - Google Patents

Abstract

A lentiviral expression vector for specifically promoting high expression of a CCR7 gene comprises a fundamental sequence, a resistance gene sequence, a multiple-cloning-sites sequence, a promoter sequence, and a CCR7 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 CCR7 gene cDNA sequence comprises an EcoR I enzyme cutting site, a CCR7 gene coding sequence and an Spe I enzyme cutting site, and the CCR7 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 CCR7 gene, and can serve as a powerful tool applied to the research and development of drugs related to CCR7.

Description

 Description: A lentiviral expression vector for increasing the expression level of CCR7 gene and its application

 [0001] The present invention belongs to the field of genetic engineering technology, and particularly relates to a chronic virus expression vector for increasing the expression level of a CCR7 gene and an application thereof.

 Background technique

 [0002] Chemokine is a class of low molecular weight (8-12 kda) cytokines secreted by different cell types that are chemotactic to immune cells and capable of chemotaxis. Directed migration, hematopoietic cells, development and differentiation of immune cells, inflammation, angiogenesis, and tumorigenesis play different roles. The function of chemokines is mediated by chemokine receptors, and the interaction of chemokines with their receptors controls the directed migration of various immune cells between the circulatory system and tissues. Chemokines can be divided into four subfamilies: CXC, CC, C and CX3C. Their corresponding receptors are called CC receptors (CCR), CXC receptors (CXCR), C and CX3C receptors (CR, CX3CR). .

 technical problem

 [0003] CCR7 is a member of the CC chemokine receptor and contains 378 amino acids. CCR7 was originally discovered as a result of B cell infection with Epstein-Barr virus and plays an important role in the maturation of dendritic cells (DC) and the homing of T cells to secondary lymphoid organs such as lymph nodes and spleen. CC chemokine receptor. There are many studies on it, but the prior art lacks a lentiviral expression vector for specifically promoting the high expression of the CCR7 gene, which hinders the progress of research in related fields.

 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, recombinant construction methods, and the like, and found that CCR7 containing an EcoR I cleavage site and an EcoR I cleavage site. The gene cDNA sequence was inserted into the multiple cloning site of the pLVX-IRES-Puro expression vector to construct a lentiviral expression vector which specifically promotes high expression of the CCR7 gene, thereby completing the present invention.

The present invention provides a lentiviral expression vector which specifically promotes high expression of a CCR7 gene, including a basic sequence of a pLVX-IRE S-puro expression vector, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence and C CR7 gene cDNA sequence; the multiple cloning site includes EcoR I cleavage site and Spe l cleavage site, and the CCR7 gene cDNA sequence includes EcoR I cleavage site, CCR7 gene coding sequence and Spe I cleavage site The CCR7 gene cDNA sequence is 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 CCR7 gene into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase CCR7. The advantages of gene expression can be used as a powerful tool in the preparation and treatment of drugs for the treatment of CCR7 gene expression in diseases such as Alzheimer's disease.

 [0007] As a further improvement of the present invention, the CCR7 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'-CAGAATTCATGTACTCCATCATTTGTTTCG-3 ', ie SEQ ID NO: 1, the sequence 歹'J of the downstream bow is: 5'- GTACTAGTCTATGGGGAGAAGGTGGTGGTG -3', ie SEQ ID NO: 2. Using the above PCR primer sequence, the CCR7 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the CCR7 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 CCR7 gene, comprising the following steps:

 [0009] A) CCR7 gene primer design: According to the CCR7 gene coding sequence, using Oligo 7 analysis, select 5'-CAGAATTCATGTACTCCATCATTTGTTTCG-3, ie SEQ ID NO: 1 as the upstream primer, select 5, - GTACTAGTCTATGGGGAGAAGGTGGTGGTG -3, ie 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 CCR7 gene cDNA sequence: PCR amplification using the upstream primer and the downstream primer to obtain a large number of CCR7 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. Preliminary identification, the preliminary identification results indicate that the CCR7 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 carrying the CCR7 gene cDNA sequence was extracted and used. Restriction enzyme EcoR I enzyme and Spe I enzyme for double digestion, electrophoresis, gelation recovery 1000

 a fragment of about bp, which is the cDNA sequence of the CCR7 gene;

[0011] C) Construction and identification of a lentiviral vector that specifically promotes high expression of the CCR7 gene: extraction of the plasmid pLVX-IR ES-Puro, double digestion with restriction endonuclease EcoR I enzyme and Spe I enzyme, electrophoresis, digestion The cDNA was recovered and the CCR7 gene cDNA sequence 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 with ampicillin. On the LB medium plate, the positive monoclonal colony culture supernatant was picked and the PC R was initially identified. The preliminary identification results indicated that the CCR7 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 CCR7 gene: The sequencing result confirms that the CCR7 gene cDNA sequence is inserted into a successful bacterial cell expansion culture, and the recombinant plasmid is extracted to obtain a specific promotion of the CC R7 gene. Expressed lentiviral expression vector.

 [0013] The present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of CCR7 gene. After successful identification, it is packaged into a virus and introduced into Jurkat cells. After screening cells with puromycin, real-time quantitative PCR and The Western Blot technique verified the change of CCR7 gene expression from mRNA and protein levels, respectively. The experimental results showed that the CCR7 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 CCR7 gene is highly expressed.

 The present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of a CCR7 gene for the preparation of a medicament for treating a disease associated with abnormal expression of a CCR7 gene.

 Advantageous effects of the invention

 Beneficial effect

 The lentiviral expression vector which specifically promotes the high expression of CCR7 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 CCR7 gene, and can be used as a powerful tool. In drug research and development related to CCR7; the present invention also provides specific promotion of CCR

A method for constructing a lentiviral expression vector with high expression of 7 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

 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 ATCC, 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 were purchased from Takara. The RNeasy Mini Kit was purchased from QIAGEN, the pGM-T carrier was purchased from Tiangen, and the Endo-Free Plasmid Mini Kit II was purchased from Omega Bio-tek.

 Example 1 Design of CCR7 Gene Primers.

 [0021] According to the CCR7 gene coding sequence (GenBank NM_001301714.1), 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 Spe l 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.

Table 1 PCR bow of the CCR7 gene |

 [] [Table 1]

 [0023] Example 2 Construction of a lentiviral vector that specifically promotes high expression of the CCR7 gene

[0024] After diluting the synthetic antibody, the coding sequence of the CCR7 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 (CCR7-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 CCR7 gene for preliminary identification. The results showed that the cultures of 8 single colonies could be successfully amplified. The CCR7 gene was exported, 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 CCR7 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 primers of CCR7 gene for preliminary identification. The results showed that all 6 cultures could successfully amplify the CCR7 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-CCR7 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-CCR7 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-CCR7 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 CCR7 gene.

 [0035] According to the GAPDH and CCR7 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 pLVX-CCR7 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 CCR7, 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 CCR7 gene in each group was detected by SYBR Primescript RT-PCR Kit. After continuous culture of pLVX-CCR7 cells for 20 passages, the above experiment was repeated. The summarized results are shown in Figure 2. It can be seen that the expression of CCR7 gene is 80-fold higher than that of Jurkat cells, whether it is just after screening or after 20 generations of pLVX-CCR7 cells, and the expression of CCR7 gene in pLVX empty vector cells. There is almost no change compared with Jurkat cells, indicating that the CCR7 gene cDNA sequence provided by the present invention is successfully inserted into the pLVX-IRES-Puro expression vector, and can specifically, stably, efficiently and stably promote the high expression of CCR7 gene.

[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 CCR7 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 CCR7 gene, and can be used as a powerful tool. In drug research and development related to CCR7; the present invention also provides specific promotion of CCR

The construction method of the lentiviral expression vector with high expression of 7 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 CCR7 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 The cDNA sequence of the CCR7 gene is ij; the multiple cloning site includes an EcoR I enzyme cut site and a Spe I enzyme cut site, and the CCR7 gene cDNA sequence includes an EcoR I enzyme cut site, the CCR7 gene coding sequence and the Spe I enzyme cut site. I enzyme cutting site, the CCR7 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 CCR7 gene according to claim 1, characterized in that: the CCR7 gene coding sequence is obtained by PCR amplification, and the PCR primers include upstream primers and downstream primers, so The sequence of the above-mentioned upstream primer is: 5'-CAGAATTCATGTACTCCATCATTTGTTTCG-3, that is, SEQ ID NO: 1, and the sequence of the above-mentioned downstream primer is: 5'- GTACTAGTCTATGGGGAGAAGGTGGTGGTG -3, i.e. SEQ ID NO: 2. [Claim 3] The construction method of a lentiviral expression vector that specifically promotes high expression of CCR7 gene according to claim 2, characterized in that: comprising the following steps: A) CCR7 gene primer design: According to the CCR7 gene coding sequence, use Oligo 7 to analyze and select 5, - CAGAATTCATGTACTCCATCATTTGTTTCG -3', that is, SEQ ID NO: 1 as the upstream primer, select 5'- GTACTAGTCTATGGGGAGAAGGTGGTGGTG -3, i.e. SEQ ID NO: 2 as the downstream primer, and then synthesize the upstream primer and the downstream primer; B) Obtaining the cDNA sequence of the CCR7 gene: Use the upstream primer and the downstream primer to perform PCR amplification to obtain a large number of CCR7 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 CCR7 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, and extract the pGM-T vector containing the CCR7 gene cDNA sequence, and use restriction endonuclease E coR I enzyme and Spe I enzyme for double enzyme Cut, electrophoresis, and gel cutting to recover a fragment of about 1000 bp. This fragment is the CCR7 gene cDNA sequence; C) Construction and identification of a lentiviral vector that specifically promotes high expression of CCR7 gene: Extract plasmid pLVX-IRES-Puro, and perform double digestion with restriction endonuclease EcoR I enzyme and Spe I enzyme. , electrophoresis, gel cutting to recover the vector, and then use T4 DNA ligase to connect the CCR7 gene cDNA sequence into the pLVX-IRES-Puro expression vector to obtain the connection product, transform the connection product into competent E. coli DH50C, and spread evenly Spread onto 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 bacterial liquid with successful insertion of the CCR7 gene cDNA sequence is sequenced and identified; D) Extraction of lentiviral vectors that specifically promote the high expression of the CCR7 gene: The sequencing results confirm that the CCR7 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 CCR7 gene. Expression vector. [Claim 4] Use of the lentiviral expression vector specifically promoting the high expression of CCR7 gene according to any one of claims 1 to 3 in the preparation of drugs for the treatment of diseases related to abnormal CCR7 gene expression