WO2017214945A1

WO2017214945A1 - Lentiviral expression vector for improving hepcidin gene expression level and application thereof

Info

Publication number: WO2017214945A1
Application number: PCT/CN2016/086069
Authority: WO
Inventors: 毛侃琅
Original assignee: 毛侃琅
Priority date: 2016-06-16
Filing date: 2016-06-16
Publication date: 2017-12-21

Abstract

A lentiviral expression vector for specifically promoting higher expression of Hepcidin genes, comprising a fundamental sequence, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence, and a Hepcidin gene cDNA sequence of a pLVX-IRES-puro expression vector; a multiple cloning site comprises an EcoR I enzyme cutting site and an Spe I enzyme cutting site; the Hepcidin gene cDNA sequence comprises the EcoR I enzyme cutting site, a Hepcidin gene coding sequence, and the Spe I enzyme cutting site; the Hepcidin gene cDNA sequence is inserted into the multiple cloning site sequence in a forward direction. The lentiviral expression vector has the advantages of high transfection efficiency, a small amount required, and specific, continuous, efficient and stable expression in a Hepcidin gene, and can be applied to drug research and development associated with Hepcidin as a powerful tool.

Description

Lentiviral expression vector for increasing Hepcidin gene expression level and application thereof

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

Background technique

[0002] Prostate cancer is a common malignant tumor in men and ranks second among male cancer deaths. New research suggests that cellular iron metabolism plays an important role in prostate cancer growth, angiogenesis and metastasis. Iron accumulation is involved in the growth and metabolism of the body and is a necessary trace element for human growth. The development of many tumors has led to an increase in the demand for iron. Iron is considered to be the basic substance for maintaining the growth and development of tumor cells. Iron metabolism regulating proteins affect tumor changes, and reducing iron metabolism can be considered as an anti-tumor research direction. . It has been suggested that proteins that regulate iron metabolism can affect tumor growth and reduce the utilization of intracellular iron as a pathway for anti-tumor therapy.

technical problem

[0003] The study found that the relationship between Hepcidin and iron metabolism, in the use of iron-rich foods or through the parenteral route to iron, resulting in increased iron content in mice, the expression of Hepcidin mRNA in mouse liver cells increased significantly High, and has a significant positive correlation with iron dose. The expression of bioactive Hepcidin in tumor population is also affected by many factors. The role of iron metabolism in the body has attracted the attention of many scholars. Therefore, Hepcidin may be a potential tumor therapeutic target with good bursting potential, but the lack of lentiviral expression vectors in the prior art that specifically promote the high expression of Hepcidin gene does not facilitate the 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 Hepcidin which contains an EcoR I cleavage site and a Spe I restriction 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 Hepcidin gene, thereby completing the present invention. The present invention provides a lentiviral expression vector which specifically promotes high expression of the Hepcidin gene, including the basic sequence of the pLVX-I RES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and the Hepcidin gene. a cDNA sequence; the multiple cloning site includes an EcoR I cleavage site and a Spe I cleavage site, and the Hepcidin gene cDNA sequence includes EcoR

The I cleavage site, the Hepcidin gene coding sequence and the Spe I cleavage site, and the Hepcidin 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 Hepcidin gene into the pLVX-IRES-Pur o expression vector has the advantages of high transfection efficiency, low dosage, sustainable, high efficiency and stable improvement. The advantages of Hepcidin gene expression can be used as a powerful tool in the preparation and treatment of Hepcidi n gene expression in the treatment of diseases such as Alzheimer's disease.

[0007] As a further improvement of the present invention, the Hepcidin gene coding sequence is obtained by PCR amplification, and the PC R primer comprises an upstream primer and a downstream primer, and the sequence of the upstream primer is: 5'-GGAATTCATGGCACTGAGCTCCCAGATCTG -3, ie SEQ ID NO: 1, the sequence of the downstream primer is: 5, - GACTAGTCTACGTCTTGCAGCACATCCC -3, ie SEQ ID NO: 2. Using the above PCR primer sequence, the Hepcidin gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the Hepcidin 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 Hepcidin gene, comprising the following steps:

[0009] A) Hepcidin gene primer design: According to the Hepcidin gene coding sequence, using 01igo 7 analysis, select 5, - GGAATTCATGGCACTGAGCTCCCAGATCTG -3, ie SEQ ID NO: 1 as the upstream bow, select 5,- GACTAGTCTACGTCTTGCAGCACATCCC -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 Hepcidin gene cDNA sequence: PCR amplification using the upstream primer and the downstream primer to obtain a large number of Hepcidin gene coding sequences, and then adding the A tail reaction to the sequence, using T4 DNA ligase Attached to the pGM-T vector to obtain the ligation product, the ligation product was transformed into competent E. coli ToplO, uniformly coated onto the ampicillin-containing LB medium plate, and the positive monoclonal bacteria were picked. The culture supernatant was cultured and the PCR was initially identified. The preliminary identification results indicated that the Hepcidin gene cDNA sequence was inserted into the successful bacterial solution for sequencing. The correct E. coli was identified by liquid LB medium, and the Hepcidin gene cDNA was extracted. The sequence of pGM-T vector was digested with restriction endonuclease Eco RI enzyme and Spe l enzyme, and the fragment of about 250 bp was recovered by electrophoresis and gel-cutting. This fragment is the Hepcidin gene cDNA sequence;

[0011] C) Construction and identification of a lentiviral vector that specifically promotes high expression of the Hepcidin gene: extraction of the plasmid pLVX-IRES-Puro, restriction enzyme digestion with EcoR I enzyme and Spe I enzyme, electrophoresis, gel extraction The vector was ligated to the pLVX-IRES-Puro expression vector by T4 DNA ligase to obtain a ligation product, which was transformed into competent E. coli ToplO and uniformly coated to ampicillin-containing LB. On the medium plate, the positive monoclonal colonies were cultured and preserved, and PC R was initially identified. The preliminary identification results indicated that the Hepcidin 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 Hepcidin gene: The sequencing result confirms that the Hepci din gene cDNA sequence is inserted into a successful bacterial cell expansion culture, and the recombinant plasmid is extracted to obtain a specific Hepcidin 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 Hepcidin gene, and after being successfully constructed, it is packaged into a virus and introduced into RWPE-2 cells, and the cells are selected by puromycin, and then quantified by real-time fluorescence. The PCR and Western Blot techniques were used to verify the expression of Hepcidin gene from mRNA and protein levels. The experimental results showed that the Hepcidin gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IRES-Puro expression vector, which was specific, sustained, efficient and stable. Promote high expression of Hepcidin gene.

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

Advantageous effects of the invention

Beneficial effect

[0015] The lentiviral expression vector which specifically promotes high expression of Hepcidin gene has the advantages of high transfection efficiency, low dosage, specific, sustained, efficient and stable promotion of high expression of Hepcidin gene, and can be used as a powerful tool. In drug research and development related to Hepcidin; the present invention also provides specific promotion The construction method of the lentiviral expression vector with high expression of Hepcidin gene has good operation effect, reduces the cost of sequence synthesis, and has low cost.

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] RWPE-2 cells were purchased from the Cell Resource Center of 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, Lenti-X GoStix reagent Boxes are 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 Hepcidin Gene Primers.

[0021] According to the Hepcidin gene coding sequence (GenBank NM_021175.3), 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), 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 Hepcidin gene PCR bow |

[] [Table 1] Sequence (5'-3') Upstream Primer GGAATTCATGGCACTGAGCTCCCAG

ATCTG

Downstream primer GACTAGTCTACGTCTTGCAGCACAT

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

[0024] After diluting the synthetic antibody, the coding sequence of the Hepcidin 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 (Hepcidin-T vector), and the ligation product was transformed into competent E. coli ToplO and uniformly coated onto ampicillin-containing LB medium plate at 37 ° C. After 12 h of culture, 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 with 100 g/ml ampicillin). 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 uniformly coated in 100 g/ mL of ampicillin on the plate). 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 Hepcidin gene for preliminary identification. The results showed that the cultures of 8 single colonies could be successfully amplified. The He pcidin gene was extracted, 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 Hepcidin 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 ToplO, 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 Hepcidin gene for preliminary identification. The results showed that all 6 culture broths could successfully amplify the Hepcidi n 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-Hepcidin 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-Hepcidin 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-Hepcidin 2 g was transfected into 293FT cells using a lentiviral packaging assistant kit. After 48 hours, the virus-containing supernatant medium was collected, and the virus solution was filtered through a sieve of 0.45 μm for infection of RWPE-2 cells, and the titer of the virus detected by Lenti-X GoStix kit was 5000000 to 50000000 IFU.

Example 4 Lentiviral transduction RWPE-2 cells

[0033] Inoculate RWPE-2 cells in a 6-well plate, 1000000 cells per well. After 12 hours, the cell density is about 50<3⁄4, and the virus solution is taken separately. The virus is diluted 10 times with DMEM complete medium, and then polyglycolamine is added. (polybrene) to a final concentration of 8 g/mL. Remove the medium from the 6-well plate and add the virus-containing DMEM complete medium.

(containing 10% fetal bovine serum), after 24 hours, the virus-containing DMEM complete medium was discarded, and fresh DME M complete medium was replaced. After 24 hours, the cells were selected with 0.5 g/ml concentration of puromycin. After 10 days of screening, the medium was changed 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 Hepcidin gene.

[0035] Based on the GAPDH and Hepcidin gene mRNA sequences, the primer design software Oligo 7.0 was used to design bows.

[] [table 3]

[0036] RWPE-2 cells, pLVX empty vector control RWPE-2 cell group, and pLVX-Hepcidin high expression cells were inoculated into 6-well plates, respectively. Cell density reached 80<3⁄4-90<3⁄4吋, total RNA was extracted from each group with RNeasy Mini Kit, and mRNA was reverse transcribed into cDNA using PrimeScrip RT reagent Kit. Reverse transcription conditions: 37 ° C, 15 min _; 85 ° C, 5s; 4°C, ∞. After the reverse transcription was completed, the cDNA was diluted with 90 μM of RNase Free dH 20 and stored at -20 ° C for later detection. The cDNA of each group of cells was used as a template, GAPDH was used as an internal reference, and the relative expression of Hepcidin was detected by real-time quantitative PCR (QPCR). The reaction conditions were set: 95. C 30s, 1 cycle, 54°C 30s 40 cycles, 95. C 5s, 60 ° C lmin, 95. C 15s , using S YBR Primescript RT-PCR

Kit detects the relative expression of Hepcidin gene in each group of cells. After continuous culture of pLVX-Hepcidin cells for 20 passages, the above experiment was repeated. The summarized results are shown in Figure 2. It can be seen that whether it is just after screening or after 20 generations of pLVX-Hepcidin cells, the expression of Hepcidin gene is about 240 times higher than that of RWPE-2 cells, and the Hepccidin gene of pLVX empty vector cells. The expression level of the Hepcidin gene was successfully inserted into the p LVX-IRES-Puro expression vector, which promoted the high expression of Hepcidin 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 high expression of Hepcidin gene provided by the invention has high transfection efficiency The utility model has the advantages of low dosage, specific, sustained, high efficiency and stable promotion of high expression of the Hepcidin gene, and can be used as a powerful tool in drug research and development related to Hepcidin; the invention also provides specific promotion of high expression of Hepcidin gene. The construction method of the lentiviral expression vector 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 Hepcidin 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 Hepcidin gene cDNA sequence; The multiple cloning site includes an EcoR I restriction site and a Spe I restriction site. The Hepcidin gene cDNA sequence includes an EcoR I restriction site, a Hepcidin gene coding sequence and a Spe I restriction site. point, the Hepcidin 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 Hepcidin gene according to claim 1, characterized in that: the Hepcidin gene coding sequence is obtained by PCR amplification, and the PCR primers include upstream primers and downstream primers, The sequence of the upstream primer is: 5'- GGAATTCATGGCACTGAGCTCCCAGATCTG -3, that is, SEQ ID NO: 1, and the sequence of the downstream primer is: 5'-

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

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

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

GACTAGTCTACGTCTTGCAGCACATCCC-3', that is, 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 Hepcidin gene: Use the upstream primer and the downstream primer to perform PCR amplification to obtain a large number of Hepcidin 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 ToplO, 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 PCR identification. The preliminary identification results show that the Hepcidin gene cDNA sequence was successfully inserted into the bacterial liquid for sequencing and identification; the liquid LB medium was used to culture and sequence the correct bacteria. Enterobacteriaceae, and extract the pGM-T vector containing the Hepcidin gene cDNA sequence, double-digest it with the restriction endonuclease EcoR I enzyme and Spe I enzyme, electrophoresis, and gel cutting to recover a fragment of about 250 bp, which is Hepcidin gene cDNA sequence;

C) Construction and identification of a lentiviral vector that specifically promotes high expression of the Hepcidin gene: Extract the plasmid pLVX-IRES-Puro, double-digest it with the restriction endonuclease EcoR I enzyme and Spe I enzyme, electrophoresis, and gel cutting to recover the vector, and then Use T4 DNA ligase to connect the Hepcidin gene cDNA sequence into the pLVX-IRES-Puro expression vector to obtain a ligation product, transform the ligation product into competent E. coli ToplO, 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 conduct preliminary PCR identification. The preliminary identification results indicate that the bacterial liquid with successful insertion of the Hepcidin gene cDNA sequence is sequenced and identified;

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

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