WO2017214942A1 - Vecteur d'expression lentiviral pour améliorer l'expression du gène tctp, et ses applications - Google Patents
Vecteur d'expression lentiviral pour améliorer l'expression du gène tctp, et ses applications Download PDFInfo
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- WO2017214942A1 WO2017214942A1 PCT/CN2016/086066 CN2016086066W WO2017214942A1 WO 2017214942 A1 WO2017214942 A1 WO 2017214942A1 CN 2016086066 W CN2016086066 W CN 2016086066W WO 2017214942 A1 WO2017214942 A1 WO 2017214942A1
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- tctp gene
- sequence
- tctp
- expression vector
- cdna sequence
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/867—Retroviral vectors
Definitions
- the present invention belongs to the field of genetic engineering technology, and particularly relates to a lentiviral expression vector for enhancing expression of a TCTP gene and an application thereof.
- Diabetes is a group of chronic non-infectious, metabolic diseases characterized by elevated blood glucose levels that are a serious hazard to human health. Whether in developed or developing countries, diabetes has become an epidemic in modern society and has become a worldwide public health problem that poses a serious threat to human health. According to the World Health Organization
- type 2 diabetes T2DM
- type 2 diabetes T2DM
- the prevalence of type 2 diabetes has increased dramatically in countries around the world, and the surge in type 2 diabetes is the leading cause of the dramatic increase in the total number of diabetic patients worldwide.
- the prevalence of diabetes in adults is close to 10%, and type 2 diabetes accounts for about 90% of all diabetic patients.
- TCTP is a protein widely expressed in eukaryotes. Its structure is highly conserved and participates in a series of cellular physiological functions such as cell cycle, apoptosis, protein translation, cell morphology, etc. Death protein. Recent studies have shown that TCTP plays an important role in the survival of pancreatic islet ⁇ cells and needs further study. However, the lack of lentiviral expression vectors that specifically promote the high expression of TCTP gene in the prior art makes the relevant studies not well developed.
- the present invention provides a lentiviral expression vector which specifically promotes high expression of a TCTP 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 a T CTP a gene cDNA sequence; the multiple cloning site comprises an EcoR I cleavage site and a Spe l cleavage site, and the TCTP gene cDNA sequence comprises an EcoR I cleavage site, a TCTP gene coding sequence, and a Spe I cleavage site The TCTP gene cDNA sequence is positively inserted into the multiple cloning site sequence.
- the lentiviral expression vector constructed by inserting the TCTP gene cDNA sequence into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase TCTP.
- the advantages of gene expression can be used as a powerful tool in the preparation and treatment of drugs for the treatment of TCTP gene expression in diseases such as Alzheimer's disease.
- the TCTP gene coding sequence is obtained by PCR amplification
- the PCR primer comprises an upstream primer and a downstream primer
- the sequence of the upstream primer is: 5'-
- GGAATTCATGATTATCTACCGGGACCTC -3 ie SEQ ID NO: 1
- sequence of the downstream primer is: 5,- GACTAGTCTAAAGATGTATTCCCCAATTC -3', ie SEQ ID NO: 2
- the TCTP gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the TCTP gene, which reduces the cost of sequence synthesis and lowers the cost.
- the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of a TCTP gene, comprising the following steps:
- A) TCTP gene primer design According to the TCTP gene coding sequence, using Oligo 7 analysis, select 5'-GGAATTCATGATTATCTACCGGGACCTC-3', ie SEQ ID NO: 1 as the upstream bow, select 5 '- GACTAGTCTAAAGATGTATTCCCCAATTC -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;
- coli was identified by liquid LB medium, and the pGM-T vector carrying the TCTP gene cDNA sequence was extracted and digested with the restriction enzymes EcoR I and Spe I, electrophoresis and gelatinization. Recover a fragment of about 500 bp, which is the TCTP gene cDNA sequence;
- the ligation product is obtained, and the ligation product is transformed into competent E. coli ToplO, uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid is picked and subjected to preliminary identification by PCR, and preliminary identification is performed.
- the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of TCTP gene, and after successful identification, packaging into a virus and transfecting into INS-1 cells, and screening cells with puromycin, using real-time fluorescence quantitative
- the PCR and Western Blot techniques were used to verify the changes of TCTP gene expression from mRNA and protein levels, respectively.
- the experimental results showed that the TCTP gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IR ES-Puro expression vector, which was specific, sustained, efficient and stable. Promote high expression of TCTP gene.
- the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of a TCTP gene for the preparation of a medicament for treating a disease associated with abnormal expression of a TCTP gene.
- the lentiviral expression vector which specifically promotes the high expression of the TCTP 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 the TCTP gene, and can be used as a powerful tool.
- the present invention also provides specific promotion of TCTP
- 1 is a plasmid map of the pLVX-IRES-Puro expression vector.
- FIG. 2 is a schematic diagram showing the results of fluorescent quantitative PCR detection after puromycin screening of cells.
- INS-1 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.
- Example 2 Construction of a lentiviral vector that specifically promotes high expression of TCTP gene
- the coding sequence of the TCTP 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 (TCTP-T vector) was obtained on the vector, and the ligation product was transformed into competent E. coli To plO, uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h.
- Negative control group 1 consistentt cells were uniformly coated on ampicillin-free plates
- negative control group 2 peripheral cells were uniformly coated in 100
- positive control group 1 the connection product of the double enzyme-cut empty vector was evenly coated in 100
- 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.
- the bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the TCTP 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 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.
- 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-TCTP 2 ⁇ ⁇ 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 ⁇ m for infection of INS-1 cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 50,000,000 IFU.
- Inoculated INS-1 cells in a 6-well plate 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 then polyglycolamine was added. (polybrene) The final concentration was 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 TCTP gene.
- INS-1 cells, pLVX empty vector control INS-1 cell group, and pLVX-TCTP high expressing cells were inoculated to a 6-well plate, respectively.
- the cell density reached 80 ⁇ 3 ⁇ 4-90 ⁇ 3 ⁇ 4 ⁇ , 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.
- the expression level of TCTP gene is 60-fold higher than that of INS-1 cells, whether it is just after screening or after 20 generations of pLVX-TCTP cells, and the TCTP gene of pLVX empty vector cells.
- the expression level of the TCTP gene was successfully inserted into the pLVX-IRES-Puro expression vector, which promoted the high expression of TCTP gene specifically, continuously, efficiently and stably.
- the lentiviral expression vector provided by the invention specifically promoting the high expression of the TCTP gene has high transfection efficiency. It has the advantages of low dosage, specific, sustained, efficient and stable promotion of high expression of TCTP gene, and can be used as a powerful tool in drug research and development related to TCTP.
- the present invention also provides specific promotion of high expression of TCTP gene.
- the construction method of lentiviral expression vector has good operation effect and reduces the cost of sequence synthesis
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Abstract
L'invention concerne un vecteur d'expression lentiviral destiné à favoriser spécifiquement l'expression élevée d'un gène TCTP, ledit vecteur comprenant une séquence fondamentale, une séquence de gène de résistance, une séquence de sites multiples de clonage, une séquence de promoteur et une séquence d'ADNc du gène TCTP d'un vecteur d'expression de pLVX-IRES-puro. Les sites multiples de clonage comprennent un site de découpe d'enzyme EcoR I et un site de découpe d'enzyme Spe I, la séquence d'ADNc du gène TCTP comprend un site de découpe d'enzyme EcoR I, une séquence de codage du gène TCTP et un site de découpe de l'enzyme Spe I, et la séquence d'ADNc du gène TCTP est insérée vers l'avant dans la séquence de sites multiples de clonage. Le vecteur d'expression lentiviral peut servir d'outil pour la recherche et le développement de médicaments associés à TCTP.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2016/086066 WO2017214942A1 (fr) | 2016-06-16 | 2016-06-16 | Vecteur d'expression lentiviral pour améliorer l'expression du gène tctp, et ses applications |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2016/086066 WO2017214942A1 (fr) | 2016-06-16 | 2016-06-16 | Vecteur d'expression lentiviral pour améliorer l'expression du gène tctp, et ses applications |
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| Publication Number | Publication Date |
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| WO2017214942A1 true WO2017214942A1 (fr) | 2017-12-21 |
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| PCT/CN2016/086066 WO2017214942A1 (fr) | 2016-06-16 | 2016-06-16 | Vecteur d'expression lentiviral pour améliorer l'expression du gène tctp, et ses applications |
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| WO (1) | WO2017214942A1 (fr) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002022170A1 (fr) * | 2000-09-11 | 2002-03-21 | Takeda Chemical Industries, Ltd. | Agents regulateurs de la secretion d'insuline |
| US20050221303A1 (en) * | 2001-02-13 | 2005-10-06 | Adam Telerman | Sequences involved in phenomena of tumour suppression, tumour reversion, apoptosis and/or virus resistance and their use as medicines |
| WO2014109728A1 (fr) * | 2013-01-11 | 2014-07-17 | The Scripps Research Institute | Procédés et compositions pour améliorer l'efficacité de transduction de vecteurs rétroviraux |
-
2016
- 2016-06-16 WO PCT/CN2016/086066 patent/WO2017214942A1/fr active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002022170A1 (fr) * | 2000-09-11 | 2002-03-21 | Takeda Chemical Industries, Ltd. | Agents regulateurs de la secretion d'insuline |
| US20050221303A1 (en) * | 2001-02-13 | 2005-10-06 | Adam Telerman | Sequences involved in phenomena of tumour suppression, tumour reversion, apoptosis and/or virus resistance and their use as medicines |
| WO2014109728A1 (fr) * | 2013-01-11 | 2014-07-17 | The Scripps Research Institute | Procédés et compositions pour améliorer l'efficacité de transduction de vecteurs rétroviraux |
Non-Patent Citations (3)
| Title |
|---|
| CHEN, K. ET AL.: "Long-Term Artificial Selection Reveals a Role of TCTP in Autophagy in Mammalian Cells", MOL. BIOL. EVOL., vol. 31, no. 8, 2 June 2014 (2014-06-02), pages 2194 - 2211, XP055451858 * |
| DATABASE NCBI [O] 15 March 2015 (2015-03-15), XP055451861, Database accession no. NM_001286272.1 * |
| KIM, D.K. ET AL.: "Translationally Controlled Tumor Protein Is Associated with Podocyte Hypertrophy in a Mouse Model of Type 1 Diabetes", DIABETOLOGIA, vol. 55, 4 February 2012 (2012-02-04), pages 1205 - 1217, XP035024364 * |
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