WO2017214936A1 - Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène abcb6, et ses applications - Google Patents
Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène abcb6, et ses applications Download PDFInfo
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- WO2017214936A1 WO2017214936A1 PCT/CN2016/086060 CN2016086060W WO2017214936A1 WO 2017214936 A1 WO2017214936 A1 WO 2017214936A1 CN 2016086060 W CN2016086060 W CN 2016086060W WO 2017214936 A1 WO2017214936 A1 WO 2017214936A1
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- abcb6
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- abcb6 gene
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- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
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- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 1
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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 which specifically promotes high expression of ABCB6 gene, and a construction method and application thereof.
- ABS transporter Adenosine triphosphate binding cassette transporter
- T P transports a variety of endogenous and exogenous biomolecules in the solute.
- the substrates for transport include: sugars, amino acids, metal ions, peptides, proteins, cellular metabolites and drugs.
- ABCB transport proteins are widely present in eukaryotic and prokaryotic organisms. To date, 49 ABC transporter superfamily members have been identified in the human genome, which are divided into seven subfamilies of A-G.
- ABCB1 is the first human ABC transporter to be discovered. Because multiple members are associated with multidrug resistance (MD R), the ABCB subfamily is also known as the ABC transporter MDR family. As one of them, A BCB6 is not only related to MDR, but also related to a variety of tumors. However, the function and role of ABCB6 in it has not been clarified so far, and further research is needed. However, the prior art lacks a specific slow promotion of high expression of ABCB6 gene. Viral expression vectors have made the relevant studies not well developed.
- the ABCB6 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 ABCB6 gene, thereby completing the present invention.
- the present invention provides a lentiviral expression vector which specifically promotes high expression of the ABCB6 gene, including pLVX-IR
- the ABCB6 gene cDNA sequence is inserted into the multiple cloning site sequence.
- the lentiviral expression vector constructed by inserting the cDNA sequence of ABCB6 gene into the pLVX-IRES-Puro expression vector has the advantages of high transfection efficiency and low dosage, and can stably, efficiently and stably increase ABCB6.
- the advantages of gene expression can be used as a powerful tool in the preparation and treatment of drugs for the treatment of ABCB6 gene expression in diseases such as Alzheimer's disease.
- the ABCB6 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'-GACTAGTATGGTGACTGTGGGCAACTACTG -3', ie SEQ ID NO: 1
- the sequence of the downstream primer is: 5'- GGCGGCCGCTCACCGTTCCATGGTCTGAGGC
- the ABCB6 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the ABCB6 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 the ABCB6 gene, and comprises the following steps:
- A) ABCB6 gene primer design According to the ABCB6 gene coding sequence, use Oligo
- B) obtaining the ABCB6 gene cDNA sequence PCR amplification using the upstream primer and the downstream primer to obtain a large number of ABCB6 gene coding sequences, and then adding the A tail reaction to the sequence, using T4 DNA ligase
- the ligation product is obtained by ligation to the pGM-T vector, 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 PCR.
- Preliminary identification the preliminary identification results indicate that the ABCB6 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification; the correct E. coli was identified by liquid LB medium, and the pGM-T vector carrying the ABCB6 gene cDNA sequence was extracted.
- the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of ABCB6 gene. After successful identification, it is packaged into a virus and introduced into A375 cells. After screening cells with puromycin, real-time quantitative PCR and The Western Blot technique verified the change of ABCB6 gene expression from mRNA and protein levels, respectively. The experimental results confirmed that the ABCB6 gene cDNA sequence provided by the present invention was successfully inserted into the pLV X-IRES-Puro expression vector, which can promote specifically, continuously, efficiently and stably. High expression of ABCB6 gene
- the present invention also provides a lentiviral expression vector which specifically promotes high expression of the ABCB6 gene in the preparation of a therapeutic ABCB
- the lentiviral expression vector which specifically promotes the high expression of ABCB6 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 ABCB6 gene, and can be used as a powerful tool.
- the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the A BCB6 gene, which has a good operation effect, reduces the cost of sequence synthesis, and has a low cost.
- 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.
- A375 cells were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, and 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.
- the coding sequence of the ABCB6 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 a ligation product (ABCB6-T vector), and the ligation product was transformed into competent E. coli ToplO and uniformly coated onto an ampicillin-containing LB medium plate.
- the same control group was set as 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 in 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 on the On a plate containing 100 g/mL ampicillin).
- 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.
- the bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the ABCB6 gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly treated with Spe I enzyme and Not I. 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 cell culture to take a good state of growth cells were seeded in six well, one million cells per well, with the lentiviral packaging aid kits, recombinant plasmids extracted taken pLVX-ABCB6 2 ⁇ ⁇ transfected into 293FT cells, After 48 h, the virus-containing supernatant medium was collected, and the virus solution was filtered through a 0.45 ⁇ m sieve for infection of A3 75 cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 50,000,000 IFU.
- A375 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 ABCB6 gene.
- the primer design software Oligo 7.0 was used to design bows.
- 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.
- ABCB6 gene is more than 110-fold higher than that of A375 cells, whether it is just after screening or after 20 generations of pLVX-ABCB6 cells, and the ABCB6 gene expression level of pLVX empty vector cells.
- the cDNA sequence of AB CB6 gene 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 ABCB6 gene.
- the lentiviral expression vector which specifically promotes the high expression of ABCB6 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 ABCB6 gene, and can be used as a powerful tool.
- the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the A BCB6 gene, which has a good operation effect, reduces the cost of sequence synthesis, and has a low cost.
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Abstract
L'invention concerne un vecteur d'expression lentiviral destiné à favoriser spécifiquement l'expression élevée d'un gène ABCB6, 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 ABCB6 d'un vecteur d'expression de pLVX-IRES-puro. Les sites multiples de clonage comprennent un site de découpe d'enzyme Spe I et un site de découpe d'enzyme Not I, la séquence d'ADNc du gène ABCB6 comprend un site de découpe d'enzyme Spe I, une séquence de codage du gène ABCB6 et un site de découpe de l'enzyme Not I, et la séquence d'ADNc du gène ABCB6 est insérée vers l'avant dans la séquence de sites multiples de clonage. Le vecteur d'expression lentiviral présente les avantages d'une efficacité de transfection élevée et d'une faible quantité requise, il est capable d'exprimer, de manière spécifique, en continu, avec efficacité et stabilité le gène ABCB6, et peut servir d'outil puissant pour la recherche et le développement de médicaments associés à ABCB6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2016/086060 WO2017214936A1 (fr) | 2016-06-16 | 2016-06-16 | Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène abcb6, et ses applications |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2016/086060 WO2017214936A1 (fr) | 2016-06-16 | 2016-06-16 | Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène abcb6, et ses applications |
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| Publication Number | Publication Date |
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| WO2017214936A1 true WO2017214936A1 (fr) | 2017-12-21 |
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| Application Number | Title | Priority Date | Filing Date |
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| PCT/CN2016/086060 WO2017214936A1 (fr) | 2016-06-16 | 2016-06-16 | Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène abcb6, et ses applications |
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| WO (1) | WO2017214936A1 (fr) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849623A (zh) * | 2012-11-28 | 2014-06-11 | 天津华大基因科技有限公司 | Abcb6基因突变体及其应用 |
| WO2014109728A1 (fr) * | 2013-01-11 | 2014-07-17 | The Scripps Research Institute | Procédés et compositions pour améliorer l'efficacité de transduction de vecteurs rétroviraux |
| CN104195137A (zh) * | 2014-08-11 | 2014-12-10 | 徐州医学院 | 抑制Six2基因表达的shRNA、慢病毒表达载体及其构建方法 |
-
2016
- 2016-06-16 WO PCT/CN2016/086060 patent/WO2017214936A1/fr active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849623A (zh) * | 2012-11-28 | 2014-06-11 | 天津华大基因科技有限公司 | Abcb6基因突变体及其应用 |
| WO2014109728A1 (fr) * | 2013-01-11 | 2014-07-17 | The Scripps Research Institute | Procédés et compositions pour améliorer l'efficacité de transduction de vecteurs rétroviraux |
| CN104195137A (zh) * | 2014-08-11 | 2014-12-10 | 徐州医学院 | 抑制Six2基因表达的shRNA、慢病毒表达载体及其构建方法 |
Non-Patent Citations (3)
| Title |
|---|
| "pLVX-IRES-Puro Protocol No. PT 4063 -5", VECTOR INFORMATION, 26 September 2008 (2008-09-26), pages 1 - 3, XP055450108 * |
| LIU, H. ET AL.: "Chronic Hypoxia-Induced Autophagy Aggravates the Neuropathology of Alzheimer's Disease through AMPK-mTOR Signaling in the APPSwe/PSldE9 Mouse Model", JOURNAL OF ALZHEIMER'S DISEASE, vol. 48, 31 December 2015 (2015-12-31), pages 1019 - 1032 * |
| ZHANG, CAIE ET AL.: "Construction of ABCB6 Expression Vector and Screening of Its Stable A375 Cell Line", CHINESE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, vol. 21, no. 4, 31 August 2012 (2012-08-31), pages 351 - 354 * |
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