CN102766654A - Slow virus expression vector specially promoting high expression of liver cell CYP1A2 gene, and construction method and application thereof - Google Patents
Slow virus expression vector specially promoting high expression of liver cell CYP1A2 gene, and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a slow virus expression vector specially promoting high expression of a liver cell CYP1A2 gene. The expression vector comprises a basic sequence of a pLVX-AcGFP-C1 expression vector, a resistance gene sequence, multiple cloning site sequences, a promoter sequence and a CYP1A2 gene cDNA sequence. The multiple cloning sites comprise an XhoI restriction site and an XmaI restriction site; the CYP1A2 gene cDNA sequence comprises an XhoI restriction site, a CYP1A2 gene coding sequence and an XmaI restriction site; and the CYP1A2 gene cDNA sequence is inserted into the multiple cloning site sequence in a forward direction. The slow virus expression vector provided by the invention has advantages of high transfection efficiency, small dosage, specificity, sustainability, efficiency, stable expression of the CYP1A2 gene, and can be applied to research and development of CYP1A2 associated medicaments as a powerful tool.
Description
Technical field
The invention belongs to gene engineering technology field, relate in particular to slow virus expression vector and the construction process and the application of special promotion liver cell CYP1A2 gene high expression.
Background technology
Cytochrome P 450 enzymes (Cytochrome P450 is one type of important mixed function oxidase system CYP450), mainly be distributed on the endoplasmic reticulum of organism with plastosome in, participate in the biotransformation of many endogenous and exogenous material.CYP1A2 belongs to the CYP1A subtribe in the CYP450 family, by the CYP1A2 genes encoding, mainly is present in the liver, accounts for 13% of liver CYP enzyme total amount, is only second to CYP3A and CYP2C subfamily, occupies the 3rd of each CYP enzyme content of liver.CYP1A2 also has a small amount of distribution in tissues such as enteron aisle, brain, lung.CYP1A2 participates in the metabolic process of more than 20 kind of medicines such as theine, warfarin, theophylline, Proprasylyte, mexiletine, verapamil, nifedipine, tacrine, also is responsible for the hydroxylation reaction of some endogenous hormones simultaneously.In addition, CYP1A2 also plays an important role in the reactivation process in the body of some procarcinogen and preceding toxicant.
In the prior art; Ruan Hao has been connected to the CYP1A2 gene cDNA fragment on the pFastBac carrier; And it is packaged into baculovirus postoperative infection sf9 cell; Obtained expressing the sf9 cell of CYP1A2 gene, this cell can be used in the in vitro study drug metabolism, but because of not being that human archeocyte is not suitable for the correlative study of allogenic material to people's toxicity.Still there is not the carrier that can in human archeocyte, continue, stablize and efficiently express the CYP1A2 gene in the prior art.
Summary of the invention
For solving the problem that exists in the prior art; The contriver has carried out a large amount of exploratory developments at aspects such as the selection of carrier, recombination to construct methods; Against expectation find; The CYP1A2 gene cDNA sequence that will comprise Xho I restriction enzyme site and Xma I restriction enzyme site inserts in the MCS of pLVX-AcGFP1-C1 expression vector can successfully make up the slow virus expression vector of special promotion people source liver cell CYP1A2 gene high expression, thereby accomplishes the present invention.
The present invention provides a kind of slow virus expression vector of special promotion liver cell CYP1A2 gene high expression, comprises basic sequence, resistant gene sequence, MCS sequence, promoter sequence and the CYP1A2 gene cDNA sequence of pLVX-AcGFP-C1 expression vector; Said MCS comprises Xho I restriction enzyme site and Xma I restriction enzyme site; Said CYP1A2 gene cDNA sequence comprises Xho I restriction enzyme site, CYP1A2 gene coded sequence and Xma I restriction enzyme site, and said CYP1A2 gene cDNA sequence forward inserts in the said MCS sequence.
Adopt technique scheme; The slow virus expression vector that CYP1A2 gene cDNA sequence insertion pLVX-AcGFP1-C1 expression vector establishment provided by the invention obtains has the transfection efficiency height; Consumption is few; Sustainable, efficiently, stably improve the advantage of CYP1A2 genetic expression, can be used as strong tool applications in the research and development of preparation treatment CYP1A2 abnormal gene expression relative disease medicine.
As further improvement of the present invention; Said CYP1A2 gene coded sequence obtains through pcr amplification; The PCR primer comprises upstream primer and downstream primer; The sequence of said upstream primer is: 5 '-CCGCTCGAGATGGCATTGTCCCAGTCTGTTC-3 ' (SEQ ID NO:1), the sequence of said downstream primer is: 5 '-ACCCGGGTCAGTTGATGGAGAAGCGCAGC-3 ' (SEQ ID NO:2).Adopt above-mentioned PCR primer sequence, can amplify the CYP1A2 gene coded sequence through PCR, and can successfully be inserted into continuous expression CYP1A2 gene in the pLVX-AcGFP1-C1 expression vector, reduced the synthetic expense of sequence, cost is lower.
Accordingly, the present invention also provides the construction process of the slow virus expression vector of special promotion liver cell CYP1A2 gene high expression, comprises the steps:
A) CYP1A2 gene primer design: according to the CDS sequence of CYP1A2 gene; Choose 5 '-CCGCTCGAGATGGCATTGTCCCAGTCTGTTC-3 ' (SEQ ID NO:1) as upstream primer after using Oligo 7 to analyze; Choose 5 '-ACCCGGGTCAGTTGATGGAGAAGCGCAGC-3 ' (SEQ ID NO:2) as downstream primer, synthetic then said upstream primer and said downstream primer; Said upstream primer and said downstream primer do not have primer dimer, and the annealing temperature gap is less;
B) acquisition of CYP1A2 gene cDNA sequence: carry out pcr amplification with said upstream primer and said downstream primer; Obtain a large amount of CYP1A2 gene coded sequences; After then this sequence being added the A end reaction; Be connected to the T4DNA ligase enzyme and obtain connecting product on the pGM-T carrier, should connect product and be transformed in the competence bacillus coli DH 5 alpha, evenly be applied to and contain on the penbritin LB culture medium flat plate; Picking positive monoclonal bacterium colony cultivate to be preserved the bacterium liquid performing PCR preliminary evaluation of going forward side by side, and preliminary evaluation presentation of results CYP1A2 gene cDNA sequence is inserted the evaluation of checking order of successful bacterium liquid; Identify correct intestinal bacteria with the order-checking of liquid LB culture medium culturing; And extracting is wherein with the pGM-T carrier of CYP1A2 gene cDNA sequence; With cutting with Xma I enzyme again after the restriction enzyme Xho I enzyme switchback receipts; Electrophoresis, cut glue and reclaim the big or small fragment of about 1600bp, this fragment is the CYP1A2 gene cDNA sequence;
C) structure and the evaluation of the lentiviral vectors of special promotion liver cell CYP1A2 gene high expression: extract plasmid pLVX-AcGFP1-C1; With cutting with Xma I enzyme again after the restriction enzyme Xho I enzyme switchback receipts; Electrophoresis, cut glue and reclaim carrier; With T4DNA ligase said CYP1A2 gene cDNA sequence is connected in the pLVX-AcGFP1-C1 expression vector again; Obtain connecting product, should connect product and be transformed in the competence bacillus coli DH 5 alpha, evenly be applied to and contain on the penbritin LB culture medium flat plate; Picking positive monoclonal bacterium colony cultivate to be preserved the bacterium liquid performing PCR preliminary evaluation of going forward side by side, and preliminary evaluation presentation of results CYP1A2 gene cDNA sequence is inserted the evaluation of checking order of successful bacterium liquid;
D) extracting of the lentiviral vectors of special promotion liver cell CYP1A2 gene high expression: sequencing result is confirmed that the CYP1A2 gene cDNA sequence inserts successful bacterium liquid amplification cultivation; Recombinant plasmid is carried out extracting, obtain the slow virus expression vector of special promotion liver cell CYP1A2 gene high expression.
The present invention utilizes genetic engineering technique to make up the slow virus expression vector of special promotion liver cell CYP1A2 gene high expression; After evaluation makes up successfully; Be packaged into virus infection L-02 liver cell; Behind the tetracycline screening cell; Use real-time fluorescence quantitative PCR and Western Blot technology respectively from the variation of mRNA and protein level checking CYP1A2 genetic expression, experimental result proves that CYP1A2 gene cDNA sequence provided by the invention successfully is inserted in the pLVX-AcGFP1-C1 expression vector, can special, lasting, efficiently, stably promote the CYP1A2 gene high expression.
The present invention also provides the purposes of slow virus expression vector in the medicine of preparation treatment CYP2E1 abnormal gene expression relative disease of special promotion liver cell CYP1A2 gene high expression.
Compared with prior art; Beneficial effect of the present invention is following: the slow virus expression vector of special promotion liver cell CYP1A2 gene high expression provided by the invention has the transfection efficiency height; Consumption is few; Can special, lasting, efficiently, stably promote the advantage of CYP1A2 gene high expression, can be used as strong tool applications in drug research relevant and exploitation with CYP1A2; The present invention also provides the construction process of the slow virus expression vector of special promotion liver cell CYP1A2 gene high expression, and operating effect is good, has reduced the synthetic expense of sequence, and cost is lower.
Description of drawings
Fig. 1 is the plasmid map of pLVX-AcGFP1-C1 expression vector.
Fig. 2 is the synoptic diagram as a result of 1A2-T carrier bacterium liquid PCR.
Fig. 3 is the synoptic diagram as a result of pLVX-CYP1A2 carrier bacterium liquid PCR.
Fig. 4 is a pLVX-CYP1A2 carrier sequencing result synoptic diagram.
Fig. 5 for tetracycline screening cell after fluorescence quantitative PCR detection synoptic diagram as a result.
Fig. 6 for tetracycline screening cell after Western blot detected result synoptic diagram.
Fig. 7 is tetracycline screening cell cultured continuously Western blot detected result synoptic diagram after 15 generations.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment the present invention is done further explanation.
The L-02 liver cell is available from Shanghai school of life and health sciences cell resource center; The 293FT cell is available from Invitrogen company; Premix PrimeSTAR HS enzyme, slow virus expression vector pLVX-AcGFP1-C1, virus packing auxiliary reagent box, Lenti-X GoStix test kit are all available from Takara company; RNeasy Mini Kit is available from Qiagen company, and the pGM-T carrier is available from sky root company, and Endo-Free Plasmid Mini Kit II is available from Omega bio-tek company.
The design of embodiment one CYP1A2 gene primer.
Encoding sequence (GenBank NM_000761.3) according to the CYP1A2 gene; Use Oligo7 that it is analyzed; Seek upstream primer and downstream primer (requirement does not have primer dimer as far as possible and the annealing temperature gap is less); 5 ' end at upstream primer and downstream primer adds protection base and restriction enzyme site Xho I and Xma I respectively then, and the primer sequence that design obtains is as shown in table 1.The PCR primer of design is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The PCR primer sequence of table 1CYP1A2 gene
The structure of the lentiviral vectors of embodiment two special promotion liver cell CYP1A2 gene high expressions.
After the dilution of synthetic primer; Increase with the encoding sequence of Premix PrimeSTAR HS enzyme to the CYP1A2 gene; After then it being added the A end reaction after electrophoresis reclaims; Be connected to the T4DNA ligase enzyme and obtain connecting product (1A2-T carrier) on the pGM-T carrier; Should connect product is transformed in the competence bacillus coli DH 5 alpha; Evenly be applied to and contain on the penbritin LB culture medium flat plate; In 37 ℃ of cultivation 12h, negative control group 1 (competent cell is uniformly coated on the flat board that does not contain penbritin), negative control group 2 (competent cell is uniformly coated on the flat board that contains 100 μ g/ml penbritins), positive controls 1 (the connection product of double digestion empty carrier is uniformly coated on the flat board that contains 100 μ g/ml penbritins), positive controls 2 (empty carrier is uniformly coated on the flat board that contains 100 μ g/mL penbritins) are set simultaneously.Experimental group has grown single bacterium colony, and negative control group 1 has grown bacterium colony; Negative control group 2, positive controls 1, positive controls 2 do not grow bacterium colony.
After the single bacterium colonies of 8 of pickings are cultivated and preserved from experimental group, respectively get 0.5 μ L nutrient solution, carry out pcr amplification with the primer of CYP1A2 gene and come preliminary evaluation.The result is as shown in Figure 2, shows that the nutrient solution of 8 single bacterium colonies all can successfully amplify the CYP1A2 gene, then recombinant vectors is delivered to Shanghai and gives birth to the order-checking of worker company.
Get the correct bacterium of sequencing result; Place liquid LB substratum to cultivate 14h; Extract the 1A2-T carrier then; 1A2-T carrier and pLVX-AcGFP1-C1 carrier are carried out enzyme with Xho I enzyme earlier to be cut; Carry out enzyme with Xma I enzyme again after electrophoresis reclaims and cut, carry out electrophoresis again and reclaim, will reclaim product at last and connect with the T4DNA ligase enzyme; Be transformed into once more in the competence bacillus coli DH 5 alpha; Evenly be applied to and contain on the penbritin LB culture medium flat plate, cultivate 12h in 37 ℃, negative control group 1 (competent cell is uniformly coated on the flat board that does not contain penbritin), negative control group 2 (competent cell is uniformly coated on the flat board that contains 100 μ g/ml penbritins), positive controls 1 (the connection product of double digestion empty carrier is uniformly coated on the flat board that contains 100 μ g/ml penbritins), positive controls 2 (empty carrier is uniformly coated on the flat board that contains 100 μ g/mL penbritins) are set simultaneously.Experimental group has grown single bacterium colony, and negative control group 1 has grown bacterium colony; Negative control group 2, positive controls 1, positive controls 2 do not grow bacterium colony.
After the single bacterium colonies of 8 of pickings are cultivated and preserved from experimental group, respectively get 0.5 μ L nutrient solution, carry out pcr amplification with the primer of CYP1A2 gene and come preliminary evaluation.The result is as shown in Figure 3, shows that wherein 6 nutrient solutions all can successfully amplify the CYP1A2 gene, then these recombinant vectors bacterium liquid is delivered to Shanghai and gives birth to the order-checking of worker company, and sequencing result is as shown in Figure 4, conforms to fully with expection.
The recombinant plasmid bacterium liquid of preserving before taking out; Getting 20 μ l is inoculated in the 15ml LB substratum (containing 100 μ g/ml penbritins); 37 ℃, 300rpm cultivates 16h, carries out extracting recombinant plasmid pLVX-CYP1A2 with Endo-Free Plasmid Mini Kit II; Survey its purity and concentration, the result is as shown in table 2.
The purity of table 2 recombinant plasmid and concentration
Embodiment three slow viruss packing.
Cultivate the 293FT cell, get the growth conditions good cell and be inoculated in six holes, every hole 10
6Individual cell; Pack the auxiliary reagent box with slow virus; Get extractive recombinant plasmid pLVX-CYP1A22 μ g transfection to the 293FT cell, collect the supernatant substratum that contains virus behind the 48h, filter viral liquid with the sieve of 0.45 μ m; Be used to infect the L-02 liver cell, the titre that Lenti-X GoStix test kit detects virus is 5 * 10
6～5 * 10
7IFU.
Embodiment four lentiviruses transduction L-02 liver cells.
Inoculate the L-02 liver cell in 6 orifice plates, every hole 10
6Individual cell, cell density is about 50% behind the 12h, gets viral liquid respectively, and with 10 times of virus dilution of RPMI-1640 perfect medium, adding polybrene (polybrene) to final concentration again is 8 μ g/mL.Remove the substratum in 6 orifice plates; Add the RPMI-1640 perfect medium (containing 10% foetal calf serum) that contains virus; Discard the RPMI-1640 perfect medium that contains virus behind the 24h, change fresh RPMI-1640 perfect medium, the tetracycline with 0.5 μ g/ml concentration behind the 24h screens cell.Screening 10d, every separated 3d changes substratum once, and constantly increases concentration to the 1.00 μ g/ml of tetracycline.
Embodiment five fluorescence quantitative PCR detection CYP1A2 gene expression amounts.
According to GAPDH and CYP1A2 gene mRNA sequence, utilize primer-design software Oligo 7.0 design primers, the result is as shown in table 3.
Table 3 fluorescence quantification PCR primer sequence
Inoculate L-02 liver cell, pLVX empty carrier contrast L-02 cell, pLVX-CYP1A2 high expressing cell to 6 orifice plate respectively.When cell density reaches 80%-90%, extract the total RNA that respectively organizes cell, utilize PrimeScrip RT reagent Kit the mRNA rt to be cDNA, the rt condition with RNeasy Mini Kit: 37 ℃, 15min; 85 ℃, 5s; 4 ℃, ∞.After reverse transcription finishes, add the RNase Free dH of 90 μ l
2O dilutes cDNA, and-20 ℃ of preservations are used so that the back is detected.Getting the cDNA 1 μ l that respectively organizes cell is template, is confidential reference items with GAPDH, and real-time fluorescence quantitative PCR (QPCR) detects the CYP1A2 relative expression quantity; Reaction conditions is set: 95 ℃ of 30s, 1 circulation, 54 ℃ of 30s 40 circulations; 95 ℃ of 5s; 60 ℃ of 1min, 95 ℃ of 15s utilize SYBR Primescript RT-PCR Kit to detect and respectively organize cell CYP2E1 gene relative expression quantity.PLVX-CYP1A2 cell cultured continuously after 15 generations, is repeated above experiment.Result after gathering is as shown in Figure 5; Can see from Fig. 5; No matter be just to have screened; Still cultivated the pLVX-CYP1A2 cell after 15 generations, its CYP1A2 expression of gene amount all has the rising about 300 times than the L-02 cell, and the unloaded somatic CYP1A2 gene expression amount of pLVX is compared with the L-02 cell and do not changed basically; Explain that CYP1A2 gene cDNA sequence provided by the invention successfully is inserted in the pLVX-AcGFP1-C1 expression vector, can special, lasting, efficiently, stably promote the CYP1A2 gene high expression.
Embodiment six Western blot detect CYP1A2 protein expression amount.
Get L-02 liver cell, pLVX empty carrier contrast L-02 cell, 1 bottle of (25cm of pLVX-CYP1A2 high expressing cell respectively
2Tissue Culture Flask), removes substratum, wash 3 times, add 200 μ l cell pyrolysis liquids with cold PBS; And scrape with cell the cracked cell is scraped from the bottle wall fast, collect in the EP pipe of albumen to 500 μ l, 4 ℃ are continued cracking 30min, 12000rpm; 4 ℃ of centrifugal 20min add 5 * SDS-PAGE Sample Loading Buffer, 100 ℃ of sex change 5min at last; Carry out the 12%SDS-polyacrylamide gel electrophoresis, the albumen electricity is gone on the pvdf membrane, 5% skim-milk sealing 1h; Add CYP1A2 antibody, GAPDH antibody respectively, 4 ℃ of incubated overnight.TBST washes film 3 times, and every 10min once adds two again and resists, and incubated at room 1h, TBST wash film 3 times, and every 10min once.Carry out imaging analysis after adding Western blot chemical illuminating reagent, the result is as shown in Figure 6.With GAPDH is confidential reference items, and the result shows that the CYP1A2 expressing quantity of pLVX-CYP1A2 cell has obvious rising, is about 5.41 times of normal L-02 cell, and expressing quantity significantly raises.
The cultured continuously cell is after 15 generations; Extract pLVX-CYP1A2 cell and cellular control unit thereof respectively; Western blotting analyzes the CYP1A2 expressing quantity, and the result is as shown in Figure 7, and is approaching with result (Fig. 6) before; This explanation pLVX-CYP1A2 cell construction success, can continue, efficiently, high expression level CYP1A2 gene stably.
Above content is to combine concrete preferred implementation to the further explain that the present invention did, and can not assert that practical implementation of the present invention is confined to these explanations.For the those of ordinary skill of technical field under the present invention, under the prerequisite that does not break away from the present invention's design, can also make some simple deduction or replace, all should be regarded as belonging to protection scope of the present invention.
Claims (4)
1. the slow virus expression vector of a special promotion liver cell CYP1A2 gene high expression is characterized in that: the basic sequence, resistant gene sequence, MCS sequence, promoter sequence and the CYP1A2 gene cDNA sequence that comprise the pLVX-AcGFP-C1 expression vector; Said MCS comprises Xho I restriction enzyme site and Xma I restriction enzyme site; Said CYP1A2 gene cDNA sequence comprises Xho I restriction enzyme site, CYP1A2 gene coded sequence and Xma I restriction enzyme site, and said CYP1A2 gene cDNA sequence forward inserts in the said MCS sequence.
2. the slow virus expression vector of special promotion liver cell CYP1A2 gene high expression according to claim 1; It is characterized in that: said CYP1A2 gene coded sequence obtains through pcr amplification; The PCR primer comprises upstream primer and downstream primer; The sequence of said upstream primer is: 5 '-CCGCTCGAGATGGCATTGTCCCAGTCTGTTC-3 ' (SEQ ID NO:1), the sequence of said downstream primer is: 5 '-ACCCGGGTCAGTTGATGGAGAAGCGCAGC-3 ' (SEQ ID NO:2).
3. the construction process of the slow virus expression vector of special promotion liver cell CYP1A2 gene high expression according to claim 2 is characterized in that: comprise the steps:
A) CYP1A2 gene primer design: according to the CDS sequence of CYP1A2 gene; Choose 5 '-CCGCTCGAGATGGCATTGTCCCAGTCTGTTC-3 ' (SEQ ID NO:1) as upstream primer after using Oligo 7 to analyze; Choose 5 '-ACCCGGGTCAGTTGATGGAGAAGCGCAGC-3 ' (SEQ ID NO:2) as downstream primer, synthetic then said upstream primer and said downstream primer;
B) acquisition of CYP1A2 gene cDNA sequence: carry out pcr amplification with said upstream primer and said downstream primer; Obtain a large amount of CYP1A2 gene coded sequences; After then this sequence being added the A end reaction; Be connected to the T4 dna ligase and obtain connecting product on the pGM-T carrier, should connect product and be transformed in the competence bacillus coli DH 5 alpha, evenly be applied to and contain on the penbritin LB culture medium flat plate; Picking positive monoclonal bacterium colony cultivate to be preserved the bacterium liquid performing PCR preliminary evaluation of going forward side by side, and preliminary evaluation presentation of results CYP1A2 gene cDNA sequence is inserted the evaluation of checking order of successful bacterium liquid; Identify correct intestinal bacteria with the order-checking of liquid LB culture medium culturing; And extracting is wherein with the pGM-T carrier of CYP1A2 gene cDNA sequence; With cutting with Xma I enzyme again after the restriction enzyme Xho I enzyme switchback receipts; Electrophoresis, cut the fragment that glue reclaims about 1600 bp size, this fragment is the CYP1A2 gene cDNA sequence;
C) structure and the evaluation of the lentiviral vectors of special promotion liver cell CYP1A2 gene high expression: extract plasmid pLVX-AcGFP1-C1; With cutting with Xma I enzyme again after the restriction enzyme Xho I enzyme switchback receipts; Electrophoresis, cut glue and reclaim carrier; With T4 DNA ligase said CYP1A2 gene cDNA sequence is connected in the pLVX-AcGFP1-C1 expression vector again; Obtain connecting product, should connect product and be transformed in the competence bacillus coli DH 5 alpha, evenly be applied to and contain on the penbritin LB culture medium flat plate; Picking positive monoclonal bacterium colony cultivate to be preserved the bacterium liquid performing PCR preliminary evaluation of going forward side by side, and preliminary evaluation presentation of results CYP1A2 gene cDNA sequence is inserted the evaluation of checking order of successful bacterium liquid;
D) extracting of the lentiviral vectors of special promotion liver cell CYP1A2 gene high expression: sequencing result is confirmed that the CYP1A2 gene cDNA sequence inserts successful bacterium liquid amplification cultivation; Recombinant plasmid is carried out extracting, obtain the slow virus expression vector of special promotion liver cell CYP1A2 gene high expression.
4. according to each described special slow virus expression vector purposes in the medicine of preparation treatment CYP2E1 abnormal gene expression relative disease that promotes liver cell CYP1A2 gene high expression in the claim 1 to 3.
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| CN105296519A (en) * | 2015-09-17 | 2016-02-03 | 长江大学 | Construction and application of mouse CRABP2 gene eukaryotic expression vector |
| WO2017214945A1 (en) * | 2016-06-16 | 2017-12-21 | 毛侃琅 | Lentiviral expression vector for improving hepcidin gene expression level and application thereof |
| WO2017214830A1 (en) * | 2016-06-14 | 2017-12-21 | 石庆学 | Lentiviral vector for specifically promoting high expression of pd-1 gene, and applications thereof |
| WO2017214829A1 (en) * | 2016-06-14 | 2017-12-21 | 石庆学 | Lentiviral expression vector for specifically promoting high expression of pd-l1 gene, and applications thereof |
| CN108949827A (en) * | 2018-07-20 | 2018-12-07 | 深圳市南山区疾病预防控制中心 | The special shRNA Lentiviral for inhibiting lung cancer PPARd gene expression and its construction method and application |
| WO2020000474A1 (en) * | 2018-06-29 | 2020-01-02 | 深圳市博奥康生物科技有限公司 | Recombinant vector promoting pou5f1 protein overexpression and construction method therefor |
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| CN105296519A (en) * | 2015-09-17 | 2016-02-03 | 长江大学 | Construction and application of mouse CRABP2 gene eukaryotic expression vector |
| WO2017214830A1 (en) * | 2016-06-14 | 2017-12-21 | 石庆学 | Lentiviral vector for specifically promoting high expression of pd-1 gene, and applications thereof |
| WO2017214829A1 (en) * | 2016-06-14 | 2017-12-21 | 石庆学 | Lentiviral expression vector for specifically promoting high expression of pd-l1 gene, and applications thereof |
| WO2017214945A1 (en) * | 2016-06-16 | 2017-12-21 | 毛侃琅 | Lentiviral expression vector for improving hepcidin gene expression level and application thereof |
| WO2020000474A1 (en) * | 2018-06-29 | 2020-01-02 | 深圳市博奥康生物科技有限公司 | Recombinant vector promoting pou5f1 protein overexpression and construction method therefor |
| CN108949827A (en) * | 2018-07-20 | 2018-12-07 | 深圳市南山区疾病预防控制中心 | The special shRNA Lentiviral for inhibiting lung cancer PPARd gene expression and its construction method and application |
| CN108949827B (en) * | 2018-07-20 | 2022-03-25 | 深圳市南山区疾病预防控制中心 | shRNA lentiviral expression vector for specifically inhibiting lung cancer PPARd gene expression and construction method and application thereof |
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