CN110982842B - Design and application of lentivirus expression vector - Google Patents

Design and application of lentivirus expression vector Download PDF

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CN110982842B
CN110982842B CN201911401286.3A CN201911401286A CN110982842B CN 110982842 B CN110982842 B CN 110982842B CN 201911401286 A CN201911401286 A CN 201911401286A CN 110982842 B CN110982842 B CN 110982842B
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lentivirus
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expression vector
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武兴康
鲁玉凡
韩晨晨
秦雪梅
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Guangzhou Jinsheng Biotechnology Co ltd
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Shanxi University
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Abstract

The invention belongs to the fields of molecular biology and cell biology, and particularly relates to design and application of a lentivirus expression vector. The invention designs a vector, and CMV enhancer and CMV promoter are used as transcription initiation elements of inserted genes; EF1a-core promoter is used as a transcription initiation element of a selection marker puromycin and a green fluorescent protein GFP, and a puromycin resistance gene and a GFP sequence are connected in series through T2A for expression; the lentivirus vector is used as the vector framework, and the two elements are inserted into the framework vector and are reasonably designed and optimized. The slow virus vector designed by the invention can efficiently express inserted genes, and the expression efficiency is superior to that of the prior vector with the same type of characteristics.

Description

Design and application of lentivirus expression vector
Technical Field
The invention belongs to the fields of molecular biology and cell biology, and particularly relates to design and application of a lentivirus expression vector.
Background
Lentiviral vectors (Lentiviral vectors) are viral vector systems which have been modified based on HIV-1 virus and which are capable of efficiently introducing genes of interest into primary cells or cell lines of animals and humans. Lentiviral vector-mediated gene expression is sustained and stable because the inserted gene integrates into the host cell genome and divides as the cell genome divides. In addition, lentiviruses have a wide host range, can infect dividing and non-dividing cells, can infect almost all types of cells, and are particularly suitable for cells with low transfection efficiency of plasmid vectors.
Currently, there are various forms of lentiviral expression vectors, all designed with a lentiviral vector backbone loaded with different gene control elements. When gene overexpression is carried out, the lentivirus expression vector consisting of different elements can be selected through different experimental purposes. The pCDH series lentivirus vector with double promoters developed by the American System Biosciences company has the vector size not more than 9000bp, allows the insertion of a gene with a larger fragment, has an antibiotic resistance gene and a fluorescent protein double-screening gene, can obtain a stable-transfected cell strain through antibiotic screening and detect the transfection efficiency through the fluorescent protein, and has unique advantageous characteristics so that the pCDH series lentivirus vector becomes one of mainstream lentivirus expression vectors. However, the inventors found in experiments that the expression level of the inserted gene of the pCDH series vector is not very high, and cannot meet some experimental requirements. However, the same type of lentiviral expression vector in the market does not have the advantages and characteristics of the pCDH vector. Therefore, it is necessary to design a lentiviral expression vector having three characteristics of small vector, dual selection marker, high level expression of inserted gene, etc.
Disclosure of Invention
Aiming at the problems, the invention provides design and application of a lentivirus expression vector.
In order to achieve the purpose, the invention adopts the following technical scheme:
a nucleotide sequence of the lentivirus expression vector is SEQ ID NO.1.
Furthermore, the map of the vector is shown in figure 1, and the sequence is shown in SEQ ID NO.1.
Further, the sequence comprises the following vector elements:
ampicillin resistance gene: a sequence allowing the vector to be screened in e.coli using Ampicillin;
PUC Ori: coli, a sequence that allows high copy replication of the vector in e.coli;
RSV promoter: sequences that promote transcription of viral RNA in packaging cells;
d.5' LTR-DeltaU3: an integration site sequence for integration of the viral genome into the host genome;
e.3' LTR-DeltaU3: an integration site sequence for integration of the viral genome into the host genome; a transcription termination sequence of the viral RNA in the packaging cell;
f. Ψ viral RNA packaging as the viral signal sequence;
RRE: the Rev protein response element sequence of HIV-1, allowing viral RNA to exit the nucleus through the Rev protein pathway;
h.CPPT: HIV-1 is rich in purine fragment sequence, promote the virus RNA to transfer into the host cell nucleus in the process of virus infection of host cell;
CMV enhancer/promoter: enhancer and promoter sequences that promote efficient expression of the gene of interest;
BamHI/SalI/XhoI/XbaI: the restriction enzyme site sequence is convenient for inserting exogenous genes;
ef-1a core promoter: a promoter sequence that facilitates expression of the screening gene;
screening expression sequences of puromycin and GFP genes, wherein EGFP is T2A and Puro;
WPRE: modifying element sequences after viral transcription to enhance the stability of viral RNA in packaging cells; the application of a lentivirus vector, wherein the vector is used for high-efficiency expression of inserted genes;
n.SV40 early pA: further enhancing the transcription termination sequence of the viral RNA in the packaging cell.
The application of a lentivirus expression vector can be applied to the following five aspects based on the nucleotide sequence of the lentivirus expression vector: the target gene can be inserted, the target gene can be packaged into a slow virus infection target cell, the target gene can be expressed and inserted at a high level, a stable transfer cell strain can be obtained by puromycin screening, and the transfection efficiency can be detected by GFP.
A method for using a lentiviral expression vector comprises the following steps:
(1) Inserting a target gene: obtaining a segment inserted with a target gene by a PCR technology, and inserting the target gene segment into a multiple cloning site of a lentivirus expression vector by adopting an enzyme digestion connection method;
(2) Packaging into lentivirus-infected cells of interest:
the lentivirus expression vector is transcribed in 293T cells to generate virus genome RNA, the packaging vector is transcribed and translated in 293T cells to generate virus proteins, then the virus genome RNA and the virus proteins are assembled into lentiviruses in the cells, the lentiviruses are released from the 293T cells to a culture medium, the lentiviruses are collected, and the lentiviruses and target cells are incubated together to infect the target cells;
(3) Obtaining a stable cell strain by screening the cells infected by the lentivirus in the step (2) through puromycin: after the target cells are infected by the lentivirus for 48 hours, adding puromycin to 2-10 mu g/mL, culturing for more than 3 generations until the cells do not die any more, obtaining stable cell strains, collecting the stable cell strains, and detecting the expression quantity of the inserted target genes by western blot;
(4) High level expression of the inserted gene of interest: through western blot detection, the expression of a target gene in a target cell or a stably transformed cell infected by the lentivirus for 48 hours is judged to determine whether the target gene is expressed at a high level;
(5) Transfection efficiency was measured by GFP: after the lentivirus transfects 293T cells or the packaged lentivirus infects target cells, the transfection efficiency can be judged by observing the intensity of GFP through a fluorescence microscope.
Furthermore, the restriction sites in the restriction and ligation are BamHI, salI, xhoI and XbaI.
Further, the packaging vector may be selected from psPAX2 and PMD2.G, and may also be selected from REV, VSV-G and PMDL.
Further, the designed vector is applied to the high-efficiency expression of the target gene in mammalian cells.
Compared with the prior art, the invention has the following advantages: the invention designs a lentivirus expression vector, which is named as pRSV-CMV-Flag-EF1-Puro-T2A-GFP and has a puromycin resistance gene and a GFP double screening gene, a stable transfer cell strain can be obtained by puromycin screening, the transfection efficiency can be detected by GFP, and compared with a lentivirus expression vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro with the same characteristics, the lentivirus expression vector can efficiently express an inserted gene.
1. The vector designed by the invention adopts CMV enhancer and CMV promoter as transcription initiation elements of the inserted gene, and can express the inserted gene at a high level.
2. The carrier designed by the invention adopts puromycin resistance gene and GFP as screening genes, is beneficial to obtaining stable cell strains, and can monitor transfection efficiency at any time by using GFP.
3. The vector designed by the invention has small length, and after a gene with larger fragment is inserted, the vector has small influence on the transfection efficiency of recombinant plasmid and the virus packaging efficiency, and is favorable for overexpression of protein with larger molecular weight.
4. The vector designed by the invention realizes the high-efficiency expression of the inserted gene in a lentivirus transfection mode on the basis of the benefits 1, 2 and 3.
Drawings
FIG. 1 is a structural map of pRSV-CMV-Flag-EF1-Puro-T2A-GFP vector;
FIG. 2 is a western blot assay for overexpression of the inserted gene Insert in HCT116 cells using lentiviral expression vectors pCDH-CMV-MCS-EF1-copGFP-T2A-Puro (pCDH) and pRSV-CMV-Flag-EF1-Puro-T2A-GFP (pRSV), respectively;
FIG. 3 is a graph of the relative quantification of the western blot bands of FIG. 2, (A) is the fold of overexpressed Insert in cells overexpressing the inserted gene relative to unloaded cells; (B) Total Insert in the cells to overexpress the gene relative to the fold in the unloaded cells.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Design and construction of vectors:
according to the vector map (FIG. 1), the individual elements required for the vector were arranged in the order of the map, and the pRSV-CMV-Flag-EF1-Puro-T2A-GFP vector was designed. According to the names of the elements, the sequence of each element is inquired in an NCBI database, and the sequence of each element is copied into the corresponding position according to the vector map information to form the sequence information (SEQ ID NO. 1) of the vector. Then, the nucleic acid sequence information of the vector was delivered to a third-party company, synthesized according to the vector sequence, and the vector construction was completed.
Example 2
The vector is used, and the vector designed according to example 1 is inserted with four restriction enzyme sites (BamHI, salI, xhoI, xbaI) and two Flag tags behind CMV promoter, and can express inserted gene expression vectors of N-terminal Flag, C-terminal Flag, two-terminal Flag or other tags according to requirements. The connection of the recombinant vector suggests various DNA cloning and assembly technologies developed by adopting the principles of atypical enzyme digestion connection, PCR, homologous recombination, single-strand annealing splicing and the like.
After the recombinant vector is successfully constructed, the recombinant lentiviral vector and a packaging vector are transfected into 293T cells together, wherein the packaging vector can be psPAX2 and PMD2.G, and can also be REV, VSV-G and PMDL. After 72 hours of transfection, the virus was collected, cells of interest were transfected, and the transfection efficiency was observed by a fluorescence microscope. Under the condition of higher cell transfection efficiency, puromycin with corresponding concentration is added according to a cell line, the cells are cultured for more than 3 generations until the cells do not die any more, and western blot is used for detecting the expression quantity of the inserted gene.
Example 3
In order to verify the advantages of the lentiviral vector designed by the invention in the aspect of high-efficiency expression of inserted genes, the vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro (pCDH) with the most popular characteristics of the same type is selected for comparison.
Obtaining a sequence of an inserted gene Insert from HCT116 of a target cell by RT-PCR, adding an HA tag at the N end of the Insert by a PCR technology to obtain an HA-Insert sequence, and inserting the HA-Insert into a multiple cloning site of pCDH-CMV-MCS-EF1-copGFP-T2A-Puro (pCDH) and pRSV-CMV-Flag-EF1-Puro-T2A-GFP (pRSV) by adopting a Gibson splicing method.
After the sequencing is correct, transfecting 293T cells with a lentivirus expression vector and a packaging vector (psPAX 2, PMD2. G), after transfecting for 72h, collecting the virus, concentrating the virus with PEG8000/NaCl, transfecting HCT116 cells, after transfecting for 48h, taking a part of the cells for detecting the protein expression level, adding puromycin into the rest cells to 2 mu g/mL, screening for more than 3 generations until the cells do not die any more, obtaining a stable cell strain, and collecting the cell detection protein expression level.
Protein expression levels were measured using western blots, overexpression levels of the inserted gene Insert in HCT116 cells were measured using HA antibodies, and overall expression levels of the inserted gene Insert in HCT116 cells were measured using Insert antibodies.
As shown in the western blot detection chart of FIG. 2, the expression level of the Insert gene Insert was significantly higher than that of pCDH when pRSV was used to express the Insert gene. Further, upon quantitative analysis of western blot bands, as shown in the relative quantitative analysis chart of FIG. 3A, the overexpression amount of the inserted gene Insert in the stably transfected cell line with pRSV was 550 times that of the unloaded one, while the overexpression amount of the inserted gene Insert in the stably transfected cell line with pCDH was 150 times that of the unloaded one. Further, the expression amount of the whole of the target gene Insert was quantitatively analyzed, and as shown in the relative quantitative analysis chart of FIG. 3B, the expression amount of the whole of the target gene Insert in the stably transfected cell line with pRSV was 7 times that of the empty cell, while the expression amount of the whole of the target gene Insert in the stably transfected cell line with pCDH was 2 times that of the empty cell. As can be seen, the expression level of the inserted gene in the stably transfected cell line with pRSV was 3.5 times that of the stably transfected cell line with pCDH, indicating that pRSV-CMV-Flag-EF1-Puro-T2A-GFP designed by the present invention is superior to pCDH-CMV-MCS-EF1-copGFP-T2A-Puro of System Biosciences, USA.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.
Sequence listing
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tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360
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agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 3300
acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 3360
acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 3420
gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 3480
tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc 3540
gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 3600
agctgtacaa gggctccgga gagggcaggg gaagtcttct aacatgcggg gacgtggagg 3660
aaaatcccgg ccccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg 3720
tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca 3780
ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc 3840
gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct 3900
ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg 3960
ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc 4020
accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg 4080
gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg 4140
tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct 4200
tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca 4260
agcccggtgc ctgacaactt tattatacat agttgatcaa ttccgataat caacctctgg 4320
attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 4380
gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 4440
tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 4500
ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg 4560
ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 4620
aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 4680
attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca 4740
cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 4800
ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 4860
agacgagtcg gatctccctt tgggccgcct ccccgcatcg ggaattcccg cggttcgctt 4920
taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg 4980
gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt 5040
ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 5100
ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 5160
actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta 5220
gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga 5280
gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 5340
atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 5400
atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct 5460
aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 5520
agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg 5580
aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc 5640
gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 5700
gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 5760
caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg 5820
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 5880
cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 5940
aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 6000
cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 6060
ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 6120
aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 6180
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt 6240
acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 6300
aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 6360
attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 6420
cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 6480
aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 6540
ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 6600
gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 6660
attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 6720
tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 6780
tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 6840
atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 6900
agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 6960
aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 7020
caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 7080
ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 7140
gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 7200
tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 7260
atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 7320
tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 7380
accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 7440
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 7500
caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 7560
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 7620
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 7680
tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 7740
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 7800
tatgagaaag cgccacgctt cccgaagaga gaaaggcgga caggtatccg gtaagcggca 7860
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 7920
gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 7980
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 8040
ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 8100
ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 8160
tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 8220
ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg 8280
caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 8340
ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc 8400
atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag 8460
ctt 8463

Claims (4)

1. A lentiviral expression vector, comprising: the nucleotide sequence of the lentivirus expression vector is SEQ ID NO.1.
2. Use of a lentiviral expression vector according to claim 1, wherein: based on its nucleotide sequence, the following five aspects can be applied: the target gene can be inserted, the target gene can be packaged into a slow virus infection target cell, the target gene can be expressed and inserted at a high level, a stable transfer cell strain can be obtained by puromycin screening, and the transfection efficiency can be detected by GFP.
3. A method of using the lentiviral expression vector of claim 1, wherein:
(1) Inserting a target gene: obtaining a segment inserted with a target gene by a PCR technology, and inserting the target gene segment into a multiple cloning site of a lentivirus expression vector by adopting an enzyme digestion connection method;
(2) Packaging into cells of interest for lentivirus infection:
the lentivirus expression vector is transcribed in 293T cells to generate virus genome RNA, the packaging vector is transcribed and translated in 293T cells to generate virus proteins, then the virus genome RNA and the virus proteins are assembled into lentiviruses in the cells, the lentiviruses are released from the 293T cells to a culture medium, the lentiviruses are collected, and the lentiviruses and target cells are incubated together to infect the target cells;
(3) Obtaining a stable cell strain by screening the cells infected by the lentivirus in the step (2) through puromycin: after the target cells are infected by the lentivirus for 48 hours, adding puromycin to 2-10 mu g/mL, culturing for more than 3 generations until the cells do not die any more, obtaining stable cell strains, collecting the stable cell strains, and detecting the expression quantity of the inserted target genes by western blot;
(4) High level expression of the inserted gene of interest: through western blot detection, expression of a target gene in a target cell or a stable transfer cell after the lentivirus is infected for 48 hours is judged to determine whether the target gene is expressed at a high level;
(5) Transfection efficiency was measured by GFP: after the lentivirus transfects 293T cells or the packaged lentivirus infects target cells, the transfection efficiency can be judged by observing the GFP intensity through a fluorescence microscope.
4. The method of claim 3, wherein the lentiviral expression vector comprises: the restriction enzyme sites in the restriction enzyme ligation are BamHI, salI, xhoI and XbaI.
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