CN102250950A - DNA level-based encephalitis B virus replicon vector system, and construction method and application thereof - Google Patents
DNA level-based encephalitis B virus replicon vector system, and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a DNA level-based encephalitis B virus replicon vector system, and a construction method and application thereof. Reverse transcription-polymerase chain reaction (RT-PCR) and fusion PCR methods are utilized, a modified low copy plasmid pOKM is taken as a skeleton, and under the condition of losing most structural genes (from the 165th nucleotide to the 2402nd nucleotide), a Japanese encephalitis virus (JEV) replicon vector system based on DNA level, which comprises a cytomegalovirus (CMV) promoter, 5'UTR (untranslated region), previous 23 amino acids (C23) at the N end of C protein, 25 amino acids (E25) at the C end of E protein, all nonstructural proteins, 3'UTR, ribozyme (HDVr) and a polyA sequence is constructed. In order to detect that whether the constructed JEV replicon can express exogenous genes, plasmids of pJEV-REP-GFP-IRES and pJEV-REP-GFP comprising reporter gene EGFP are constructed.
Description
Technical field
The present invention relates to gene engineering technology field, be specifically related to a kind of encephalitis b virus replicon carrier system and construction process and application based on dna level.
Background technology
Rna replicon is that the structure gene in the rna virus cdna group is deleted, and keep the cis-acting elements of its nonstructural gene and non-coding region, replicon utilizes the self-replicating characteristic of positive chain RNA virus, can express the Nonstructural Protein and the foreign protein of virus in cell.Rna replicon sublist based on positive chain RNA virus reaches system, has been widely used in fields such as new generation vaccine, oriented carrier preparation and gene therapy.
Application number is that 200910236832.2 Chinese invention patent application discloses a kind of Japanese encephalitis virus JEV replicon vector and application thereof, and the clone and the packaging system that are used for this replicon carrier packing.But it is wherein also inequality aspect the structure gene disappearance, inequality at the restriction enzyme enzyme recognition site in the multiple clone site zone of inserting foreign gene in addition, the restriction enzyme that this research is selected is an isocaudarner comparatively commonly used, can avoid foreign gene to insert some specific DNA fragments and bring a lot of difficulties.In addition, also inequality in the sequence that keeps structure gene and other eukaryotic expression original paper, this respect has important decisive action to duplicating with expression efficiency of carrier itself.
Summary of the invention
The objective of the invention is to according to the deficiencies in the prior art, a kind of encephalitis b virus replicon carrier system based on dna level is provided.
Another purpose of the present invention is to provide the construction process of above-mentioned encephalitis b virus replicon carrier system based on dna level.
A further object of the invention is to provide the application of above-mentioned encephalitis b virus replicon carrier system based on dna level.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
A kind of encephalitis b virus replicon carrier system based on dna level, it is the method for utilizing RT-PCR and merging PCR, with transformed low copy plasmid pOKM is skeleton, under the situation of the most structure genes of disappearance (165-2402 Nucleotide), make up the JEV replicon carrier system based on dna level of the proteic signal peptide of NS1 (E25), all Nonstructural Proteins, 3 ' UTR, ribozyme (HDVr) and the polyA sequence of preceding 23 amino acid (C23) comprise CMV promotor, 5 ' UTR, C albumen n end, E PROTEIN C end.
The construction process of above-mentioned encephalitis b virus replicon carrier system based on dna level, comprise the steps: to design primer, its sequence is shown in SEQ ID NO:1 ~ 18, and amplified fragments A from plasmid pWF-GFP amplifies fragment B, C, D and fragment again from the geneome RNA of JEV
, be template with Segment A and B again, amplify fragment by the method that merges PCR
With fragment C and D is template, merges pcr amplification and goes out fragment
, with fragment
,
With
Directed cloning obtains the encephalitis b virus replicon carrier system based on dna level in low copy plasmid.
As a kind of preferred version, in the above-mentioned construction process, described low copy plasmid is pOKM.
By the encephalitis b virus replicon carrier system based on dna level of the present invention, also can further construct the encephalitis b virus replicon carrier system based on dna level that contains reporter gene, this system has introduced Spe in the position of the disappearance structure gene of the described system of claim 1
And Sal
Two restriction enzyme sites.
By the above-mentioned encephalitis b virus replicon carrier system that contains reporter gene based on dna level, can also further construct the encephalitis b virus replicon carrier system that contains IRES element and reporter gene based on dna level, this system be contain reporter gene based on the encephalitis b virus replicon carrier system of dna level in Sal
3 ' end of restriction enzyme site inserts the IRES element, by inserting the IRES element, thereby allows can begin inner translation in the starting point of NS1 signal sequence, can improve albumen cutting and working (machining) efficiency after the translation.In addition, the expression of foreign protein is not only to rely on the subgenomic expression of JEV in this case, and it obtains the ability of the transient expression that the CMV promotor gives simultaneously, thereby expresses foreign protein better.
As a kind of preferred version, the translational control element that above-mentioned IRES element is an encephalomyocarditis virus.
Can insert foreign gene in the above-mentioned two kinds of encephalitis b virus replicon carrier systems of the present invention, obtain recombinant expression vector, behind the transfecting eukaryotic cells, express foreign protein again.
The IRES sequence is shown in SEQ ID NO:19.
Compared with prior art, the present invention has following beneficial effect:
Application number is that 200910236832.2 Chinese invention patent application discloses a kind of Japanese encephalitis virus JEV replicon vector and application thereof.The replicon carrier that this patent relates to is lacking structure gene prM and E gene, and has kept the coding region of C gene and the signal peptide coding region of NS1; And lacking the entire infrastructure gene, the present invention comprises that (C, prM and E gene) only kept preceding 23 amino acid (C23) of C albumen n end and 25 amino acid (E25) of E PROTEIN C end. and article (Engineering the Japanese encephalitis virus RNA genome for the expression of foreign genes of various sizes:Implications for packaging capacity and RNA replication efficiency) that Sang-Im Yun delivers at Journal of Neurovirology magazine have shown that the JEV replicon is at transfectional cell after 48 hours; The result of real-time quantitative RT-PCR R shows that the replicon rna that the JEV replicon that lacks three structural genes produces is to keep the C gene more than 6 times, then exceeds about 6 times at the expression of luciferase. Therefore, the JEV replicon is higher, more superior in the output and the obvious efficient of exogenous protein expression amount of JEV replicon rna in the output and the exogenous protein expression amount of JEV replicon rna at disappearance entire infrastructure gene (C, prM and E gene) .Simultaneously, the present invention also adds IRES (internal ribosome entry site) before the signal peptide coding region of NS1, not only has the function of replicon aspect expression alien gene, possesses the function of transient expression simultaneously, but the expression amount of energization foreign protein.The present invention is having the different of essence at the JEV replicon of delivering based on people such as the JEV replicon of dna level and Sang-Im Yun aspect the transmission; In addition, the present invention bases oneself upon a kind of vaccine carrier based on the DNAI level of research and development, and DNA is more stable than RNA aspect preservation and transmission, operates also more aspect.
Description of drawings
Fig. 1 is a pcr amplification product, and wherein, 1 is DL2,000DNA Marker; 2 is the A fragment; 3 is the B fragment; 4 is fragment
5 is fragment
6 is fragment
7 is the D fragment; 8 is the C fragment; 9 is DL15,000 DNA Marker;
Fig. 2 cuts evaluation for the enzyme that the JEV replicon makes up, and wherein, 1 is DL15,000 DNA Marker; 2 cut product (NotI/BamHI) for the pOK-JEV-REP enzyme; 3 cut product (BamHI/ XbaI) for the pOK-JEV-REP enzyme; 4 cut product (XbaI/KpnI) for the pOK-JEV-REP enzyme; 5 is λ-EcoT14 I digest Marker;
Fig. 3 is the pcr amplification product of GFP and GFP-IRES, and wherein, 1 is the GFP-IRES fragment PCR products; 2 is the segmental PCR product of EGFP; 3 for being the PCR product of template with water; 4 for being the PCR product of template with water; 5 is DL2,000 DNA Marker;
Fig. 4 cuts evaluation for the enzyme of pJEV-REP-GFP-IRES and pJEV-REP-GFP, and wherein, 1 is DL2,000DNA Marker; 2 cut product for the pJEV-REP-GFP enzyme; 3 cut product for the pJEV-REP-GFP-IRES enzyme;
Fig. 5 identifies JEV replicon replication for RT-PCR;
Fig. 6 identifies the synthetic of the positive and negative adopted chain of JEV replicon for RT-PCR, and wherein, 1-4. is that the cDNA after the reverse transcription does 10 times of dilutions; 5-8 is that the cDNA after the reverse transcription does 20 times of dilutions; 9 is DL2,000 DNA Marker;
Fig. 7 is the expression (* 400) that contains Nonstructural Protein behind the reporter gene JEV-REP recombinant plasmid transfection 293T cell, and wherein, A is a pJEV-REP-GFP transfection 293T cell; B is a pJEV-REP-GFP-IRES transfection 293T cell; C is the 293T cell;
Fig. 8 is the result (* 200) of pJEV-REP-GFP-IRES and pJEV-REP-GFP transfectional cell 48h, and wherein, A is a pJEV-REP-GFP transfection BHK-21 cell; B is a pJEV-REP-GFP-IRES transfection BHK-21 cell; C is a pJEV-REP-GFP transfection 293T cell; D is a pJEV-REP-GFP-IRES transfection 293T cell.
Embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
Amplified fragments A from plasmid pWF-GFP, the about 0.7Kb of size; Reverse transcription product cDNA with Japanese B encephalitis virus vaccine strain SA14-14-2 geneome RNA is that template amplifies B, C and fragment respectively then
, size is respectively 3.3Kb, 1.8 Kb and 3.6Kb; Be template then, amplify the D fragment, big or small 0.8Kb with plasmid pWF-GFP; Be template with A and B fragment again, amplify fragment by the method that merges PCR
, size is about 4KB, and is that template merges pcr amplification and goes out fragment with C and D fragment
, size is about 2.7Kb(and sees Fig. 1).
Clone and the evaluation of total length JEV replicon cDNA:
Fragment
,
,
Three fragment directed clonings are to carrier pOKM, the JEV replicon plasmid called after pJEV-REP that obtains.Carry out enzyme respectively with restriction enzyme NotI and BamHI, BamHI and XbaI, XbaI and KpnI then and cut evaluation, the result shows that the fragment that obtains after the pJEV-REP enzyme is cut is about 4 Kb and 7 Kb, 3.6 Kb and 7.4 Kb and 2.7 Kb and 8.3 Kb(respectively and sees Fig. 2), clip size is consistent with expection.Then carry out sequencing, the plasmid that determine to make up does not have and suddenlys change, and shows fragment
,
,
Be inserted among the low copy plasmid pOKM, the clone of this explanation JEV replicon cDNA successfully constructs.
Amplify the EGFP fragment from the method for plasmid pWF-GFP by PCR, size is about 720bp.In addition, be the method amplification IRES sequence that template is passed through PCR with plasmid pIRES1-neo, amplify the EGFP-IRES fragment with fragment EGFP by the method that merges PCR again, size is about 1, and 300bp is inserted into respectively in the pJEV-REP plasmid then.Carry out enzyme by restriction enzyme SpeI and SalI and cut evaluation, the result shows that the fragment that obtains after plasmid pJEV-REP-GFP enzyme is cut is about 720bp and 11,000bp, and the pJEV-REP-GFP-IRES enzyme obtains about 1310bp and 11 after cutting, 000bp(sees Fig. 3), be consistent with expection, through the order-checking certified correct, this shows that the JEV replicon that contains reporter gene GFP successfully constructs (structure iron is as 4).
With the transfection BHK-21 cell of pJEV-REP-GFP-IRES and pJEV-REP-GFP plasmid, collecting cell carries out total RNA extraction behind the 48h, utilizes different counter-rotating primers to detect it again and duplicates situation.RT-PCR result shows (as Fig. 5), two groups of JEV replicons that contain the EGFP reporter gene are the same with JEV, normal chain and minus strand all can amplify the specific fragment of about 300bp, and can't amplify specific fragment without the RNA of counter-rotating, show the pollution that does not have plasmid in the RNA of counter-rotating; And after 10 times and 20 times dilutions of cDNA do, the result shows that normal chain synthetic amount is than minus strand synthetic amount many (as Fig. 6).
PJEV-REP-GFP-IRES and pJEV-REP-GFP plasmid detect two kinds of JEV-REP with IIF method behind the transfection 293T cell 48h respectively all the proteic expression (see figure 7) of JEV NS1.
The expression of embodiment 5 efficiently expressing exogenous gene EGFP
In the transfection bhk cell of transfection pJEV-REP-GFP-IRES plasmid and the result behind the 293T cell, along with the expression amount of the increase fluorescence of time increases (see figure 8) gradually.
SEQUENCE?LISTING
＜110〉Agricultural University Of South China
＜120〉a kind of encephalitis b virus replicon carrier system and construction process and application based on dna level
<130>
<160> 18
<170> PatentIn?version?3.2
<210> 1
<211> 40
<212> DNA
<213> PCMVp1
<400> 1
tttttggcgg?ccgctagtta?ttaatagtaa?tcaattacgg 40
<210> 2
<211> 44
<212> DNA
<213> PCMVP1L
<400> 2
tttttggcgg?ccgctagtta?ttaatagtaa?tcaattacgg?ggtc 44
<210> 3
<211> 47
<212> DNA
<213> PJEV164olR
<400> 3
ctcggtcgac?ggtggtaaca?ctagtgcggg?gtaggccgcg?tttcagc 47
<210> 4
<211> 43
<212> DNA
<213> PJEV2403olF
<400> 4
actagtgtta?ccaccgtcga?ccgagaccga?tcaattgctt?tgg 43
<210> 5
<211> 20
<212> DNA
<213> PJEVC5706R
<400> 5
ttacgctcgc?cacaaaccac 20
<210> 6
<211> 20
<212> DNA
<213> CJEV-5557F
<400> 6
cgaccccgcc?tggaaccacg 20
<210> 7
<211> 25
<212> DNA
<213> CJEV-9155R
<400> 7
gaaccccaaa?gcttcaaact?ctaga 25
<210> 8
<211> 25
<212> DNA
<213> pJEV9117F
<400> 8
cttggagcac?ggtatctaga?gtttg 25
<210> 9
<211> 24
<212> DNA
<213> pJEV10976R
<400> 9
agatcctgtg?ttcttcctca?ccac 24
<210> 10
<211> 25
<212> DNA
<213> pJEV10395F
<400> 10
tgtgatttaa?ggtagaaaag?tagac 25
<210> 11
<211> 36
<212> DNA
<213> SPAP2L
<400> 11
cgaggtacct?accacatttg?tagaggtttt?acttgc 36
<210> 12
<211> 29
<212> DNA
<213> GFP1F
<400> 12
ccgcactagt?atggtgagca?agggcgagg 29
<210> 13
<211> 30
<212> DNA
<213> GFP717R
<400> 13
ctcggtcgac?cttgtacagc?tcgtccatgc 30
<210> 14
<211> 42
<212> DNA
<213> EGFP720Rol
<400> 14
gggggaggga?gaggggcgtt?acttgtacag?ctcgtccatg?cc 42
<210> 15
<211> 18
<212> DNA
<213> IRES?1F
<400> 15
cgcccctctc?cctccccc 18
<210> 16
<211> 38
<212> DNA
<213> IRES?588R
<400> 16
ctcggtcgac?catgttgtgg?caagcttatc?atcgtgtt 38
<210> 17
<211> 22
<212> DNA
<213> 2403F
<400> 17
cgagaccgat?caattgcttt?gg 22
<210> 18
<211> 27
<212> DNA
<213> 2609R
<400> 18
cttcgctagg?gatctgggcg?tttctgg 27
<210> 19
<211> 588
<212> DNA
<213> IRES
<400> 19
CGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTGATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAAGCTTGCCACAAC 588
Claims (9)
1. the encephalitis b virus replicon carrier system based on dna level is characterized in that this system comprises preceding 23 amino acid (C23) of CMV promotor, 5 ' UTR, C albumen n end, the proteic signal peptide of NS1, all Nonstructural Proteins, 3 ' UTR, ribozyme and the polyA sequence of E PROTEIN C end.
2. the construction process of the described encephalitis b virus replicon carrier system based on dna level of claim 1, it is characterized in that comprising the steps: to design primer, its sequence is shown in SEQ ID NO:1 ~ 18, amplified fragments A from plasmid pWF-GFP amplifies fragment B, C, D and fragment again from the geneome RNA of JEV
, be template with Segment A and B again, amplify fragment by the method that merges PCR
With fragment C and D is template, merges pcr amplification and goes out fragment
, with fragment
,
With
Directed cloning obtains the encephalitis b virus replicon carrier system based on dna level in low copy plasmid.
3. the construction process of the encephalitis b virus replicon carrier system based on dna level according to claim 2 is characterized in that described low copy plasmid is pOKM.
4. the encephalitis b virus replicon carrier system based on dna level that contains reporter gene is characterized in that this system has introduced Spe in the disappearance structure gene position of the described system of claim 1
And Sal
Two restriction enzyme sites.
5. the encephalitis b virus replicon carrier system based on dna level that contains IRES element and reporter gene is characterized in that the IRES element is arranged in the described encephalitis b virus replicon carrier Sal of system of claim 4
3 ' end of restriction enzyme site.
6. the encephalitis b virus replicon carrier system based on dna level that contains IRES element and reporter gene according to claim 5 is characterized in that the translational control element that described IRES element is an encephalomyocarditis virus.
7. a recombinant expression vector is characterized in that containing any described encephalitis b virus replicon carrier of claim system in the claim 1 ~ 6.
8. the application of the described recombinant expression vector of claim 7 in protein expression.
9. the described recombinant expression vector of claim 7 is expressed the application in the reporter gene in eukaryotic cell.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2013188918A1 (en) * | 2012-06-20 | 2013-12-27 | The Australian National University | Improved recombinant viruses |
| CN103540604A (en) * | 2013-10-23 | 2014-01-29 | 浙江农林大学 | Building method of deoxyribonucleic acid (DNA) replicor vaccine vector of swine Japanese encephalitis virus and use thereof |
| CN107937438A (en) * | 2017-11-29 | 2018-04-20 | 四川农业大学 | The duck tembusu virus for carrying renilla luciferase transiently transfects replicon and its construction method and application |
| CN108040484A (en) * | 2014-12-11 | 2018-05-15 | 巴斯德研究院 | Encephalitis B immunogenic composition based on slow virus carrier |
| CN111334482A (en) * | 2020-03-31 | 2020-06-26 | 华南农业大学 | Replication-enhanced attenuated JEV (Japanese encephalitis Virus) and preparation method and application thereof |
| CN118127078A (en) * | 2024-01-29 | 2024-06-04 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | PJEV-CMV-p54 replicon plasmid, preparation method and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013188918A1 (en) * | 2012-06-20 | 2013-12-27 | The Australian National University | Improved recombinant viruses |
| CN103540604A (en) * | 2013-10-23 | 2014-01-29 | 浙江农林大学 | Building method of deoxyribonucleic acid (DNA) replicor vaccine vector of swine Japanese encephalitis virus and use thereof |
| CN108040484A (en) * | 2014-12-11 | 2018-05-15 | 巴斯德研究院 | Encephalitis B immunogenic composition based on slow virus carrier |
| CN108040484B (en) * | 2014-12-11 | 2022-04-26 | 巴斯德研究院 | Lentiviral vector-based Japanese encephalitis immunogenic compositions |
| CN107937438A (en) * | 2017-11-29 | 2018-04-20 | 四川农业大学 | The duck tembusu virus for carrying renilla luciferase transiently transfects replicon and its construction method and application |
| CN107937438B (en) * | 2017-11-29 | 2021-03-16 | 四川农业大学 | Duck tembusu virus transient transfection replicon carrying renilla luciferase and construction method and application thereof |
| CN111334482A (en) * | 2020-03-31 | 2020-06-26 | 华南农业大学 | Replication-enhanced attenuated JEV (Japanese encephalitis Virus) and preparation method and application thereof |
| CN118127078A (en) * | 2024-01-29 | 2024-06-04 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | PJEV-CMV-p54 replicon plasmid, preparation method and application thereof |
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Application publication date: 20111123 |