CN116875621B - Method for improving IRES sequence mediated screening gene expression efficiency in transfer vector - Google Patents
Method for improving IRES sequence mediated screening gene expression efficiency in transfer vector Download PDFInfo
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- 239000013598 vector Substances 0.000 title claims abstract description 74
- 238000012546 transfer Methods 0.000 title claims abstract description 70
- 238000012216 screening Methods 0.000 title claims abstract description 46
- 230000014509 gene expression Effects 0.000 title claims abstract description 36
- 230000001404 mediated effect Effects 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 19
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 57
- 241000700605 Viruses Species 0.000 claims abstract description 51
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 claims abstract description 32
- 108010048367 enhanced green fluorescent protein Proteins 0.000 claims abstract description 21
- 101150072564 gE gene Proteins 0.000 claims abstract description 11
- 101100382437 Porcine circovirus 2 Cap gene Proteins 0.000 claims abstract description 6
- 238000003780 insertion Methods 0.000 claims abstract description 6
- 230000037431 insertion Effects 0.000 claims abstract description 6
- 230000003612 virological effect Effects 0.000 claims description 10
- 230000006801 homologous recombination Effects 0.000 claims description 7
- 238000002744 homologous recombination Methods 0.000 claims description 7
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- 108091081024 Start codon Proteins 0.000 claims description 4
- 239000003550 marker Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000013518 transcription Methods 0.000 claims description 4
- 230000035897 transcription Effects 0.000 claims description 4
- 108020005038 Terminator Codon Proteins 0.000 claims 1
- 241000725681 Swine influenza virus Species 0.000 abstract description 5
- 230000006798 recombination Effects 0.000 abstract description 2
- 238000005215 recombination Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 17
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 6
- 238000001890 transfection Methods 0.000 description 5
- 229960005486 vaccine Drugs 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- 101150044789 Cap gene Proteins 0.000 description 2
- 241001673669 Porcine circovirus 2 Species 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
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- 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
- C12N15/67—General methods for enhancing the expression
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16741—Use of virus, viral particle or viral elements as a vector
- C12N2710/16743—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
Abstract
The invention discloses a method for improving IRES sequence mediated screening gene expression efficiency in a transfer vector, which belongs to the technical field of molecular biology, and the method is characterized in that the transfer vector is transfected into target cells and then is sensed by viruses. According to the invention, the gE gene of the pseudorabies virus is used as an insertion site, the inserted exogenous gene is the porcine circovirus 2 Cap gene or the swine influenza virus HA1 gene, the IRES sequence mediated EGFP screening gene is connected to the downstream of the exogenous gene, the transfer vector is transfected into target cells, and then the virus is used for sensing, so that the expression efficiency of the screening gene can be improved after the virus senses, the occurrence of the condition of weak IRES sequence mediated screening gene expression signal and even false negative is avoided, the validity judgment of the transfer vector is facilitated, and the occurrence of virus recombination failure caused by invalid transfer vector is avoided.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a method for improving IRES sequence-mediated screening gene expression efficiency in a transfer vector.
Background
The virus rescue provides an important platform for researching the functions of virus genes, developing vaccine vectors and the like. Homologous recombination plays an important role in the viral rescue process. The transfer vector carries exogenous genes and/or screening genes, the two sides of the exogenous genes and/or the screening genes are provided with homologous sequences at the upstream and downstream of the insertion site, and the exogenous genes and/or the screening genes are integrated into the viral genome by the principle of recombination between the homologous sequences of the transfer vector and the viral genome, so that the expected purpose of virus rescue is realized.
The expression efficiency of the exogenous gene and/or the screening gene in the transfer vector directly affects the screening of the recombinant virus, therefore, the verification work of the expression efficiency of the exogenous gene, especially the screening gene, in the transfer vector is objectively carried out before the homologous recombination of the transfer vector and the viral genome is carried out, and the subsequent virus rescue work can be carried out only when the exogenous gene or the screening gene can be normally expressed. The validity verification of the transfer vector is a key link in the virus rescue process.
The Internal Ribosome Entry Site (IRES) or similar functional sequences enable the exogenous gene and the screening gene to be integrated into the viral genome at the same time, so that the exogenous gene and the screening gene are expressed at the same time, and great convenience is provided for virus purification and verification work. However, since IRES-mediated screening genes are less efficient in expression than their 5-terminal exogenous genes, screening genes typically have an expression efficiency of only 1/10 of their 5-terminal exogenous genes. Therefore, when screening genes are detected, the effectiveness verification of the transfer vector is seriously affected due to weak signals and even false negative conditions caused by low expression efficiency of the screening genes. There is currently no effective solution to this problem. Therefore, whether the expression efficiency of the selection gene in the transfer vector required for virus rescue can be improved is a common key technical problem about whether the virus can be successfully rescued.
Disclosure of Invention
The invention aims at: in order to solve the problems in the background art, a method for improving the expression efficiency of IRES sequence-mediated screening genes in a transfer vector is provided.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for improving IRES sequence mediated screening gene expression efficiency in transfer vector, which is characterized in that the transfer vector is transfected into target cells and then is sensed by viruses.
As a further description of the above technical solution:
the virus is a virus corresponding to a transfer vector.
As a further description of the above technical solution:
the virus is pseudorabies virus, and the pseudorabies virus is living virus.
As a further description of the above technical solution:
the virus particles take the gE gene of pseudorabies virus as an insertion site, and a CMV promoter is preferably added before a gE start codon so as to enhance the transcription and expression of a target gene PCV2 Cap.
As a further description of the above technical solution:
and (3) connecting an IRES-mediated EGFP gene after the exogenous gene stop codon, and screening the recombinant virus by taking EGFP as a screening marker.
As a further description of the above technical solution:
the transfer vector screening gene is any one of EGFP gene, YFP gene and RFP gene.
As a further description of the above technical solution:
the transfer vector is a viral homology arm sequence with two ends carrying the requirement of homologous recombination.
As a further description of the above technical solution:
the sequences of 1081bp upstream and 1246bp downstream of the gE gene are used as the left and right arms of the homology of the transfer vector and the pseudorabies virus (PRV) genome.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
the invention aims to solve the problem of low expression efficiency of IRES mediated screening markers (or other mediated proteins), namely, after transferring a transfer vector plasmid into a cell, the expression efficiency of the screening markers is found to be low, and EGFP is taken as an example, the EGFP expression quantity is often about 1/10 of that of an upstream exogenous gene, so that screening signals are weak, and whether the transfer vector is effective or not is not judged. The method comprises the steps of firstly transfecting a transfer vector plasmid into cells, and then using virus sense as the cells (infecting host cells by viruses), and finding that the expression efficiency of IRES-mediated screening markers in the transfer vector plasmid can be greatly enhanced, which is favorable for subsequent observation of the screening markers, for judgment of the effectiveness of the transfer vector, and for the transfer vector to enter into formal experiments (the transfer vector and a viral genome are transfected into the host cells together, and recombinant viruses are hoped to be obtained, namely, target genes of interest in the transfer vector are integrated into specific positions of the viral genome in a homologous recombination mode to obtain new recombinant viruses), so that the transfer vector plasmid is ensured to be effective, and the problem of recombinant virus failure caused by invalidation of the transfer vector plasmid is avoided.
Drawings
FIG. 1 is a schematic diagram of the construction of a homologous recombination transfer vector for a method for improving IRES sequence-mediated screening of gene expression efficiency in a transfer vector according to the present invention;
FIG. 2 is a schematic diagram showing the expression of the screened gene EGFP in a transfer vector after the transfer vector of the porcine circovirus type 2 Cap protein is transfected into cells, and the strain is or is not sensed by the pseudorabies virus HD/c strain;
FIG. 3 is a schematic diagram showing the expression of the screening gene EGFP in a transfer vector after cells are transfected by the transfer vector expressing the swine influenza virus HA1 protein and being or not infected by the pseudorabies virus HD/c strain.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Referring to fig. 1-2, the present invention provides a technical solution:
a method for improving IRES sequence mediated screening gene expression efficiency in transfer vector, after transfer vector transfects target cells, virus is used for sensing, the infected virus is virus corresponding to the transfer vector, the virus is pseudorabies virus, the pseudorabies virus is live virus (inactivated virus cannot infect cells and must be live virus), the construction of the gene transfer vector is screened by expressing recombinant pseudorabies virus band EGFP of porcine circovirus type 2 Cap protein in the embodiment, and the inactivation gE of PRV exploded after 2011 is utilized in the embodiment - TK - PRV type II vaccine strain (PRV HD/c strain, NCBI serial number MZ063026, patent number ZL 201710774869.5) as vaccine vector for expressing PCV2 Cap (Cap, SEQ ID NO. 1) of the enucleated localization signal, i.e., replacing TK with Cap gene - /gE - The gE gene of PRVII vaccine strain (NCBI serial number MZ 063026), the protein sequence of gE protein is shown in SEQ ID NO.3 (see sequence table), the Cap gene is shown in SEQ ID NO.1 (see sequence table), and the translated Cap protein sequence is shown in SEQ ID NO.2 (see sequence table);
the virus particle takes the gE gene of pseudorabies virus as an insertion site, and a CMV promoter is preferably added before a gE start codon so as to enhance the transcription and expression of a target gene PCV2 Cap; connecting IRES-mediated EGFP genes after gE stop codons, and screening recombinant viruses by taking EGFP as a screening marker; on the basis, the sequences of 1081bp upstream and 1246bp downstream of the gE gene are used as a transfer vector to form a transfer vector of the invention with homologous left and right arms of the PRV genome: SEQ ID NO.4: left arm (1-1081 bp) -CMV promoter (1082-1670 bp) -Kozak sequence (1671-1676 bp) -integrated PCV2 Cap (1677-2964 bp) -IRES (2965-3549 bp) -EGFP gene (3550-4303 bp) -right arm (4304-5549 bp), protein sequence of SEQ ID No.4 (see sequence Listing), which is synthesized by Zhejiang Shang Biotechnology Co., ltd, and finally constructed onto pUC18 vector, to obtainpUC18-CMV/gE - PCV2Cap + /EGFP + And (3) transferring the carrier.
Respectively transfecting the same amount of blank pUC18 plasmid and recombinant transfer vector into PK15 cells, wherein one recombinant transfer vector transfection group is subjected to the infection of PRV HD/c live strain at 37 ℃ after 24 hours of transfection, the infection is performed for 1 hour, the supernatant is discarded, and the liquid is changed for continuous culture; EGFP fluorescent protein expression was observed 24 hours after further culturing. The result is shown in figure 2, after the transfer vector transfects cells, the quantity of green fluorescent signals expressed by EGFP genes is obviously stronger after the cells are infected by PRV HD/c live viruses than that of the cells not infected by viruses, which shows that the expression efficiency of screening genes can be obviously improved after the cells are infected by the live viruses.
Example 2
Referring to fig. 1 and 3, the present invention provides a technical solution:
a method for improving IRES sequence mediated screening gene expression efficiency in transfer vector, after transfer vector transfects target cells, virus is used for sensing, the infected virus is the virus corresponding to the transfer vector, the virus is pseudorabies virus, the pseudorabies virus is live virus, the recombinant pseudorabies virus with EGFP (enhanced green fluorescent protein) screening gene transfer vector for expressing swine influenza virus HA1 in the embodiment is constructed, and in the embodiment, the inactivated gE of PRV exploded after 2011 is utilized - TK - PRV type II vaccine strain (PRV HD/c strain, NCBI serial number MZ063026, patent number ZL 201710774869.5) as viral vector for expressing swine influenza virus HA1 protein (HA 1, SEQ ID No.5 (see sequence listing)), i.e., replacing TK with HA1 gene fragment of swine influenza virus SIV - /gE - -the gE gene of the strain of PRVII (NCBI serial number MZ 063026). The HA1 gene (SEQ ID NO.5: the translated HA1 protein sequence is shown in SEQ ID NO.6 (see sequence table);
in the virus particle, the gE gene of pseudorabies virus is taken as an insertion site, and a CMV promoter is preferably added before a gE start codon so as to enhance the transcription and expression of a target gene SIV HA 1; connecting IRES-mediated EGFP genes after exogenous gene stop codons, and screening recombinant viruses by taking EGFP as a screening marker; on the basis, the sequences of 1081bp upstream and 1246bp downstream of the gE gene are used as a transfer vector to form a transfer vector of the invention with homologous left and right arms of the PRV genome: SEQ ID NO.7 (see sequence Listing): left arm (1-1081 bp) -CMV promoter (1082-1670 bp) -Kozak sequence (1671-1676 bp) -integrated SIV HA1 (1677-2816 bp) -IRES (2817-3984 bp) -EGFP gene (3985-4738 bp) -right arm (4739-5984 bp);
the above sequences are synthesized by Zhejiang Shangya biotechnology Co., ltd, and finally constructed on pUC18 vector to obtain pUC18-CMV/gE - SIV HA1 + /EGFP + And (3) transferring the carrier. The strategy for the recombinant viral transfer vector mediated homologous recombination is shown in FIG. 1. Respectively transfecting equivalent recombinant transfer vectors expressing the HA1 protein into Vero cells, wherein one recombinant transfer vector transfection group is subjected to the infection of PRV HD/c live strains at 37 ℃ after 24 hours of transfection, the infection is performed for 1 hour, the supernatant is discarded, and the liquid is changed for continuous culture; EGFP fluorescent protein expression was observed 24 hours after further culturing. The results are shown in FIG. 3, and the green fluorescence signal is obviously stronger after PRV HD/c virus infection after cell transfection by the transfer vector than that of the experimental group without virus infection, which shows that the expression efficiency of the screening gene can be obviously improved after the live virus infection.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (6)
1. A method for improving the expression efficiency of IRES sequence-mediated screening genes in a transfer vector, which is characterized in that after the transfer vector is transfected into target cells, virus is used for sensing;
the infected virus is a virus corresponding to a transfer vector, the virus is pseudorabies virus, and the pseudorabies virus is a live virus.
2. The method for improving the expression efficiency of IRES sequence-mediated screening genes in a transfer vector according to claim 1, wherein the virus particles use the gE gene of pseudorabies virus as an insertion site, and a CMV promoter is added before the gE initiation codon to enhance the transcription and expression of the target gene PCV2 Cap.
3. The method for improving the expression efficiency of IRES sequence-mediated selection gene in a transfer vector according to claim 1, wherein IRES-mediated transfer vector selection gene is ligated after the foreign gene termination codon, and recombinant virus is selected using the transfer vector selection gene as a selection marker.
4. The method for improving the expression efficiency of IRES sequence-mediated screening genes in a transfer vector according to claim 3, wherein the transfer vector screening gene is any one of EGFP gene, YFP gene and RFP gene.
5. The method of claim 4, wherein the transfer vector comprises viral homology arm sequences at both ends for homologous recombination.
6. The method for improving IRES sequence-mediated screening gene expression efficiency in a transfer vector according to claim 5, wherein the sequences 1081bp upstream and 1246bp downstream of the gE gene are used as the left and right arms of homology of the transfer vector with the pseudorabies virus genome.
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