CN116904509A - Method for improving expression level of firefly luciferase gene by blocking cleavage site forming circRNA - Google Patents
Method for improving expression level of firefly luciferase gene by blocking cleavage site forming circRNA Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, in particular to a method for improving the expression level of an exogenous gene, such as a firefly luciferase gene, by sealing a cleavage site forming circRNA. The method comprises the steps of obtaining a cleavage site of a firefly luciferase gene luc to form a circular RNA through searching and identifying, synthesizing a DNA sequence of the luc gene with the cleavage site blocked to form the circular RNA, further expressing the luc gene with the cleavage site mutated to the circular RNA in silkworm cells, and increasing the abundance of luc mRNA by reducing the formation of the luc circular RNA, thereby improving the expression level of the exogenous gene. Thereby achieving an increase in the expression level of the luc gene. The technical scheme disclosed by the invention provides a brand-new method for improving the expression level of the exogenous gene.
Description
Technical Field
The invention relates to the field of gene engineering expression exogenous genes, in particular to a method for improving the expression level of firefly luciferase genes by blocking cleavage sites forming circRNA.
Background
Increasing the expression level of exogenous genes is one of the most important targets in the field of genetic engineering. The existing researches show that the expression level of the exogenous gene is influenced by various factors such as the sequence of the expressed gene, elements for controlling the expression of the gene, the type of the expressed cell, the physiological state, the environmental condition of the expressed cell and the like. In practice of increasing the expression level of exogenous genes, one often adopts strategies such as optimizing codons, selecting strong promoters, introducing Kozak sequences, controlling the distance between the promoters and the initiation codon, and adopting secretion and/or fusion expression. In addition, people also improve the expression level of the exogenous gene by selecting different expression systems (cells), modifying cell chassis, optimizing expression conditions and other strategies. More and more studies have demonstrated that cells have the ability to recognize foreign RNA, DNA, and have a variety of mechanisms for degrading and eliminating foreign nucleic acids, e.g., miRNA, siRNA, piRNA, CRISPR mechanisms. Thus, recombinant expression vector DNA expressing a foreign gene, once it enters a cell, may be recognized by DNA sensors and NOD-like receptors in the cell and thus be rejected by the immune system in the cell; exogenous RNA may be detected by RIG-I-like receptors (RLRs), retinoic acid-inducing gene I (RIG-I) and melanoma differentiation-associated gene 5 of the cells, and thus cleared by the cells.
Recent studies have shown that primary transcripts of genes endogenous to eukaryotic cells can be processed by splicing systems to form mature linear mRNA, and that, in addition, loop RNA (circRNA) can be formed by reverse splicing, so that formation of circRNA by reverse splicing can be in competitive relationship with formation of linear mRNA. In addition, it has also been reported that reverse splicing to form circRNA can also be used to feedback regulate the level of linear transcripts in its parent gene. However, there is no report that cDNA of the gene forms circRNA when expressed in a heterologous cell, and there is no report that the expression level of firefly luciferase gene in a heterologous cell is improved by blocking the cleavage site forming the circRNA.
Disclosure of Invention
The invention aims to provide a method for blocking a cleavage site forming circRNA to improve the expression level of firefly luciferase luc gene. It has now been found that the primary transcript of a gene endogenous to eukaryotic cells can be spliced to form either linear mRNA or circRNA, but that there is no report of the formation of circRNA by the cDNA of the gene when expressed in heterologous cells, and no report of increasing the expression level of the firefly luciferase gene in heterologous cells by blocking the cleavage site forming the circRNA.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for blocking cleavage sites forming circRNA to increase expression levels of a foreign gene, comprising the steps of:
(1) Searching a cleavage site of the exogenous gene forming the circRNA;
(2) Determining the relative level of each formed circRNA and assessing the use of the cleavage sites forming the circRNA;
(3) Synthesizing a DNA sequence of the exogenous gene forming the cleavage site closure of the circRNA;
(4) An exogenous gene-M gene expressing a cleavage site mutation of the circRNA;
thereby realizing the blocking of the cleavage site forming the circRNA and improving the expression level of the exogenous gene.
In the present invention, the step (1) includes the steps of: (1) transfecting the exogenous gene transcribed in vitro into a cell with an expression system; then extracting total RNA of the cells, and digesting the linear RNA; then reverse transcription is carried out to synthesize cDNA by using the digested RNA as a template and using a random primer; (2) PCR amplification is carried out by using the cDNA as a template and using a divergent primer dPrimers; (3) cloning PCR amplified products with different molecular weights into a conventional T-vector, and performing Sanger sequencing; and comparing the sequencing result with the open reading frame sequence of the exogenous gene to obtain the circRNA sequence formed by the exogenous gene transcript, and the information of the cleavage site and the conjugation site of the formed circRNA.
In the present invention, in step (1), the exogenous gene-RNA of the RNA sequence of the open reading frame of the exogenous gene transcribed in vitro is transfected into the cells of the selected expression system; or cloning the exogenous gene sequence transcribed in vitro into an expression vector pVector to construct a recombinant plasmid pVector-exogenous gene, and transfecting cells of a selected expression system; after 24 hours, total RNA from the cells was extracted and linear RNA was digested with RNase R. Preferably, the divergent primer pair dPrimers is designed based on the sequence of the foreign gene-RNA.
In the present invention, the step (2) includes the steps of: determining the relative level of each circRNA by quantitative qPCR (quantitative polymerase chain reaction) by taking the cDNA as a template, qprimers as primers and housekeeping genes as internal references; the use of each cleavage site was assessed based on the relative levels of each circRNA. Preferably, the corresponding divergent primer pair qprimers is designed based on the flanking sequences of the respective circRNA junction sites formed.
In the present invention, the step (3) includes the steps of:
(1) determining codons corresponding to cleavage sites forming the circRNA according to the amino acid sequence encoded by the exogenous gene;
(2) on the basis of ensuring that the amino acid sequence encoded by the exogenous gene is unchanged, the codon corresponding to the break site is mutated, the DNA sequence exogenous gene-M of the exogenous gene with the break site mutation of the circRNA is chemically synthesized, and sequencing verification is carried out.
In the present invention, the step (4) includes the steps of: transfecting or infecting exogenous gene-M and exogenous gene respectively; realizing the blocking of the cleavage site forming the circRNA and improving the expression level of the exogenous gene. Further: (1) cloning the exogenous gene-M into an expression vector pvactor to construct a recombinant plasmid pvactor-exogenous gene-M; or the exogenous gene-M is cloned into a virus vector Rvirus to construct recombinant virus Rvirus-exogenous gene-M. Similarly, cloning exogenous genes into the rvrus to construct recombinant virus rvrus-luc; (2) transfecting the pvactor-exogenous gene-M, pVector-exogenous gene into cells respectively; or recombinant virus Rvirus-exogenous gene-M, rvirus-exogenous gene to infect cell; (3) after transfecting cells with plasmids or infecting cells with recombinant viruses for 48 hours, extracting total RNA of the cells, and respectively carrying out reverse transcription by using random primers to synthesize cDNA; designing convergence primer cPrimers for synthesizing and quantifying the expression level of the luc gene linear transcript; (4) qPCR is carried out by taking cDNA as a template and qPrimers, cPrimers as a primer and housekeeping genes as internal references, and the relative expression level of linear transcripts and cyclic transcripts of the exogenous genes in a pvactor-exogenous gene-M, pVector-exogenous gene transfected cell group and an Rvirus-exogenous gene-M, rvirus-exogenous gene infected cell group is calculated according to Ct values, so that the formation of the exogenous gene mRNA is reduced and the capability of the exogenous gene mRNA is improved by evaluating the breaking sites of mutant circRNA; (5) detecting the translation level of the exogenous gene in transfected cells or recombinant virus infected cells, and evaluating the cleavage site of mutant circRNA to reduce the formation of cyclic transcripts and improve the expression level of the exogenous gene protein.
In the invention, the exogenous genes comprise firefly luciferase genes, green fluorescent protein genes and viral protein genes; the cells comprise silkworm BmN cultured cells, hepG2 cells and CIK cells.
The invention discloses application of blocking a cleavage site forming circRNA in improving the expression level of an exogenous gene.
The method for blocking the cleavage site forming the circRNA to increase the expression level of the firefly luciferase gene by using the exogenous gene luc as an illustration comprises the following steps: (1) Searching for cleavage sites of the firefly luciferase gene luc forming circRNA, and mainly comprising the following steps: (1) transfecting the RNA sequence luc-RNA of the open reading frame of the in vitro transcribed luc gene into cells of the selected expression system; or cloning luc gene sequence into expression vector pVector to construct recombinant plasmid pVector-luc, and transfecting the cells of the selected expression system; (2) after 24 hours, total RNA of the cells was extracted, and linear RNA was digested with RNase R; (3) reverse transcription is carried out to synthesize cDNA by using the RNA in the step (2) as a template and a random primer; (4) designing and synthesizing a divergent primer pair dPrimers according to the sequence of the luc-RNA; (5) carrying out PCR amplification by using the cDNA in the step (3) as a template and using the divergent primer dPrimers in the step (4); (6) cloning PCR amplified products with different molecular weights into a conventional T-vector, and performing Sanger sequencing; (7) comparing the sequencing result with the open reading frame sequence of the luc gene to obtain the information of the sequence of the circRNA formed by the luc gene transcript, the cleavage site and the junction site formed by the circRNA.
Preferably, in the above step (1), the RNA sequence luc-RNA of the open reading frame of the luc gene is prepared by in vitro transcription using the T7 promoter or the SP6 promoter to control the cDNA sequence of the luc gene; when the luc gene sequence is cloned into an expression vector pvactor to construct a recombinant plasmid pvactor-luc, an insect cell expression vector pZT-V5/His (Invitrogen company) is selected, and cells of the selected expression system are silkworm BmN culture cells. In the step (4), one or more pairs of divergent primers may be randomly designed and synthesized based on the sequence of the luc-RNA.
(2) The relative level of each formed circRNA was determined and the use of the cleavage sites forming the circRNA was assessed, essentially comprising the steps of: (1) designing a corresponding divergent primer pair qprimers according to the flanking sequences of the formed respective circRNA junction sites; (2) determining the relative level of each circRNA by quantitative qPCR with the cDNA in the step (3) as a template, qprimers as primers and housekeeping genes as internal references; the housekeeping gene selected can be actin A3 gene, translation initiation factor TIF4A gene or other genes; (3) the use of each cleavage site was assessed based on the relative levels of each circRNA.
(3) The synthesis of the DNA sequence of the luc gene, which forms the cleavage site block of the circRNA, essentially comprises the following steps:
(1) determining codons corresponding to cleavage sites forming the circRNA according to the amino acid sequence encoded by the luc gene; (2) on the basis of ensuring that the amino acid sequence encoded by the luc gene is unchanged, mutating codons corresponding to the rupture sites, chemically synthesizing DNA sequence luc-M of the luc gene with mutated rupture sites of the circRNA, and carrying out sequencing verification.
To verify the effect of blocking the cleavage site forming the circRNA on the formation of the corresponding circRNA, in vitro transcribed luc-RNA, luc-M RNA, was transfected into cultured cells, and after extraction of RNA, the relative levels of the individual circRNAs were determined by quantitative qPCR using qprimers as primers and housekeeping genes as internal reference. The effect of blocking the cleavage site forming the circRNA on the circRNA was assessed by comparing the levels of RNA formation of corresponding circRNA from luc and luc-M.
(4) The luc-M gene expressing the cleavage site mutation of the circRNA mainly comprises the following steps: (1) cloning luc-M into an expression vector pVector to construct a recombinant plasmid pVector-luc-M; or cloning Luc-M into viral vector Rvirus to construct recombinant virus Rvirus-Luc-M. Likewise, cloning the luc gene into the rvrus to construct a recombinant virus rvrus-luc; (2) transfecting the cells with pvactor-luc-M, pVector-luc, respectively; or recombinant virus Rvirus-luc-M, rvirus-luc infected cells; (3) after transfecting cells with plasmids or infecting cells with recombinant viruses for 48 hours, extracting total RNA of the cells, and respectively carrying out reverse transcription by using random primers to synthesize cDNA; designing convergence primer cPrimers for synthesizing and quantifying the expression level of the luc gene linear transcript; (4) qPCR is carried out by taking cDNA as a template and qPrimers, cPrimers as a primer and housekeeping genes as internal references, and the relative expression level of the linear transcript and the circular transcript of the luc gene in a pvactor-luc-M, pVector-luc transfected cell group and an Rvirus-luc-M, rvirus-luc infected cell group is calculated according to Ct values, so that the cleavage site of the mutant circRNA reduces the formation of the luc gene circRNA and improves the mRNA capacity of the luc gene; preferably, the expression vector pVector is selected from insect cell expression vector pIZT-V5/His (Invitrogen corporation), namely: cloning Luc-M, luc genes into pZT-V5/His respectively to construct pZT-Luc-M and pZT-Luc respectively; viral vector rvrus selection baculovirus expression vector pFastBac TM Dual, i.e.cloning of the Luc-M, luc Gene into pFastBac, respectively TM Dual constructs pFastBac-Luc-M and pFastBac-Luc respectively; (5) detecting the translation level of the luc gene in the transfected cell or recombinant virus-infected cell of step (3) of claim 4, assessing the cleavage site of the mutant circRNA to reduce loop transcript formation and increase the expression level of the foreign gene protein.
Preferably, the mature Bac-to-Bac strategy is used in the construction of recombinant viruses Rvirus-Luc-M and Rvirus-Luc. Namely: the method comprises the steps of respectively converting DH10/Bac competent cells by pFastBac-Luc-M and pFastBac-Luc, coating on LB agar medium plates with tetracycline, kanamycin, gentamicin, IPTG and X-gal contents of 10 mug/ml, 50 mug/ml, 7 mug/ml, 40 mug/ml and 100 mug/ml respectively, culturing at 37 ℃, picking white colonies, extracting recombinant Bacmid-Luc-M and Bacmid-Luc DNA, further respectively infecting silkworm culture cells by the Bacmid-Luc-M and Bacmid-Luc DNA, and collecting culture cell supernatants after cell morbidity.
Preferably, the translation level of the luc gene determined as described above can be measured by Western blot for the protein expression level of the luc gene or by measuring luciferase activity.
In the present invention, luc is an exemplary exogenous gene, but the present invention is not limited to luc gene, and other exogenous genes may be used, for example: green fluorescent protein gene, viral protein gene, etc. In the present invention, the cells are exemplified by silkworm BmN cultured cells, but the present invention is not limited to silkworm cells, and may be HepG2 cells or CIK cells.
The expression level of the exogenous gene is improved through the technical scheme, and compared with the prior art, the invention has the following advantages due to the application of the technical scheme:
1. the invention not only proves that the cDNA of the exogenous gene forms the circRNA when being transcribed and expressed by the heterologous cell for the first time, but also improves the expression level of the exogenous gene by sealing to form the cleavage site of the circRNA of the exogenous gene.
2. In the conventional method, people often optimize the expression element by optimizing the codon, selecting a strong promoter, introducing a Kozak sequence, controlling the distance between the promoter and the initiation codon, adopting strategies such as secretion and/or fusion expression and the like, and improving the expression level of the exogenous gene. The technical scheme disclosed by the invention provides a new choice for improving the expression level of the exogenous gene.
Drawings
FIG. 1 shows the sequencing results of 8 kinds of circRNA formed from luc RNA detected after in vitro transcribed luc RNA transfects cultured cells of Bombyx mori in example one. Red arrows show the circRNA junction sites.
FIG. 2 shows the sequencing results of luc genes in examples two and three, which block transcription to form the circRNA site. A. B is a sequence after the formation sites of the circluc_1 and the circluc_2 are blocked, and C is a sequence after the formation sites of the circluc_1 and the circluc_2 are blocked at the same time. The nucleotide after mutation of the corresponding site of the luc gene is underlined.
FIG. 3 shows the effect of the mutant circluc_1 reverse splice-like site on luc gene transcript levels in example two. A is the effect of the mutant circluc_1 reverse splice-like site on the level of circluc_1 transcription; b, effect of the mutant circluc_1 reverse splice-like site on luc mRNA levels.
FIG. 4 shows the effect of the mutant circluc_2 reverse splice-like site on luc gene transcript levels in example two. A is the effect of the mutant circluc_2 reverse splice-like site on the level of circluc_2 transcription; b, effect of the mutant circluc_2 reverse splice-like site on luc mRNA levels.
FIG. 5 is the effect of simultaneous mutation of the reverse splice-like sites of circluc_1, circluc_2 on luc gene transcription level in example three. A, simultaneously mutating the effect of the reverse splice-like sites of the cicluc_1, cicluc_2 on the transcription level of the cicluc_1 and cicluc_2; b, effect of simultaneous mutation of the circluc_1, circluc_2 reverse splice-like sites on luc mRNA levels.
FIG. 6 shows the sequencing result of the luc gene of example four, in which the two-nucleotide sequence of the Pic-1-Pic-8 8-nucleotide sequence of the Pic-RNA luc reverse splice-like site were simultaneously mutated. The nucleotide after mutation of the corresponding site of the luc gene is underlined.
FIG. 7 is a schematic representation of the luc gene lucmut with simultaneous mutation of the Pic_1-Circluc_ 8 8 Pic RNA luc reverse splice-like sites in example IV 1-8 Sequence alignment with wild-type luc gene sequence.
FIG. 8 is a schematic representation of the luc gene lucmut with simultaneous mutation of the Pic_1-Circluc_ 8 8 Pic RNA luc reverse splice-like sites in example IV 1-8 Sequence alignment with wild-type luc gene sequence.
FIG. 9 is a diagram of a fourth embodimentSimultaneously mutating luc gene lucmut of the luc-1-8 8 luc reverse splice-like sites of the circRNA 1-8 Sequence alignment with wild-type luc gene sequence.
FIG. 10 is a schematic representation of the luc gene lucmut with simultaneous mutation of the Pic_1-Circluc_ 8 8 Pic RNA luc reverse splice-like sites in example IV 1-8 Sequence alignment with wild-type luc gene sequence.
FIG. 11 shows the effect of the mutation of the birc_1-birc_ 8 8 birc RNA luc reverse splice-like sites on luc gene transcript levels in example four. A, effect of the simultaneous mutation of the reverse splice-like site of circluc_1-circluc_8 on the transcription level of circluc_1-circluc_8. B, effect of simultaneous mutation of the reverse splice-like site of circluc_1-circluc_8 on luc mRNA levels.
FIG. 12 is the effect of the mutation of the birc_1-birc_ 8 8 birc RNA luc reverse splice-like sites on luciferase activity in example four.
FIG. 13 is a recombinant virus BmNPV-luc, bmNPV-luc of example five mut1 、BmNPV-luc mut2 、BmNPV-luc mut1-2 、BmNPV-luc mut1-8 Detection of expression luc levels in cultured cells of silkworms. A, fluorescent quantitative PCR (polymerase chain reaction) detection of luc mRNA level after different recombinant viruses infect cultivated cells of silkworms. B, western blotting detects the luc protein level after different recombinant viruses infect cultivated cells of silkworms. The upper graph shows the Western blot result, and the lower graph shows the gray scanning analysis result of the signal strip. (0.01<*P<0.05，0.001<**P<0.01，***P<0.001）
Detailed Description
The specific operation method related to the invention is a conventional method, such as cloning and specific operation of enzyme digestion; the test method involved is also conventional, for example, PCR amplification is carried out under conditions of 95℃for 4 minutes followed by denaturation at 95℃for 45 seconds, annealing at 50℃for 45 seconds, extension at 72℃for 30 seconds, amplification for 35 cycles, and then holding at 72℃for 10 minutes; except for the sequences and vectors specifically designed, the starting reagents involved are all conventional products. The invention is further described below with reference to the accompanying drawings and examples:
EXAMPLE luc RNA is capable of forming circRNA in cultured cells of Bombyx mori
(1) Carrying out PCR amplification by taking plasmid DNA with luc gene cDNA sequence (SEQ ID NO: 1) as a template and taking T7-luc-F (SEQ ID NO: 2) and T7-luc-R (SEQ ID NO: 3) as primers;
(2) The PCR product is electrophoresed by 1% agarose gel, and the target PCR product is recovered from the agarose gel;
(3) Using the recovered PCR product as a template, and performing in vitro transcription by using T7 RNA polymerase;
(4) Digesting the DNA template of the in vitro transcription product with DNase I enzyme, extracting with phenol and chloroform, and removing the enzyme;
(5) Precipitating RNA with isopropanol, washing the precipitate with 75% ethanol, air-drying at room temperature, dissolving with DEPC (diethyl carbonate) water solution, and adjusting the concentration to 1 μg/μl;
(6) 2. Mu.g of RNA from step (5) transcribed in vitro was mixed with 2. Mu.L of liposomes (FuGENE HD transfection reagent, roche Co.) and transfected 2X 10 6 Culturing cells of the silkworm BmN;
(7) After 48 hours of transfection, the cultured cells were collected and total RNA was extracted with RNAiso Plus kit (TaKaRa Co.);
(8) Linear RNA was removed by digestion with RNase R enzyme (Epicentre) at 37 ℃ for 15 min (2 μg RNA-formulated reaction system with 1U enzyme digestibility);
(9) Using the RNA in the step (8) as a template, and performing reverse transcription by using a random primer according to the specification of a reverse transcription kit (full gold company); PCR amplification was performed using the reverse transcribed cDNA as a template, with divergent primers dluc-F (SEQ ID NO: 4) and dluc-R (SEQ ID NO: 5) designed from the luc cDNA sequence; (10) The PCR product is electrophoresed by 1% agarose gel, and the target PCR product is recovered from the agarose gel;
(11) Cloning the recovered PCR product into a pMD19-T vector (TaKaRa Co.) and entrusting the commercial company to perform Sanger sequencing;
(12) The sequencing results were aligned with the cDNA sequence of the luc gene, and 8 circRNAs formed from the luc gene RNA were detected, designated as circluc_1-circluc_8, respectively, the sequences corresponding to 290-858 nt (circluc_1), 360-850 nt (circluc_2), 419-696 nt (circluc_3), 438-728 nt (circluc_4), 439-709 nt (circluc_5), 441-816 nt (circluc_6), 452-764 nt (circluc_7) and 460-728 nt (circluc_8), respectively. FIG. 1 shows the sequencing result of the middle junction site (junction site) region of each circRNA, and the arrow represents the junction site of the circRNA.
EXAMPLE two blocking of the sites formed by circluc_1, circluc_2 increased the expression level of the luc Gene
(1) T of luc Gene based on the binding site sequence of circluc_1 282 Mutating to A, C 285 Mutant into A, G 288 Mutant to C, C 291 Mutation to T, blocking the 5' -cleavage site (split site) of circluc_1; will G 858 Mutation to C, blocking the 3' -cleavage site (split site) of the circluc_1, obtaining the luc gene sequence with the circluc_1 forming site blocked, designated luc mut1 . The amino acid sequence encoded by the luc gene was not altered by the relevant site mutation FIG. 2-A is luc mut1 Sequencing results of the corresponding sites.
(2) C of luc Gene based on the binding site sequence of circluc_2 357 Mutating to G, and C 360 Mutation to a, blocking the 5' -cleavage site (split site) of circluc_2; will A 849 Mutating to G, and transforming T 852 The mutation to C-blocked fragment site (split site) of the circluc_2 to obtain the luc gene sequence blocked by the circluc_2 forming site, designated luc mut2 . FIG. 2-B is luc mut2 Sequencing results of the corresponding sites.
(3) Luc, luc mut1 And luc mut2 Cloning of the sequences into the pIZT-V5/His vector, respectivelyEcoRI/XbaI site, pIZT-luc mut1 And pIZT-luc mut2 An expression plasmid.
(4) Taking 2 μg pIZT-luc and pIZT-luc mut1 And pIZT-luc mut2 Expression plasmids were mixed with 2. Mu.L of liposomes (FuGENE HD transfection reagent, roche Co.) and transfected 2X 10, respectively 6 The cells were cultured with silkworm BmN.
(5) After 48 hours of transfection, the cultured cells were collected and total RNA was extracted with RNAiso Plus kit (TaKaRa Co.);
(6) Designing quantitative primers luc-F (SEQ ID NO: 6) and luc-R (SEQ ID NO: 7) according to the luc mRNA sequence, designing quantitative primers circluc_1-F (SEQ ID NO: 8) and circluc_1-R (SEQ ID NO: 9) according to the binding site sequence of circluc_1, designing quantitative primers circluc_2-F (SEQ ID NO: 10) and circluc_1-R (SEQ ID NO: 9) according to the binding site sequence of circluc_2, and designing quantitative primers TIF4A-F (SEQ ID NO: 11) and TIF4A-R (SEQ ID NO: 12) for synthesizing the translation initiation factor TIF4A of the reference gene;
(7) Determining the relative expression levels of luc mRNA, circluc_1, circluc_2 by fluorescent quantitative PCR using the RNA of step (5) as a template and the primers of step (6). The results showed that the level of circluc_1 in the cells after blocking the circluc_1 formation site was reduced (FIG. 3-A) and the luc mRNA level was increased (FIG. 3-B); likewise, the level of circluc_2 in the cells after blocking the circluc_2 formation site was decreased (FIG. 4-A), and the luc mRNA level was increased (FIG. 4-B).
EXAMPLE three Simultaneous blocking of the sites formed by circluc_1, circluc_2 to increase the expression level of the luc Gene
(1) The simultaneous blocking of the formation sites of circluc_1 and circluc_2 was performed according to the protocol of example two steps (1) and (2), and the luc gene sequence with the simultaneous blocking of the formation sites of circluc_1 and circluc_2 was obtained, designated luc mut1-2 。
(2) Luc is added with mut1-2 Cloning of the sequence into pIZT-V5/His vectorEcoRI/XbaI site, obtain pIHT-luc mut1-2 An expression plasmid.
(3) Mu.g of pIZT-luc (example II construction) and pIZT-luc were taken mut1-2 Expression plasmids were mixed with 2. Mu.L of liposomes (FuGENE HD transfection reagent, roche Co.) and transfected 2X 10, respectively 6 The cells were cultured with silkworm BmN.
(4) After 48 hours of transfection, the cultured cells were collected and total RNA was extracted with RNAiso Plus kit (TaKaRa Co.);
(5) Determining the relative expression levels of luc mRNA, circluc_1, circluc_2 by fluorescent quantitative PCR using the RNA of step (4) as template and the primers of step (6) of example two. The results showed that the levels of both circluc_1 and circluc_2 in the cells after simultaneous blocking of the formation sites of circluc_1, circluc_2 were reduced (FIG. 5-A) and that the luc mRNA levels were increased (FIG. 5-B).
Example four Simultaneous blocking of the site formed by circluc_1-circluc_8 to increase the expression level of the luc Gene
(1) The site formed by the nucleic acid sequence of nucleic acid sequence 1-nucleic acid sequence 8 was mutated according to the sequence of nucleic acid sequence 1-nucleic acid sequence 8, obtaining a sequence simultaneously blocking the site sequence formed by the circluc_1-circluc_8, designated luc mut1-8 。luc mut1-8 The sequencing result of the corresponding site in the sequence is shown in FIG. 6, luc mut1-8 The results of the alignment with luc sequences are shown in FIGS. 7, 8, 9 and 10.
(2) Luc is added with mut1-8 Cloning of the sequence into pIZT-V5/His vectorEcoRI/XbaI site, obtain pIHT-luc mut1-8 An expression plasmid.
(3) Mu.g of pIZT-luc (example II construction) and pIZT-luc were taken mut1-8 Expression plasmids were mixed with 2. Mu.L of liposomes (FuGENE HD transfection reagent, roche Co.) and transfected 2X 10, respectively 6 The cells were cultured with silkworm BmN.
(4) After 48 hours of transfection, the cultured cells were collected and total RNA was extracted with RNAiso Plus kit (TaKaRa Co.);
(5) The primers were designed according to the binding site sequence of the binding site 3-8. The primers were designed according to the binding site 3-F (SEQ ID NO: 13) and the binding site 1-R (SEQ ID NO: 9), the primers were designed according to the binding site 4-F (SEQ ID NO: 14) and the binding site 1-R (SEQ ID NO: 9), the primers were designed according to the binding site 5-binding site sequence and the primers were designed according to the binding site 1-R (SEQ ID NO: 15) and the binding site 1-R (SEQ ID NO: 9), the primers were designed according to the binding site 6-F (SEQ ID NO: 16) and the binding site 1-R (SEQ ID NO: 9), and the primers were designed according to the binding site 7-binding site sequence and the binding site 18-R (SEQ ID NO: 17) and the binding site 18-R (SEQ ID NO: 9);
(6) Determining the relative expression levels of luc mRNA, and cicluc_1-cicluc_8 by fluorescent quantitative PCR using the RNA of step (4) as a template and the primers of step (5) and step (6) of example two. The results showed that after simultaneous blocking of the sites forming the circluc_1-circluc_8, the level of circluc_1-circluc_8 in the cells was decreased (FIG. 11-A) and the luc mRNA level was increased (FIG. 11-B).
(7) Taking pIZT-luc and pIZT-luc mut1-8 Plasmid (1, 2, 3. Mu.g) was co-transfected with 2 ng internal pRL-TK plasmid (Promega) (2 ng) into BmN cells;
(8) Cells were collected after transfection of 60 h, and specific activities of 100. Mu.g of two enzymes were detected according to the instructions of firefly luciferase and Renilla luciferase detection kit (Promega Corp.). As a result, as shown in FIG. 12, after the mutation of the circular_1-circular_ 8 8 circular RNA luc reverse splice-like sites, the specific activity of firefly luciferase and Renilla luciferase increased, and the transfection agent amount dependence was exhibited.
EXAMPLE five blocking of the site formed by circluc_1-circluc_8 increases the level of recombinant baculovirus expressed luc genes
(1) Luc, luc mut1 、luc mut2 、luc mut1-2 、luc mut1-8 Cloning of the sequences into pFastBac TM -Dual (Invitrogen Co.)XbaI/EcoR I the recombinant baculovirus transfer vector was obtained and named pFast-luc, pFast-luc mut1 、pFast-luc mut2 、pFast-luc mut1-2 、pFast-luc mut1-8 ；
(2) pFast-luc, pFast-luc was expressed according to Bac-to-Bac (Bacteria to Baculovirus) expression System mut1 、pFast-luc mut2 、pFast-luc mut1-2 、pFast-luc mut1-8 E.coli DH10/Bac competent cells are respectively transformed, the competent cells are coated on LB agar culture plates containing tetracycline (10 mug/ml), kanamycin (50 mug/ml), gentamicin (7 mug/ml), IPTG (40 mug/ml) and X-gal (100 mug/ml), cultured for 48 hours at 37 ℃, white colony culture is selected, recombinant Bacmid genome DNA is extracted, PCR is carried out on the recombinant Bacmid genome DNA by using M13 forward primer (SEQ ID NO: 19) and M13 reverse primer (SEQ ID NO: 20), and the recombinant Bacmid genome DNA can be amplified from the recombinant Bacmid DNAThe target bands consistent with the theoretical molecular weight show that the recombinant Bacmid DNA is correctly constructed according to the requirement and respectively named as Bacmid-luc and Bacmid-luc mut1 、Bacmid-luc mut2 、Bacmid-luc mut1-2 、Bacmid-luc mut1-8 ；
(3) Taking 3 μg of Bacmid-luc and Bacmid-luc mut1 、Bacmid-luc mut2 、Bacmid-luc mut1-2 、Bacmid-luc mut1-8 Respectively transfecting BmN cells into a 6-pore plate, collecting culture medium after 5 days, obtaining P1 generation recombinant virus when the cell shape becomes round to a disease state, collecting supernatant after infection of BmN cells by a proper amount of P1 generation virus for 5 days, obtaining P2 generation recombinant virus, repeating the above operations, and obtaining P3 generation recombinant viruses BmNPV-luc and BmNPV-luc mut1 、BmNPV-luc mut2 、BmNPV-luc mut1-2 、BmNPV-luc mut1-8 ；
(4) By 4.0X10 5 P3-generation recombinant virus BmNPV-luc with several copies and BmNPV-luc mut1 、BmNPV-luc mut2 、BmNPV-luc mut1-2 、BmNPV-luc mut1-8 Infection with 1X 10 6 After 60. 60 h of BmN cells, the cells were collected, total RNA was extracted, and the results of fluorescent quantitative PCR showed that BmNPV-luc was compared with BmNPV-luc group mut1 、BmNPV-luc mut2 、BmNPV-luc mut1-2 、BmNPV-luc mut1-8 The level of luc mRNA expression in the group showed an increase in which BmNPV-luc was expressed mut1-8 The rise of the group was most pronounced (fig. 13-a).
(5) And (3) taking the cells collected in the step (4), splitting the cells by using a cell lysate, separating proteins by SDS-PAGE, transferring the proteins in the gel to a PVDF membrane, using a mouse anti-Luc protein antibody as a primary antibody, using horseradish peroxidase-labeled goat-mouse IgG as a secondary antibody, and simultaneously using alpha-tubulin as an internal reference for Western blot detection. The results showed that BmNPV-luc compared to BmNPV-luc group mut1 、BmNPV-luc mut2 、BmNPV-luc mut1-2 、BmNPV-luc mut1-8 The Luc of the group showed an increase in relative expression levels, wherein BmNPV-Luc mut1-8 The rise of the group was most pronounced (fig. 13-B).
The sequence related to the invention is as follows:
SEQ ID NO.1:
atggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaa
SEG ID NO.2:
taatacgactcactataggaattccatggaagatgccaaaaacattaag
SEG ID NO.3:
TCTAGACACGGCGATCTTGCCGCCC
SEQ ID NO.4:
CCCATCTTCGGCAACCAGAT
SEQ ID NO.5:
GCTTTGGAAGCCCTGGTAGT
SEQ ID NO.6:
CCTAAGCAACTTGCACGAGA
SEQ ID NO.7:
GATCAGAATGGCGCTGGTTG
SEQ ID NO.8:
TGCCCTGTCATCGGTGTGCC
SEQ ID NO.9:
GATGGTTTTGTCCCGGTCGA
SEQ ID NO.10:
GATTCAATCAGCCAGCCCAC
SEQ ID NO.11:
GAATGGACCCTGGGACACTT
SEQ ID NO.12:
CTGACTGGGCTTGAGCGATA
SEQ ID NO.13:
CTCGTTGAAGCCGGGTGGCA
SEQ ID NO.14:
GGCAACCAGATCATCAAAAG
SEQ ID NO.15:
CGACACCGCTACAAAAGATC
SEQ ID NO.16:
GGAGGAGCTAAAAGATCATC
SEQ ID NO.17:
CGCTGGGCTATCATGGATAG
SEQ ID NO.18:
CAGCGTGGTGCCATTAGCAA
SEQ ID NO.19:
CCCAGTCACGACGTTGTAAAACG
SEQ ID NO.20:
AGCGGATAACAATTTCACACAGG。
Claims (10)
1. a method for blocking cleavage sites forming circRNA to increase expression levels of a foreign gene, comprising the steps of:
(1) Searching a cleavage site of the exogenous gene forming the circRNA;
(2) Determining the relative level of each formed circRNA and assessing the use of the cleavage sites forming the circRNA;
(3) Synthesizing a DNA sequence of the exogenous gene forming the cleavage site closure of the circRNA;
(4) An exogenous gene-M gene expressing a cleavage site mutation of the circRNA;
thereby realizing the blocking of the cleavage site forming the circRNA and improving the expression level of the exogenous gene.
2. The method for blocking the cleavage site forming circRNA to increase the expression level of the foreign gene according to claim 1, wherein the step (1) comprises the steps of:
(1) transfecting the exogenous gene transcribed in vitro into a cell with an expression system; then extracting total RNA of the cells, and digesting the linear RNA; then reverse transcription is carried out to synthesize cDNA by using the digested RNA as a template and using a random primer;
(2) PCR amplification is carried out by using the cDNA as a template and using a divergent primer dPrimers;
(3) cloning PCR amplified products with different molecular weights into a conventional T-vector, and performing Sanger sequencing; and comparing the sequencing result with the open reading frame sequence of the exogenous gene to obtain the circRNA sequence formed by the exogenous gene transcript, and the information of the cleavage site and the conjugation site of the formed circRNA.
3. The method for blocking the cleavage site forming circRNA to increase the expression level of the foreign gene according to claim 2, wherein in step (1), the RNA sequence of the open reading frame of the foreign gene transcribed in vitro is transfected into the cells of the selected expression system; or cloning the exogenous gene sequence transcribed in vitro into an expression vector pVector to construct a recombinant plasmid pVector-exogenous gene, and transfecting cells of a selected expression system; after 24 hours, total RNA from the cells was extracted and linear RNA was digested with RNase R.
4. The method for blocking the cleavage site forming the circRNA to increase the expression level of the foreign gene according to claim 2, wherein the synthetic divergent primer pair dPrimers is designed according to the sequence of the foreign gene-RNA.
5. The method for blocking the cleavage site forming circRNA to increase the expression level of the foreign gene according to claim 2, wherein the step (2) comprises the steps of: determining the relative level of each circRNA by quantitative qPCR (quantitative polymerase chain reaction) by taking the cDNA as a template, qprimers as primers and housekeeping genes as internal references; the use of each cleavage site was assessed based on the relative levels of each circRNA.
6. The method of blocking the cleavage site forming circRNA to increase the expression level of the foreign gene according to claim 5, wherein the corresponding divergent primer pair qprimers is designed based on the flanking sequences of the respective circRNA junction site formed.
7. The method for blocking the cleavage site forming circRNA to increase the expression level of the foreign gene according to claim 1, wherein the step (3) comprises the steps of:
(1) determining codons corresponding to cleavage sites forming the circRNA according to the amino acid sequence encoded by the exogenous gene;
(2) on the basis of ensuring that the amino acid sequence encoded by the exogenous gene is unchanged, the codon corresponding to the break site is mutated, the DNA sequence exogenous gene-M of the exogenous gene with the break site mutation of the circRNA is chemically synthesized, and sequencing verification is carried out.
8. The method for blocking the cleavage site forming circRNA to increase the expression level of the foreign gene according to claim 1, wherein the step (4) comprises the steps of: transfecting or infecting exogenous gene-M and exogenous gene respectively; realizing the blocking of the cleavage site forming the circRNA and improving the expression level of the exogenous gene.
9. The method of blocking the cleavage site forming circRNA to increase the expression level of the foreign gene according to claim 1, wherein the foreign gene comprises a firefly luciferase gene, a green fluorescent protein gene, a viral protein gene; the cells comprise silkworm BmN cultured cells, hepG2 cells and CIK cells.
10. The application of blocking the cleavage site forming the circRNA in improving the expression level of the exogenous gene.
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|---|---|---|---|
| CN202310563109.5A CN116904509A (en) | 2023-05-18 | 2023-05-18 | Method for improving expression level of firefly luciferase gene by blocking cleavage site forming circRNA |
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| CN202310563109.5A CN116904509A (en) | 2023-05-18 | 2023-05-18 | Method for improving expression level of firefly luciferase gene by blocking cleavage site forming circRNA |
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|---|---|---|---|
| CN202310563109.5A Pending CN116904509A (en) | 2023-05-18 | 2023-05-18 | Method for improving expression level of firefly luciferase gene by blocking cleavage site forming circRNA |
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| Country | Link |
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| CN (1) | CN116904509A (en) |
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2023
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