阅读说明：本技术 新型基因治疗载体pIRES-Rsirt2/4-Tet-nap的制备方法及应用 (Preparation method and application of novel gene therapy vector pIRES-Rsirt2/4-Tet-nap ) 是由 孙晗笑 利时雨 于 2019-01-28 设计创作，主要内容包括：本发明公开了一种pIRES-Rsirt2/4-Tet-nap重组质粒,其沿载体转录方向依次连接有<I>R-</I><I>Sirt2</I>基因、IRES序列、<I>R-Sirt4</I>基因、HSV TK polyA序列、tTA序列、TRE序列和nap序列,或者反向连接。其中,<I>R-Sirt2</I>和<I>R-Sirt4</I>基因的功能是重构线粒体网络并逆转衰老细胞表型；IRES序列则是连接<I>sirtuin2</I>基因和<I>sirtuin4</I>基因,使两者反向共表达；tTA和TRE是四环素诱导表达元件,调控DNA结合和分子伴侣介导自噬的肽段nap序列表达。本发明所述的自噬重组载体,采用IRES序列连接<I>sirtuin2</I>和<I>sirtuin4</I>基因,能在同一载体中共表达Sirtuin2和Sirtuin4蛋白,表达后由四环素元件调控质粒DNA细胞内自噬。故该重组质粒可用于基因治疗线粒体功能障碍相关疾病的药物中的应用。(The invention discloses a pIRES-Rsirt2/4-Tet-nap recombinant plasmid, which is connected with a vector transcription direction in sequence R‑ Sirt2 Genes, IRES sequences, R‑Sirt4 Genes, HSV TK polyA sequence, tTA sequence, TRE sequence and nap sequence, or reverse connection. Wherein the content of the first and second substances, R‑Sirt2 and R‑Sirt4 the function of the gene is to reconstitute the mitochondrial network and reverse the senescent cell phenotype; IRES sequence is then ligated sirtuin2 Genes and sirtuin4 genes, which are co-expressed in opposite directions; tTA and TRE are tetracycline-inducible expression elements, regulatory DNA binding and moleculesChaperone mediated autophagy peptide segment nap sequence expression. The autophagy recombinant vector is connected by an IRES sequence sirtuin2 And sirtuin4 the gene can express Sirtuin2 and Sirtuin4 protein in the same carrier, and after expression, the tetracycline element regulates plasmid DNA intracellular autophagy. Therefore, the recombinant plasmid can be applied to the medicines for treating diseases related to mitochondrial dysfunction.)

1. The invention constructs pIRES-Rsirt2/4-Tet-nap double-gene co-expression autophagy vector, and realizes the high-efficiency controllable expression of exogenous plasmid DNA vector of target gene in mammalian cells; which is connected with a CMV promoter in sequence along the transcription direction of the vector,R-Sirt2Genes, IRES sequences,R-Sirt4Gene, HSV TK polyA sequence, tTA sequence, TRE sequence and nap sequence, Kana resistance gene and PUC plasmid skeleton.

2. The pIRES-Rsirt2/4-Tet-nap double-gene co-expression autophagy vector of claim 1, wherein: the above-mentionedsirtuin2The nucleotide sequence of the gene is shown as SEQ ID No.1 in the sequence table, and the gene is expressed assirtuin4The nucleotide sequence of the gene is shown as SEQ ID No.2 in the sequence table, the nucleotide sequence of the IRES sequence is shown as SEQ ID No.3 in the sequence table, the HSV TK polyA sequence is shown as SEQ ID No.4 in the sequence table, the tTA sequence is shown as SEQ ID No.5 in the sequence table, the TRE sequence is shown as SEQ ID No.6 in the sequence table, and the nap sequence is shown as SEQ ID No.7 in the sequence table.

3. The pIRES-Rsirt2/4-Tet-nap double-gene co-expression autophagy vector of claim 1, wherein: under the regulation and control of tetracycline-induced expression elements tTA and TRE, the start nap sequence consists of two action element peptide fragments, including:

(1) molecular chaperone autophagy recognition motif "KFERQ" sequence: TTCAAACTTGCAGTT, respectively;

(2) DNA binding peptide fragment (nucleic acid peptide) sequence: TCGCAGA TCAAGTTGTC CATCAAGCGC CTGGTCACC, amino acid sequence is "SQIKL SIKRLVT".

4. The initiation nap sequence under tetracycline-induced regulation of claim 3 consists of two acting element peptides, and the induction expression of nap peptide can automatically bind to plasmid DNA and induce lysosome autophagy of molecular chaperones, so that the exogenous plasmid DNA molecular vector disappears in cells.

5. The method for constructing pIRES-Rsirt2/4-Tet-nap double-gene co-expression autophagy vector of claim 1, which comprises the following steps:

(1) obtaining a product containing specific cleavage sitessirtuin2Gene fragments andsirtuin4a gene fragment;

(2) subjecting the product obtained in step (1)sirtuin2Gene fragments andsirtuin4the gene fragment is ligated to a vector to construct a vector containingsirtuin2-IRES-sirtuin4Recombinant vectors of genes;

(3) the compound constructed in the step (2) containssirtuin2-IRES-sirtuin4The recombinant vector of the gene is connected with pTet-On and pTRE-light vectors through double enzyme digestion to obtain a recombinant vector containing the genesirtuin2-IRES-sirtuin4-rtTA-TREThe carrier of；

(4) Inserting synthetic nap sequence into the sequence containingsirtuin2-IRES-sirtuin4-rtTA-TREThe pIRES-Rsirt2/4-Tet-nap double-gene co-expression autophagy recombinant vector is constructed.

6. The method of claim 4, wherein the PCR primers are:

sirtuin2gene PCR primers:

an upstream primer F: 5'-ATGTCCGCACCTTCGTCCAAGCCGA-3', respectively;

a downstream primer R: 5'-TCGGCTTGGACGAAGGTGCGGACAT-3', respectively;

sirtuin4gene PCR primers:

an upstream primer F: 5'-ATGAGGACATCGCTGCGGATATCGG-3', respectively;

a downstream primer R: 5'-CCGATATCCGCAGCGATGTCCTCAT-3', respectively;

P_Tight+ MCS gene fragment PCR primers:

an upstream primer F: 5'-GGATCCTTACTCCCTATCAGTGATAGAGAAC-3', respectively;

a downstream primer R: 5'-TCTAGAGATATCGTCGAC-3', respectively;

rTetR-minVP16-sv40polyA fragment PCR primers:

the upstream primer F1: 5'-AAGCTTATGTCTAGACTGGACAAGAGC-3', respectively;

the downstream primer R1: 5'-CATCAATGTATCTTATTACCCGGGGAGC-3', respectively;

the upstream primer F2: 5'-ATGCTCCCCGGGTAATAAGATACATTGAT-3', respectively;

the downstream primer R2: 5'-GGATCCATACTTCCCGTCCGCCAG-3' are provided.

7. The pIRES-Rsirt2/4-Tet-nap double-gene co-expression autophagy recombinant vector of any one of claims 1, 2, 3 and 4 has the effects of reconstructing a mitochondrial network, activating cell metabolism and reversing cell phenotype, and is applied to the drugs for preventing and treating diseases related to mitochondrial dysfunction such as aging-related diseases or metabolic syndrome.

8. Use of the pIRES-Rsirt2/4-Tet-nap double-gene co-expressed autophagy recombinant vector of any one of claims 1, 2, 3, 4 in Rsir2, Rsir4 and/or gene therapy related vectors including any gene of interest, pharmaceuticals, foods, nutraceuticals.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant vector capable of reconstructing a mitochondrial network and reversing the phenotype of an aged cell, and a preparation method and application thereof.

Background

With the continuous and deep research of IRES structure and function, the IRES structure is utilized to research disease treatment drugs, construct virus models which can be transferred into cells, and has more and more applications in gene therapy. Through patent inquiry, the IRES is found to be widely applied to the construction of vectors nowadays. IRES (internal ribosome) sequences were found in the RNA genomes of Poliovirus (PV) and encephalomyocarditis virus (EMCV) in 1988 by the Nahum Sonenberg and Eckard Wimmer laboratories, respectively. IRES are typically located in the 5'UTR of RNA viruses, allowing RNA translation in a 5' cap-independent manner. The two genes are linked by IRES, and the IRES and the two genes are transcribed into the same mRNA under the control of an upstream promoter. During translation, the translation initiation of the IRES upstream gene follows a cap-dependent mode of eukaryotic genes, and the translation of the downstream gene into the initiation gene is initiated by ribosome recruitment of the IRES, so that two genes connected by the IRES sequence share the same transcription unit for expression.

The current pharmaceutical research using IRES as a target has mainly focused on the therapeutic drugs for hepatitis c virus infection. Dash et al demonstrated that ifn.ii, ifn.b and IFN-t could target the 5' terminal IRES of HCV, inhibiting protein translation. Prabhu et al demonstrated that siRNA targeting IRES neck ring structure II was able to inhibit six genotypes of HCV in vitro, and thus neck ring structure II could be used as a target study drug for inhibiting HCV. Ray et al found an RNA molecule corresponding to IRES-loop structure 11I, capable of dose-dependent inhibition of IRES-mediated translation, but not cap-dependent translation. The GCAC sequence in the neck and fourth ring (SLrV) region and near the initiation codon has an important function for mediating the aggregation of ribosome on HCV RNA, and experiments of Subramanian and the like prove that SLIV with a short hairpin structure plays a remarkable role in inhibiting the replication of HCV RNA in Huh7 cells. Das et al have found that a small yeast RNA specifically inhibits Poliovirus (PV) IRES-mediated translation. Meanwhile, the IRNA can specifically inhibit translation mediated by HCV IRES in vivo and in vitro, and can compete for the combination of La autoantigen and HCV IRES, thereby providing a new basis for anti-HCV infection research medicaments. According to the characteristics of the IRES structure, different RNA aptamers are designed aiming at the structural domain II, the structural domains III-IV and the structural domain IV, and the RNA aptamers all prove to have the activity of inhibiting IRES-mediated translation. In addition, artificial ribonuclease, DNAzyme (Dz) molecules, antisense oligonucleotide, synthetic peptide (LAP), Peptide Nucleic Acid (PNA) and the like can inhibit the activity of IRES in a targeted manner, and the research of the molecules lays a foundation for the research of anti-HCV virus drugs.

The basic principle of tetracycline (Tet) inducible regulation of expression systems is that the conformation of a regulatory protein is altered by an inducing drug such as Tet, thereby controlling the expression of a target protein. Initially, a Tet-inducible regulatory gene expression system was established based on the Tet-resistant operon on the transposon of e.coli Tn 10. The Tet repressor (TetR) has specific affinity with the DNA sequence of a Tet operator (TetO), and when no Tet exists in cells, the TetR can be combined with the TetO, so that the expression of a downstream resistance gene is blocked. When Tet exists, the drug changes the conformation of TetR, so that the TetR is separated from TetO, the inhibition of resistance genes is relieved, and the resistance protein expression enables bacteria to generate drug resistance. By utilizing the specific combination characteristic of TetR and TetO, researchers develop various Tet regulation systems, but can be classified into two categories according to the expression characteristics of the TetR regulation systems: the inhibiting system Tet-off and the activating system Tet-on.

The Tet-off gene regulated expression system is an induced expression system established by Gossen and the like at the earliest time, and a tetracycline transcriptional activator (tTA) is a fusion protein. The Tetracycline Response Element (TRE) is formed by connecting a virus IE promoter micro-promoter sequence (Pcmv) of human cytomegalovirus, a target gene and a Tet longitudinal subsequence of escherichia coli. When Doxycycline (Dox) or tetracycline is not added, the gene of interest is transcribed. The Tet-on system differs from the Tet-off system in that its regulatory protein is an antisense tetracycline transcriptional activator (rtTA). Its transcription regulatory element rtTA is formed by the fusion of antisense Tet repressor protein (reverse TetR, rTetR) and VP 16. The response element TRE is similar to that of the sense tetracycline-inducible expression control system, but the mechanism of action is opposite to that of the sense tetracycline-inducible expression control system.

In order to effectively control the expression of a target gene in a cell, a novel gene therapy vector pIRES-Rsirt2/4-Tet-nap is constructed, a nucleic acid autophagy peptide containing a pentapeptide motif for molecular chaperone autophagy is introduced into the vector, the vector is induced to autophagy by exogenously administering 4-epidoxycycline, and the feasibility of the novel expression vector for controlling the effective and controllable expression of the target gene is evaluated by detecting the expression level of the target gene in the cell.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a pIRES-Rsirt2/4-Tet-nap recombinant plasmid. The recombinant plasmid is transfected into cells, can effectively reconstruct a mitochondrial network and reverse the senescence phenotype of senescent mammalian cells.

The invention also aims to provide a construction method of the pIRES-Rsirt2/4-Tet-nap recombinant plasmid.

The invention also aims to provide application of the pIRES-Rsirt2/4-Tet-nap recombinant plasmid.

pIRES-Rsirt2/4-Tet-nap double-gene co-expression autophagy vector, which is connected with vector transcription direction in sequenceR-Sirt2Genes, IRES sequences,R-Sirt4Genes, HSV TK polyA sequence, tTA sequence, TRE sequence and nap sequence, or the reverse connection. Wherein the content of the first and second substances,R-Sirt2andR-Sirt4the function of the gene is to reconstitute the mitochondrial network and reverse the senescent cell phenotype; IRES sequence is then ligatedsirtuin2Genes andsirtuin4genes, which are co-expressed in opposite directions; tTA and TRE are tetracycline-inducible expression elements that regulate DNA binding and chaperone-mediated autophagy peptide fragment nap sequence expression.

The autophagy recombinant vector is connected by an IRES sequencesirtuin2Andsirtuin4the gene can co-express Sirtuin2 and Sirtuin4 proteins in the same vector, and after expression, the tetracycline element induces and regulates the intracellular autophagy of the plasmid DNA vector, so that the high-efficiency controllable expression of the exogenous plasmid DNA vector in mammalian cells is realized. pIRES-Rsirt2/4-Tet-nap double geneThe co-expression autophagy recombinant vector has obvious effects in reconstructing a mitochondrial network, activating cell metabolism and reversing cell phenotype, so that the co-expression autophagy recombinant vector has wide application in medicines for preventing and treating aging-related diseases or metabolic syndrome and other mitochondrial dysfunction-related diseases. Therefore, the recombinant plasmid DNA vector can be applied to vectors related to mitochondrial dysfunction related diseases for gene therapy, and medicines in medicines, foods and health products.

Drawings

FIG. 1 shows the information of the vector pAcGFP1-C3 in example 1.

FIG. 2 shows the pTRE-light vector information of example 1.

FIG. 3 shows the information of the pTet-On vector of example 1.

FIG. 4 shows the pIRES-Rsirt2/4-Tet-nap vector information constructed in example 1.

FIG. 5 shows Rhodotorula glutinis of example 1sirtuin2Andsirtuin4the PCR amplification result of the gene (lane 1: DL2000 Marker; lane 2:sirtuin2a gene amplification product; lane 3:sirtuin4gene amplification product).

FIG. 6 shows the results of the cleavage of the vector of example 1 pMD18-T-Rsirt2/4 (M: DL5000 Marker; lane 1: EcoRI and EcoRV the results of the double cleavage of pMD18-T-Rsirt 2/4).

FIG. 7 shows the cleavage results of the vector of example 1 pIRES-Rsirt 2/4-C3. A. The DL2000Marker identifies the double cleavage results (M: DL2000 Marker; lane 1: EcoRV and EcoRI); B. the DL5000 Marker identified the double cleavage results (M: DL5000 Marker; lane 1: EcoRV and EcoRI double cleavage results).

FIG. 8 shows the results of PCR amplification of the promoter regulatory region fragment in example 1 (M: DL2000 Marker; 1: amplified product of promoter regulatory region fragment).

FIG. 9 shows the results of PCR amplification of the transcription element fragment in example 1 (M: DL2000 Marker; 1: transcription element fragment amplification product).

FIG. 10 shows the results of the double restriction of the recombinant plasmid pIRES-Rsirt2/4-Tet-nap in example 1 (M1: DL2000 Marker; 1: the result of the double restriction of the recombinant plasmid pIRES-Rsirt2/4-Tet-nap with Nhe I and Bgl II; 2: the result of the double restriction of the recombinant plasmid pIRES-Rsirt2/4-Tet-nap with Bgl II and HindIII; 3: the result of the double restriction of the recombinant plasmid pIRES-Rsirt2/4-Tet-nap with HindIII and Pst I; 4: the result of the double restriction of the recombinant plasmid pIRES-Rsirt2/4-Tet-nap with Kpn I and BamHI; M2: DL10000 Marker).

FIG. 11 shows the identification of expressed proteins by SDS-PAGE in example 1 (M: protein Marker; lane 1: E.coli strain expressing Rt-sirt2/4 plasmid; lane 2: E.coli strain expressing empty plasmid; lane 3: untransfected E.coli strain).

FIG. 12 shows the expression of the target gene detected by Western blot in example 1. A. Detecting the expression of the target gene in HEK293T cells on day 1 post-transfection; B. detecting the expression of the target gene in HEK293T cells on day 2 post-transfection; C. detecting the expression of the target gene in HEK293T cells at day 4 post-transfection; D. expression of the gene of interest in HEK293T cells was examined on day 8 post-transfection (lane 1: expression)Rt-sirt2/4HEK293T cells of plasmid; lane 2: HEK293T cells expressing the empty plasmid; lane 3: untransfected HEK293T cells).

FIG. 13 shows the expression of the target gene after autophagy in example 1. A. Detecting the expression of the target gene in HEK293T cells at 6 h after the administration of 4-epidoxycycline; B. detecting the expression of a target gene in HEK293T cells at 12 h after the administration of 4-epidoxycycline; C. detecting the expression of the target gene in HEK293T cells at 24 h after the administration of 4-epidoxycycline; D. detecting the expression of the target gene in HEK293T cells at 48 h after the 4-epidoxycycline is given; E. expression of the target Gene in HEK293T cells 72h after 4-Epinomycin administration (1: plasmid not administered 4-Epinomycin)Rt-sirt2/4(ii) expression of (a); 2: expression of plasmid Rt-sirt2/4, which confers 4-epidoxycycline; 3: HEK293T cells transfected with the empty plasmid).

Detailed Description

In the following examples, experimental techniques such as PCR technique, primer design technique, vector construction technique, detection technique, electrophoresis technique, etc. are conventional techniques in genetic engineering, and can be implemented by those skilled in the art according to the prior art (for example, refer to molecular cloning instruction manual, third edition of scientific publishing agency, translated by Huangpetang, et al, J. SammBruke, et al, or according to the product description). The equipment, reagents, carriers, strains and the like used in the operation process are all conventional products purchased from the market. The present invention will be described in further detail with reference to examples.