CN115068632A - Application of Ago2 in preparation of medicine for treating heart failure, protein, gene, transformant, medicine and preparation method thereof - Google Patents
Application of Ago2 in preparation of medicine for treating heart failure, protein, gene, transformant, medicine and preparation method thereof Download PDFInfo
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Abstract
The invention relates to application of Ago2 in preparing a medicament for treating heart failure, and a protein, a gene, a transformant, a medicament and a preparation method thereof, belonging to the field of biological medicines. The invention provides application of Ago2 in preparation of a medicine for treating heart failure, provides a medicine for treating heart failure and a preparation method thereof based on new application of Ago2, and provides a protein, a gene, a recombinant expression vector and a transformant with an effect of treating heart failure. Animal experiments prove that the anti-heart failure medicine based on Ago2 can obviously improve the heart function of heart failure animals and has an effective anti-heart failure effect.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to application of Ago2 in preparing a medicine for treating heart failure, a protein, a gene, a transformant, a medicine and a preparation method thereof.
Background
Cardiovascular diseases have become the leading cause of death in our country, and heart failure is a clinical syndrome in which various cardiovascular diseases progress to the terminal stage. Although some new drugs have been clinically used in recent years, about 50% of patients with heart failure die within 5 years after diagnosis. Poor prognosis and high mortality in heart failure are among the leading causes of threatened human health and increased medical burden.
At present, adenovirus and lentivirus are mostly adopted as expression vectors for gene therapy, but the lentivirus is mostly transformed from leukemia virus or HIV, and the adenovirus vector has short expression time and immunogenicity to organisms, and is not suitable for future clinical application. The recombinant adeno-associated virus (rAAV) overcomes the defects which are difficult to overcome by other gene expression vectors, has no immunogenicity, and can drive a target gene to express in vivo for a long time, thereby becoming the most promising vector for gene therapy.
microRNAs (miRNAs) are a class of non-coding small RNAs with important gene expression regulation function newly discovered in recent years. Mature miRNAs are generally bound fully or incompletely complementary to the 3 'untranslated region (3' UTR) sequences of their target mRNAs by the principle of base complementary pairing. miRNA is found to be involved in heart failure caused by various causes. In recent years, the human body safety and tolerance of the drug CDR132L designed aiming at the miR-132 as the target are good, and no obvious toxic reaction exists (Eur Heart J.2021; 42(2): 178). Meanwhile, the CDR132L can obviously reduce the heart failure marker NT-proBNP of the patient, obviously narrow the electrocardiogram QRS wave and show the reduction trend of the myocardial fibrosis marker. However, the improvement of EF value of heart failure patients by CDR132L is not significant, so it is necessary to find miRNA and key regulatory factor for miRNA function, which play more important roles in heart failure. Ago2 is reported as the core protein of miRNAs (science.2014; 346(6209): 608-13; cell.2014; 158(3):607) for performing the regulation function, but no report about the association between Ago2 and heart failure is found so far, and the report about the therapeutic effect of Ago2 in heart failure and the related therapeutic drug use are blank.
Disclosure of Invention
According to the problems and the defects objectively existing in the field, the inventor accidentally discovers the relationship between the mitochondria Ago2 and the heart failure generation mechanism, further performs molecular experiments, develops a heart failure resisting medicine taking mitochondria Ago2 as a medicine target, simultaneously performs animal experiments to verify the effect, and discovers that the high-expression mitochondria-targeted Ago2 can obviously improve the heart function of mice and treat heart failure.
The technical purpose of the invention is realized by the following technical scheme:
use of Ago2 in the preparation of a medicament for the treatment of heart failure.
The action target of the medicine for treating heart failure is Ago 2;
preferably, the Ago2 is mitochondrially targeted Ago 2;
preferably, said Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2.
A medicament for treating heart failure comprising: a pharmaceutically active ingredient; characterized in that the pharmacodynamically active ingredients comprise: a substance highly expressing mitochondrially targeted Ago 2.
The high expression mitochondrially targeted Ago2 is selected from the group consisting of: an expression vector connected with a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence, a transformant transformed with an expression vector connected with a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence, an expression vector sequentially connected with a myocardial specific promoter, a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence, and a transformant transformed with an expression vector sequentially connected with a myocardial specific promoter, a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence;
preferably, said Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2;
preferably, the expression vector is an adeno-associated virus expression vector pAAV-D (+);
preferably, the mitochondrial localizable leader peptide sequence is selected from: cox8, Su9, Sod 2;
preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, alpha-MHC, MLC-2v, Desmin;
preferably, the expression vector linking the mitochondrially-localizable leader peptide sequence and the Ago2 gene sequence is selected from the group consisting of: the recombinant adeno-associated virus expression vector pAAV-D (+) -Cox8-Ago2 linked with a Cox8 gene sequence and an Ago2 gene sequence, or the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-Cox8-Ago2 linked with a myocardial specific promoter tnt, a Cox8 gene sequence and an Ago2 gene sequence in sequence;
preferably, the host cell of the transformant is selected from the group consisting of: 293 cells;
preferably, the transformant transformed with the expression vector linked to the mitochondrial-localizable leader peptide sequence and the Ago2 gene sequence is selected from the group consisting of: the recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -cox8-Ago2, or the recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-cox8-Ago 2;
preferably, the Cox8 gene sequence is shown in SEQ ID NO. 1;
preferably, the Ago2 gene sequence is shown in SEQ ID NO. 2.
A protein having an effect of treating heart failure, comprising: the mitochondrial leader peptide sequence and Ago2 sequence can be located.
The mitochondrial homing peptide is selected from: cox8, Su9, Sod 2;
preferably, the protein having the effect of treating heart failure refers to a fusion protein consisting of Cox8 and Ago 2.
A gene having an effect of treating heart failure, comprising: the mitochondrial leader peptide gene sequence and Ago2 gene sequence can be located.
The gene having the effect of treating heart failure further comprises: a myocardial-specific promoter;
preferably, the mitochondrial-localizable leader peptide is selected from: cox8, Su9, Sod 2;
preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, alpha-MHC, MLC-2v, Desmin;
preferably, said Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2;
preferably, the gene having the effect of treating heart failure is selected from the group consisting of: a gene consisting of a Cox8 gene sequence and an Ago2 gene sequence, or a gene consisting of a myocardial specific promoter tnt sequence, a Cox8 gene sequence and an Ago2 gene sequence;
preferably, the Cox8 gene sequence is shown in SEQ ID NO. 1;
preferably, the Ago2 gene sequence is shown in SEQ ID NO. 2.
A recombinant expression vector with the effect of treating heart failure is characterized in that the recombinant expression vector is an expression vector connected with the sequence of the gene with the effect of treating heart failure.
The expression vector is selected from: is an adeno-associated virus expression vector pAAV-D (+);
preferably, the recombinant expression vector having the effect of treating heart failure is selected from the group consisting of: the recombinant adeno-associated virus expression vector pAAV-D (+) -Cox8-Ago2 is connected with a Cox8 gene sequence and an Ago2 gene sequence, or the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-Cox8-Ago2 is sequentially connected with a myocardial specific promoter tnt, a Cox8 gene sequence and an Ago2 gene sequence.
A transformant having an effect of treating heart failure, which is a host cell transformed with the recombinant expression vector having an effect of treating heart failure.
The host cell is selected from: 293 cells;
preferably, the transformant having the effect of treating heart failure is selected from the group consisting of: the recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -cox8-Ago2 or the recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-cox8-Ago 2.
A method for preparing a medicament for treating heart failure is characterized by expressing a fusion protein consisting of a mitochondrion-localizable leader peptide and Ago 2.
Connecting the gene sequence of the mitochondrion-localizable leader peptide and the gene sequence of Ago2 with an expression vector to obtain a recombinant expression vector;
preferably, the myocardial specific promoter, the gene sequence of the mitochondrion-localizable leader peptide and the gene sequence of Ago2 are sequentially connected with an expression vector to obtain a recombinant expression vector;
preferably, the host cell is transformed with the recombinant expression vector to obtain a transformant;
preferably, the host cell is transformed by the recombinant expression vector by adopting a calcium-phosphorus co-transfection method to obtain a transformant;
preferably, said Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2;
preferably, the expression vector is an adeno-associated virus expression vector pAAV-D (+);
preferably, the mitochondrial localizable leader peptide sequence is selected from: cox8, Su9, Sod 2;
preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, alpha-MHC, MLC-2v, Desmin;
preferably, the recombinant expression vector is selected from the group consisting of: the recombinant adeno-associated virus expression vector pAAV-D (+) -Cox8-Ago2 linked with a Cox8 gene sequence and an Ago2 gene sequence, or the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-Cox8-Ago2 linked with a myocardial specific promoter tnt, a Cox8 gene sequence and an Ago2 gene sequence in sequence;
preferably, the transformant is selected from the group consisting of: the recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -cox8-Ago2 or the recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-cox8-Ago 2;
preferably, the Cox8 gene sequence is shown in SEQ ID NO. 1;
preferably, the Ago2 gene sequence is shown in SEQ ID NO. 2.
Under the premise of permission of patent laws of some countries or regions, the invention also claims a substance for highly expressing mitochondrially targeted Ago2, or a fusion protein consisting of a mitochondrial leader peptide sequence and Ago2 sequence can be positioned, or a fusion protein consisting of Cox8 and Ago2, or a recombinant adeno-associated virus expression vector pAAV-D (+) -Cox8-Ago2 linked with a Cox8 gene sequence and Ago2 gene sequence, or a recombinant adeno-associated virus expression vector pAAV-D (+) -Cox-8-Ago 2 linked with a myocardial specific promoter tnt, a Cox8 gene sequence and a Ago2 gene sequence in sequence, or a recombinant adeno-associated virus rAAV-Cox-8-Ago 2 transformed with a recombinant adeno-associated virus expression vector pAAV-D (+) -Cox8-Ago2, or a recombinant adeno-associated virus rAAV-Cox-8-Ago-2 transformed with a recombinant adeno-associated virus expression vector pAAV-D (+) -8-Ago-2-Ago-related adenovirus 8-2 -Ago2 for use in the treatment of heart failure.
The invention provides a medicine for treating heart failure, which is characterized in that the effective components of the medicine comprise a substance which takes mitochondrion targeted Ago2 as a medicine target point and can increase the expression of mitochondrion targeted Ago 2.
The drug effective component of the drug comprises a substance highly expressing mitochondrion targeted Ago 2.
The mitochondrion targeted Ago2 substance comprises a mitochondrion targeted signal sequence fragment and an Ago2 sequence fragment, and the sequence fragments of cox8-Ago2 are shown as SEQ ID NO.1 and SEQ ID NO. 2.
The medicament also comprises pharmaceutically acceptable auxiliary materials and/or reagents for buffering, synthesizing and/or purifying the sequence fragments.
A method for preparing a medicament for treating heart failure, comprising: the substance which can highly express the mitochondrion targeted Ago2 is used as an active ingredient of a medicament for treating heart failure.
Inserting the sequence fragment capable of expressing Cox8-Ago2 into an expression vector, thereby preparing a recombinant plasmid capable of stably expressing mitochondrion targeted Ago 2. The sequence fragment of the Cox8-Ago2 is shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention aims to provide a medicine for treating heart failure, the effective components of the medicine comprise a medicine target of mitochondrion targeted Ago2, and the medicine effect of treating heart failure is achieved by highly expressing a substance of the mitochondrion targeted Ago 2.
High expression: specifically and highly expressing Ago2 containing a mitochondrial localization sequence to mitochondria through various vectors, and specifically and highly expressing Ago2 to mitochondria through a mitochondrial localization sequence such as Cox8, Su9, Sod2 and other leading signals; or delivering Ago2 into mitochondria via a mitochondrial targeting nanoparticle;
any of the above approaches may serve to increase the amount of mitochondrially targeted Ago2 expression. The act of loading each of the above-described substances into commercial packages labeled for anti-heart failure use on any scale falls within the scope of the claimed invention.
Further, the drug effective component of the drug comprises a substance highly expressing mitochondrion-targeted Ago 2.
Further, the substance for highly expressing mitochondrially targeted Ago2 comprises a leader peptide sequence fragment of mitochondrion localization cox8 and a sequence fragment of Ago2 shown as SEQ ID No.1 and SEQ ID No. 2.
Further, the medicament also comprises pharmaceutically acceptable auxiliary materials and/or reagents for buffering, synthesizing and/or purifying the sequence fragments. The technicians in the field can add various pharmaceutically acceptable auxiliary agents/auxiliary materials into the heart failure resisting medicine according to objective requirements to prepare various dosage forms, and the sale or popularization is convenient.
Another object of the present invention is to provide a method for preparing a medicament for treating heart failure, comprising: the substance of high expression mitochondrion targeted Ago2 is used as the active component of the medicine for treating heart failure.
In a further embodiment, the method of making comprises: a leader peptide sequence containing the mitochondrion positioning cox8 and a sequence fragment of Ago2 are inserted into an expression vector, so that a recombinant plasmid of the mitochondrion targeting Ago2 capable of being stably expressed is prepared.
In a specific embodiment, the expression vector comprising the leader peptide sequence of mitochondrial localization cox8 and the sequence fragment of Ago2 is adeno-associated viral expression vector pAAV-D (+).
In order to realize the gene therapy aim of heart failure, the invention recombines and constructs the cox8-Ago2 targeted by mitochondria and a recombinant adeno-associated virus vector containing a myocardial specific promoter tnt, obtains high titer meeting the treatment requirement through detection, and proves that the cardiac function of a heart failure mouse can be effectively improved in an animal experiment. Therefore, based on the findings and results, the invention provides a medicine for treating clinical heart failure, which takes the mitochondrial Ago2 as a treatment target.
The invention relates to a medicament for treating heart failure. The heart failure resisting medicine relates to a construction and preparation method of a recombinant adeno-associated virus recombinant (rAAV-cox8-Ago2) of Ago2 capable of specifically targeting mitochondria, and plays a role in treating heart failure by highly expressing the mitochondrial targeted rAAV-Ago 2. The invention constructs pAAV-D (+) -tnt-cox8-Ago2 expression plasmid by using a chemical synthesis method, and prepares and purifies recombinant adeno-associated virus containing target fragments by using a calcium phosphate cotransfection method for three plasmids. Animal experiments prove that the heart failure resisting medicine provided by the invention can obviously improve the heart function of heart failure animals and has an effective heart failure resisting effect.
The invention designs and synthesizes a sequence of mitochondrion targeting Ago2 based on a cox8 leader base sequence and an Ago2 base sequence of targeting mitochondrion, and successfully inserts the sequence into a eukaryotic expression vector pAAV-D (+) to form a recombinant plasmid pAAV-D (+) -cox8-Ago 2. Thereafter, the following three plasmids were prepared: 1) pXX9 plasmid, 2) phepper plasmid, 3) pAAV-D (+) -cox8-Ago2 plasmid, which are respectively transferred into 293 cells by a calcium-phosphorus cotransfection method to be packaged to prepare recombinant adeno-associated virus (rAAV9) capable of expressing mitochondria-targeted cox8-Ago2, and the titer is determined by a real-time PCR method after purification. And finally, injecting the packaged recombinant adeno-associated virus (rAAV-cox8-Ago2) of the same serotype into a heart failure model mouse caused by aortic ligation (TAC) through tail vein injection, and finding that the long-term expression of mitochondria Ago2 in the heart of the mouse is obviously changed, which indicates that the recombinant adeno-associated virus can mediate the long-term effective expression of mitochondria Ago 2. Ultrasonic and catheter results show that the expression of the recombinant adeno-associated virus mediated mitochondrion targeted Ago2 can obviously improve the cardiac function of a TAC mouse, and further support the anti-heart failure effect of the mitochondrion targeted Ago 2.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 shows a significant increase in the level of Ago2 localized by cardiac mitochondria in heart failure mice; wherein A is a western blot result graph of the content of Ago2 in the sham mouse and the TAC mouse; b is a bar graph of the Ago2 content of sham and TAC mice; the notations in the figures have the following meanings: VDAC1 is expressed as an internal reference protein of mitochondria; sham represents sham control group; TAC represents a TAC-induced heart failure model mouse.
FIG. 2 shows the plasmid construct of pAAV-D (+) -tnt-cox8-Ago 2; wherein tnt promoter represents a cardiomyocyte-specific promoter, and m-Ago2 represents mouse-derived Ago 2.
FIG. 3 shows the Western blotting detection of Ago2 expression in the hearts of TAC mice treated differently in Experimental example 2; the meanings of the symbols in the figures are listed below: the rAAV-tnt-GFP represents a TAC mouse injected with a recombinant adeno-associated virus vector which is specifically targeted to a myocardial cell and expresses GFP, the rAAV-tnt-cox8-Ago2 represents a TAC mouse injected with a recombinant adeno-associated virus vector which is specifically targeted to the myocardial cell and highly expresses Ago2 in mitochondria, and the VDAC1 represents an internal reference protein of mitochondria; the detection result shows that the rAAV-tnt-cox8-Ago2 can obviously increase the expression of TAC mouse heart mitochondria Ago 2.
FIG. 4 is a bar graph of ejection fraction, B shortening fraction, C maximum rate of rise of left ventricular pressure during isovolumetric contraction, and D minimum rate of rise of left ventricular pressure during isovolumetric contraction of TAC mice as determined by cardiac ultrasound and catheter monitoring of rAAV-tnt-cox8-Ago2 in Experimental example 2; the result shows that the rAAV-tnt-cox8-Ago2 can obviously increase the systolic function and the diastolic function of the TAC mouse.
The meanings of the symbols in the figures are listed below: the rAAV-tnt-GFP represents a TAC mouse injected with a recombinant adeno-associated virus vector which is specifically targeted to the myocardial cells and expresses GFP, and the rAAV-tnt-cox8-Ago2 represents a TAC mouse injected with a recombinant adeno-associated virus vector which is specifically targeted to the myocardial cells and highly expresses Ago2 in mitochondria; control represents TAC mice as a blank control injected with physiological saline; sham stands for sham-operated control mice, TAC stands for TAC-induced heart failure model mice.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without any inventive step, are within the scope of the present invention.
The following examples or experimental examples refer to the following sources of instrumentation, reagents and consumables and biological material:
1) instrumentation and equipment
2) reagent and consumable
RNasey Mini Kit (from Qiagen), TRIZOL (from Invitrogen), TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Agarose Gel DNA recovery Kit from TaKaRa),
Endo-Free Plasmid Maxi Kit (Plasmid extraction Kit, available from OMEGA), EasyPure Plasmid MiniPrep Kit (Plasmid extraction Kit, available from beijing sumikagai); eukaryotic expression vector pAAV-D (+) was constructed and presented by the cooperative professor Shouwa, and a cox8-Ago2 DNA sequence fragment was obtained from the Wuhan KoreaLimited company synthesis.
Sources of biological material:
c57 background mice were purchased from beijing waukang;
293T cells were from ATCC company, USA.
Group 1 example, pharmaceutical use of Ago2
The present set of embodiments provides for the use of Ago2 in the preparation of a medicament for the treatment of heart failure.
In specific embodiments, the action target of the drug for treating heart failure is Ago 2;
preferably, the Ago2 is mitochondrially targeted Ago 2;
preferably, said Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2.
The present group of embodiments provides a medicament for treating heart failure. All embodiments of this group share the following common features: the medicament comprises: a pharmaceutically active ingredient; the pharmacodynamically active ingredient comprises: a substance highly expressing mitochondrially targeted Ago 2.
In some embodiments, the agent highly expressing mitochondrially targeted Ago2 is selected from the group consisting of: an expression vector connected with a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence, a transformant transformed with an expression vector connected with a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence, an expression vector sequentially connected with a myocardial specific promoter, a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence, and a transformant transformed with an expression vector sequentially connected with a myocardial specific promoter, a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence;
preferably, said Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2;
preferably, the expression vector is an adeno-associated virus expression vector pAAV-D (+);
preferably, the mitochondrial localizable leader peptide sequence is selected from: cox8, Su9, Sod 2;
the leader peptide Cox8 is Cox8 described in "Double deletion of PINK1 and Parkin improvirs lipidic mitophagy and exarbates acetylaminophen-induced peptide in micro";
the leader peptide Su9 is Su9 described in "MicroRNA direct amino transformation and mutation polypeptide muscle differentiation";
the leader peptide Sod2 is Sod2 described in "Specific immunization of biological genes in particulate-derived cells by mitalores".
Preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, alpha-MHC, MLC-2v, Desmin;
tnt is tnt recorded in the text "Nuclear miR-320media Diabetes-Induced Cardiac Dysfunction by y Activating transformation of fat Acid metabolism Genes to mouse lipid activity in the Heart";
the alpha-MHC is the alpha-MHC described in the Deletion of BCATm involved in insulin-stimulated glucose oxidation in the heart;
MLC-2v is MLC-2v described In "Molecular In Vivo Imaging Using a nonactive cardio-Specific MLC-2v Promoter drive Dual-Gene Recombinant expression Monitoring System";
desmin is Desmin described in "Muscle-Specific reagents for Gene Therapy".
Preferably, the expression vector linking the mitochondrially-localizable leader peptide sequence and the Ago2 gene sequence is selected from the group consisting of: the recombinant adeno-associated virus expression vector pAAV-D (+) -Cox8-Ago2 linked with a Cox8 gene sequence and an Ago2 gene sequence, or the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-Cox8-Ago2 linked with a myocardial specific promoter tnt, a Cox8 gene sequence and an Ago2 gene sequence in sequence;
preferably, the host cell of the transformant is selected from the group consisting of: 293 cells;
preferably, the transformant transformed with the expression vector linked to the mitochondrial-localizable leader peptide sequence and the Ago2 gene sequence is selected from the group consisting of: the recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -cox8-Ago2, or the recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-cox8-Ago 2;
preferably, the Cox8 gene sequence is shown in SEQ ID NO. 1;
preferably, the Ago2 gene sequence is shown in SEQ ID NO. 2.
The group of embodiments provide a drug for treating heart failure, the drug effective component of the drug for treating heart failure takes mitochondrion Ago2 as a drug target, and the drug effect of treating heart failure is achieved by highly expressing the mitochondrion targeted Ago 2. According to the invention, experiments show that the content of Ago2 positioned by heart mitochondria of a heart failure mouse is obviously increased (figure 1), which shows that Ago2 in mitochondria can play a role in regulation and control in the pathophysiological process of heart failure, and further mice experiments are carried out, and the content of the mitochondria Ago2 is increased, so that the heart ejection fraction of a TAC mouse can be obviously improved (figure 4A), and the functions of enhancing the heart function and achieving the purpose of treating heart failure are achieved.
Based on years of research experience in the field of clinical treatment of heart failure, the inventor conjectures that Ago2 is likely to play a crucial role in the occurrence and development of heart failure, and carries out related experiments.
According to the invention, experiments show that the Ago2 level in TAC heart failure mice is remarkably high, and the Ago2 is overexpressed in the TAC heart failure mice and can resist heart failure, and based on years of experience of an inventor in clinical research and development, the logical association between the Ago and the later can be presumed as follows: in heart failure, the increase in Ago2 protein suggests that Ago2 may play an important role in heart failure. However, whether this increase is compensatory protective or destructive, requires subsequent functional studies. In functional research, the invention discovers that Ago2 overexpression can treat heart failure, and the raised Ago2 in the heart failure plays a compensatory protective role, and the protective role can be amplified only by overexpression with insufficient strength.
Further, the drug effective component of the drug comprises a substance of Ago2 which can increase the mitochondria targeting. It is well known to those skilled in the art that a mitochondrial localization signal can be added in front of the Ago2 sequence to specifically increase the expression of the mitochondrial Ago2 to achieve a therapeutic effect, and other means can be used, for example, some mitochondrial targeting nanoparticles can be used to directly deliver Ago2 to mitochondria, and the purpose of increasing the level of Ago2 mitochondria can also be achieved.
Preferably, the pharmacodynamic component of the drug comprises a mitochondrially targeted fragment of Ago2 sequence. The most common mode in molecular biology is to add a mitochondrial localization signal sequence in front of a target sequence fragment so that Ago2 is transported to mitochondria to play a therapeutic role. Cox8 mitochondrial signal peptide is a commonly used mitochondrial localization signal, and the sequence of the co8-Ago2 is shown as SEQ ID No.1 and SEQ ID No. 2.
In some alternative embodiments of the present invention, other mitochondrial localization signal sequences, such as leader sequences of Su9, Sod2, etc., can also specifically deliver Ago2 to mitochondria, and recombinant plasmids of Ago2 containing these leader sequences have similar or equivalent anti-heart failure effects to those of experimental example 2 below, and are not repeated herein in detail in view of limited space.
In some alternative embodiments of the invention, fragments of Ago2 sequences of some other species, such as human Ago2, rat Ago2, etc., which are highly homologous to the mouse Ago2 gene sequence, and recombinant plasmids containing Ago2 of these species have anti-heart failure effects similar to or equivalent to those of experimental example 2 below, which are not described herein in detail in view of limited space.
In some alternative embodiments of the present invention, other promoters of pAAV-D (+) of some recombinant adeno-associated virus plasmids, such as α -MHC, MLC-2v, Desmin, etc., which can achieve specific targeting of the recombinant adeno-associated virus vectors to myocardium as with tnt, and the recombinant plasmid of pAAV-D (+) -cox8-Ago2 containing these promoter sequences has similar or equivalent heart failure resistance to Experimental example 2, which is not repeated herein in detail in view of limited space.
In a most specific embodiment of the invention, the drug has the drug effect component of recombinant adeno-associated virus plasmid pAAV-D (+) -tnt-cox8-Ago2 expressing mitochondrion targeted mouse Ago 2; the recombinant adeno-associated virus (rAAV) vector overcomes the defects which are difficult to overcome by other gene expression vectors, can carry target genes to transfect cells in a division stage and a non-division stage (namely, has a wide transgene range), has no side effect (no immunogenicity), has high infection efficiency, can drive the target genes to express in vivo for a long time, and successfully solves the problem of in vitro mass replication without adenovirus pollution, thereby becoming the most promising vector for gene therapy.
More specifically, the cox8 mitochondrial leader sequence and the mus-Ago2 sequence are inserted into an adenovirus expression vector pAAV-D (+) as shown in SEQ ID NO.1 and SEQ ID NO.2 to construct a pAAV-D (+) -tnt-cox-Ago2 plasmid for expression. The above-mentioned double-stranded nucleotide sequence was synthesized by Wuhan Kerist Biometrics Ltd and inserted into the vector pAAV-D (+).
Further, the medicine also comprises pharmaceutically acceptable auxiliary materials and/or reagents for buffering, culturing and/or expanding the recombinant adeno-associated virus plasmid pAAV-D (+) -cox8-Ago 2; the technicians in the field can add various pharmaceutically acceptable auxiliary agents/auxiliary materials into the heart failure resisting medicine according to objective requirements to prepare various dosage forms, and the sale or popularization is convenient.
The present group of embodiments provides a protein having an effect of treating heart failure. All embodiments of this group share the following common features: the protein with the effect of treating heart failure comprises: the mitochondrial leader peptide sequence and Ago2 sequence can be located.
In some embodiments, the mitochondrial-localizable leader peptide is selected from the group consisting of: cox8, Su9, Sod 2;
preferably, the protein having the effect of treating heart failure refers to a fusion protein consisting of Cox8 and Ago 2.
Group 4 examples, genes of the present invention
The present group of embodiments provides a gene having an effect of treating heart failure. All embodiments of this group share the following common features: the gene comprises: the mitochondrial leader peptide gene sequence and Ago2 gene sequence can be located.
In a further embodiment, the gene having an effect of treating heart failure further comprises: a myocardial-specific promoter;
preferably, the mitochondrial-localizable leader peptide is selected from: cox8, Su9, Sod 2;
preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, alpha-MHC, MLC-2v, Desmin;
preferably, said Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2;
both m-Ago2 and mus-Ago2 herein represent mouse-derived Ago 2.
Preferably, the gene having the effect of treating heart failure is selected from the group consisting of: a gene consisting of a Cox8 gene sequence and an Ago2 gene sequence, or a gene consisting of a myocardial specific promoter tnt sequence, a Cox8 gene sequence and an Ago2 gene sequence;
preferably, the Cox8 gene sequence is shown in SEQ ID NO. 1;
preferably, the Ago2 gene sequence is shown in SEQ ID NO. 2.
Group 5 examples recombinant expression vectors of the invention
The present group of embodiments provides a recombinant expression vector having an effect of treating heart failure. All embodiments of this group share the following common features: the recombinant expression vector with the effect of treating heart failure is an expression vector connected with a sequence of a gene with the effect of treating heart failure, which is provided by any one of the group 4 embodiments.
In specific embodiments, the expression vector is selected from the group consisting of: is an adeno-associated virus expression vector pAAV-D (+);
preferably, the recombinant expression vector having the effect of treating heart failure is selected from the group consisting of: the recombinant adeno-associated virus expression vector pAAV-D (+) -Cox8-Ago2 is connected with a Cox8 gene sequence and an Ago2 gene sequence, or the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-Cox8-Ago2 is sequentially connected with a myocardial specific promoter tnt, a Cox8 gene sequence and an Ago2 gene sequence.
Example of group 6, transformant of the present invention
The present group of embodiments provides a transformant having an effect of treating heart failure. All embodiments of this group share the following common features: the transformant having the effect of treating heart failure is a host cell transformed with the recombinant expression vector having the effect of treating heart failure provided in any one of the embodiments of group 5.
In some embodiments, the host cell is selected from the group consisting of: 293 cells;
preferably, the one transformant having the effect of treating heart failure is selected from the group consisting of: the recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -cox8-Ago2 or the recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-cox8-Ago 2.
EXAMPLE 7 preparation of the medicament of the invention
The present group of embodiments provides a method for preparing a medicament for treating heart failure. All embodiments of this group share the following common features: a fusion protein consisting of a mitochondrially localizable leader peptide and Ago2 was expressed.
In some embodiments, the gene sequence of the mitochondrial-localizable leader peptide and the gene sequence of Ago2 are linked to an expression vector to obtain a recombinant expression vector;
preferably, the myocardial specific promoter, the gene sequence of the mitochondrion-localizable leader peptide and the gene sequence of Ago2 are sequentially connected with an expression vector to obtain a recombinant expression vector;
preferably, the host cell is transformed with the recombinant expression vector to obtain a transformant;
preferably, a calcium-phosphorus co-transfection method is adopted to transform the recombinant expression vector into a host cell to obtain a transformant;
preferably, said Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2;
preferably, the expression vector is an adeno-associated virus expression vector pAAV-D (+);
preferably, the mitochondrial localizable leader peptide sequence is selected from: cox8, Su9, Sod 2;
preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, alpha-MHC, MLC-2v, Desmin;
preferably, the recombinant expression vector is selected from the group consisting of: the recombinant adeno-associated virus expression vector pAAV-D (+) -Cox8-Ago2 linked with a Cox8 gene sequence and an Ago2 gene sequence, or the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-Cox8-Ago2 linked with a myocardial specific promoter tnt, a Cox8 gene sequence and an Ago2 gene sequence in sequence;
preferably, the transformant is selected from the group consisting of: the recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -cox8-Ago2, or the recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-cox8-Ago 2;
preferably, the Cox8 gene sequence is shown in SEQ ID NO. 1;
preferably, the Ago2 gene sequence is shown in SEQ ID NO. 2.
In some specific embodiments of the group, the recombinant adeno-associated virus plasmid pAAV-D (+) -cox8-Ago2, which can highly express mitochondrial targeting substances, namely Ago2 sequence fragment containing cox8 mitochondrial localization signal, is used as the active ingredient of the anti-heart-failure drug.
Furthermore, a total gene synthesis technology is adopted to synthesize the mus-cox8-Ago2 double-stranded nucleotide and clone the double-stranded nucleotide on a pAAV-D (+) vector, so that the Ago2 recombinant plasmid capable of stably expressing the mitochondrion target is prepared. In a further specific scheme, the cox8-Ago2 sequence fragment capable of expressing mitochondrial targeting is shown as SEQ ID NO.1 and SEQ ID NO. 2; the expression vector is an adeno-associated virus expression vector pAAV-D (+).
The specific experimental procedures of this group of examples can be seen in example 1.
The technical effects of the present invention are further verified and explained below by experimental examples.
Experimental example 1 construction of recombinant adeno-associated Virus
Construction of pAAV-D (+) vector
A Cox8-Ago2 double-stranded nucleotide was synthesized by a whole gene synthesis technique based on the base sequences of the mitochondrial leader sequences Cox8 and mus-Ago2 (mouse-derived Ago2) and cloned on a pAAV-D (+) vector (FIG. 2), and the double-stranded nucleotide was synthesized by Wuhan Kerister BioLimited. The sequences of cox8 and Ago2 are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.
2. Plasmid transformation
The pAAV-D (+) -cox8-Ago2 plasmid is added into 100 μ l DH5 alpha competent cells and placed in ice for 30 min; heating at 42 deg.C for 45sec, and standing in ice for 1 min; adding 500 μ l of antibiotic-free LB culture medium, and performing shake culture at 37 deg.C and 100rpm for 60 min; culturing on an Amp + LB plate culture medium, and selecting a white monoclonal colony for identification.
3. Plasmid small grape
Single colonies were picked and added to 3ml of Amp + LB liquid medium and cultured overnight at 37 ℃ with shaking at 280 rpm. The method comprises the following specific steps of extracting plasmids by using an easy pure Plasmid MiniPrep Kit of Beijing Quanji corporation: 1. 1.5ml of overnight cultured bacteria 10,000g are centrifuged for 1min, and the supernatant is aspirated as far as possible; 2. adding 250 μ l of colorless solution RB (containing RNase A), and shaking to suspend the bacterial precipitate; 3. adding 250 μ l blue solution LB, gently turning and mixing for 4-6 times to crack thallus fully to form blue bright solution; 4. adding 350 μ l yellow solution NB, mixing gently for 5-6 times until compact yellow aggregate is formed, standing at room temperature for 2 min; 5.15,000g, centrifuging for 5min, carefully sucking the supernatant, and adding into an adsorption column; 6.15,000g, centrifuging for 1min, and discarding the effluent; 7. adding 650 μ l of WB solution, centrifuging at 15,000g for 1min, and discarding the effluent; 8.15,000g, centrifuging for 2min, and completely removing residual WB; 9. placing the adsorption column in a new Ep tube, adding 20 μ l of 70 deg.C preheated EB in the center of the column, standing at room temperature for 1 min; 10.10,000g, centrifuging for 1min, eluting DNA, and storing the eluted DNA at-20 ℃.
4. Plasmid major grape
A1L sterile Erlenmeyer flask was prepared and 300ml of sterile LB medium was added, plus ampicillin solution to a final concentration of 100. mu.g/ml. 50. mu.l of the desired plasmid (pXX9, phepper, pAAV-D (+) -GFP, pAAV-D (+) -cox8-Ago2) was added thereto, and the mixture was cultured overnight at 37 ℃ at 280 rpm. The Plasmid was extracted according to the instructions of the Endo-Free Plasmid Maxi Kit of OMEGA, the specific procedure was as follows: 1. centrifuging at room temperature for 10min at 5000g to collect bacteria; 2. removing the culture medium, adding 10ml of Solution I/RNase A mixed Solution, and shaking in a vortex to completely resuspend; 3. adding 10ml Solution II into the re-suspended mixed Solution, slightly reversing and mixing for 10-15 times, and standing at room temperature
rAAV-mediated viral packaging
293T cells grow to 90%, 1-2hr before calcium and phosphorus transfection, 12-15ml of fresh culture medium (containing serum) is replaced for each culture dish, calcium chloride (CaCl2) is firstly added into a 50ml centrifugal tube, then plasmids are added to form Ca-DNA mixed solution, the Ca-DNA mixed solution is fully mixed, 2XHEBS BUFFER is slowly dripped into the Ca-DNA mixed solution to form Ca-DNA-P mixed solution, the centrifugal tube is shaken while 2XHEBS is added, and the Ca and the P particles are fully mixed to form. After 8-12hr, changing 18-20ml serum-free medium, after 72hr, removing the medium by aspiration, washing with PBS 3 times, adding 1ml Tris + NaCl (pH 8.5) into each culture dish, scraping cells with a spatula, collecting in a clean centrifuge tube, and freezing at-80 deg.C.
6. Virus purification
Taking out the cells frozen at-80 deg.C, thawing at 37 deg.C for dissolving, repeatedly freezing and thawing for 4 times, centrifuging for 15min at 8,000g, placing the supernatant into a clean centrifuge tube, and removing the cell precipitate. Mixing with-20 deg.C pre-cooled anhydrous ethanol and rAAV at volume ratio of 3: 1, standing in refrigerator at-20 deg.C for 2hr, centrifuging at 4 deg.C and 13,000rpm for 15min, and removing supernatant; after ethanol evaporation, the pellet was dissolved by adding the corresponding volume of Tris + NaCl (pH 8.5). Filtration was performed using Millipore small filters (0.22 μm).
7. Viral titer determination
Sample treatment:
40 mul rAAV virus liquid
Proteinase K (20mg/ml) 5. mu.l
Reacting at 55 deg.C for 1 hr;
phenol: chloroform: isoamyl alcohol 45. mu.l
Centrifuging at 4 deg.C for 5min at 12,000g to recover water phase;
chloroform 45. mu.l
The aqueous phase was recovered by centrifugation at 12,000g for 5min at 4 ℃.
Real-time PCR：
Primer 1(10μm)0.4μl
Primer 2(10μm)0.4μl
SYBR Green I Mix 10μl
ddH 2 O 8.2μl
Template 1μl
95 ℃ 30sec- - - (95 ℃ 5sec- - -60 ℃ 5sec- - -72 ℃ 20 sec). times.40 cycles- - -Multi cut
Experimental example 2, the therapeutic effect on heart failure was examined by using recombinant adeno-associated virus expressing cox8-Ago2 of rAAV9 type as an example
Detection of TAC mouse cardiac mitochondrial Ago2 expression:
TAC surgery was performed using 8 week old C57 mice: when the experiment is finished (after 4 weeks), a Biyunnan tissue mitochondria extraction kit is adopted to extract myocardial mitochondria to detect the expression condition of mitochondria Ago2 in the heart of a TAC mouse by a Western blot method, and the result shows that: the TAC mouse cardiac mitochondrial Ago2 content was significantly increased (fig. 1).
Adopting C57 mouse of 8 weeks age, injecting rAAV-tnt-cox8-Ago2 and control rAAV-tnt-GFP virus through tail vein, the virus titer is 1X 10 11 PFU/each, TAC surgery was performed 2 weeks later: when the thoracic aorta ligation (TAC) is reached to the end point of the experiment (after 4 weeks), extracting myocardial mitochondria, detecting the expression condition of the mitochondria Ago2 in the heart of a TAC mouse by using a Western blot method, and displaying the result: rAAV-tnt-cox8-Ago2 treatment significantly increased expression of cardiac mitochondrial Ago2 (FIG. 3).
TAC mouse heart function detection: and (3) detecting the heart function of the TAC mouse by adopting heart ultrasonic at the end point of the experiment, wherein the method comprises the following steps:
the instrument used was an ultrasound instrument equipped with a 30MHz high frequency probe. After a mouse is anesthetized by isoflurane, the mouse is placed on a detection platform in a supine mode, a two-dimensional image of a left ventricle is collected along a horizontal short axis and a long axis section of papillary muscle of the left ventricle near a sternum of the mouse, more than 5 continuous cardiac cycle M-type ultrasonic images are obtained under the guidance of the two-dimensional image, a cardiac ultrasonic detection cardiac hemodynamic index is obtained according to the analysis result of the collected images by using software, and the following indexes are calculated after the analysis of related software: including Heart Rate (HR), Left Ventricular diastolic inner diameter (Left Ventricular diastolic inner diameter, diastole, LVIDd), Left Ventricular systolic inner diameter (Left Ventricular systolic inner diameter, systole, LVIDs), Left Ventricular Posterior diastolic thickness (Left Ventricular diastolic Wall, LVPWd), Left Ventricular Posterior systolic thickness (Left Ventricular systolic Wall, systole, LVPWs), Ventricular interval diastolic thickness (interstitial Ventricular systolic), Ventricular interval systolic thickness (interstitial systolic, Sd), Ventricular interval systolic thickness (interstitial Fraction, system, IVsj), Ejection Fraction (Ejef, Frajef), and Fractional Shortening (FS, etc.). The results show that: rAAV-cox8-Ago2 treatment significantly improved cardiac function in TAC mice (FIG. 4).
SEQUENCE LISTING
<110> affiliated Tongji hospital of Tongji medical college of Huazhong university of science and technology
Application of <120> Ago2 in preparation of heart failure treatment drugs, and protein, gene, transformant, drug and preparation thereof
Preparation method
<130> P220591/HZT
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 108
<212> DNA
<213> Artificial Sequence
<220>
<223> COX8
<400> 1
atgtccgtcc tgacgccgct gctgctgcgg ggcttgacag gctcggcccg gcggctccca 60
gtgccgcgcg ccaagatcca ttcgttgggg gatccaccgg tcgccacc 108
<210> 2
<211> 2580
<212> DNA
<213> Artificial Sequence
<220>
<223> Mus-Ago2
<400> 2
atgtactcgg gagccggccc cgttcttgct tctcctgctc cgacaacatc acccatccca 60
ggatatgcct tcaaacctcc acctcggccg gacttcggca ccaccgggag aacaatcaaa 120
ctacaggcca atttctttga aatggacatc cccaaaattg acatctatca ctatgaattg 180
gacatcaaac ctgagaaatg ccctcggaga gtgaacaggg aaattgtgga gcacatggtc 240
cagcacttta aaactcagat cttcggggac cggaagccag tgtttgatgg aaggaagaat 300
ctgtacacag caatgcccct tccgatcggc agggacaagg tggagctgga ggtcacgctc 360
ccgggagaag gcaaagatcg catctttaag gtatccatca agtgggtgtc gtgcgtgagc 420
ctgcaggcgt tacacgatgc actttcgggg cggctgccca gcgtcccctt cgagacgatc 480
caggccctgg acgttgtcat gaggcactta ccatccatga ggtacacccc tgttggccgt 540
tccttcttca ctgcatctga aggctgttcc aaccctctgg gtgggggcag agaagtgtgg 600
tttggcttcc atcagtccgt ccgaccttct ctttggaaaa tgatgctgaa tattgatgta 660
tcggcaacag cgttttacaa ggcacagcca gtgatcgagt ttgtttgtga agttttggat 720
tttaaaagta ttgaagaaca acaaaaacct ctgacagatt cccaaagggt aaagtttacc 780
aaagaaatca aaggtctaaa ggtggagata acgcactgtg gtcagatgaa gaggaagtac 840
cgtgtctgca atgtgacccg gcggcctgcc agtcaccaaa cgttcccact gcagcaggag 900
agtgggcaga cagtggagtg tacagtggcc cagtacttca aggacaggca caagctggtt 960
ctgcgctacc cccacctccc gtgtttacaa gtcggacagg agcagaaaca cacctacctt 1020
cctttggagg tctgtaacat agttgctgga cagagatgta taaaaaaatt aacagacaat 1080
cagacctcaa ccatgatcag agcaactgcc aggtcagcac ctgatcgcca agaggagatc 1140
agcaaactga tgcgaagtgc aagtttcaat acagacccat atgttcgtga atttggaatc 1200
atggtgaaag atgagatgac agatgtgact gggcgggttc tgcagccgcc ctccatcctc 1260
tacgggggca ggaacaaagc aattgccacc cctgtccagg gtgtgtggga catgcgaaac 1320
aagcagttcc acacgggcat cgagatcaag gtgtgggcca tcgcctgctt cgctccccag 1380
cgccagtgta cagaagtcca tctcaagtcc ttcacagagc aactgaggaa gatctcgaga 1440
gatgctggga tgccgatcca gggtcagccc tgcttctgta aatacgcaca gggtgcagac 1500
agtgtggagc ccatgttccg acacctgaag aacacatacg ctggcctcca gctggtggtg 1560
gtcatcctgc ctggcaaaac tcctgtgtat gcggaagtca agcgtgtggg agacacagtg 1620
ctggggatgg ccacacagtg cgtccagatg aagaacgtgc agaggaccac gccacagacc 1680
ctatccaatc tctgcttaaa gatcaatgtc aaactgggag gcgtcaacaa catcctgctg 1740
ccacagggca ggcctccagt gttccagcaa cctgtcatct tcctgggagc cgatgtcacc 1800
cacccaccag ctggggatgg gaagaaacct tctattgctg ccgtcgtggg cagcatggac 1860
gcacacccca accgctactg tgccaccgtg cgcgtgcagc agcaccgaca ggagatcatc 1920
caggacctgg ctgccatggt acgagagttg ctcattcagt tctacaagtc cacccgcttc 1980
aagcccaccc gcatcatctt ctaccgcgat ggcgtctccg agggccagtt ccagcaggtt 2040
ctccaccatg agctcctggc catcagagag gcctgcatca agctggagaa ggactatcag 2100
ccaggaatca cgttcatcgt ggtgcagaag cggcaccaca cacgcctctt ctgcacagac 2160
aaaaatgagc gggttgggaa gagtgggaac attcccgcag gcacaaccgt ggacacgaag 2220
atcacccacc ccactgagtt tgacttctac ctgtgcagtc acgccggcat ccaggggaca 2280
agccgtcctt cccactacca cgtgctttgg gatgacaatc gtttttcttc tgatgagctg 2340
cagattctga cctaccagct gtgtcacacc tacgtgcgct gcacacgctc tgtgtcaata 2400
cccgcgccag cttactatgc tcacctggtg gccttccgag ccaggtacca cctggtggat 2460
aaagaacatg acagcgctga aggaagccat acctctgggc agagcaacgg gcgagatcac 2520
caagcgctgg ccaaggcggt ccaggtccac caggacacac tgcgcaccat gtactttgct 2580
Claims (10)
- Use of Ago2 in the manufacture of a medicament for the treatment of heart failure.
- 2. The use of Ago2 in the preparation of a medicament for the treatment of heart failure according to claim 1, wherein the target of action of said medicament for the treatment of heart failure is Ago 2;and/or, the Ago2 is mitochondrially targeted Ago 2;and/or, the Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2.
- 3. A medicament for treating heart failure comprising: a pharmaceutically active ingredient; characterized in that the pharmacodynamically active ingredients comprise: a substance highly expressing mitochondrially targeted Ago 2.
- 4. The drug for treating heart failure according to claim 3, wherein the substance highly expressing mitochondrially targeted Ago2 is selected from the group consisting of: an expression vector connected with a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence, a transformant transformed with an expression vector connected with a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence, an expression vector sequentially connected with a myocardial specific promoter, a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence, and a transformant transformed with an expression vector sequentially connected with a myocardial specific promoter, a mitochondrion-localizable leader peptide sequence and an Ago2 gene sequence;and/or, the Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2;and/or the expression vector is an adeno-associated virus expression vector pAAV-D (+);and/or, the mitochondrial localizable leader peptide sequence is selected from: cox8, Su9, Sod 2;and/or, the myocardium-specific promoter is selected from: tnt, alpha-MHC, MLC-2v, Desmin;and/or, the expression vector connecting the mitochondrially-localizable leader peptide sequence and the Ago2 gene sequence is selected from the group consisting of: the recombinant adeno-associated virus expression vector pAAV-D (+) -Cox8-Ago2 linked with a Cox8 gene sequence and an Ago2 gene sequence, or the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-Cox8-Ago2 linked with a myocardial specific promoter tnt, a Cox8 gene sequence and an Ago2 gene sequence in sequence;and/or, the host cell of the transformant is selected from: 293 cells;and/or, the transformant transformed with the expression vector linked to the mitochondrially localizable leader peptide sequence and the Ago2 gene sequence is selected from the group consisting of: the recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -cox8-Ago2, or the recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-cox8-Ago 2;and/or the Cox8 gene sequence is shown in SEQ ID NO. 1;and/or the sequence of the Ago2 gene is shown as SEQ ID NO. 2.
- 5. A protein having an effect of treating heart failure, comprising: the mitochondrial leader peptide sequence and Ago2 sequence can be located.
- 6. The protein having an effect of treating heart failure according to claim 5, wherein the mitochondrial targeting leader peptide is selected from the group consisting of: cox8, Su9, Sod 2;and/or, the protein with the effect of treating heart failure refers to a fusion protein consisting of Cox8 and Ago 2.
- 7. A gene having an effect of treating heart failure, comprising: the mitochondrial leader peptide gene sequence and Ago2 gene sequence can be located.
- 8. The gene having an effect of treating heart failure according to claim 7, further comprising: a myocardial-specific promoter;and/or, the mitochondrial-localizable leader peptide is selected from: cox8, Su9, Sod 2;and/or, the myocardium-specific promoter is selected from: tnt, alpha-MHC, MLC-2v, Desmin;and/or, the Ago2 is selected from: human Ago2, rat Ago2 and mouse Ago 2;and/or, the gene with the effect of treating heart failure is selected from the group consisting of: a gene consisting of a Cox8 gene sequence and an Ago2 gene sequence, or a gene consisting of a myocardial specific promoter tnt sequence, a Cox8 gene sequence and an Ago2 gene sequence;and/or the Cox8 gene sequence is shown in SEQ ID NO. 1;and/or the sequence of the Ago2 gene is shown as SEQ ID NO. 2.
- 9. A recombinant expression vector having an effect of treating heart failure, which is an expression vector to which the sequence of a gene having an effect of treating heart failure according to claim 7 or 8 is ligated.
- 10. The recombinant expression vector with heart failure treatment effect according to claim 9, wherein the expression vector is selected from the group consisting of: is an adeno-associated virus expression vector pAAV-D (+);and/or, the recombinant expression vector with the effect of treating heart failure is selected from the group consisting of: the recombinant adeno-associated virus expression vector pAAV-D (+) -Cox8-Ago2 is connected with a Cox8 gene sequence and an Ago2 gene sequence, or the recombinant adeno-associated virus expression vector pAAV-D (+) -tnt-Cox8-Ago2 is sequentially connected with a myocardial specific promoter tnt, a Cox8 gene sequence and an Ago2 gene sequence.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210708828.7A CN115068632A (en) | 2022-06-22 | 2022-06-22 | Application of Ago2 in preparation of medicine for treating heart failure, protein, gene, transformant, medicine and preparation method thereof |
| CN202211079886.4A CN116019935A (en) | 2022-06-22 | 2022-09-05 | Application of Ago2 in preparing medicines for treating heart failure or diabetic cardiomyopathy and protein, gene and transformant thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210708828.7A CN115068632A (en) | 2022-06-22 | 2022-06-22 | Application of Ago2 in preparation of medicine for treating heart failure, protein, gene, transformant, medicine and preparation method thereof |
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| CN115068632A true CN115068632A (en) | 2022-09-20 |
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| CN202210708828.7A Withdrawn CN115068632A (en) | 2022-06-22 | 2022-06-22 | Application of Ago2 in preparation of medicine for treating heart failure, protein, gene, transformant, medicine and preparation method thereof |
| CN202211079886.4A Pending CN116019935A (en) | 2022-06-22 | 2022-09-05 | Application of Ago2 in preparing medicines for treating heart failure or diabetic cardiomyopathy and protein, gene and transformant thereof |
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| CN202211079886.4A Pending CN116019935A (en) | 2022-06-22 | 2022-09-05 | Application of Ago2 in preparing medicines for treating heart failure or diabetic cardiomyopathy and protein, gene and transformant thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111630168A (en) * | 2017-12-15 | 2020-09-04 | 汉诺威医学院 | Improved compounds for the treatment of heart failure |
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2022
- 2022-06-22 CN CN202210708828.7A patent/CN115068632A/en not_active Withdrawn
- 2022-09-05 CN CN202211079886.4A patent/CN116019935A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111630168A (en) * | 2017-12-15 | 2020-09-04 | 汉诺威医学院 | Improved compounds for the treatment of heart failure |
| CN111630168B (en) * | 2017-12-15 | 2023-09-22 | 汉诺威医学院 | Improved compounds for treating heart failure |
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| CN116019935A (en) | 2023-04-28 |
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