175 results about "Replicon" patented technology

The invention provides a production strain of an antibiotic-free plasmid, the production strain is a gene editing strain of a PIR strain and is named as PIR1-WN:: 0636 or PIR1-PR: 0636, the production strain contains a nucleotide sequence for coding toxin protein and the antibiotic-free plasmid, and the antibiotic-free plasmid contains a nucleotide sequence for coding antitoxin protein; and preferably, the replicon DNA element of the nonreactive plasmid is R6K-gamma. The toxin protein gene of the production strain disclosed by the invention can be stably passaged, has lethality after being induced and can be used for plasmid screening; according to the invention, the positive rate of transforming the nonreactive plasmid into the PIR1-WN:: 0636 strain is more than 80%, and stable production of the plasmid with a high superhelix ratio can be realized.

The invention provides a construction method and application of a high-efficiency expression system based on a Seali forest virus replicon. The method comprises the following steps: constructing a saRNA system for expressing a reporter gene; constructing to obtain replicase providing plasmids in the taRNA system; constructing to obtain an antigen providing plasmid in the taRNA system; the method comprises the following steps: respectively linearizing pSFV-CMV-T7-X, pSFV-CMV-T7-TR-X and pSFV-CMV-T7-replicase, and then carrying out in vitro transcription to prepare mRNA (Messenger Ribonucleic Acid); transfecting the prepared mRNA into a host eukaryotic cell for expression according to a certain proportion to obtain an antigen protein; and detecting the antigen protein to evaluate the expression efficiency of the saRNA and taRNA system based on the SFV replicon. According to the self-amplification DNA and RNA vaccine based on the virus replicon, the development period can be shortened, and the vaccine antigen can be expressed at a high level.

The invention provides a sphingomonas paucimobilis CRISPRi / dCas12a gene regulation system as well as a construction method and application thereof, and particularly belongs to the technical field of gene engineering. The gene regulation and control system disclosed by the invention comprises a pBBR1-dCas12a-crRNA plasmid, wherein the plasmid comprises a pBBR1 replicon, a dCas12a gene, a crRNA expression cassette and a chloramphenicol resistance gene; the crRNA expression cassette comprises a Ptac promoter, two DR fragments and an rrnB T1 terminator, and is characterized in that the crRNA expression cassette comprises a Ptac promoter, two DR fragments and an rrnB T1 terminator. The system disclosed by the invention can stably express in sphingomonas paucimobilis and inhibit the transcription of genes so as to reduce the expression of specific genes without modifying the DNA (Deoxyribose Nucleic Acid) sequence of the specific genes, has lower toxicity to S.paucimobilis, is high in host growth speed and can realize the efficient expression of dcas12a.

The invention provides a polygene base editing carrier, an editing method and application, and belongs to the technical field of gene editing. According to the polygene base editing vector provided by the invention, the mode that the replicon element is connected with U6 and sgRNA in series is utilized, so that the plasmid containing the replicon element can continuously exist in a cell, and other elements on the plasmid can be continuously expressed in the cell, so that the polygene silencing efficiency is improved. The invention also provides a tandem carrier, namely an epi element in the carrier is removed. The two multi-gene editing vectors constructed in the embodiment of the invention have silence editing effects on targeting sites, and the editing efficiency of the vector containing the epi element is higher. According to the invention, a 5-gene silent homozygous cell line is obtained through a drug enrichment screening method, and a material and a method are provided for disease-resistant breeding.

The invention provides a system and a method for screening Cas protein mutants capable of identifying different PAM sequences, and belongs to the technical field of bioengineering. The system comprises a Cas protein mutant screening vector and an sgRNA expression vector, the Cas protein mutant screening vector comprises the following expression elements: a Cas protein coding gene, a plasmid replicon and a first resistance screening tag expression cassette; the sgRNA expression vector comprises the following expression elements: a plasmid replicon, a second resistance screening tag expression cassette, a third resistance screening tag expression cassette and an sgRNA expression cassette. According to the system and the method, aiming at the problem that the efficiency of identifying PAM sequences except NGG by wild type spCas9 protein is low, an active Cas9 mutant capable of identifying PAM sites except NGG is screened out, and the application field and the editing efficiency of a CRISPR gene editing system can be greatly expanded.

The present invention relates to the field of in vitro transcription, and particularly to a plasmid backbone capable of improving the stability of polyadenylate tails. The present invention provides an expression cassette, which sequentially includes: the rop gene, the replicon ori, and the resistance gene; the rop gene, the replicon ori, and the resistance gene are connected by a linking fragment. The present invention has established a low-copy pmRVacSL and a medium-copy pmRVacM plasmid vector system through artificial modification. Since pmRVacSL is a low-copy vector, the yield of plasmid DNA is relatively low. The pmRVacM plasmid backbone solves the problem of relatively low plasmid DNA yield of pmRVacSL. This vector system can not only maintain the stable replication of polyA tail sequences of more than 150 bp, but also quickly add genes of interest and polyadenylate (polyA) sequences, and efficiently and rapidly obtain the DNA template for in vitro transcription of the target mRNA.

The invention discloses a gene editing tool based on CRISPR-Cas12f1 and application of the gene editing tool. The gene editing tool comprises a plasmid pET-Cas12f1 for expressing endonuclease Cas12f1, and the plasmid comprises a PgrcA promoter, a Cas12f1 nuclease gene, a lambda-RED recombination system gene, an araC protein gene and a ParaC promoter; the invention discloses an auxiliary plasmid pACYC-sgRNA-sacB for guiding Cas12f1, and the plasmid comprises a PJ23119 promoter, an sgRNA sequence, a sacB gene and a replicon P15A. The genome editing tool can realize gene editing in Escherichia coli, and the efficiency of gene knockout after multiple rounds of editing of strains is remarkably improved. The gene editing system is used for gene knockout, and the problem that the efficiency is greatly reduced after a traditional gene editing tool is used for multiple rounds of editing is solved.

The invention discloses a method for constructing an engineered bacterium of Clostridium aeruginosa, comprising the following steps: preparing a desired gene in a biosynthetic pathway of a target product, namely a pathway gene, to form a pathway gene cluster; embedding the pathway gene cluster into a transposase recognition site of a mariner transposon to form a pathway gene expression cassette; selecting a constitutive strong promoter commonly used in Clostridium jundal for driving transposase expression, loading the pathway gene expression cassette into a multiple cloning site of a plasmid, and inserting an inducible repressor element upstream of the replicon to obtain a transposition plasmid; transforming the transposition plasmid into Clostridium aeruginosa to obtain an engineered bacterium of Clostridium aeruginosa. The yield of 3-hydroxybutyric acid expressed by the engineered bacterium constructed by the above method is as high as 3g / L.

The present invention comprises compositions comprising at least two RNA replicons (self-amplifying RNA vector (saRNA or rRNA)) that can be replicated by a replicase of a self-replicating virus (e.g., a replicase of alphavirus origin). In the at least two replicons, at least one of the replicons optionally comprises an open reading frame encoding an RNA-dependent RNA polymerase or replicase, the RNA-dependent RNA polymerase or replicase being capable of replicating each of the at least two replicons. Furthermore, each replicon comprises an open reading frame encoding a different antigen of interest, such as a different antigen derived from the same or different pathogenic organism, such as glycoproteins and nucleoproteins of Ebola virus.

The invention discloses a construction method and application of a Uusu figure virus report replicon. According to the invention, a low-copy plasmid pACYC-177B is used as a carrier, all non-structural proteins and non-coding region genes of USUV are retained, 38 amino acids before the N terminal of C protein are retained in the structural protein part, 45 amino acids behind the C terminal of E protein are retained in the structural protein part, and a NanoLuc luciferase tag is inserted between the retained C and E protein genes to obtain a pACYC-TMN-Nlucc-Rep report replicon. The Uu figure virus report replicon pACYC-TMN-Nluc-Rep constructed in the invention can normally work on cells such as BHK21, Vero, DF-1, Hela and the like, and can be effectively translated and replicated. Therefore, the USUV report replicon pACYC-TMN-Nlucc-Rep constructed in the invention can be used as an important research tool to be applied to the research of the USUV.

The invention relates to RNA constructs encoding (i) at least one therapeutic biomolecule; and (ii) at least one innate inhibitor protein (IIP). The constructs are RNA replicons and saRNA molecules, and the invention includes genetic constructs or vectors encoding such RNA replicons. The invention extends to the use of such RNA constructs and replicons in therapy, for example in treating diseases and / or in vaccine delivery. The invention extends to pharmaceutical compositions comprising such RNA constructs, and methods and uses thereof.

The present invention pertains to the use of a vaccine based on an alphavirus RNA replicon particle (αRP) vector encoding an antigen of an IAV-S for the passive vaccination of piglets against a pathogenic infection with swine influenza virus.

The present invention relates to vaccination against animal pathogens using alpha virus-replicon RNA particles and oil adjuvants. To vaccines and kits comprising said replicon particles and said oil adjuvant. The invention also relates to methods and uses for preparing and using the vaccine and the components of the kit.

The present invention discloses an application of an insect virus FHV RNA1 replicon in exogenous gene amplification, wherein the application amplifies the exogenous gene at the mRNA level by utilizing the autonomous replication ability of the insect virus FHV RNA1 replicon; the present invention discloses a plasmid system based on the insect virus FHV RNA1 replicon and a method for constructing the plasmid system; the present invention also discloses a method and application of the plasmid system for expressing proteins in cells, wherein the plasmid system is used to obtain a cloning vector after double enzyme digestion, and after homologous recombination with an exogenous gene, the animal or plant protein is expressed in the cell.

A cassette for the delivery of one or more mRNA is provided, the cassette comprising a 5' UTR, a Nodamura replicon, a ribosomal skipping polynucleotide, the one or more mRNA, a plant virus assembly origin polynucleotide ("OAS"), and a 3' UTR. In one aspect, the cassette further comprises a 5'mRNA cap. In a yet further aspect, the cassette does not comprise a poly A tail.

A nucleic acid construct encoding multiple polypeptide antigens or immunogens or other polypeptides of interest. The construct expresses a protein that comprises immunogenic or antigenic sequences of two, three or more polypeptides of interest. Once expressed by the nucleic acid construct, the protein, is processed by cleavage of an N-terminal signal peptide and cleavage of 6K, 6K-like, or internal signal peptide cleavage sites to release each polypeptide of interest. Vectors, including replicons and live virus vectors, comprising the construct may be used to deliver it to a subject in need of vaccination. Vaccines and methods of treatment using the nucleic acid construct are also described.

The invention relates to a CRISPR-Cpf1-based single plasmid gene editing system and application thereof, and belongs to the technical field of gene editing. According to the invention, a plasmid pcr19NM is used as a carrier, a crRNA sequence insertion region, a gene for coding Cpf1 protein, a gene for coding NgAgo protein and a replicon pE194ts are connected to the pcr19NM, and expression of the Cpf1 protein and the NgAgo protein is regulated and controlled through a promoter Pm1, so that a recombinant plasmid pWLBE-Pm1Cpf1Ng is obtained. The crRNA of a target gene is introduced into a crRNA sequence insertion region, and a homologous arm is connected to a recombinant plasmid pWLBE-Pm1Cpf1Ng, so that gene knockout, base modification or integration can be realized. Based on the single plasmid gene editing system provided by the invention, accurate gene editing can be efficiently completed by transforming a single plasmid, including complete knockout of two genes, base modification of six genes or integration of one gene. According to the single plasmid gene editing system disclosed by the invention, the complexity of operation is reduced, the gene editing period is shortened, and the method for applying the bacillus subtilis to genetic manipulation is expanded.

Provided herein are methods and compositions related to the finding that specific mutations in nonstructural proteins of self-replicating RNA molecules including RNA replicons, such as Sindbis RNA replicons or Venezuelan equine encephalitis (VEE) replicons, can stabilize the longevity of the RNA replicon and can stabilize the expression of the cargo proteins encoded by the self-replicating RNA molecule or RNA replicon. Also disclosed herein are compositions and methods directed to the discovery that certain viral proteins, including viral proteins that repress internal cellular innate immunity, can stabilize the payload expression and replication of the replicons. Technologies provided herein provide stabilization mechanisms that can improve vaccines, gene therapies, and / or other gene delivery methods.

The present application relates to a kind of 2'-fucosyllactose producing recombinant escherichia coli and its construction method and application, belong to the field of bioengineering technology.The present application expresses 2'-fucosyllactose remediation synthesis pathway related gene in recombinant escherichia coli, including the gene fkp of coding GDP-fucose pyrophosphorylase FKP and the gene futC of coding fucosyltransferase FutC, respectively in the upstream of fkp gene and futC gene expression macromolecular coagulation material protein FUSLCD after the modification of coagulation tag GCN4, and replaces replicon, constructs a strain of high-yield 2'-fucosyllactose recombinant escherichia coli, yield and production intensity reach 38.68g / L and 0.35g / L / h respectively.It has good application prospect, has the potential of industrial application.

To provide: a synthetic oncolytic virus which, upon injection at a tumor site, triggers anti-cancer immune response of local tumors and enables systemic immunity against distal tumors; a pharmaceutical composition comprising the virus; and a method of producing the pharmaceutical composition.SOLUTION: The present disclosure provides synthetic oncolytic viruses comprising a lipid nanoparticle comprising one or more types of lipid and a self-amplifying replicon RNA comprising a sequence that encodes an interleukin (IL)-12 molecule. The lipid nanoparticle is capable of triggering immunogenic cell death. The IL-12 molecule is expressed by the self-amplifying replicon RNA.SELECTED DRAWING: Figure 1

The present disclosure relates to synthetic oncolytic viruses comprising a lipid nanoparticle comprising one or more types of lipid and a self-amplifying replicon RNA comprising a sequence that encodes an immunomodulatory molecule.

The invention relates to the technical field of biology, in particular to a nucleic acid molecule, a carrier containing the nucleic acid molecule, a host cell, a pharmaceutical composition and application of the nucleic acid molecule and the carrier. The nucleic acid molecule provided by the invention is particularly suitable for application scenes of gene therapy and treatment of various diseases.

The invention relates to a plasmid vector capable of easily detecting the copy number of plasmids and a detection kit thereof. According to the plasmid vector, a plasmid replicon ORI gene and a single copy conserved gene of an escherichia coli genome are constructed on the same plasmid. The detection kit contains the plasmid vector or a single-copy quality control product prepared from the plasmid vector. The method has the advantages of high detection result accuracy, high reproducibility and simple detection process, and effectively solves the related problems existing in the traditional escherichia coli plasmid copy number detection method.

The present invention relates to a method for increasing prime editing efficiency in plants. More specifically, the present invention relates to a recombinant vector for plant prime editing with enhanced editing efficiency, and a use thereof, wherein the vector comprises a virus-based replicon including: a prime editing guide RNA (pegRNA) expression cassette regulated by a U6 composite promoter; and a prime editor expression cassette.

The invention relates to a circular vector for plant transformation comprising: a first tethering nucleic acid and a second tethering nucleic acid; a nucleic acid encoding a polynucleotide of interest (POI); a nucleic acid comprising an origin of replication; and two or more nucleic acids encoding replicon proteins and methods for using the same. Also provided are plants produced by the methods of the invention and products produced from the plant.

The invention provides a self-replicating RNA (Ribonucleic Acid) construct as well as a preparation method and application thereof, and in particular relates to a self-replicating RNA construct derived from an alphavirus replicon as well as a preparation method and application thereof. According to the present invention, the expression of the target gene is enhanced through the specific sequence mutation, and the cytotoxicity of the self-replicating RNA construct is effectively reduced.

The present invention embraces compositions comprising at least two RNA replicons (self-amplifying RNA vectors (saRNAs or rRNAs)) that can be replicated by a replicase of a self-replicating virus, e.g., a replicase of alphavirus origin. Of the at least two replicons, at least one of which optionally comprises an open reading frame encoding for the RNA-dependent RNA polymerase or replicase that is able to replicate each of the at least two replicons. Further, each replicon comprises an open reading frame encoding for different antigens of interest, e.g., different antigens derived from the same or from different pathogenic organisms, for example the glycoprotein and nucleoprotein of Ebola virus.

This invention discloses a plasmid for gene overexpression in non-pattern *Bacillus laterosporus*. Its leader region contains a methyltransferase expression cassette derived from the *Bacillus laterosporus* i83 genome, enabling stable existence in specific hosts containing the RM system. Based on this, a PidR2 promoter is added to obtain the pMCPidR2 shuttle plasmid. The PidR2 promoter effectively drives target gene expression, the pWV01 replicon ensures plasmid replication in non-pattern hosts, and the chloramphenicol resistance gene is used for positive colony selection. The plasmid contains a TraJ element, allowing it to be introduced into non-pattern microbial hosts via conjugation transfer. This invention also provides a target gene expression strategy in non-pattern microorganisms, achieving high transformation efficiency and high strain yield through conjugation transfer transformation of donor bacteria, conjugation transfer helper bacteria, and recipient bacteria, resulting in stable expression of the target protein. This invention provides an effective vector for efficient and stable gene overexpression in specific non-pattern microorganisms, helping to solve the problem of difficult genetic manipulation of industrial production strains.

The disclosure provides methods for an in vitro evolution technique to identify and characterize mutations in the non-structural genes of an alphavirus replicon that increase the strength and persistence of expression of the replicon genome. Also provided herein are in vivo methods for administering to an animal model a mutated alphavirus replicon that codes for a gene of experimental or therapeutic interest in the subgenome of the alphavirus replicon. The mutations identified herein improve the therapeutic potential of self-replicating RNA, which may have implications for cancer immunotherapy and beyond, e.g., for vaccination or gene therapy.

Provided herein are nucleic acids and vectors including a recombinant soybean mild mottle virus (SbMMV) replicon. Also disclosed are plant cells, whole plants, and plant parts including the nucleic acids or vectors. Further provided are methods of producing or expressing a nucleic acid, or modifying the genetic material of a plant cell.