49 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 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.

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.

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.

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 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.

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.

A nucleic acid construct encoding a plurality of polypeptide antigens or immunogens or other polypeptides of interest. The constructs express proteins comprising immunogenic or antigenic sequences of two, three or more polypeptides of interest. Once expressed by the nucleic acid construct, the protein is processed by cleaving the N-terminal signal peptide and cleaving the 6K, 6K-like or internal signal peptide cleavage site to release each polypeptide of interest. Vectors comprising the constructs can be used to deliver them to subjects in need of vaccination, the vectors comprising a replicon and a live viral vector. Vaccines and methods of treatment using the nucleic acid constructs are also described.

The present disclosure provides novel self-amplifying RNA (saRNA) constructs that demonstrate enhanced protein expression, prolonged durability, reduced immunogenicity, and the ability to express multiple therapeutic proteins homogeneously. The saRNA constructs comprise a 5' untranslated region (5'UTR), non-structural protein genes derived from alphaviruses, at least one gene of interest encoding a therapeutic protein, a 3' untranslated region (3'UTR), and one or more modified nucleosides. Also disclosed are dual construct systems comprising a first construct encoding non-structural proteins and a second construct encoding one or more genes of interest. Methods of producing and using the saRNA constructs for engineering cells, particularly immune cells, for treatment of various conditions including cancer, inflammatory conditions, and infectious diseases are provided. The saRNA constructs enable the generation of "armored" immune cells expressing multiple therapeutic proteins, thereby providing a multi-pronged approach to complex diseases.

Provided are a self-replicating RNA construct, and a preparation method therefor and the use thereof. More specifically, the present invention relates to a self-replicating RNA construct derived from an alphavirus replicon, and a preparation method therefor and the use thereof. The cytotoxicity of the self-replicating RNA construct is effectively reduced by enhancing the expression of a target gene by means of a specific sequence mutation.

A geminivirus replicon, an in planta directed evolution / selection system based thereon, and use. Provided is a geminivirus-based controllable artificial replicon, which comprises two LIRs derived from BeYDV; the two LIRs are located at two flanks of the artificial replicon, respectively. Also provided is an in planta directed evolution / selection system, which comprises the geminivirus-based controllable artificial replicon, a Rep and / or RepA protein, and a mutant or mutant library of a genetic element. The replication of the geminivirus-based controllable artificial replicon is configured to be associated with the desired function of the mutant of the genetic element. The provided in planta directed evolution / selection system is a universal, rapid, and efficient in planta directed evolution system, and can be used for generating new excellent alleles and assisting future agricultural breeding.

The application discloses a pSY1A plasmid vector for a wheat germ cell-free expression system, which is constructed by introducing an ampicillin resistance gene, an Ori replicon, a T7 promoter, a translation enhancer, a MCS (multiple cloning site), a 3'-UTR sequence and a T7 terminator sequence on the basis of part of a sequence of an E. coli plasmid pUC19. The pSY1A plasmid vector has a MCS into which an exogenous gene can be inserted, and the MCS and a transcription assembly constitute a transcription module, so that the transcription module can be preserved by preserving the plasmid, and in-vitro transcription of the wheat germ cell-free expression system becomes more convenient and faster.

This invention discloses an antisense oligonucleotide targeting and inhibiting the RdRp gene and its applications. This study designed and screened a series of antisense oligonucleotides (ASOs) targeting the highly conserved SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), and systematically evaluated their antiviral effects against various SARS-CoV-2 variants. Experimental results show that the designed ASO molecules, especially RDRP-5, exhibit significant viral inhibitory activity at multiple experimental levels (including reporter gene systems, viral replicon models, and true viral infection experiments).

The invention discloses a bacillus subtilis genome integrated plasmid for efficient and stable expression of genes and application of the bacillus subtilis genome integrated plasmid in production of beta-galactosidase. The plasmid comprises a bacillus subtilis promoter, an RBS sequence, a terminator, a bacillus subtilis resistance gene, a genome integration homologous arm, an escherichia coli replicon and a resistance gene. The homologous arm is selected from upstream and downstream sequences of sites behind termination codons such as genes spxA, tatCY, rlmCD and the like, so that exogenous genes are accurately integrated to a plurality of non-essential sites on the premise of not damaging essential genes. The invention also provides a method for constructing a bacillus subtilis engineering strain for producing beta-galactosidase by using the plasmid. The integration system is high in expression level, stable expression can be achieved without adding antibiotics and inducers, the fermentation cost is remarkably reduced, the enzyme production efficiency is improved, and the integration system is suitable for industrial enzyme preparation production.

The present invention relates to an expression vector that encodes all or a portion of replicon proteins from a positive stranded virus, wherein expression of the replicon proteins is under the control of CMV and T7 promoters, and wherein expression of a pay load is under the control of a sub-genomic promoter. Also provided are methods of using the vector in therapeutics and vaccines.

The application discloses a replicon capable of autonomously replicating in trichophaea piniformis cells and application thereof, and belongs to the technical field of biology.The nucleotide sequence of the replicon tpARSrp is shown in SEQ ID NO.9.The application constructs a replicon tpARSrp capable of playing a replication function in a trichophaea piniformis expression system, the replicon has higher stability than AMA1 replicon, enriches a replicon element library, can be used for constructing a non-integrated expression vector of trichophaea piniformis, is used for transient expression of an exogenous gene or gene editing, provides a tool for gene editing of trichophaea piniformis, and expands the selection diversity of filamentous fungi using replicons.

The application provides an optimized CRISPRi / dCas12a gene regulation system of Sphingomonas scs, a recombinant engineering strain and method and application, and specifically belongs to the technical field of genetic engineering. The application provides an optimized CRISPRi / dCas12a gene regulation system of Sphingomonas scs, which comprises a recombinant plasmid carrying a replicon capable of stable replication in Sphingomonas scs, a PJDP promoter driving dcas12a gene transcription, dCas12a a gene, a resistance gene and a crRNA expression cassette. The optimized gene regulation system is a high-efficiency CRISPRi system suitable for S. paucimobilis Based on the optimized gene regulation system, the PHB synthesis pathway can be accurately regulated, the production of gellan gum is improved without affecting the normal growth of the strain, and gellan gum with higher transparency is directly produced.

The invention discloses a pSY1A plasmid vector for a wheat germ cell-free expression system, which is characterized in that on the basis of partial sequence of escherichia coli plasmid pUC19, an ampicillin resistance gene, an Ori replicon, a T7 promoter, a translation enhancer, MCS multiple cloning sites, a 3 '-UTR sequence and a T7 terminator sequence are introduced to construct the plasmid vector pSY1A. According to the pSY1A plasmid vector provided by the invention, the vector has multiple cloning sites into which exogenous genes can be inserted, and the multiple cloning sites and a transcription component form a transcription module, so that the transcription module can be stored by storing plasmids, and the in-vitro transcription of a wheat germ cell-free expression system becomes more convenient and quicker.

The present invention relates to the field of in-vitro transcription, in particular to a plasmid backbone capable of improving the stability of the polyadenylic acid tail. Provided is an expression cassette, sequentially comprising: a rop gene, a replicon ori, and a resistance gene, wherein the rop gene, the replicon ori, and the resistance gene are connected by means of linker fragments. Low-copy pmRVacSL and medium-copy pmRVacM plasmid vector systems are established by means of artificial modification. Since pmRVacSL is a low-copy vector, the plasmid DNA yield is relatively low. The pmRVacM plasmid backbone addresses the problem of low plasmid DNA yield of pmRVacSL. The vector system not only enables stable replication of polyA tail sequences of 150 bp or more, but also allows for rapid addition of genes of interest and polyadenylic acid (polyA) sequences, efficiently and rapidly obtaining the target DNA template for mRNA in vitro transcription.

The application belongs to the technical field of biological medicine, and discloses a method for preparing porcine epidemic diarrhea virus (PEDV) epitope nanoparticles by using Kluyveromyces marxianus and application thereof. The applicant assembles and prepares a double replicon expression vector suitable for the Kluyveromyces marxianus through screening, and the vector achieves the effect of high conversion rate and high expression efficiency in the Kluyveromyces marxianus. The double replicon expression vector pGKD32 is shown as SEQ ID NO. 2. The Kluyveromyces marxianus expression system provided by the application is used to successfully express the PEDV epitope in the form of nanoparticles, thereby providing a new idea for the research and development of the PED oral immunization strategy.

The present application relates to the construction and application of the EcORep-based continuous directed evolution reporter system of aminoacyl-tRNA synthetase, and specifically, the reporter system comprises: a green fluorescent protein mutant sfGFP containing amber stop codon TAG N150TAG ; a chloramphenicol acetyltransferase mutant CAT containing amber stop codon TAG D112TAG ; a linear replicon containing aminoacyl-tRNA synthetase; a reporter plasmid containing a plus-strand mismatch DNA polymerase to continuously mutate the linear replicon, thereby generating an in-vivo library of aminoacyl-tRNA synthetase mutants; and a tRNA, which is orthogonal to the aminoacyl-tRNA synthetase and enables the incorporation of unnatural amino acids and the complete expression of proteins. The present application proves that the evolution reporter system can realize the efficient directed evolution of aminoacyl-tRNA synthetase, and further expands the application range of genetic code expansion technology.

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

The present invention relates to a polypeptide composition, comprising a nucleic acid encoding a mutant pp65 polypeptide, the nucleic acid being mRNA, viral RNA, or replicon RNA. The composition is capable of expressing the mutant pp65 polypeptide in vivo, eliciting a specific humoral or cellular immune response against the pp65 polypeptide, and producing an effector cell having a tumor cell killing effect and a specific antibody.

Provided herein are trans amplifying ribonucleic acids (taRNA) comprising replicase constructs and / or trans replicon constructs (trRNA) with target sites for microRNA (e.g., cell- type enriched microRNA), for example, in a 3' untranslated region (UTR) of the replicase construct and / or trRNA, and methods of use thereof. These taRNA may be useful for mitigating taRNA- specific immune responses or obstacles to expression in subjects.

Crimean-Congo hemorrhagic fever (CCHF) virus replicon particles (VRP) are described. These VRP are capable of undergoing a single round of virus replication, but are unable to produce new particles or spread to neighboring cells due to the lack of the glycoprotein-encoding M genome segment. In some instances, the VRP contains one or more mutations in the viral ovarian tumor domain protease encoded by the L genome segment or heterologous antigens within its S genome segment. These VRP are shown to elicit a protective immune response against lethal CCHF virus challenge in an animal model.

Systems, compositions, devices, methods, etc., provide improved anti-malaria immunological responses comprising making, providing and administering vaccines comprising specific RNA molecules such as self-replicating replicon RNA (repRNA) encoding proteins from Plasmodium such as the P. yoelii (Py) CS protein (CSP), including in some embodiments substantially target proteins encoding target antigens, for example a whole or substantially whole CSP in the repRNA. The prime-and-trap intervals for the administration of the vaccine can comprise administration of only a single dose of a repRNA-Non-encapsulating oil-in-water emulsion nanocarriers (e.g., LION™) component followed by administration of as few as 3 or 2 doses, or even just a single dose, of the WO component (e.g., RAS or genetically attenuated WO) at 0 day (same day), or 1, 2, 3, 4, 5, 10, 14, 15 days or 28 days later.

The invention relates to molecular biology and industrial enzymes, and provides a system for randomly integrating and expressing D-psicose 3-epimerase (DAEase) in a bacillus subtilis chromosome and application of the system. The preparation method comprises the following steps: integrating an expression cassette containing PgsiB / PnprE / PHpaII / PmyE, 7-20bp RBS and SEQ ID NO: 2 by adopting mariner / Himar1 or Tn7; a temperature-sensitive replicon is removed at 42-51 DEG C, and a resistance marker can be deleted. A KpRD / NADH 340nm continuous spectrum is used for screening, and HPLC (High Performance Liquid Chromatography) rechecking is performed. The crude enzyme activity is greater than or equal to 3000U / mL within 68-72 hours by supplementing 3L of materials. Under the conditions that the temperature is 55-70 DEG C, 0.1 mM Co < 2 + > and the substrate is 300 g / L, the D-psicose is obtained by catalyzing D-fructose through whole cells / crude enzyme, the conversion rate is larger than or equal to 25%, stability and compliance are both considered, and the method is suitable for industrialization.