145 results about "Replicon" patented technology

A Mob⁻ plasmid having a RSF1010 replicon, comprising a gene coding for Rep protein and said plasmid has been modified to inactivate gene related to mobilization ability. The present invention also describes a bacterium having an ability to produce useful metabolites, comprising the plasmid and said bacterium lack active thymidylate synthase coded by thyA gene and thymidine kinase coded by tdk gene, and a method for producing useful metabolites, such as native or recombinant proteins, enzymes, L-amino acids, nucleosides and nucleotides, vitamins, using the bacterium.

Improved molecular vaccines comprise nucleic acid vectors that encode a fusion polypeptide that includes polypeptide or peptide physically linked to an antigen. The linked polypeptide is one that (a) promotes processing of the expressed fusion polypeptide via the MHC class I pathway and/or (b) promotes development or activity of antigen presenting cells, primarily dendritic cells. These vaccines employ one of several types of nucleic acid vectors, each with its own relative advantages: naked DNA plasmids, self-replicating RNA replicons and suicidal DNA-based on viral RNA replicons. Administration of such a vaccine results in enhance immune responses, primarily those mediated by CD8+ cytotoxic T lymphocytes, directed against the immunizing antigen part of the fusion polypeptide. Such vaccines are useful against tumor antigens, viral antigens and antigens of other pathogenic microorganisms and can be used in the prevention or treatment of diseases that include cancer and infections.

The invention generally relates to recombinant polynucleotides, positive-strand RNA virus (psRNAV) recombinant expression vectors, and packaging systems. The packaging systems are based on the expression of helper functions by coinfecting re-combinant poxvirus vectors comprising recombinant polynucleotides. Methods for obtaining psRNAV replicon particles using these packaging systems are disclosed. Immunogenic compositions and pharmaceutical formulations are provided that comprise replicon particles of the invention. Methods for generating an immune response or producing a pharmaceutical effect are also provided.

The invention discloses a construction method and an application of a corynebacterium gene continuous knockout system, belonging to the technical field of genetic engineering. The corynebacterium gene continuous knockout system comprises a template plasmid and a temperature-sensitive expression plasmid, wherein the template plasmid provides a kan fragment (also known as a kan box) with loxp or variant loxL/R sites at two ends; and the temperature-sensitive expression plasmid carries a Cm resistance gene cat and a temperature-sensitive C. glutamicum replicon, expresses a Cre recombinant enzyme and is used for removing a Km resistance gene kan. The corynebacterium continuous knockout system disclosed by the invention can be generally used for continuous gene transformation of corynebacterium and has the advantages of simplicity and convenience in operation and high efficiency. By utilizing the knockout system, gene (continuous) knockout of three major typical subspecies genomes of the corynebacterium can be successfully completed, and the system is proved to be applicable to research and production of corynebacterium metabolites.

The invention relates to methods for genetic analysis of DNA sequences containing complementary duplicons which are duplicated and linked DNA sequences separated by an intermediate sequence. Furthermore, the invention relates to the Bidirectional Amplification of Complementary Duplicons (BACD) using a single primer. The amplification of complementary duplicons can be used for a variety of purposes such as, but not limited to, determining the species from which a sample comprising genomic DNA is derived, or used as a marker for a trait of interest. Also provided are means for analysing large datasets which can be used to identify primers useful for the genetic analysis methods of the invention.

The invention discloses an integrative plasmid pOPHI and a resistance screening marker-free self-luminescent mycobacterium. The integrative plasmid pOPHI comprises a promoter, an enzyme gene (LuxCDABE) needed for luminescence, a fusion gene of an integrase gene (Int) and a resistance screening gene, direct repeat sequences DifR and DifL positioned on two ends of the fusion gene, a phage integration site (attP) and a replicon. After the integrative plasmid pOPHI is transferred into the mycobacteria, a photobacterium of which the resistance gene and the integrase gene are lost is screened out by subculturing, and the photobacterium is the resistance screening marker-free self-luminescent mycobacterium. Whether the resistance screening marker-free self-luminescent mycobacterium obtained by construction is dead or not is judged according to whether the bacterium is luminescent or not, the mycobacterium can be used for a plurality of detections of experiments, and credible experimental data can be quickly obtained.

A process for replicating or for replicating and expressing a sequence of interest in a plant, comprising: (i) an RNA replicon or a precursor thereof, said RNA replicon being derived from a plus-sense single stranded RNA virus and comprising at least one sequence of interest; and (ii) a helper replicon, or a precursor thereof, wherein said helper replicon is (a) incapable of systemic movement in said plant both in the presence and in the absence of said RNA replicon (i) and (b) capable of expressing in a plant one or more proteins necessary for systemic movement of said RNA replicon (i), whereby said RNA replicon (i) is capable of replicating or replicating and expressing said sequence of interest in said plant, but unable to move systemically in said plant in the absence of said one or more proteins expressed by said helper replicon (ii).

The invention relates to a carrier capable of showing and expressing a heterologous gene on the surface of lactococcus lactis, and a preparation method and application of the carrier. The carrier has the nucleotide sequence which is shown as SEQ ID NO.1 in a sequence table, and contains promoter Pnis and lysM genes, a signal peptide secretion sequence ssusp45, a multiple cloning endonuclease site, and replicon repA and repC components. A method for constructing the carrier has the following steps of: amplifying a lactococcus lactis NZ3900 strain autolysin (AcmA) anchoring area gene lysM by applying a polymerase chain reaction (PCR) technology, performing enzyme digestion on the lysM and a carrier pNZ8110 and connecting, wherein a connection product is used for transforming a E.coli MC1061 strain, and extracting recombinant plasmid from positive transformed bacteria which are subjected to enzyme digestion and sequencing identification to obtain the expression carrier. The exogenous gene and the carrier are connected and then transferred into lactococcus lactis NZ3900, and the expression of the exogenous gene and the combination of expression protein and host cell walls can be realized by induction of nisin. The carrier can be used for expressing foreign proteins including vaccine antigens and has a wide application range.

The invention relates to a pTerm-SC plasmid as well as a construction method and application thereof. The pTerm-SC plasmid comprises a repE gene, a sopA gene, a sopB gene, a sopC gene, an ori2 replicon, an oriV replicon, prokaryote transcription terminators and antibiotics resistance genes. The pTerm-SC plasmid has the characteristics of very low copy number, high volume and stability and the like, can be applied to gene engineering fields such as the cloning, the screening, sequencing and the genome construction of complex-structure genes including repetitive-sequence genes, instable genes and long-fragment genes and has important role in the high-efficiency and high-quality synthesis of genes.

Chimeric alphaviruses and alphavirus replicon particles are provided including methods of making and using same. Specifically, alphavirus particles are provided having nucleic acid molecules derived from one or more alphaviruses and structural proteins (capsid and/or envelope) from at least two or more alphaviruses. Methods of making, using, and therapeutic preparations containing the chimeric alphavirus particle, are disclosed.

The invention discloses Japanese encephalitis virus like particles as well as a preparation method and an application thereof. The Japanese encephalitis virus like particles are prepared by assembling a PrM/E gene of a Japanese Encephalitis virus (JEV) and a JEVRNA replicor with a nucleotide sequence as shown in SEQ ID NO: 1. The preparation method of the Japanese encephalitis virus like particles comprises the steps of: connecting the JEV PrM/E gene amplified by an RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) to a plasmid pTRE-Tight of a Tet-on advance expression system to form a recombination vector, transfecting a Hela Tet-on advanced cell by using an Xfect method, after screening and identifying through HygB, obtaining a Hela cell line for controllably and stably expressing the PrM/E gene through a limiting dilution process, inducing for expressing a PrM/E protein, and packaging to obtain the conventional virus like particles; and transfecting the Hela cell line by using a JEV replicor RNA vector and packaging to obtain the virus like particles with a capacity of transient infection. The Japanese encephalitis virus like particles can be used for preparing a vaccine or used as a detection agent, has better immunogenicity and reactivity, and is safe and reliable.

The invention discloses a bacillus gene traceless knockout/knockin plasmid and method, and a kit. The genome of the bacillus gene traceless knockout/knockin plasmid contains only one kind of resistance gene marked with a positive selection marker, a replicon capable of being duplicated in escherichia coli, a thermo-sensitive type replicon capable of being duplicated in bacillus, at least one I-Scel enzyme cutting site and only one kind of chromogenic protein gene, wherein promoters in front of the resistance gene and the chromogenic protein gene are both constitutive promoters in bacillus. The invention further provides a helper plasmid used for improving the knockout/knockin efficiency of the bacillus gene traceless knockout/knockin plasmid, and resistance gene and chromogenic protein gene in the genome of the helper plasmid are different from those of the knockout/knockin plasmid. By the adoption of the two plasmids, one or more target DNA sequences in the genome of bacillus can be modified continuously or iteratively, and the plasmids can be applied to multiple fields including bacillus genetic modification, metabolic process researching, functional genome researching and industrial application researching.

The invention provides a replicon vector taking Japanese encephalitis virus genome as a framework, as well as a cell line for packaging the same and a packaging system. The JEV replicon vector has the capability of efficiently expressing foreign protein. Mice are immunized with the replicon vector, and the titer of an anti-JEV antibody reaches 1:1280 after three immunizations so as to protect 75 percent of suckling mice from virus attack. The cell line provided can produce 1.6*105 U/ml of pseudovirus particles after packaging JEV replicon, and the titer of the anti-JEV antibody reaches 1:2560 after two immunizations so as to protect 73 percent of suckling mice from virus attack. The invention establishes a technical platform for a JEV replicon vector system for the first time, and explores the feasibility of researching replicon vaccines and pseudovirus vaccines through the JEV replicon vector system, thereby laying a solid foundation for developing and researching a plurality of novel vaccines used to prevent and treat tumors and viral diseases in future.

The invention provides a recombined sheep poxvirus transfer vector. A vector containing resistance genes and a plasmid replicon sequence and adopting Hind III and Avr II for digestion, such as a pVAX1 vector, is adopted, a DNA segment is inserted via introducing Hind III and Avr II enzyme cutting sites; the DNA segment comprises a gene sequence of a homologous arm with bidirectional poxvirus promoters and a sheep poxvirus TK gene. The invention further provides a recombined sheep poxvirus containing the recombined sheep poxvirus transfer vector and application of the recombined sheep poxvirus in the transfer of an exogenous antigen. Exogenous genes can be conveniently inserted into the recombined sheep poxvirus transfer vector, provided by the invention; moreover, the recombined sheep poxvirus transfer vector is improved according to a recombination result, and finally higher recombination efficiency is realized, accordingly, a good basis for the research and preparation of such recombination vaccines is laid.

The invention relates to general expression vector pSK-E-P/T construction for plant mitochondria and promoter activity identification. Cucumber mitochondria plasmids pC3 (sequence 1) are discovered for the first time, a gene cob upstream promoter sequence (sequence 2) and a gene atp9 downstream terminator sequence (sequence 3) of the cucumber mitochondria obtained through PCR (polymerase chain reaction) are constructed into a general expression vector pSK-E-P/T (sequence 4) of the plant mitochondria, and the vector also carries replicons copied in escherichia coli and Ampr resistance genes so as to facilitate cloning of exogenous genes and recon screening. Measurement of enzyme activity of promoter -1acZ report gene fusion plasmids in the escherichia coli and fluorescence microscope observation of psk-E-GFP for transforming the green fluorescence of the escherichia coli prove that the promoter on the expression vector has transcriptional activity. The invention provides a novel molecular biology technology tool for researching important life science hotspot problems such as cell apoptosis and the like related with the mitochondria by a mitochondria transformation path.

The invention discloses a novel coronavirus SARS-CoV-2 replicon as well as a construction method and application thereof, and belongs to the technical field of virology and molecular biology. The novel coronavirus SARS-CoV-2 replicon is an SARS-CoV-2-GFP replicon with a single reporter gene or an SARS-CoV-2-GFP-Luc replicon with double reporter genes. The invention also discloses the constructionmethod and application of the novel coronavirus SARS-CoV-2 replicon. According to the novel coronavirus SARS-CoV-2 replicon disclosed by the invention, infectious virus particles are not generated, sothat the biological safety risk in the process of screening antiviral drugs by utilizing the infectious virus particles is effectively reduced. Besides, a reporter gene carried by the replicon can allow high-throughput screening to be performed, so that the screening efficiency of anti-novel coronavirus drugs is greatly improved.

The invention belongs to the field of antiviral medicines, and discloses application of a receptor tyrosine kinase inhibitor in preparation of a medicine for preventing and/or treating novel coronavirus infection. The receptor tyrosine kinase inhibitor comprises mosatinib, alectinib, roxatidine or pharmaceutically acceptable salts of the small molecular compounds. The receptor tyrosine kinase inhibitor such as mosatinib, alectinib and roxatidine can strongly inhibit synthesis of replicon RNA of a novel coronavirus SARS-CoV-2 and reduce the RNA copy amount of a wild type novel coronavirus SARS-CoV-2 in cells, so that replication of the novel coronavirus SARS-CoV-2 is inhibited, and the receptor tyrosine kinase inhibitor has the effect of preventing and/or treating novel coronavirus SARS-CoV-2 infection.

The invention provides an expression system for a nonintegrated, long-time and erasable expression vector. The expression vector of the expression system adopts human herpes virus replication origin (Orip), an EBNA-1 expression box is introduced, and both ends of the Orip, both end of EBN-1 or both ends of Orip-EBNA-1 are provided with recombinase recognition sequences. The expression vector is not integrated in a cell genome, can stably express for a long time, can controllably erase vector plasmid by instantaneously inducing (or expressing) recombinase from a hose cell, can be applied to most kinds of cells by using a strong initiator and can distinguish a cell with the vector plasmid from a cell without the vector plasmid by using a real-time marker. The expression system can widely applied to mammal cell expression and biomedicine engineering for realizing cell reprogramming by expressing a foreign gene.