62 results about "Pseudovirion" patented technology

A method of disassembly/reassembly of papillomavirus VLPs is provided. The resultant VLPs have enhanced homogeneity, present conformational, neutralizing PV epitopes, and therefore are useful prophylactic and diagnostic agents. Further, these VLPs can be used to encapsulate desired moieties, e.g., therapeutic or diagnostic agents, or "marker" DNAs, and the resultant VLPs used as in vivo delivery vehicles or as pseudovirions for evaluating vaccine efficacy.

The present invention discloses Moloney murine leukemia virus (MoMLV)-based viral packaging cell line for the production of anti-viral vaccines. The invention also includes methods of making, administering and formulating pseudovirions and replicon deficient viral particles of the invention and methods of inducing immunity.

A method of disassembly/reassembly of papillomavinis VLPs is provided. The resultant VLPs have enhanced homogeneity, present conformational, neutralizing PV epitopes, and therefore are useful prophylactic and diagnostic agents. Further, these VLPs can be used to encapsulate desired moieties, e.g., therapeutic or diagnostic agents, or marker" DNAs, and the resultant VLPs used as in vivo delivery vehicles or as pseudovirions for evaluating vaccine efficacy.

The invention discloses an animal pathogen microbial pseudovirion vector and a preparation technology thereof. The gene coding sequences of a maturase protein and a coat protein of an MS2 phage and a cDNA (complementary Deoxyribose Nucleic Acid) sequence which corresponds to a 5' non-coded sequence of a gene regulating element sequence included by a genome part are connected to the downstream of a pTrcHis2A vector promoter, so that a pseudovirion vector is constructed. A 6His purification tag is added between the fifteenth and sixteenth amine acids of the coat protein in the MS2 phage througha gene insertion method, a pseudovirion particle comprising an exogenous gene is expressed into an RNA (Ribonucleic Acid)-protein complex, and the pseudovirion particle is captured with a protein purifying method, so that the complex operating process for preparing the pseudovirion particle can be simplified, and the purification quality of the pseudovirion particle is enhanced simultaneously. The pseudovirion vector is pTrcMS of which the nucleotide sequence is shown as SEQ ID No.1.

The invention discloses pseudovirions; a capsid protein of MS2 bacteriophage is wrapped with a DNA-protein complex of a detection sequence formed by series connection of three base fragments of influenza A virus, influenza B virus and beta-actin successively. The invention also provides a preparation method of the pseudovirions and an application of the pseudovirions as quality control products for detecting the influenza A virus and the hepatitis B virus. The preparation method of the pseudovirions is simple and is easy to operate; the obtained pseudovirus is high in purity, can be used as the quality control products in an influenza A virus and influenza B virus nucleic acid detection kit, and has the characteristics of no infectivity, good stability, ribonuclease resistance and the like.

The invention provides a pseudovirion containing hepatitis c virus (HCV) RNA (Ribonucleic Acid) fragment and preparation method thereof which are applied in the field of biomedical clinical vitro diagnostic. The pseudovirion is a RNA-protein complexes that MS2 bacteriophage capsid protein wraps the hepatitis c virus, and the complexes is icosahedral; the method comprises the following steps of designing and synthesizing primer to obtain target gene MS2 by overlapping and splicing PCR (Polymerase Chain Reaction) method; connecting the target gene MS2 to plasmid pET-32a (+) to obtain recombinant plasmid pET-MS2; connecting the recombinant plasmid pET-MS2 and the HCV fragment to obtain pET-MS2-HCV recombinant plasmid; the obtained pET-MS2-HCV recombinant plasmid is introduced into escherichia coli for prokaryotic expression; releasing virus-like particles by adopting ultrasonication and the obtained virus-like particles are the pseudovirion containing the hepatitis c virus RNA fragment. The pseudovirion preparation method provided by the invention has the advantages that the preparation method is simple, the operation is easy, the obtained pseudovirus is high in purity, and the pseudovirion can be used as standard and quality control material of detection of RT-PCR (Reverse Transcription-Polymerase Chain Reaction), is good in stability, has no infectivity, has RNase-resistant, and the like.

A method of disassembly/reassembly of papillomavirus VLPs is provided. The resultant VLPs have enhanced homogeneity, present conformational, neutralizing PV epitopes, and therefore are useful prophylactic and diagnostic agents. Further, these VLPs can be used to encapsulate desired moieties, e.g., therapeutic or diagnostic agents, or “marker” DNAs, and the resultant VLPs used as in vivo delivery vehicles or as pseudovirions for evaluating vaccine efficacy.

The invention provides a pseudoviral particle containing a hepatitis e virus (HEV) RNA (Ribose Nucleic Acid) fragment and a preparation method thereof. The pseudoviral particle is an RNA-protein complex formed by coating hepatitis e virus RNA by an MS2 bacteriophage coat protein, and the RNA-protein complex is spherical. The preparation method comprises the steps of designing and artificially synthesizing a primer to obtain a target gene MS2 through a PCR (Polymerase Chain Reaction) method, connecting the target gene MS2 to a plasmid pET (polyethylene glycol terephthalate)-28b(+) to obtain a recombinant plasmid pET-28b/MS2/HEV, guiding the recombinant plasmid into escherichia coli for prokaryotic expression, and settling a virus-like particle by adopting a polyethylene glycol method, wherein the virus-like particle is the pseudoviral particle containing the hepatitis e virus RNA fragment. The pseudoviral particle provided by the invention can be used as a standard substance and a quality control product of general HEV gene I-IV type RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection, and has the characteristics no infectivity, safety, reliability, high stability and resistance to ribonuclease.

The invention discloses a beta-globin recombinant lentiviral vector. A gene sequence of the vector comprises a modified beta-globin full-length gene, a beta-globin gene control region HS2-HS3, a beta-globin gene 5' promoter, a beta-globin gene 3' enhancer, a chromatin insulator CTCF and pSIN4-CMV-K2M plasmids; and a nucleotide sequence is as shown in SEQ ID No.1. The invention further discloses an application of the recombinant lentiviral vector in transfection of hematopoietic stem/progenitor cells to obtain normal function beta-globin. An experiment proves that specific, efficient and stable expression of the lentiviral vector in erythroid cells can be achieved by using a vector expression system, pseudoviral particles with relatively high virus titer are obtained and the normal function beta-globin is obtained after transfection of the hematopoietic stem/progenitor cells.

The invention relates to a preparation method of an artificial dual false virus particle comprising HCV (hepatitis C virus) and HIV (human immunodeficiency virus) nucleic acid fragments, belonging to the field of the clinical diagnosis of the biomedicine. The preparation method comprises the steps of designing and synthesizing the forward and reverse primers of coliphage MS2 mature protein gene, envelope protein gene and Pac site, taking MS2 bacteriophage RNA as a template, carrying out RT-PCR (reverse transcription-polymerase chain reaction) amplification by the primers, and finally connecting an amplified product onto a pMD18-T carrier for sequencing to test and verify the clone correctness; extracting the PMD 18-T plasmid comprising the MS2 mature protein gene, the envelope protein gene and the Pac site, separating the inserted mature protein gene, envelope protein gene and Pac site by Ncol and BamHI restriction enzymes, and connecting to a Ncol and BamHI bi-digested pET-28b carrier to obtain a connected product. The preparation method of the artificial dual false virus particle comprising HCV and HIV nucleic acid fragments is simple and easy to operate, and the obtained false virus is high in clone purity, and is an ideal raw material of a job standard product and a quality control product of an HCV and HIV diagnostic kit.

The invention relates to a pseudovirus standard substance for nucleic acid detection of a novel coronavirus (2019-nCoV or called SARS-CoV-2) and a preparation method of the pseudovirus standard substance. Novel coronavirus nucleic acid is integrated into a lentiviral expression plasmid vector, then constructed lentiviral expression plasmids and packaging plasmids are jointly transfected into cells, and a large number of pseudoviral particles packaged with novel coronavirus RNA are obtained after cell expression. After plasmid DNA is removed through DNase I digestion and sucrose density gradient ultracentrifugation, freeze drying is performed to obtain a pseudovirus particle freeze-dried product. The pseudovirus standard substance has the partial RNA sequence packaged with the virus and also has a pseudovirus standard substance packaged with all the sequences, can completely simulate the whole process of virus RNA extraction and nucleic acid detection, and can be used for the verification evaluation and the laboratory quality control of the novel coronavirus nucleic acid qualitative and quantitative detection method. The obtained standard substance has the characteristics of high purity, good safety, accurate quantity value and the like, is good in stability, can be transported at normal temperature, and provides guarantee for quantity value traceability and transmission of thestandard substance.

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.

A method of disassembly/reassembly of papillomavirus VLPs is provided. The resultant VLPs have enhanced homogeneity, present conformational, neutralizing PV epitopes, and therefore are useful prophylactic and diagnostic agents. Further, these VLPs can be used to encapsulate desired moieties, e.g., therapeutic or diagnostic agents, or "marker" DNAs, and the resultant VLPs used as in vivo delivery vehicles or as pseudovirions for evaluating vaccine efficacy.

The invention belongs to the technical field of biology, and particularly discloses a novel coronavirus pseudovirus, and a preparation method and application thereof. The prepared pseudovirus has no replication nor infection activity, and the biological safety is reliable. The suspension can reach about 10<6> copies/microliter, the indexes of uniformity and stability have no significant difference, and the requirements of positive reference substances can be met. Pseudovirus particles are used as a positive reference substance, and a triple fluorescent RT-PCR method is developed. The linear fitting coefficients between the nucleic acid content and the amplification Ct values of three target genes are all 0.99 or above, and the positive reference substance is calibrated at 1.2*10<4> copies/microliter. An RT-RPA constant-temperature isothermal amplification method is developed, and the detection limit can reach 10 copies/microliter.

The invention relates to a dengue virus and Japanese encephalitis virus embedded pseudo virus particle vaccine and a preparation method thereof. The embedded pseudo virus particles are formed by transfectting and expressing recombinant expression plasmids of a cloned dengue virus and Japanese encephalitis virus structural gene in target cells. The preparation method of the embedded pseudo virus comprises the following steps of: (1) constructing the recombinant expression plasmids containing a dengue virus-Japanese encephalitis virus structural protein coding sequence; (2) introducing the recombinant plasmid into the target cells and sieving stably-transfectted cells for cloning; (3) culturing the stably-transfectted cells so as to generate dengue virus-Japanese encephalitis virus embedded pseudo virus particles; and (4) extracting and purifying the embedded pseudo virus particles from cell culture supernatant fluid. The vaccine embedded with the dengue virus-Japanese encephalitis virus antigens can stimulate the organism to generate immune response, further prevents dengue virus and/or Japanese encephalitis virus infectious diseases, and has the advantages of good immune effect, safety and industrial production suitability.

The invention provides a novel microbicide anhydride modified anti-SEVI (semen-derived enhancer of viral infection) polyclonal antibody for preventing HIV (human immunodeficiency virus) sexual transmission. The novel microbicide anhydride modified anti-SEVI polyclonal antibody comprises an anti-SEVI polyclonal antibody, wherein the anti-SEVI polyclonal antibody can be used for immunizing rabbit or other tested animals by a conventional method by synthesizing a polypeptide fragment PAP248-286 of SEVI, antiserum of anti-SEVI is collected, and IgG (immunoglobulin G) of the antibody is purified to obtain the polyclonal antibody. The anhydride modified anti-SEVI polyclonal antibody is obtained by performing anhydride modification on the antibody. The anhydride modified anti-SEVI polyclonal antibody has excellent anti-virus activity to HIV-1 pseudovirion strain, zoo virus strain and anti-HIV medicinal HIV-1 drug-resistance strain, and the anhydride modified anti-SEVI polyclonal antibody can specifically bind SEVI, so that the reinforcing effect of the SEVI in seminal fluid to HIV infection can be blocked. The antibody has the beneficial effects that a dual-functional HIV sexual transmission preventing candidate microbicide can be used for resisting the strengthening capability of SEVI to HIV infection and has high-effective HIV activity.

The invention discloses a preparation method of H7N9 pseudoviruses and applications of H7N9 pseudoviruses, which belong to the field of bio-medicine techniques. The preparation method comprises the following steps: cloning HA and NA gene sequences of H7N9 avian influenza viruses to mammalian expression vectors pVAX-1; and then, co-transfecting 293T cells by using pVAX-HA and retrovirus vectors pNL-4.3. Luc. E-R-isoplasmids, so that H7N9 pseudoviruses are generated. Cytostatics and neuraminidase inhibitors for inhibiting the entry of H7N9 avian influenza viruses are screened by using H7N9 pseudoviruses and a pseudovirus cell model, studies on influenza virus neutralizing antibodies are performed by using H7N9 pseudoviruses, and influenza virus infections are prevented and treated by using H7N9 pseudoviruses as anti-influenza immunological preparations.

The invention discloses a method for forming nerve cells by induction and a composition. The invention provides a method for forming nerve cells by specially inducing colloid cells to differentiate, and specifically the method comprises the following steps: firstly, constructing a NeuroD1 gene on a virus vector, and packaging to obtain virus-like particles with NeuroD1; and after the NeuroD1 virus-like particles are infected with the colloid cells, cultivating for a certain time to differentiate the colloid cells into the nerve cells. The obtained never cells and the composition thereof can have potential value in treating neurodegenerative diseases such as Parkinsonism.

The invention discloses a pseudovirion particle and a preparation method and application thereof. The pseudovirion particle comprises RNA fragments of a monkey type D retrovirus encapsulated by MS2 phage capsid protein. The preparation method of the pseudovirion particle is simple, operation is easy, fineness of the obtained pseudovirion particle is high, and the pseudovirion particlecan be used as an external standard quality control product of a nuclein detecting method of monkey type D retrovirus detection. The method has the characteristics of high stability and nuclease resistance, a target template sequence exists only in the form of RNA, infectivity is avoided, and security is high.

The invention discloses a preparation method for a pseudovirus particle containing a human immunodeficiency virus RNA segment. The preparation method comprises the following steps: 1) preparing a clone plasmid containing a MS2 gene sequence; 2) screening an HIV gene conserved sequence and preparing an HIV clone plasmid; 3) performing double-plasmid cotransfection to induce the expression of the pseudovirus particle. The pseudovirus particle prepared according to the preparation method is obviously increased, is high in stability, can be stored for a long time, is high in purity, contains high-purity RNA, contains no DNS and is suitable for large-scale production and application.

The invention provides a false virus granule vaccine with dengue fever virus recombination replicon as a core and method for preparation, wherein the vaccine can express antigens with high efficiency in the affected cells, the antigen can be extracted effectively with good immunity effect. The vaccine can be used for preventing and treating tumor and virosis.

The invention discloses a method for preparing lentiviral particle packaged EBOV RNA as a positive reference substance for EBOV nucleic acid detection. The preparation method comprises the following steps: connecting part of segments of three genes NP, GP and L of EBOV in series and then integrating the segments into an expression vector of lentivirus, separately amplifying the constructed lentiviral expression plasmid and packaging plasmid in an engineering escherichia coli strain to obtain a large number of plasmids, then jointly transfecting the constructed lentiviral expression plasmid andpackaging plasmid into HEK293T cells, and carrying out cell expression to obtain a large number of lentiviral particles packaged with RNA genes of EBOV. The invention relates to the technical field of gene engineering. According to the method for preparing lentiviral particle packaged EBOV RNA as a positive reference substance for EBOV nucleic acid detection. The obtained particles are used as apositive reference substance for EBOV nucleic acid detection and have the advantage of well simulating the whole process from virus nucleic acid extraction to nucleic acid detection in an actual sample, and freeze-dried powder obtained by freeze-drying pseudovirus particles of EBOV through a freeze-drying protective agent is very stable.

The invention provides high-stability pseudoviral particles as well as a plasmid vector and a method for preparing the pseudoviral particles. The pseudoviral particles contain a nucleic acid fragment of norovirus, wherein the nucleic acid fragment can serve as a target for detecting the norovirus. The plasmid vector contains a maturase coding gene of a Qbeta phage, a capsid protein coding gene, a packaging site sequence and a DNA segment including a cDNA sequence in correspondence with the nucleic acid fragment. The method comprises the following steps: constructing the plasmid vector, then transcribing and/or translating the plasmid vector in escherichia coli cells, and conducting separating and purifying, so that the pseudoviral particles are obtained. With the application of the method disclosed by the invention, the pseudoviral particles, which contain the norovirus nucleic acid fragment functioning as the detection target, can be prepared; and the pseudoviral particles have the advantages of being free from infectivity, high in copy number, good in stability and the like.

The invention provides a method for detection and evaluation of photochemical virus inactivation effect of methylene blue, which adopts HCV pseudovirion constructed by enveloped RNA technology as a quality control, and performs methylene blue photochemical virus inactivation of the quality control and the blood or blood products needing to be subjected to virus inactivation under a same conition. The method has no virus infection risk to human body, and can reflect the disappearance condition of HCV infection by the damage degree of pseudovirion nucleic acid.

The invention discloses a medicine composition for treating neuron degeneration disease. The invention provides a method for forming neurons by specifically inducing spongiocyte differentiation. The method disclosed by the invention specifically comprises the following steps: firstly establishing a Myt1L (Myelin transcription factor 1-like) gene on a virus vector and packaging to obtain pseudovirus particles with Myt1L; and infecting spongiocyte by the pseudovirus particles with the Myt1L, and culturing for a period of time, thus differentiating the spongiocyte into neuron cells. The neuron cells and the composition thereof have a potential value in treatment of neuron degeneration diseases, such as Parkinson disease.