576results about How to "High titer" patented technology

The invention relates to a method for screening drug target genes based on a CRISPR/Cas9 high-throughput technology. The method comprises the steps of firstly establishing a sgRNA library; secondly packaging the sgRNA library by using slow viruses, and collecting the viruses; thirdly screening the sgRNA library in a cancer cell line; fourthly extracting cells obtained by screening and genome DNA of the cells before screening; and finally enriching sgRNA in genome DNA. Compared with the prior art, the method has the advantages that a CRISPR/Cas cell screening process is improved, the virus infection efficiency is determined with a simple and convenient method by utilizing puromycin resistance of infected cells, and MOI values of the viruses are determined; more importantly, a virus packaging method is greatly optimized, so that the virus packaging efficiency is improved to be more than 5 times that of a conventional method, and large-scale drug target screening cost can be greatly reduced; and the method is used for promoting industrialization of cancer drug target screening.

The invention relates to a serum free culture medium for growing various cells derived from the kidney tissue. In the serum free culture medium, dulbecco's modified eagle medium (DMEM)/ nutrient mixture F-12(F12)(1:1) is used as a basic culture medium and comprises the components including yeast autolysate, soy protein hydrolysate, amino acids, a fatty acid combining agent, an antioxidant reagent, reduced glutathione, a polyamine mixture, ethanolamine, beta-mercaptoethanol, glycerol, minerals, Pluronic F-68 and the like, and the pH value is 7.2. The culture medium of the invention can be used for culturing various cell lines derived from the kidney tissue, such as HEK293 cells, Marc145 cells, Vero cells and BHK21 cells and for propagating viruses.

The invention discloses a method for inhibiting HIV-1 infectious agent from infecting primary lymphocyte by utilizing CRISPR/Cas9 and relates to genetic engineering and an anti-virus infection technology. According to the method, by utilizing the latest CRISPR/Cas9 nuclease system, through the combination of Ad5F35 mosaic type adenovirus vectors of efficient targeted primary T lymphocyte, by editing CCR5 expression of human CD4+T lymphocyte, HIV-1 virus infection is effectively inhibited. According to the method, the CRISPR/Cas9 system is packaged by utilizing the Ad5F35 type adenovirus vectors, the advantages of the CRISPR/Cas9 system and the Ad5F35 type adenovirus vectors are organically combined, the novel method for resisting HIV-1 virus infection is provided, and the method has the potential for being applied for HIV-1 gene treatment research; meanwhile, the Ad5F35 type adenovirus vectors carrying with the CRISPR/Cas9 system can be used for conducting targeted editing to other genes in the primary lymphocyte or gene editing research of other hematopoietic system cells.

Peptide constructs comprised of multideterminant T helper peptides from the envelope glycoprotein of HIV previously identified to induce proliferative responses in four different haplotypes of mice and IL-2 responses in 52-73% of HIV positive, flu positive patients (cluster peptides), were co-linearly synthesized with the peptide 18 of the V3 loop of HIV-1 gp 160, corresponding to the principal neutralizing determinant of HIV-IIIB and also shown to contain a dominant CTL epitope. Cognate help for peptide 18 antibody was elicited following a single immunization in all strains of mice which had previously responded to a T cell epitope encompassed by the peptides. In two strains of mice, the level of neutralizing antibody achieved was comparable to levels adequate for protection from homologous viral challenge in chimpanzees. After a single boost, much higher antibody titers for 90% neutralization in the range of 1:1000 to 1:16,000 were achieved. Spleen cells from mice of three distinct MHC haplotypes sharing the D^d class I MHC molecule but with different class II molecules, immunized with the compound peptides, exhibited enhanced gp160-specific CTL activity.

The invention provides a novel coronavirus antigen epitope and application thereof. A super computer is used for simulating S, E, M and N protein structures of the novel coronavirus, and novel coronavirus epitopes SEQ ID NO 1-38 are obtained through calculation. The epitopes have very good immunogenicity; an antibody generated by inducing a part of epitope polypeptide has the effect of neutralizing the novel coronavirus; the antigen epitope can be combined with antibodies in serum of novel coronavirus patients at home and abroad, has the potential of resisting various novel coronavirus strains, and also has the potential of identifying and applying different strains. The epitope provided by the invention can be used for (1) research and development of novel coronavirus vaccines and universal coronavirus vaccines such as MERS viruses and SARS viruses; and (2) preparing a novel coronavirus antibody, and further detecting and typing viruses and treating diseases caused by the viruses. Thenovel coronavirus antigen epitope has a wide application prospect in the aspects of prevention of coronavirus and propagation and outbreak thereof, and detection and diagnosis of virus strains.

The invention discloses a porcine pseudorabies virus strain as well as an inactivated vaccine and applications thereof, belonging to the field of separation and application of the porcine pseudorabies virus strain. The invention firstly provides a porcine pseudorabies virus BJ strain separated from diseased pig tissues, and the microbial preservation number of the porcine pseudorabies virus BJ strain is CGMCC (China General Microbiological Culture Collection Center) No.7351. The invention discloses a method for preparing the inactivated vaccine by applying the porcine pseudorabies virus BJ strain. The method comprises the steps of culturing a virus strain to obtain a virus solution; adding an inactivator, and inactivating and concentrating the virus solution; and evenly mixing an adjuvant and the virus solution, and emulsifying to obtain the inactivated vaccine. The technological parameters of the inactivated vaccine preparation method are further optimized, and the immune protection efficacy and safety of the inactivated vaccine can be improved. Shown by the immune protection efficacy and safety tests, the porcine pseudorabies inactivated vaccine prepared has good immune protection efficacy and safety, and can be clinically used for preventing or treating porcine pseudorabies.

Chimeric parainfluenza viruses (PIVs) incorporate a PIV vector genome or antigenome and one or more antigenic determinant(s) of a heterologous PIV or non-PIV pathogen. These chimeric viruses are infectious and attenuated in humans and other mammals and are useful in vaccine formulations for eliciting an immune responses against one or more PIVs, or against a PIV and non-PIV pathogen. Also provided are isolated polynucleotide molecules and vectors incorporating a chimeric Ply genome or antigenome which includes a partial or complete PIV vector genome or antigenome combined or integrated with one or more heterologous gene(s) or genome segment(s) encoding antigenic determinant(s) of a heterologous PIV or non-PIV pathogen.

The invention discloses a broad-spectrum vibrio subunit vaccine for preventing fish pathogenic vibrio infection and a preparation method, belonging to the technical field of biology. The fusion protein OmpK-FlaA of an outer membrane protein OmpK with a conserved structure and a flagellin protein FlaA on the vibrio parahaemolyticus surface serves as an antigen component of the vaccine. The method is characterized by comprising the following steps of: superposing and extending Ppolymerase chain reaction (PCR) to the ompK and flaA genes of the vibrio parahaemolyticus to obtain a fusion protein gene flaA-ompK; constructing flaA-ompK-pET-28a recombinant plasmids; and obtaining the fusion FlaA-OmpK with high purity through exogenous induction expression and purification. The vaccine prepared bythe invention is safe and nontoxic and has no side effects; injection immune can be adopted; and the immune can also be realized by taking enteric microsphere vaccine orally; cross immunity protection can be acted to fish pathogenic vibrio (vibrio parahaemolyticus, vibrio alginolyticus, vibro harveyi, vibrio anguillarum and vibrio vulnificus).

The invention discloses a method for preparing porcine parvovirus inactivated vaccines. The preparation method comprises the following steps of: recovery culture of ST cells or IBRS-2 cells, bioreactor micro-carrier culture of the ST cells or the IBRS-2 cells, continuous culture of bioreactor micro-carriers of the ST cells or the IBRS-2 cells, inoculated porcine parvovirus culture, collection of virus liquid, and virus inactivation for preparing the porcine parvovirus inactivated vaccines. The method for preparing the porcine parvovirus inactivated vaccines is a porcine parvovirus process for culturing the ST cells by adopting the bioreactor micro-carriers; the micro-carriers provide larger surface area, and can improve the density of the ST cells and the virus titer; the reactors can automatically monitor the growth of the cells or the optimal biochemical conditions during propagation of viruses; and the viruses produced on each batch of reactors have uniform titer, small batch and stable quality.

The invention relates to construction and application of recombinant nucleocapsids of Cap genes of porcine circovirus expression type 2 nucleocapsid protein, belonging to the genetic engineering bacterin field. C-terminal gene fragments of porcine parvovirus (PPV) VP2 genes are cloned into a type 5 adenovirus shuttle vector of the human beings, and recombinant adenovirus rAd-deltaVP2 is obtained; deltaVP2 proteins are expressed successfully and highly efficiently and can be self-assembled into the nucleocapsids [PPV:VLPs]; the PPV VP2 nucleocapsids are used as antigen transport vectors and 165 to 200 sites of amino acid (deltaCap) genes of the porcine circovirus type 2 (PCV2) nucleocapsid proteins (Cap) are embedded into an N-terminal (deltaVP2) of the PPV VP2, and then recombinant adenovirus rAd-deltaCap-deltaVP2 is obtained; embedded VP2 (deltaCap-deltaVP2) proteins are expressed successfully and highly efficiently and can be self-assembled into nucleocapsids [PPV:VLP(PCV2)]. The invention also relates to application of the recombinant virus and recombinant PPV VP2 nucleocapsids of the expression Cap genes of the recombinant virus in the aspects of bacterin immunity and so on.

The present invention relates to human papillomavirus (HPV) type hybrid virus-like particles and a preparation method thereof. The virus-like particles can be used for preventions two or more HPV infections and diseases caused by HPV infections. The present invention further relates to uses of the protein and the virus-like particles in preparations of drug compositions or vaccines, wherein the drug compositions or the vaccines are provided for preventions of HPV infections and diseases caused by HPV infections, and the diseases comprise cervical cancer, condyloma acuminatum, and the like.

The invention aims at providing a new castle disease and H9 subtype bird flu bivalent vaccine. The new castle disease and H9 subtype bird flu bivalent vaccine contains antigens and adjuvant. The antigens are inactivated H9 subtype bird flu viruses and new castle disease viruses. The H9 subtype bird flu viruses are QDY strains, and the preservation number of the H9 subtype bird flu viruses is CCTCC v201517. The QDY strains of the H9 subtype bird flu viruses and a Lasota strain of the new castle disease viruses are inoculated to chick embryos respectively, and then virus liquid is collected; the virus liquid and the oil adjuvant are mixed and emulsified into the vaccine after the virus liquid is inactivated through a formaldehyde solution. The new castle disease and bird flu bivalent inactivated vaccine is good in immunogenicity, antibody production is fast after immunity, the produced antibody titer is high, the produced antibody holding time is long, the retention period is long, the immunizing dose is small, the selected adjuvant is easy to inject, and two kinds of diseases can be prevented through one-time injection. The vaccine has the advantages of being efficient and good in safety.

The invention comprises, fusing exogenous antigen genes and filobactivirus III gene proteins and expressed onto the tail of filobactivirus, fusing antigen epitopes and filobactivirus III gene proteins and expressed onto the surface of filobactivirus, preparing antibodies taking filobactivirus with exogenous antigen and antigen epitopes as the immunogen. The invention claims:1)a method of preparing monoantibodies and polyantibodies based on filobactivirus showing technology;2) a method of preparing cTnI monoantibody and polyantibody based on filobactivirus showing technology showing antigen and antigen epitopes of human troponin; 3) a PIII filobactivirus showing carrier pFD5; 4) a filobactivirus showing fusion expressing carrier pFD5-cTnI;5) a PIII filobactivirus showing fusion expressing carrier PC89-cTnI[95??08];6)taking filobactivirus particles showing cTnI and cTnI[95í½108] as immunogens, immunizing new zealand white rabbit to get cTnI antibody.

The invention provides a porcine epidemic diarrhea virus inactivated vaccine. The porcine epidemic diarrhea virus inactivated vaccine contains the inactivated porcine epidemic diarrhea virus and an adjuvant, wherein the adjuvant is prepared from the following components in percentage by weight: 5% of squalane, 1% of oleic acid, 1% of Tween 80, 92% of 0.005M sodium citrate buffer solution, and 1% of beta-glucan, the porcine epidemic diarrhea virus is prepared by inactivating the porcine epidemic diarrhea virus strain PEDV-KB2013-4, is assigned with the microbial accession number of CGMCC No.12663, has the classification name of Porcine Epidemic Diarrhea Virus (PEDV), and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on Aug 23, 2016, and the preservation address is the Institute of Microbiology of Chinese Academy of Sciences located in 3, Courtyard 1, West Beichen Road, Chaoyang District, Beijing.

The invention provides a canine parvovirus strain CPV-YH and applications thereof, and belongs to the technical field of microbes. The preservation number of the provided canine parvovirus strain CPV-YH is CGMCC No.12990. The invention also provides applications of the canine parvovirus strain CPV-YH in the preparation of canine parvovirus vaccines and canine parvovirus diagnostic reagents. Based on the canine parvovirus strain CPV-YH, the invention also provides a vaccine composition for preventing and/or treating diseases caused by canine parvovirus and a reagent for detecting a canine parvovirus strain and/or diagnosing diseases caused by canine parvovirus. A novel content and direction are provided for the research on the variation of epidemic virus; a novel strain is provided for the canine parvovirus vaccine development; and a high quality raw material and choice are provided for the vaccine development.

The invention relates to a process for preparing a veterinary rabies inactivated vaccine, in particular to a process for preparing a veterinary rabies inactivated and freeze-dried vaccine through a suspension culture cell. The method has the key point that: a bioreactor is used for large-scale suspension culture of baby hamster kidney BHK21-C13 cells and rabies virus is inoculated, and the rabiesvirus is subjected to mass propagation through a fed-batch and perfusion technology, so that a high-concentration and high-titer rabies antigen is obtained and concentrated, inactivated and purified to prepare the veterinary rabies inactivated vaccine; and thus, technical problems such as complexity, low antigen content, low effectiveness, large dosage, poor batch-to-batch variation and the like existing in the conventional process are solved.

The invention provides a method for preparing a rabies vaccine stock solution from serum-free Vero cells. The method comprises the following steps: culturing Vero cells with a serum-free culture medium to obtain a serum-free culture medium adaptive cell line; establishing a serum-free Vero cell seed bank by utilizing the serum-free culture medium adaptive cell strain; recovering, culturing, performing passage and amplifying cells of the Vero cell working seed bank by using the serum-free culture medium to serve as basal cells for bioreactor culture, and continuously perfusing to culture high-density Vero cells with the serum-free culture medium by applying a bioreactor and a microcarrier after cell expansion; inoculating virus seeds of a rabies vaccine virus strain working seed bank, performing bioreactor microcarrier serum-free culture, starting to continuously perfuse to obtain a virus fluid when the virus is amplified to peak to obtain titer of the virus fluid; and performing clarifying, ultrafiltration concentration, inactivation and purification treatment to obtain the serum-free human rabies vaccine stock solution.

The invention relates to a preparation method of an inactivated newcastle disease vaccine. The preparation method comprises the steps of virus liquid inactivation, preparation of a compound traditional Chinese medicine extract, preparation of an aqueous phase, preparation of an oil phase and emulsification. According to the preparation method, the compound traditional Chinese medicine extract serving as an immunopotentiator is added to the inactivated newcastle disease vaccine so as to well cause cellular immunologic response and enhance the immunologic function of an organism, so that an antibody is generated in advance, and the antibody titer is obviously improved.

The invention discloses a complete antigen for detecting tetrahydrocannabinol and preparing an antibody. The invention also discloses an anti-tetrahydrocannabinol monoclonal antibody prepared by the complete antigen and a tetrahydrocannabinol monoclonal antibody immunity detection plate through a collaurum tag for detecting the tetrahydrocannabinol in medicine, a urine specimen or other human specimens. Compared with HPLC and other methods, the detection plate is simple, portable and easy to carry, can carry out field detection and does not need expensive equipment. When the detection plate is used to detect the tetrahydrocannabinol, the whole test can be completed within 1.5 minutes; the detecting sensitivity can reach 0.5ng; and the detection plate has no crossed reaction with more than 60 kinds of common medicaments, drugs and internal metabolins of the tetrahydrocannabinol.