23 results about "Sf9" patented technology

The invention relates to a development method of an insect culture medium, which comprises the following steps: S1, constructing a double-promoter tandem vector by taking pFastBac plasmid as a skeleton; s2, establishing a baculovirus expression system; S21, transforming the double-promoter tandem vector into DH10Bac escherichia coli, so as to obtain recombinant Bacmid; s22, transfecting an Sf9 cell by using the recombinant Bacmid, so as to obtain a P0-generation baculovirus; s23, transfecting an Sf9 cell by using the P0-generation baculovirus to obtain a P1-generation baculovirus; s3, screening insect culture media: S31, providing culture media to be detected, and respectively inoculating Sf9 cells into the culture media to be detected; s32, respectively inoculating the P0-generation baculovirus or the P1-generation baculovirus into each to-be-detected culture medium for culturing; and S33, after culture is finished, detecting virus antibodies in the culture media to be detected, and screening the culture media. A comprehensive and quantitative evaluation system is established for development of the insect culture medium.

The invention discloses chicken infectious anemia virus-like particles as well as a preparation method and application thereof. The invention discloses a virus-like particle vaccine for preventing chicken infectious anemia. The virus-like particle vaccine comprises VP1 and VP2 proteins of chicken infectious anemia viruses. Chicken infectious anemia VP1 and VP2 proteins are expressed by using a baculovirus expression system, immunoblotting shows that the two proteins are successfully expressed in sf9 cells, electron microscope observation finds that the expressed proteins can be autonomously assembled into complete chicken infectious anemia virus-like particles, and the chicken infectious anemia virus-like particles have a space structure similar to that of an original virus and can be used for preparing chicken infectious anemia virus-like particles. Meanwhile, the virus-like particles have the advantages of high titer, high safety, capability of stimulating humoral immunity and cellular immunity and the like. The preparation method disclosed by the invention is simple, can be used for preparing the antigen protein of the chicken infectious anemia virus on a large scale, is high in expression quantity and short in time consumption, greatly reduces the production cost, and is suitable for large-scale production.

The invention discloses a recombinant subunit vaccine for resisting trichomonas pigeonae infection and a preparation method of the recombinant subunit vaccine. The vaccine comprises two recombinant proteins with sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively and a pharmaceutically acceptable carrier. According to the invention, Sf9 cells are used for respectively expressing trichomonas pigeonae adhesion protein 33 and adhesion protein 65, the immunogenicity of the obtained recombinant protein is similar to that of natural protein, the expression level is high, the immunogenicity is strong, a very small amount of recombinant protein can provide a better immune protection effect, and the recombinant protein has no pathogenicity to pigeons, and can be used for preparing the recombinant protein for preventing and treating trichomonas pigeonae. Meanwhile, large-scale serum-free suspension culture can be carried out through a bioreactor, so that the vaccine production cost is greatly reduced.

The present invention belongs to the field of insect cell culture technology and discloses a method for preparing an Sf9 cell feeder layer. The method mainly comprises two steps: step S1 is subculture of Sf9 cells, in which Sf9 cells are cultured in a suitable culture medium to a suitable cell confluence; step S2 is mitomycin C treatment of the Sf9 cells, in which a specific concentration of mitomycin C is used to treat the subcultured Sf9 cells at a specific time and temperature to inhibit their cell division ability, followed by washing to remove mitomycin C and replacing with fresh culture medium. The feeder layer cells obtained by the present invention can effectively inhibit their own proliferation while maintaining good cell activity and structural integrity. They can survive stably and provide necessary support for the co-culture system. They can be used for the cultivation of insect symbiotic bacteria and other difficult-to-culture insect cells. The method is simple to operate, has mild conditions, and good reproducibility. The prepared feeder layer has a stable effect, providing a powerful tool for related research fields.

The present application provides a primer pair for detecting bovine genomic DNA, a detection reagent or kit comprising the primer pair of the present application, and a method for detecting bovine genomic DNA using the primer pair, wherein the primer pair specifically binds to the sequence shown in SEQ ID NO: 1. The PCR detection method using the primer pair is simple and fast to operate, has high sensitivity, and can distinguish interfering DNAs such as pig, CHO, Vero, human, NS0, MDCK, E. coli, Pichia and Sf9.

This application provides a rhabdovirus-free Sf9-PT cell line and its applications. This cell line was deposited on March 15, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C202334. The Sf9-PT cell line screened in this application is free from rhabdovirus contamination, allows for high-density culture, exhibits higher viral proliferation levels, and demonstrates more durable viral packaging stability. This cell line can serve as a modified alternative host for the development and commercial production of biological products and vaccines, and can more safely avoid the potential dangers posed by Sf-RV virus contamination.

The invention discloses a micropterus salmoides rhabdovirus gene engineering vaccine preparation method, which comprises: S1, recombinant plasmid construction: carrying out PCR amplification on a micropterus salmoides rhabdovirus G protein gene with a 6 * His tag, cloning the amplified gene between BamH I and Hind III restriction enzyme cutting sites of a pVL1393 vector to obtain a pVL1393-G-His recombinant plasmid, S2, preparing a transfection system, and S3, carrying out purification on the transfection system to obtain the micropterus salmoides rhabdovirus gene engineering vaccine. S3, recombinant baculovirus preparation and identification, S31, cell transfection, S32, P1-generation virus harvesting, and S33, virus identification, the G protein of the micropterus salmoides rhabdovirus (MSRV) is directionally expressed through a genetic engineering technology, a large number of pathogenic viruses do not need to be cultured, the biological safety risk is greatly reduced, meanwhile, the defect that a prokaryotic expression system lacks protein post-translational modification is overcome, and the method is suitable for industrial production. The sf9 insect cell is used as an expression host, so that the recombinant G protein can be correctly folded to form a functional structure domain, and the immunogenicity is remarkably improved.

Provided herein are invertebrate cell lines for production of AAV viral proteins (such as capsid proteins) and AAV viral particles that are rhabdovirus-negative or have rhabdovirus genomes below the limit of detection, and methods of deriving such cell lines. Certain cell lines described herein, and methods of deriving such cell lines, achieve significantly greater AAV production over the cell lines and methods known in the art.

The invention provides a detection method for detecting Sf9 host DNA residues by using a fluorescent probe and application, and belongs to the technical field of biological detection.According to the method, a specific primer pair (such as SEQIDNO: 1 / 2) and the fluorescent probe (such as SEQIDNO: 11) are designed according to a high-copy conserved sequence of Sf9 host DNA, a kit containing a gene amplification solution, a standard substance and a quality control substance is matched, DNA is extracted through a liquid nitrogen grinding-column extraction method, and the detection result is accurate. And carrying out real-time fluorescent quantitative PCR amplification and then carrying out detection. According to the method, the detection sensitivity reaches 0.003 pg / mu L, the linear range is 300 pg / mu L-0. 003 pg / mu L, cross reaction to irrelevant DNA is avoided, the repeatability CV% is smaller than or equal to 10%, the reagent is still stable after being frozen and thawed for 10 times, and the whole process is completed within 1.5 hours; the method solves the problems of insufficient specificity and sensitivity, tedious operation and the like of the existing method, and can be used for quality control of biological products such as gene therapy drugs and cell vaccines.

The present invention relates to a method, nucleotide sequence and expression vector for producing human serum albumin using insect cells, and belongs to the field of the production of human serum albumin. The nucleotide sequence of the human serum albumin of the present invention is shown as SEQ ID NO.1. According to the codon preference of insect cells, the present invention optimizes the codons of the human serum albumin gene and combines it with a signal peptide sequence shown as SEQ ID NO.2, so as to achieve the expression of human serum albumin in Sf9 cells. The present invention further discloses a method for producing human serum albumin using insect cells. By adding small molecule additives during the production process, the expression level of human serum albumin in Sf9 cells can be effectively increased.

The invention discloses preparation of an akabane disease virus N protein monoclonal antibody and a double-antibody sandwich ELISA (Enzyme-Linked Immunosorbent Assay) detection method, in order to prepare the akabane disease virus nucleocapsid protein monoclonal antibody, an eukaryotic recombinant plasmid pFastBac-AKAV-N is constructed and is converted into a DH10bac competent cell, and a recombinant stem Bacmid-AKAV-N for expressing AKAV N is successfully obtained. After the recombinant baculovirus is transfected to an sf9 insect cell, the recombinant baculovirus capable of stably expressing the AKAV N protein is rescued. A mouse and a rabbit are immunized with the identified and purified recombinant N protein, and a monoclonal antibody and a polyclonal antibody are prepared respectively. The titer of the polyclonal antibody is measured through an ELISA method, and two positive cell strains are obtained through screening and are named as 2G4 and 6C7 respectively. Furthermore, the prepared polyclonal antibody is used as a coating antigen, an HRP-labeled 2G4 monoclonal antibody is used as a detection antibody, and a double-antibody sandwich ELISA detection method for AKAV N protein antigen detection is optimized and established. According to the method, the AKAV N antigen can be specifically recognized, and reliable technical support is provided for clinical antigen monitoring of AKAV. The monoclonal antibody preparation system and the double-antibody sandwich ELISA detection method established by the invention lay an important foundation for pathogen detection and epidemiological investigation of akabane disease.

The invention provides a stirring type reactor culture process of a porcine circovirus type 2 recombinant baculovirus, which comprises the following steps: preparing a seed virus of the porcine circovirus type 2 recombinant baculovirus by using an insect cell Sf9 full-suspension culture process, and inoculating an insect cell High Five by using the seed virus prepared by using an Sf9 cell to perform antigen expression of the porcine circovirus type 2 recombinant baculovirus. A full-suspension culture mode is utilized, so that the labor intensity is reduced, the production cost is reduced, and pollution is not easily caused; 2) the risk of porcine virus pollution can be reduced by using insect cells and a baculovirus expression system; and 3) the characteristics (Sf9 and High Five) of different insect cells are simultaneously utilized to respectively carry out seed virus preparation and antigen expression, so that higher virus titer can be obtained, the virus titer is greater than or equal to 7.50 Log (TCID50 / mL), higher recombinant protein expression is obtained, and the protein expression quantity is greater than or equal to 150 mu g / mL.

The application discloses a rhabdovirus-free Sf9 cell screening method of double pretreated conditioned medium, relates to the technical field of biology, and obtains the rhabdovirus-free Sf9 cell by double pretreatment of the conditioned medium through cross-flow filtration and beta-propiolactone inactivation and hydrolysis, and combination of single cell cloning screening. The method realizes efficient virus interception and high-activity retention of nutritional factors, the single cell cloning formation rate is greater than 40%, and is suitable for virus-free cell bank construction and biological product production.

The present disclosure describes methods and systems for use in the production of adeno-associated virus (AAV) particles, including recombinant adeno-associated virus (rAAV) particles. In certain embodiments, the production process and system use Spodoptera frugiperda insect cells (such as Sf9 or Sf21) as viral production cells (VPCs).

The invention discloses a baculovirus expression vector as well as a construction method and application thereof. The vector takes pFastBacDual as a skeleton, 2-9 repetitive BS sequence modification promoters are connected in series to the downstream of a polh promoter, an extremely advanced transcription factor VLF-1 gene is introduced, and the two components synergistically enhance the transcriptional activity. According to the invention, three types of recombinant vectors pBSX-eGFP, pBSX-eV and pBSX-GV are constructed, and the optimal combination is determined as overexpression of two repeated BS sequences and VLF-1, so that the expression quantity of the target protein can be increased. The vector construction process is standardized, enzyme cutting sites such as BamHI and EcoRI are reserved, and high-expression protein can be obtained after Sf9 cells are transfected and cultured for 4-5 days. The recombinant protein expression efficiency of a baculovirus expression system is improved, the advantages of correct folding and post-translational modification of the recombinant protein are reserved, and the method is suitable for large-scale industrial recombinant protein production scenes such as biopharmacy and vaccine research and development and has important application value.

The invention belongs to the technical field of insect cell culture, and discloses a preparation method of an Sf9 cell feeder layer, which mainly comprises the following two steps: S1, subculturing Sf9 cells: culturing the Sf9 cells in a suitable culture medium to a suitable cell confluence degree; and S2, mitomycin C treatment of the Sf9 cells: treating the subcultured Sf9 cells at specific time and temperature by using mitomycin C with specific concentration to inhibit the cell division ability of the Sf9 cells, then washing to remove the mitomycin C, and replacing a fresh culture medium. The obtained feeder layer cells can effectively inhibit self proliferation, maintain good cell activity and structural integrity, can stably survive and provide necessary support for a co-culture system, and can be used for culture of insect symbiotic bacteria and other insect cells difficult to culture; the method is easy and convenient to operate, mild in condition and good in repeatability, the prepared feeding layer is stable in effect, and a powerful tool is provided for the related research field.

The present invention relates to the technical field of exogenous RNA virus detection and control of biological products. Provided are a primer and probe combination for detecting multiple RNA viruses capable of infecting Sf9 cells, and the use thereof. Provided is a primer and probe combination capable of simultaneous detection of multiple RNA viruses, which are capable of infecting Sf9 cells, in insect cells and biological products thereof. The primer and probe combination contains seven forward primers, seven reverse primers, and six probes; can cover multiple RNA viruses capable of infecting Sf9 cells; has the advantages of high sensitivity, good specificity, high applicability, a small sample amount, etc.; and can be widely used in the safety evaluation of exogenous RNA viruses in insect cells and biological products thereof.

The invention relates to a primer pair for detecting the size distribution of Sf9 cell DNA fragments and a detection method. The primer pair at least comprises four groups of primer pairs; a forward primer and a reverse primer in each primer pair are respectively and specifically combined with a section shown as SEQ ID NO: 17 on the Sf9 cell genome DNA; and the lengths of amplification products obtained by amplification of each group of primer pairs are respectively less than 100bp, 100-199bp, 200-399bp and more than 400bp. The method can be used for quantitatively analyzing the size distribution of the Sf9 residual DNA fragment in the biological product, and is beneficial to improving the process and improving the product quality. The primer pair can truly and accurately reflect the size and distribution of residual DNA and fragments of the Sf9 cells, and has very high application value for quality control of gene therapy products, vaccines and protein products produced by an insect cell-baculovirus expression system.

The invention discloses an optimized construction method of a protein interaction verification system BiFC-Venus for spodoptera frugiperda and application of the optimized construction method of the protein interaction verification system BiFC-Venus for spodoptera frugiperda. The design principle of the Spodoptera frugiperda protein interaction verification system BiFC-Venus provided by the invention is as follows: plasmids of a pIZ-to-be-tested gene A-VN173-His and a pIZ-to-be-tested gene B-VC155-His are co-transfected into an Sf9 cell through the method, so as to judge whether the two proteins have interaction or not. The protein researched by the invention is eukaryotic expression and has natural conformation; consumed time is short, and data processing is simple; the background is clean, the fluorescence has high brightness and long service life, and the detection is sensitive; the temperature tolerance is high, and observation can be carried out under the physiological temperature condition. Therefore, the system can verify the interaction between the proteins, and a convenient and rapid research method is provided for the research of the insect protein function.

The present invention discloses the application of the transcription factor SfGATAe in the green prevention and control of the Spodoptera frugiperda population. The present invention discovers that overexpressing SfGATAe of Spodoptera frugiperda in the Sf9 cell line insensitive to the Cry1Ac toxin can significantly improve the sensitivity of Sf9 cells to the Cry1Ac toxin; interfering with the expression of SfGATAe in the larvae of Spodoptera frugiperda can significantly increase the resistance of the larvae of Spodoptera frugiperda to the Cry1Ac toxin. Therefore, the transcription factor SfGATAe of Spodoptera frugiperda can be applied to the green prevention and control of the Spodoptera frugiperda population.

The invention discloses a method for improving rAAV (recombinant adeno-associated virus) production of a baculovirus system, namely, sf9 cells are infected with a third-generation recombinant baculovirus solution containing an AAV (adeno-associated virus) genome without removing the cells by controlling the algebraic number, the infection mode and the sf9 cell density of the recombinant baculovirus, and the yield of rAAV can be further improved when the cell density is 2.5-4.0 * 10 cells / ml; better technical support is provided for cost reduction and benefit improvement in the fields of neuroscience research, disease model establishment, gene therapy and the like, and the application value and the market prospect are wide.

The invention provides a method for calculating the optimal amplification proportion of baculovirus and application thereof, and the method comprises the following steps: (1) culturing SF9 cells until the cell density reaches (3-4) * 10 < 6 > cells / mL, inoculating a series of titers of baculovirus stock solutions into an SF9 cell culture solution, and culturing to promote virus amplification; (2) the cell viability of the SF9 cells is tested, the virus amplification efficiency is judged based on the cell viability on the fourth day, and the judgment standard is that the inoculation titer of a baculovirus stock solution corresponding to a culture solution with the cell viability of 75-85% on the fourth day is the optimal virus addition amount, and the efficiency is the highest after virus amplification; and (3) after the culture is finished, detecting the virus titer of the culture medium corresponding to the optimal virus addition amount, and calculating the amplification proportion of the baculovirus.

The invention relates to a construction method of a recombinant baculovirus shuttle vector Bacmid '. The method comprises the following steps: cloning a lacZ alpha-attTn7 sequence into a baculovirus transfer vector subjected to reverse amplification, so as to obtain a recombinant plasmid pBacPAK8-lacZ alpha-attTn7; the recombinant virus AcNPV-lacZalpha-attTn7 is obtained by co-transfecting Sf9 cells with a defective virus Bacmid, and a recombinant virus AcNPV-lacZalpha-attTn7 is generated; carrying out amplification and ultra-high-speed centrifugation on the obtained recombinant virus, and extracting through phenol chloroform to obtain virus DNA (Deoxyribonucleic Acid); the recombinant baculovirus shuttle vector Bacmid'is obtained by co-transforming competent cells of recombinant virus DNA (deoxyribonucleic acid) and pBvBAC (obtained by cloning Vcat, miniF and Chitinase to pUC19), and carrying out recombinant cloning by utilizing blue-white spot screening. According to the present invention, the miniF replicon co-localized with lacZalpha-attTn7 is separated, such that the recombinant baculovirus shuttle vector Bacmid'with the high gene cloning stability is successfully constructed; according to the technology, the expression stability of the exogenous gene is remarkably improved, and a powerful research tool is provided for vaccine development and virology application based on the baculovirus vector.