CN117305365B - Insect cell-mammal cell expression shuttle vector SmartBM-1 and application thereof - Google Patents

Insect cell-mammal cell expression shuttle vector SmartBM-1 and application thereof Download PDF

Info

Publication number
CN117305365B
CN117305365B CN202311601025.2A CN202311601025A CN117305365B CN 117305365 B CN117305365 B CN 117305365B CN 202311601025 A CN202311601025 A CN 202311601025A CN 117305365 B CN117305365 B CN 117305365B
Authority
CN
China
Prior art keywords
vector
protein
smartbm
cells
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202311601025.2A
Other languages
Chinese (zh)
Other versions
CN117305365A (en
Inventor
翟宇佳
孙飞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Biophysics of CAS
Original Assignee
Institute of Biophysics of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Biophysics of CAS filed Critical Institute of Biophysics of CAS
Priority to CN202311601025.2A priority Critical patent/CN117305365B/en
Publication of CN117305365A publication Critical patent/CN117305365A/en
Application granted granted Critical
Publication of CN117305365B publication Critical patent/CN117305365B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/36—Vector systems having a special element relevant for transcription being a transcription termination element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Virology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses an insect cell-mammal cell expression shuttle vector SmartBM-1 and application thereof. The nucleotide sequence of the vector is shown in SEQ ID NO: 1. Experiments prove that the vector provided by the invention can simultaneously carry out high-efficiency protein expression in insect cells and mammal cells, so that the experimental efficiency is greatly improved; meanwhile, the target protein in the vector is fused with the cleavable sfGFP-TS protein for expression, and the sfGFP-TS protein indicates the expression status of the target protein, so that an experimenter can know the cell expression status at early stage of culture, evaluate the expression level and terminate the problematic experiment in advance, thereby saving material resources and manpower and further saving the experiment cost. The carrier provided by the invention has important application value.

Description

Insect cell-mammal cell expression shuttle vector SmartBM-1 and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an insect cell-mammal cell expression shuttle vector SmartBM-1 and application thereof.
Background
The baculovirus expression system (Baculovirus Expression System, BVES) is a powerful tool for efficiently expressing exogenous proteins in insect cells, and has the advantages of good safety, high expression level, post-translational processing and the like. Because the genome of the baculovirus is huge, the cloning of the exogenous gene cannot be directly inserted by an enzyme digestion connection method, people reform the genome of the baculovirus and construct a transfer vector matched with the genome of the baculovirus, so that the baculovirus and the transfer vector are recombined into the recombinant baculovirus which can infect insect cells and contains the exogenous gene. In the Bac to Bac system widely used at present, a baculovirus shuttle vector (Bacmid) can replicate in escherichia coli and can infect lepidopteran insect cells, tn7 site-specific recombination can be carried out on the baculovirus shuttle vector in escherichia coli and a pFastbac series vector containing exogenous genes, and the obtained recombinant baculovirus shuttle vector can replicate in escherichia coli efficiently, and can be used for transfecting the insect cells after extraction and purification.
The mammalian cell expression system can provide post-translational modification of recombinant human proteins that is closer to the native state, and during protein expression, protein folding and polymerization that approximates the native protein is formed, with the spatial structure and modification necessary for the active protein. The exogenous protein produced by post-translational reprocessing modification of mammalian cells is more similar to the native protein, and is also higher in activity than proteins expressed by prokaryotic expression systems, yeast, insect cells, and other eukaryotic expression systems. Protein expression using mammalian cells generally takes place in three ways: firstly, using plasmid to make transient expression; secondly, using a stably expressed mammalian cell line; thirdly, the expression is carried out by infecting mammalian cells with baculovirus with high titer, such as BacMAM system. The establishment of stable expression mammalian cell lines has a longer cycle, so that during the initial phase of protein expression, researchers have a greater propensity to complete the construction screen using transient expression or baculovirus infection. The expression pattern of transient expression is rapid and flexible, but the infection efficiency is far lower than that of baculovirus-mediated infection. Therefore, the expression of mammalian cells by baculovirus infection is increasing.
The advantages of mammalian cell expression systems, while evident, are high cost and high requirements on the skilled artisan. Moreover, some proteins are expressed by insect cells, resulting in higher yields. Therefore, it is often necessary to try both insect cell expression systems and mammalian cell expression systems simultaneously when expressing proteins, whether in the scientific or industrial world. Existing insect cell-mammalian cell expression shuttle vectors are pQE-TriSystemvector (Qiagen) and Merck under Novagen brand pTriEX series vectors, which contain CAG promoter and CMV promoter that can promote expression of foreign proteins in mammalian cells, respectively, which use p10 promoter to promote expression of foreign proteins in insect cell expression. These vectors do not contain the Tn7L, tn7R site and cannot recombine with baculovirus molecules in DH10Bac competent cells, and thus cannot obtain recombinant baculoviruses by the traditional Bac to Bac approach. However, these vectors contain the ORF603 and ORF1629 elements of baculovirus, and recombinant baculovirus containing exogenous fragments can be obtained by recombination with linearized baculovirus DNA. However, the general laboratory cannot prepare linearized baculovirus DNA, and can only purchase it at a very low price from the company selling these vectors. These vectors cannot be widely used because of the high cost of linearizing baculovirus DNA and the long ordering cycle.
In addition, efficient mammalian cell expression using baculoviruses places high demands on baculovirus titer. In general, baculovirus titers of up to 1X 10 9 pfu/ml, MOI=10 at the time of infection, can obtain better effect. For stable expression, detection of baculovirus titer prior to infection is necessary. However, the classical plaque assay for baculovirus titer takes 5-8 days; baculovirus titer is usually measured by observing fluorescence of co-expressed fluorescent proteins independent of kit, usually 3-4 days, because expression vectors use very late promoters such as p10 or ph promoters to initiate expression of fluorescent fusion proteins.
Disclosure of Invention
The invention aims to provide an insect cell-mammal cell expression shuttle vector.
The insect cell-mammal cell expression shuttle vector provided by the invention is named as a vector SmartBM-1, and can comprise a fusion promoter, a target protein expression region, an element for increasing the expression of a protein in the mammal cell, a termination sequence, an element for recombining with baculovirus and a resistance gene expression cassette;
the fusion promoter efficiently promotes protein expression in insect cells and mammalian cells;
the target protein expression region sequentially comprises the following elements: a multiple cloning site, an enzyme cutting site of HRV-3C-protease and a fluorescent protein gene; the multiple cloning site has more than one cleavage recognition sequence therein.
In the vector SmartBM-1, the fusion promoter may include the following elements: the humanized CMV promoter, the white spot syndrome virus IE1 promoter and the p10 promoter.
The fusion promoter can specifically consist of a humanized CMV promoter, an white spot syndrome virus IE1 promoter and a p10 promoter.
The nucleotide sequence of any of the humanized CMV promoters is shown in SEQ ID NO:1 from the 5' end at position 2005-2892.
The nucleotide sequence of any one of the White Spot Syndrome Virus (WSSV) IE1 promoters is shown in SEQ ID NO:1 from the 5' end at positions 2899-3055.
The nucleotide sequence of any p10 promoter is shown as SEQ ID NO:1 from the 5' end at positions 3062-3175.
In the vector SmartBM-1, the element that increases expression of the protein in the mammalian cell may be a WPRE element. The nucleotide sequence of the WPRE element is shown as SEQ ID NO:1 from the 5' end at positions 4169-4757.
In the vector SmartBM-1, the termination sequence may be an SV40 polyA sequence. The termination sequence is used to terminate expression of the protein. The nucleotide sequence of the SV40 polyA sequence is shown as SEQ ID NO:1 from the 5' end at positions 4784-5024.
In the vector SmartBM-1, the elements recombined with baculovirus are Tn7L element and Tn7R element. Specifically, the recombination with baculovirus may be recombination with empty bacmid in DH10bac, to obtain a recombinant bacmid. The nucleotide sequence of the Tn7L element is shown in SEQ ID NO:1 from the 5' end at positions 5053-5218. The nucleotide sequence of the Tn7R element is shown in SEQ ID NO:1 from the 5' end at positions 817-1040.
In the vector SmartBM-1, the resistance gene expression cassette may be a gentamicin resistance gene expression cassette. The gentamicin resistance gene expression cassette can make the transformed bacteria have gentamicin resistance. The nucleotide sequence of the gentamycin resistance gene expression cassette is shown in SEQ ID NO:1 from the 5' end at positions 1041-1857.
The target protein expression region can also comprise a Twin-strep label and an enterokinase enzyme cutting site. The Twain-strep tag is located upstream, i.e., 5' of the cleavage site of enterokinase. Enterokinase is located upstream, i.e. 5' to the multiple cloning site.
The nucleotide sequence of the Twin-strep tag is shown in SEQ ID NO:1 from the 5' end at positions 3180-3269.
The nucleotide sequence of the enzyme cutting site of the enterokinase is shown as SEQ ID NO:1 from the 5' end at positions 3270-3284.
The multiple cloning site is used for inserting the target gene. The nucleotide sequence of the multiple cloning site is shown as SEQ ID NO:1 from the 5' end at positions 3285-3314.
The nucleotide sequence of the enzyme cutting site of the HRV-3C protease is shown as SEQ ID NO:1 from the 5' end 3315-3338.
The fluorescent protein gene may specifically be an sfGFP-TS gene. The nucleotide sequence of the sfGFP-TS gene is shown as SEQ ID NO:1 from the 5' end, 3357-4151. The target gene can be fused with sfGFP-TS gene, and the expression condition of target protein can be judged by observing the fluorescence of cells.
Any of the vectors SmartBM-1 may further contain loxP site for recombination with other vectors containing loxP site. The nucleotide sequence of the loxP sequence is shown as SEQ ID NO:1 from the 5' end at positions 1928-1961.
Any of the vectors SmartBM-1 described above may also contain a pUC origin of replication that allows for high copy number replication in E.coli. The nucleotide sequence of the pUC replication origin is shown as SEQ ID NO:1 from the 5' end at positions 137-725.
The nucleotide sequence of any one of the vectors SmartBM-1 is specifically shown as SEQ ID NO: 1.
The use of the vector SmartBM-1 as described in any of the above as a shuttle vector for expression of insect cells-mammalian cells is also within the scope of the present invention.
The use of the vector SmartBM-1 of any of the above in insect cell expression systems and/or mammalian cell expression systems to express proteins is also within the scope of the invention.
The use of the vector SmartBM-1 of any of the above in the detection of baculovirus titer is also within the scope of the present invention.
The invention also provides a method for expressing target protein, which comprises the following steps:
(1) Inserting a target gene into a multiple cloning site of the vector SmartBM-1, and then transforming baculovirus to obtain recombinant baculovirus;
(2) Infecting insect cells or mammalian cells with the virus solution of the recombinant baculovirus obtained in the step (1) to obtain recombinant cells capable of expressing fusion proteins; the fusion protein comprises target protein and fluorescent protein coded by target genes;
(3) Crushing the recombinant cells expressing the fusion protein obtained in the step (2), collecting the fusion protein, and then carrying out enzyme digestion by using HRV-3C-protease to separate the target protein.
In the above method, the target gene may be a nucleotide sequence as shown in SEQ ID NO:3 is shown in the figureCOLD6And (3) a gene.
In the above method, the target protein may be an amino acid sequence as shown in SEQ ID NO:2, a COLD6 protein.
In the above method, in the step (1), the "inserting the target gene into the multiple cloning site of any one of the vectors SmartBM-1" may specifically be a substitution of a small DNA fragment between restriction enzymes BspEI and NotI of any one of the vectors SmartBM-1 with SEQ ID NO:3, and the obtained recombinant plasmid SmartBM-1-COLD6.
In the above method, in the step (1), the "inserting the target gene into the multiple cloning site of the vector SmartBM-1 to transform baculovirus to obtain recombinant baculovirus" may specifically be transforming DH10Bac competent cells with recombinant plasmid SmartBM-1-COLD6, and then screening with antibiotics (specifically gentamicin, tetracycline and kanamycin) and screening with blue-white spots to obtain recombinant baculovirus.
In the above method, in the step (2), the fusion protein may be composed of a COLD6 protein and an sfGFP-TS protein.
In the above method, in the step (2), the insect cell may be sf9 cell. The mammalian cell may be an Expi293F cell.
In the above method, in the step (3), the "disrupting the recombinant cells expressing the fusion protein obtained in the step (2), and collecting the fusion protein" may specifically be: firstly adding a lysis buffer solution containing protease inhibitor, performing ultrasonic disruption, centrifuging and collecting supernatant; adding a lysis buffer solution, and homogenizing to obtain a membrane homogenate; then adding a lysis buffer and LMNG, stirring for 2 hours at 4 ℃, centrifuging, and collecting a supernatant; finally, the supernatant is subjected to Strep self-loading, nonspecifically bound hybrid protein is washed off, and fusion protein is obtained through elution.
In the above method, in the step (3), the "enzyme-cutting with HRV-3C-protease to isolate the target protein" may specifically be that the recombinant HRV 3C-protease is used to enzyme-cut the fusion protein, and then the fusion protein is purified by passing through a molecular sieve Superdex200 to obtain the target protein.
The invention also provides a method for detecting the titer of the baculovirus to be detected, which can comprise the following steps:
(a1) Transforming the recombinant vector of which any one of the vector SmartBM-1 or the vector of which any one of the vector SmartBM-1 is inserted with genes into a recombinant vector of which any one of the vector SmartBM-1 is inserted with genes into a baculovirus to be tested to obtain the recombinant baculovirus to be tested; then preparing virus liquid of the recombinant baculovirus to be detected;
(a2) Diluting the virus liquid of the recombinant baculovirus to be tested prepared in the step (a 1) to obtain virus diluents with different dilution factors;
(a3) Taking a culture container, adding insect cells, and culturing by adherence; then respectively infecting the virus dilutions with different dilution factors prepared in the step (a 2), and culturing the virus dilutions for 24 to 36 hours at a temperature of between 25 and 30 ℃ in a dark place; 3 or more replicates per dilution;
(a4) After completion of step (a 3), the number of cells (pfu) showing fluorescence per dilution of the virus dilution was counted and the concentration thereof in the culture vessel (pfu/cm) was calculated 2 ) If the concentration of fluorescent cells in more than 3 replicates of a viral dilution of a dilution factor is 5-15pfu/cm 2 The dilution ratio is the optimal dilution ratio; counting the concentration of fluorescent cells in more than 3 repeats of the virus diluent with the optimal dilution and averaging, namely, the average concentration of the fluorescent cells in the virus diluent with the optimal dilution; then, the titer of virus liquid of the recombinant baculovirus to be tested is calculated according to the following formula:
titer of virus solution of recombinant baculovirus to be tested (pfu/ml) = (average concentration of virus dilution fluorescent cells at optimal dilution factor (pfu/cm) 2 ) X optimal dilution x culture vessel surface area (cm) with viral dilutions 2 ) Viral dilution volume (ml) at optimal dilution of infected adherent cultured insect cells in step (a 3);
the titer of the virus liquid of the recombinant baculovirus to be tested is the titer of the baculovirus to be tested.
It can be seen that the invention provides an insect cell-mammalian cell expression shuttle vector SmartBM-1 based on the Bac to Bac system. Compared with the traditional BacMAM system which utilizes baculovirus to express protein in mammalian cells (the system can not express protein in insect cells), the classical Bac to Bac system which can only express in insect cells, and the pQE-TriSystemand pTriEX series vectors which rely on linearization of viral DNA to generate recombinant baculovirus, the vector SmartBM-1 can simultaneously express high-efficiency protein in insect cells and mammalian cells, and greatly improves experimental efficiency. Meanwhile, in the vector SmartBM-1, the target protein and the cleavable sfGFP-TS protein are fused and expressed, and the sfGFP-TS protein indicates the expression condition of the target protein, so that an experimenter can know the cell expression condition at early stage of culture, evaluate the expression level and terminate the experiment with problems in advance, thereby saving material resources and manpower and saving the experiment cost. In addition, the fusion promoter region of vector SmartBM-1 contains the WSSV IE1 promoter, which can not only promote expression in mammalian cells, but also early expression of the gene of interest in insect cells, as compared to vectors that rely solely on the p10 promoter or the ph promoter to promote expression of the protein of interest in insect cells; by utilizing the characteristics of the WSSV IE1 promoter, an experimenter can finish the detection of baculovirus titer within 24-36 hours, so that the experimental efficiency is greatly improved. The vector SmartBM-1 provided by the invention has important application value.
Drawings
FIG. 1 is a map of vector SmartBM-1.
FIG. 2 shows the fluorescence observed after transfection of sf9 cells with the 5 recombinant baculovirus of step two of example 2.
FIG. 3 shows fluorescence observed after infection of the Expi293F cells with 5 and 6 virus fluids P2C in step four of example 2.
FIG. 4 shows the purification pattern and SDS-PAGE results of Superdex200 of COLD6 protein expressed by sf9 cells.
FIG. 5 shows the Superdex200 purification profile and SDS-PAGE results of COLD6 protein expressed by the Expi293F cells.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 construction of insect cell-mammalian cell expression shuttle vector SmartBM-1
Through a great deal of experiments, the inventor constructs an insect cell-mammal cell expression shuttle vector SmartBM-1, hereinafter referred to as vector SmartBM-1.
The vector SmartBM-1 (circular) consists of 5218 nucleotides, and the nucleotide sequence of the vector is shown in SEQ ID NO: 1. The map of vector SmartBM-1 is shown in FIG. 1.
The vector SmartBM-1 is characterized by the following specific features:
1. comprises a humanized CMV promoter (shown as 2005-2892 from the 5' end of SEQ ID NO: 1), a White Spot Syndrome Virus (WSSV) IE1 promoter (shown as 2899-3055 from the 5' end of SEQ ID NO: 1) and a p10 promoter (shown as 3062-3175 from the 5' end of SEQ ID NO: 1); and the humanized CMV promoter, the WSSV IE1 promoter and the p10 promoter are connected in series to form a fusion promoter, and the fusion promoter can efficiently promote protein expression in insect cells and mammalian cells;
2. the target protein expression region of the vector contains a Twin-strep tag (shown in 3180-3269 th from 5 'end of SEQ ID NO: 1), followed by an enterokinase cleavage site (shown in 3270-3284 th from 5' end of SEQ ID NO: 1), followed by a multiple cloning site (for inserting the target gene, the nucleotide sequence is shown in 3285-3314 th from 5 'end of SEQ ID NO: 1), followed by an HRV-3C protease cleavage site (shown in 3315-3338 th from 5' end of SEQ ID NO: 1), followed by a sfGFP-TS gene (i.e., the 3 'end of sfGFP gene is fused with the Twin-strep tag, and the nucleotide sequence is shown in 3357-4151 th from 5' end of SEQ ID NO: 1). The target gene can be fused with sfGFP-TS gene, and the expression condition of the target protein can be judged by observing the fluorescence of cells;
3. the vector contains a WPRE element (shown in positions 4169-4757 from the 5' end of SEQ ID NO: 1) that increases the expression of the protein in mammalian cells;
4. the vector terminates the expression of the protein with the SV40 polyA sequence (SEQ ID NO:1, positions 4784-5024 from the 5' end);
5. the vector contains Tn7L element (shown in 5053-5218 from the 5 'end of SEQ ID NO: 1) and Tn7R element (shown in 817-1040 from the 5' end of SEQ ID NO: 1), and can be recombined with NO-load bacmid in DH10bac to obtain recombined bacmid;
6. the vector contains loxP site (1928-1961 from 5' end of SEQ ID NO: 1) and can be recombined with other vectors containing the sequence;
7. the vector contains pUC replication origin (SEQ ID NO:1, 137-725 from the 5' end), which can make high copy number replication in E.coli;
8. the vector contains a gentamicin resistance gene expression cassette (shown in 1041-1857 positions from the 5' end of SEQ ID NO: 1) and can make the transformed bacteria have gentamicin resistance.
EXAMPLE 2 use of vector SmartBM-1 for expression of the plant protein COLD6 in insect and mammalian cells
The vegetable protein COLD6 (namely COLD6 protein) is a membrane protein containing 6 times of transmembrane regions, has a molecular weight of 50kDa, and has an amino acid sequence shown in SEQ ID NO: 2.
The gene encoding COLD6 protein isCOLD6The nucleotide sequence of the gene is shown as SEQ ID NO: 3.
1. Construction of recombinant plasmid SmartBM-1-COLD6
The small DNA fragment between restriction enzymes BspEI and NotI of vector SmartBM-1 was replaced with SEQ ID NO:3 (i.e.COLD6Genes) to obtain a recombinant plasmid SmartBM-1-COLD6. In the recombinant plasmid SmartBM-1-COLD6,COLD6the gene and sfGFP-TS gene are fused, and a fusion protein COLD6-sfGFP-TS consisting of COLD6 protein and sfGFP-TS protein is expressed.
2. Preparation of virus liquid by recombinant plasmid SmartBM-1-COLD6
1. Recombinant plasmid SmartBM-1-COLD6 was transformed into DH10Bac competent cells (product of ThermoFisher company, catalog number 10361012), followed by screening for antibiotics (specific antibiotics are gentamicin, tetracycline and kanamycin) and screening for blue-white spots (specific procedures refer to the Bac to Bac operating manual of Invitrogen company), to obtain several baculoviruses.
2. Respectively extracting plasmids of a plurality of baculoviruses obtained in the step 1 and taking the plasmids as templates, and respectively carrying out PCR amplification by adopting a primer pair 1 (consisting of a primer COLD6-F:5'-ATGGCTGCAGCTTCTCCTTTG-3' (SEQ ID NO: 4) and a primer COLD6-R:5'-CAACTCGAATTCGTCGTCC-3' (SEQ ID NO: 5)) and a primer pair 2 (consisting of a primer M13 Forward:5'-GTTTTCCCAGTCACGAC-3' (SEQ ID NO: 6) and a primer M13 Reverse:5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 7)) to sequentially obtain a PCR amplification product 1 and a PCR amplification product 2; then, the following judgment is made: if a baculovirus is obtained that has a band of about 1350bp in PCR amplification product 1 and no band of about 300bp in PCR amplification product 2, the baculovirus is a recombinant baculovirus (see Bac to Bac manual for specific explanation).
3. Dilution of sf9 cells with a viability of greater than 98% and in the logarithmic phase with Grace's Insect Cell Culture Medium, unsupplemented (Invitrogen) gives a sf9 cell density of 5.0X10 5 /ml sf9 cell sap.
4. Mixing the solution A and the solution B uniformly, and standing at room temperature for 25min to obtain a mixture.
Solution A consisted of 100 μl Grace's Insect Cell Culture Medium, unsupplemented and 2.5 μg recombinant baculovirus.
Solution B consisted of 100 μl Grace's Insect Cell Culture Medium, unsupplmented and 8 μl cellfectin II (Invitrogen).
5. Taking a plate (35 mm in specification), paving 2ml of sf9 cell sap obtained in the step 3, and performing wall-attached culture at 28 ℃ for 4 hours; the mixture from step 4 was then added dropwise to a plate, incubated at 28℃for 4 hours with adherence, the medium was discarded, and then fresh 2ml of SFM-900 II medium (product of ThermoFisher Co., ltd., catalog number 10902088) was added and incubated at 28℃for 4 days. At this point the transfection plates were observed with a fluorescence microscope.
The results are shown in FIG. 2, which shows green fluorescence after transfection of sf9 cells with recombinant baculovirus. The results indicate that the fusion protein COLD6-sfGFP-TS is expressed after transfection of sf9 cells by recombinant baculoviruses.
6. After completion of step 5, the supernatant in the dish was collected and stored at 4℃in the dark as virus liquid P1.
7. After completion of step 6, 200 μl of virus liquid P1 was inoculated into a solution containing 180ml of sf9 cell liquid (sf 9 cell density 2.0X10 6 Per ml) of a flask (specification: 2L), 28℃at 150rpm for 96 hours; after that, centrifugation was carried out at 4℃and 2000g for 5min, and the supernatant was collected and stored at 4℃in the dark.
3. Expression of fusion protein COLD6-sfGFP-TS by infection of insect cells with virus liquid P2
1. 10ml of virus liquid P2 was inoculated into a cell suspension containing 500ml of sf9 cells (the sf9 cell density was 2.0X10) 6 A flask (2L) was incubated at 28℃and 150rpm for 72 hours. At this time, the cells were observed with a fluorescence microscope.
The results showed that all cells showed green fluorescence, and that the fusion protein COLD6-sfGFP-TS was expressed after infection of insect cells with viral fluid P2.
2. Cells were collected by centrifugation at 2000rpm at 4℃for 5 minutes.
4. Expression of fusion protein COLD6-sfGFP-TS by mammalian cells infected with virus liquid P2
1. Adding 30% PEG6000 (solvent is water) into virus liquid P2, mixing uniformly, and standing overnight at 4deg.C (for precipitating virus); centrifuging at 3500rpm for 30min at 4deg.C, and collecting precipitate; finally, the pellet was resuspended in Freestyle293 medium (product of thermo fisher company, catalog number 12338018) (the amount of Freestyle293 medium used was 0.1 times the volume of virus liquid P2) to obtain virus liquid P2C.
2. After the step 1 is completed, taking virus liquid P2C, diluting 10000 times by using SFM-900 II culture medium to obtain diluent 1. Dilution 1 was diluted 10-fold with SFM-900 II medium to give dilution 2. Dilution 2 was diluted 10-fold with SFM-900 II medium to give dilution 3.
3. After step 2 was completed, 24 well plates were taken and 2.2X10 were added to each well 5 Individual sf9 cells and 450-550 μl of SFM-900 II culture medium, and performing stationary culture at 28 ℃ for 2 hours; afterwards, 10 μl of diluent (diluent 1, diluent 2 or diluent 3) was added to each well, and the culture was allowed to stand in a wet and dark state at 28℃with 2-3 wells inoculated continuously per virus gradient.
Cells exhibiting green fluorescence (i.e., expressing EGFP) were counted and their concentration per well (pfu/cm) was calculated at 24-36 hours of culture 2 ). At a certain dilution, the concentration of fluorescent cells in each well is 5-15pfu/cm 2 (i.e., the number of fluorescent cells per well is between 10-30), then the dilution factor is the optimal dilution factor for subsequent calculations. The concentrations of EGFP-expressing cells in each well of the optimal dilution were counted and averaged, after which the titer of virus liquid P2C was calculated according to the following formula:
titer of virus liquid P2C (pfu/ml) =average concentration of fluorescent cells in dilution of optimal dilution (pfu/cm) 2 ) X optimal dilution x culture vessel surface area (cm) with viral dilutions 2 ）/（10µl×10 -3 ）
4. After completion of step 3, 500ml of the cells were packed at a density of 2.0X10 6 To/ml of the Expi293F cell fluid (product of ThermoFisher company, catalog number A14527) was added virus liquid P2C (infectious dose MOI=10), and the mixture was cultured at 37℃for 24 hours at 150 rpm.
5. After completion of step 4, an aqueous sodium butyrate solution was added at a concentration of 100mM and the sodium butyrate was allowed to incubate in the system at 37℃for 24 hours at 150 rpm.
The Expi293F cells were observed with a fluorescence microscope. The results are shown in the left panel of FIG. 3.
6. After completion of step 5, the culture was continued at 37℃and 150rpm for 24 hours.
The Expi293F cells were observed with a fluorescence microscope. The results are shown in the right panel of FIG. 3.
The results showed that all of the virus liquid P2C showed green fluorescence after infection of the Expi293F cells. Namely, the fusion protein COLD6-sfGFP-TS is expressed after infection of the Expi293F cells with virus liquid P2C.
7. After completion of step 6, cells were collected by centrifugation at 2000g for 5min at 4 ℃.
5. Purification of COLD6 protein
1. Taking the cells collected in the third step (obtained by insect cell expression), re-suspending in a lysis buffer containing a protease inhibitor (product of Thermo company, catalog number A32965) in a ratio of 1:10 (w/v), and then homogenizing the cells with a Dounce homogenizer for 40 times in an ice bath; then transferred to a beaker, and subjected to ultrasonic disruption (power 400 w; ultrasonic 3s, gap 5s, total 5 min) on ice to obtain a cell disruption solution.
Lysis buffer: tris-HCl buffer at pH8.0, 50mM containing 100mM NaCl, 10% glycerol and 1mM DTT.
2. After the step 1 is completed, taking the cell disruption solution, centrifuging at 4 ℃ and 35000rpm for 1h, and discarding the supernatant; about 10ml of lysis buffer was then added and homogenized with a Dounce homogenizer in an ice bath to give a membrane homogenate.
3. After completion of step 2, a lysis buffer and 10% LMNG (product of Anatrace company, catalog number NG 310) were added to the membrane homogenate to give a system with a total volume of 26 ml; wherein the concentration of LMNG in the system is 1%. Then put into a rotor and stirred with a magnetic stirrer for 2 hours at 4 ℃.
4. After completion of step 3, the mixture was centrifuged at 35000rpm at 4℃for 40min, and the supernatant was collected.
5. After completion of step 4, the supernatant was passed through Strep self-loading column (Strep affinity medium available from IBA company under the product catalog No. 2-1201-010), followed by washing off nonspecifically bound heteroproteins with washing buffer (Tris-HCl buffer at ph8.0 containing 100mM NaCl and 0.01% LMNG, 50 mM), and then eluting with elution buffer (Tris-HCl buffer at ph8.0 containing 100mM NaCl, 0.004% LMNG and 50mM biotin) to obtain a fusion protein COLD6-sfGFP-TS solution.
6. After the step 5 is completed, taking a fusion protein COLD6-sfGFP-TS solution, and performing post digestion (cutting sfGFP-TS protein) by using recombinant HRV 3C protease (product of manufacturing company, product catalog number is C510303) according to the mass ratio of 1:200; then purifying with molecular sieve Superdex200 (product of Cytiva company, catalog number 17-5175-01) (Tris-HCl buffer solution of pH8.0 containing 100mM NaCl and 0.075% Digitonin, 50 mM) to obtain COLD6 protein solution; the peak was then concentrated with a 50kDa concentration tube (product catalog number UFC 805008) and finally subjected to SDS-PAGE.
The purification profile of Superdex200 and SDS-PAGE results are shown in FIG. 4 (left panel shows the purification profile of Superdex200, right panel shows the SDS-PAGE results). The result shows that the COLD6 protein obtained by insect cell expression shows a more uniform protein peak on the molecular sieve, and the protein purity is higher.
According to the above procedure, the cells collected in step three (obtained by insect cell expression) were replaced with the cells collected in step four (obtained by mammalian cell expression), and the other steps were unchanged. The purification profile of Superdex200 and SDS-PAGE results are shown in FIG. 5 (left panel shows the purification profile of Superdex200, right panel shows the SDS-PAGE results). The results show that the COLD6 protein obtained by mammalian cell expression also shows more uniform protein peaks on the molecular sieve, and the protein purity is also higher.
The results show that the cell expressing the target protein can be obtained by infecting insect cells or mammalian cells with the virus liquid prepared by the vector SmartBM-1; after the completion of the cell collection work, the purification method of the COLD6 protein may be exactly the same. From this, it was found that the vector SmartBM-1 constructed in example 1 can express the target protein in both insect cells and mammalian cells. Compared with mammalian cell expression, the experimental cost of expressing the target protein by using insect cells is lower.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims (9)

1. The vector SmartBM-1 comprises a fusion promoter, a target protein expression region, an element for increasing the expression of a protein in a mammalian cell, a termination sequence, an element for recombining with baculovirus and a resistance gene expression cassette;
the fusion promoter efficiently promotes protein expression in insect cells and mammalian cells;
the target protein expression region comprises the following elements from the 5' end: a multiple cloning site, an enzyme cutting site of HRV-3C-protease and a fluorescent protein gene; the multiple cloning site is provided with more than one enzyme digestion recognition sequence;
the fusion promoter sequentially comprises a humanized CMV promoter, a white spot syndrome virus IE1 promoter and a p10 promoter from the 5' end;
the nucleotide sequence of the humanized CMV promoter is shown in SEQ ID NO:1 from the 5' end at positions 2005-2892;
the nucleotide sequence of the white spot syndrome virus IE1 promoter is shown in SEQ ID NO:1 from the 5' end at positions 2899-3055;
the nucleotide sequence of the p10 promoter is shown in SEQ ID NO:1 from the 5' end at positions 3062-3175.
2. The vector SmartBM-1 of claim 1, wherein: the element that increases the expression of a protein in a mammalian cell is a WPRE element.
3. The vector SmartBM-1 of claim 1, wherein: the elements recombined with baculovirus are Tn7L element and Tn7R element.
4. The vector SmartBM-1 of claim 1, wherein: the resistance gene expression cassette is a gentamicin resistance gene expression cassette.
5. The vector SmartBM-1 of claim 1, wherein: the nucleotide sequence of the vector SmartBM-1 is shown in SEQ ID NO: 1.
6. Use of the vector SmartBM-1 of any one of claims 1 to 5 as a shuttle vector for insect cell-mammalian cell expression.
7. Use of the vector SmartBM-1 of any one of claims 1 to 5, A1) or A2):
a1 Expression of proteins in insect cell expression systems and/or mammalian cell expression systems;
a2 Detection of baculovirus titers for non-disease diagnosis and treatment purposes.
8. A method of expressing a protein of interest, comprising the steps of:
(1) Inserting a target gene into a multiple cloning site of the vector SmartBM-1 of any one of claims 1 to 5, and then transforming baculovirus to obtain recombinant baculovirus;
(2) Infecting insect cells or mammalian cells with the virus solution of the recombinant baculovirus obtained in the step (1) to obtain recombinant cells capable of expressing fusion proteins; the fusion protein comprises target protein and fluorescent protein coded by target genes;
(3) Crushing the recombinant cells expressing the fusion protein obtained in the step (2), and collecting the fusion protein; then, the target protein is separated by enzyme digestion with HRV-3C-proteinase.
9. A method for detecting the titer of a baculovirus to be tested for non-disease diagnosis and treatment purposes, comprising the steps of:
(a1) Transforming the vector SmartBM-1 of any one of claims 1 to 5 or a recombinant vector with genes inserted into the multiple cloning sites of the vector SmartBM-1 of any one of claims 1 to 5 into a baculovirus to be tested to obtain a recombinant baculovirus to be tested; then preparing virus liquid of the recombinant baculovirus to be detected;
(a2) Diluting the virus liquid of the recombinant baculovirus to be tested prepared in the step (a 1) to obtain virus diluents with different dilution factors;
(a3) Taking a culture container, adding insect cells, and culturing by adherence; then respectively infecting the virus dilutions with different dilution factors prepared in the step (a 2), and culturing the virus dilutions for 24 to 36 hours at a temperature of between 25 and 30 ℃ in a dark place; 3 or more replicates per dilution;
(a4) After completion of step (a 3), the number of cells (pfu) showing fluorescence per dilution of the virus dilution was counted and the concentration thereof in the culture vessel (pfu/cm) was calculated 2 ) If the concentration of fluorescent cells in more than 3 replicates of a viral dilution of a dilution factor is 5-15pfu/cm 2 The dilution ratio is the optimal dilution ratio; counting the concentration of fluorescent cells in more than 3 repeats of the virus diluent with the optimal dilution and averaging, namely, the average concentration of the fluorescent cells in the virus diluent with the optimal dilution; then, the titer of virus liquid of the recombinant baculovirus to be tested is calculated according to the following formula:
titer of virus solution of recombinant baculovirus to be tested (pfu/ml) = (average concentration of virus dilution fluorescent cells at optimal dilution factor (pfu/cm) 2 ) X optimal dilution x culture vessel surface area (cm) with viral dilutions 2 ) Viral dilution volume (ml) at optimal dilution of infected adherent cultured insect cells in step (a 3);
the titer of the virus liquid of the recombinant baculovirus to be tested is the titer of the baculovirus to be tested.
CN202311601025.2A 2023-11-28 2023-11-28 Insect cell-mammal cell expression shuttle vector SmartBM-1 and application thereof Active CN117305365B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311601025.2A CN117305365B (en) 2023-11-28 2023-11-28 Insect cell-mammal cell expression shuttle vector SmartBM-1 and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311601025.2A CN117305365B (en) 2023-11-28 2023-11-28 Insect cell-mammal cell expression shuttle vector SmartBM-1 and application thereof

Publications (2)

Publication Number Publication Date
CN117305365A CN117305365A (en) 2023-12-29
CN117305365B true CN117305365B (en) 2024-03-19

Family

ID=89255611

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311601025.2A Active CN117305365B (en) 2023-11-28 2023-11-28 Insect cell-mammal cell expression shuttle vector SmartBM-1 and application thereof

Country Status (1)

Country Link
CN (1) CN117305365B (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007091624A1 (en) * 2006-02-09 2007-08-16 Educational Foundation Jichi Medical University Novel viral vector
KR20120128730A (en) * 2011-05-17 2012-11-28 충북대학교 산학협력단 Method for Enhancing the Expression of Foreign Recombinant Protein Using Fusion Expression with Partial Polyhedrin of Baculovirus
CN107304432A (en) * 2016-04-20 2017-10-31 中国科学院生物物理研究所 It is a kind of while expressing the method and its dedicated system of n albumen or protein protomer
CN107988259A (en) * 2018-01-12 2018-05-04 中国科学院生物物理研究所 SmartBac baculovirus expression systems and its application
CN114072510A (en) * 2019-06-27 2022-02-18 X-化学有限公司 Recombinant transfer vectors for protein expression in insect and mammalian cells
CN114773487A (en) * 2022-05-31 2022-07-22 湖南大学 Influenza virus and novel coronavirus fusion recombinant protein vaccine immunogen and preparation method thereof
CN116034160A (en) * 2020-06-12 2023-04-28 旗舰先锋创新V股份有限公司 Baculovirus expression system

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007091624A1 (en) * 2006-02-09 2007-08-16 Educational Foundation Jichi Medical University Novel viral vector
KR20120128730A (en) * 2011-05-17 2012-11-28 충북대학교 산학협력단 Method for Enhancing the Expression of Foreign Recombinant Protein Using Fusion Expression with Partial Polyhedrin of Baculovirus
CN107304432A (en) * 2016-04-20 2017-10-31 中国科学院生物物理研究所 It is a kind of while expressing the method and its dedicated system of n albumen or protein protomer
CN107988259A (en) * 2018-01-12 2018-05-04 中国科学院生物物理研究所 SmartBac baculovirus expression systems and its application
CN114072510A (en) * 2019-06-27 2022-02-18 X-化学有限公司 Recombinant transfer vectors for protein expression in insect and mammalian cells
CN116034160A (en) * 2020-06-12 2023-04-28 旗舰先锋创新V股份有限公司 Baculovirus expression system
CN114773487A (en) * 2022-05-31 2022-07-22 湖南大学 Influenza virus and novel coronavirus fusion recombinant protein vaccine immunogen and preparation method thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
A 96-well format for a high-throughput baculovirus generation, fast titering and recombinant protein production in insect and mammalian cells;Hanna-Riikka Kärkkäinen等;BMC Research Notes;第2卷(第63期);第1页左栏第1段、第3页左栏第2段 *
BacMam System for High-Level Expression of Recombinant Soluble and Membrane Glycoproteins for Structural Studies;Abhiram Dukkipati等;Protein Expr Purif.;第62卷(第2期);第7页第2段、图1 *
High yield and efficient expression and purification of the human 5-HT3A receptor;Zhong-shan Wu等;Acta Pharmacol Sin.;第36卷(第8期);第1024-1032页 *
含对虾白斑综合症病毒ie1启动子的重组杆状病毒介导的哺乳动物细胞基因转移;高辉;中国优秀硕士学位论文全文数据库 基础科学辑(第2008/02期);摘要 *
杆状病毒介导外源基因在哺乳动物细胞中表达的研究进展;田飞鹏等;微生物学通报;第36卷(第12期);第1876-1881页 *

Also Published As

Publication number Publication date
CN117305365A (en) 2023-12-29

Similar Documents

Publication Publication Date Title
US6225060B1 (en) Baculovirus expression system and method for high throughput expression of genetic material
Kitts et al. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors
Costes et al. Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi
CN104788554B (en) Cat omega interferon mutant and its preparation method and application
EP0155476A1 (en) Production of polypeptides in insect cells
KR101563583B1 (en) Method for Enhancing the Expression of Foreign Recombinant Protein Using Fusion Expression with Partial Polyhedrin of Baculovirus
CN101245350A (en) Encoding nucleotide sequence of codons optimizing rotavirus protein, recombinant and uses thereof
CN108018264A (en) A kind of structure and expression using multi-copy gene coexpression baculoviral foreign protein
CN113637705B (en) Monkey type 1 adenovirus (SAdV-1) vector system and application thereof
CN104178514B (en) A kind of method of foreign protein content inside raising AcMNPV polyhedral bodies
Zheng et al. Construction of a highly efficient display system for baculovirus and its application on multigene co-display
CN117305365B (en) Insect cell-mammal cell expression shuttle vector SmartBM-1 and application thereof
CN105296538A (en) In-fusion cloning method for recombinant baculovirus
CN102286534B (en) Insect bioreactor capable of expressing multiple exogenous genes, and construction method and application thereof
CN103436557A (en) Visual tracking method for rapid expression of NS1 protein by bombyx mori bioreactor
WO2019136826A1 (en) Smartbac baculovirus expression system and use thereof
MXPA04008753A (en) Engineered baculoviruses and their use.
CN116042720A (en) Transfer carrier
Zhu Update on production of recombinant therapeutic protein: transient gene expression
US11655482B2 (en) Recombinant transition vector for increasing foreign protein expression
Sridhar et al. Baculovirus vector-mediated expression of heterologous genes in insect cells
CN101429524A (en) Viral vectors for improving carrier transduction and gene expression efficiency, and uses thereof
CN103667348B (en) A kind of double-tagging recombinant Bombyx mori baculovirus and its preparation method and application
CN112359064B (en) Application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells
CN108192923B (en) Production method of polyhedra capable of reducing internal virus particles and embedding foreign proteins

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant