CN106047739B - A kind of recombinant yeast pichia pastoris bacterium that expressing VSV-G and its preparation and application - Google Patents

A kind of recombinant yeast pichia pastoris bacterium that expressing VSV-G and its preparation and application Download PDF

Info

Publication number
CN106047739B
CN106047739B CN201610488661.2A CN201610488661A CN106047739B CN 106047739 B CN106047739 B CN 106047739B CN 201610488661 A CN201610488661 A CN 201610488661A CN 106047739 B CN106047739 B CN 106047739B
Authority
CN
China
Prior art keywords
vsv
recombinant
pichia pastoris
preparation
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
CN201610488661.2A
Other languages
Chinese (zh)
Other versions
CN106047739A (en
Inventor
魏炽炬
王靖荃
董莹
刘鑫
李龙
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.)
Shantou University
Original Assignee
Shantou University
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 Shantou University filed Critical Shantou University
Priority to CN201610488661.2A priority Critical patent/CN106047739B/en
Publication of CN106047739A publication Critical patent/CN106047739A/en
Application granted granted Critical
Publication of CN106047739B publication Critical patent/CN106047739B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/066—Lysis of microorganisms by physical methods
    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2800/102—Plasmid DNA for yeast

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a kind of recombinant yeast pichia pastoris bacterium that can express VSV-G and its preparation and application, recombinant yeast pichia pastoris bacterium has form, heredity and the physiological and biochemical property of Pichia yeast KM71, and can express VSV-G by fermentation.Preparation method includes that (1) uses PCR to react clone's VSV-G genetic fragment from plasmid pLP/VSV-G；(2) preparation and reorganization plasmid pGM-T-VSV-G；(3) preparation and reorganization plasmid pPIC3.5K-VSV-G；(4) recombinant expression carrier converts Pichia pastoris KM71 by electrotransformation, screens positive recombinant using histidine defect plate, Geneticin plate and PCR；(5) taking positive recombinant to carry out culture induction, thalline were collected by centrifugation and is cracked with acidic glass pearl.The expression strategy that the present invention takes is intracellular expression, effectively eliminates pollution of the methanol to cell pyrolysis liquid.Vesicular stomatitis virus-G protein is prepared using pichia yeast expression system, lysate can be directly used for cell transfecting after filtration sterilization.It provides the foundation for being prepared on a large scale for VSV-G, is also of great significance to the research of VSV-G and corresponding disease treatment.

Description

A kind of recombinant yeast pichia pastoris bacterium that expressing VSV-G and its preparation and application
Technical field
The present invention relates to synthetic biology technology, technique for gene engineering, more particularly to a kind of recombination that can express VSV-G are finished Red saccharomycete and its preparation and application.
Background technique
Vesicle stomatitis viral glycoprotein G(calls VSV-G in the following text) be anchoring with vesicle Stomatovirus (calling VSV in the following text) surface, And the tripolymer virus membrane antigen containing C-terminal transmembrane structure.The study found that VSV-G is mediating vesicular stomatitis virus to enter It plays a major role on host cell, and the generality of vesicular stomatitis virus infection species is also related with this albumen.VSV-G compared with Meeting occurred conformation variation in the environment of low ph value, the variation lead to can promote virion into a series of anti-of infected cell It answers, including recipient cell identification and adherency, activated receptor cell endocytic and fusion receptors cell membrane, virus is finally in cell Releasing virus inhereditary material, and then expand.
Due to the diversity and stability of VSV infection species, surface fusion protein VSV-G has been studied and with rehearsing The preparation of virion is transmitted with mediating to intracellular substance.In addition, when VSV-G is integrated on artificial liposome film, meeting The carrier for forming a kind of similar virus can be used for into target cell transporting biological macromolecular such as antibody, DNA and some complicated Organelle, it is experimentally confirmed that the mass transfer efficiency of the carrier is higher.
Currently, the preparation method of VSV-G, which is predominantly directly extracted or prepared from VSV virion, has transfected VSV-G gene Cell conditioned medium.However, the step of some complicated and time consumptions, such as sucrose density gradient centrifugation；And the height of cell culture medium Cost limits the mass production of VSV-G.
Pichia pastoris (P. pastoris) expression system was successfully established in recent years, more mature reliable protein Mass production tool.Currently, many bioactive substances are successful by the system production, such as antibody and enzyme.It is complete due to being transferred to Expression vector in red yeast can be incorporated on Yeast genome in a manner of homologous recombination, so that the system has foreign gene The stability of heredity.In addition, Pichia pastoris also has mature protein modified system, molecule manipulation is simple and easy and low in cost Etc. advantages.Before this, have several viral memebrane protein successful expression in such a system, such as HIV-1 surface protein gp120, HBsAg and Dengue fever virus E protein.
Since vesicular stomatitis virus-G protein is of great significance in basic research and disease treatment, VSV-G can be expressed by constructing one kind Recombinant yeast pichia pastoris engineering bacteria lay a good foundation to the research of VSV-G and large scale preparation.
Summary of the invention
The technical problem to be solved by the embodiment of the invention is that providing a kind of recombinant yeast pichia pastoris that can express VSV-G Bacterium and its preparation and application solve the problems, such as of the existing technology.
In order to achieve the above purpose, it adopts the following technical scheme that:
A kind of recombinant yeast pichia pastoris bacterium for expressing VSV-G, form, heredity and physiology with Pichia yeast KM71 are raw Change feature, and VSV-G can be expressed by fermentation.
Further, the VSV-G is present in the lysate of the Pichia yeast KM71 in a manner of bioactivity.
Contain the gene order that can express vesicular stomatitis virus-G protein, the sequence in the recombinant yeast pichia pastoris bacterium for expressing VSV-G It is as follows:
atgaagtgccttttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttcc acacaaccaaaaaggaaactggaaaaatgttccttctaattaccattattgcccgtcaagctcagatttaaattgg cataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaaggctattcaagcagacggttgga tgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccg atccttcactccatctgtagaacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggc ttccctcctcaaagttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactcctcaccatg tgctggttgatgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccc cactgtccataactctacaacctggcattctgactataaggtcaaagggctatgtgattctaacctcatttccatg gacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacagggttcagaagtaactact ttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgt ctggttcgagatggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctct gctccatctcagacctcagtggatgtaagtctaattcaggacgttgagaggatcttggattattccctctgccaag aaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaacccagg aaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagatacatcagagtcgatattgct gctccaatcctctcaagaatggtcggaatgatcagtggaactaccacagaaagggaactgtgggatgactgggcac catatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgat tggacatggtatgttggactccgatcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagac gctgcttcgcaacttcctgatgatgagagtttattttttggtgatactgggctatccaaaaatccaatcgagcttg tagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggactatt cttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacata gagatgaaccgacttggaaagtaa。
VSV-G gene must encode following amino acid sequences:
Mkcllylaflfigvnckftivfphnqkgnwknvpsnyhycpsssdlnwhndligtalqvkmpkshkai qadgwmchaskwvttcdfrwygpkyithsirsftpsveqckesieqtkqgtwlnpgfppqscgyatvtdaeavivq vtphhvlvdeytgewvdsqfingkcsnyicptvhnsttwhsdykvkglcdsnlismditffsedgelsslgkegtg frsnyfayetggkackmqyckhwgvrlpsgvwfemadkdlfaaarfpecpegssisapsqtsvdvsliqdverild yslcqetwskiraglpispvdlsylapknpgtgpaftiingtlkyfetryirvdiaapilsrmvgmisgttterel wddwapyedveigpngvlrtssgykfplymighgmldsdlhlsskaqvfehphiqdaasqlpddeslffgdtglsk npielvegwfsswkssiasfffiigliiglflvlrvgihlciklkhtkkrqiytdiemnrlgk。
Further, the preparation method of the above-mentioned recombinant yeast pichia pastoris bacterium for expressing VSV-G, mainly comprises the steps that
(1) VSV-G genetic fragment is cloned from plasmid pLP/VSV-G using PCR reaction；
(2) VSV-G genetic fragment obtained by recycling step (1), and the VSV-G genetic fragment and pGM-T carrier are carried out T-A connection is transformed into Escherichia coli Top 10, amplification cultivation, and digestion is detected and is sequenced, and obtains recombinant plasmid pGM-T-VSV-G, To obtain EcoRI restriction enzyme site；
(3) expression vector is connected into VSV-G segment is obtained after pGM-T-VSV-G digestion obtained by step (2) using EcoRI PPIC3.5K is transformed into Escherichia coli Top 10, amplification cultivation, and digestion is detected and is sequenced, and obtains recombinant expression carrier pPIC3.5K-VSV-G；
(4) recombinant expression carrier pPIC3.5K-VSV-G obtained by step (3) is converted into Pichia pastoris by electrotransformation KM71 is screened using histidine defect plate, Geneticin plate and PCR, with positive recombinant；
(5) it takes positive recombinant obtained by step (4) to be inoculated in BMGY fluid nutrient medium culture 18-24 h, then is transferred to BMMY 96 h of culture medium Fiber differentiation, thalline were collected by centrifugation and is cracked with acidic glass pearl, and Western Blot is recycled to detect albumen Expression confirms the expression of vesicular stomatitis virus-G protein.
Further, the primer and reaction condition of the PCR reaction are as follows:
Primer 1(F): 5 '-CCATGGCCTGTTGCTC CACCAGCTT-3 '
Primer 2 (R): 5 '-AAGCTTTCAGCAGTGGCTCACAGCAG-3 '
Reaction condition are as follows: 94 DEG C of 3 min of initial denaturation；94 DEG C of unwindase 13 0 s, 59.5 DEG C of 1 min, 60 DEG C of extension 30 s, 30 A circulation；72 DEG C of 10 min of heat preservation.
Further, 1% methanol is contained in step (5) the BMMY culture medium；Every 24 h when the Fiber differentiation process It is primary to add methanol.
A kind of lysate of the above-mentioned recombinant yeast pichia pastoris bacterium for expressing VSV-G can promote DNA in mammalian cells to turn Dye.
A kind of above-mentioned cracking liquid and preparation method thereof is specifically included that the recombinant yeast pichia pastoris bacterium for expressing VSV-G after induction It is resuspended in PBS(20 mM, pH 7.4 containing 1 mM PMSF) in, after isometric pickling glass pearl is added, by mixture play Strong vortex shakes 1 min, is immediately placed on 1 min on ice；Supernatant is collected by centrifugation in mixture at 4 DEG C after repeatedly, then uses 0.22 μm of membrane filtration.
A kind of application of above-mentioned lysate, in mediated dna transfection and other related fieldss.The lysate, which contains, to be had The vesicular stomatitis virus-G protein of bioactivity can effectively improve the transfection efficiency of DNA.
The detection of Yeast expression protein product: expression product is detected using Western Blot technology, specific steps As follows: yeast lysate is mixed with PAGE Gel Loading Buffer, and 10min is heated in boiling water bath, takes 10-20 μ l mixed Liquid is closed, PAGE gel (10% separation gel, 5% concentration glue) is splined on.After electrophoresis in the way of half-dried turn by protein delivery extremely On pvdf membrane.1 h is incubated for confining liquid at room temperature after film is rinsed；1:3000 rabbit-anti VSV-G primary antibody solution is transferred to after rinsing In, 4 DEG C of overnight incubations；1 h is incubated in the secondary antibody of room temperature and 1:50000 mouse anti-rabbit after rinsing.It is exposed after rinsing with ECL developing solution Colour developing.
Compared with prior art, the method that the present invention prepares VSV-G has operation relative ease, does not need complicated experiment Step, recombinant yeast pichia pastoris bacterium can long-term preservations, Reusability after being successfully prepared；There is low-cost advantage simultaneously, be not required to Want the cell culture medium of complicated component and valuableness.And only need to crack saccharomycete after Fiber differentiation can output have The lysate of VSV-G bioactivity, can be directly used for cell experiment.In DNA transfection, plasmid DNA transfection can be effectively improved The efficiency of zooblast.The expression strategy that the present invention takes is intracellular expression, effectively eliminates dirt of the methanol to cell pyrolysis liquid Dye.The present invention prepares vesicular stomatitis virus-G protein using pichia yeast expression system in a creative way, this is not only being prepared on a large scale for VSV-G It provides the foundation, is also of great significance to the research of VSV-G and corresponding disease treatment.
Detailed description of the invention
Fig. 1 is the preparation method schematic diagram of the recombinant yeast pichia pastoris bacterium for expressing VSV-G of the invention；
The fluorescence photo of Fig. 2 detection that albumen transmits between yeast lysate mediated cell of the invention and functional verification；
Fig. 3 is that the effect after 48 h of Ad293 cell is transfected after yeast lysate of the present invention is incubated for plasmid pGL3-control Fruit statistical chart；
Fig. 4 is the effect after yeast lysate of the present invention is incubated for plasmid pGL3-control after 48 h of transfection HeLa cell Statistical chart；
Fig. 5 is that the fluorescence after 24 h of Ad293 cell is transfected after yeast lysate of the present invention is incubated for plasmid pCMV-DsRed Photo；
Fig. 6 is that the positive after 24 h of Ad293 cell is transfected after yeast lysate of the present invention is incubated for plasmid pCMV-DsRed Cell quantity statistical chart；
Fig. 7 is that the fluorescence after yeast lysate of the present invention is incubated for plasmid pCMV-DsRed after 24 h of transfection HeLa cell shines Piece；
Fig. 8 is that the positive after yeast lysate of the present invention is incubated for plasmid pCMV-DsRed after 24 h of transfection HeLa cell is thin Born of the same parents' quantity statistics figure.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing Step ground detailed description.
Embodiment 1
1, preparation can express the method and steps of the recombinant yeast pichia pastoris bacterium of VSV-G, as shown in Figure 1, mainly including following Step:
(1) VSV-G genetic fragment is cloned from plasmid pLP/VSV-G using PCR reaction, primer and reaction condition are as follows；
Primer 1(F): 5 '-CCATGGCCTGTTGCTC CACCAGCTT-3 '
Primer 2 (R): 5 '-AAGCTTTCAGCAGTGGCTCACAGCAG-3 '
Reaction condition are as follows: 94 DEG C of 3 min of initial denaturation；94 DEG C of unwindase 13 0 s, 59.5 DEG C of 1 min, 60 DEG C of extension 30 s, 30 A circulation；72 DEG C of 10 min of heat preservation
(2) DNA fragmentation is recycled, and segment and pGM-T carrier are subjected to T-A connection, Escherichia coli Top 10 is transformed into, expands Increase culture, digestion is detected and is sequenced, and obtains recombinant plasmid pGM-T-VSV-G；
(3) it is connected into expression vector pPIC3.5K to VSV-G segment is obtained after pGM-T-VSV-G digestion using EcoRI, turned Escherichia coli Top 10, amplification cultivation are dissolved into, digestion is detected and is sequenced, and obtains recombinant plasmid pPIC3.5K-VSV-G；
(4) recombinant expression carrier converts Pichia pastoris KM71 by electrotransformation, mould using histidine defect plate, heredity Plain plate and PCR screen positive recombinant；
(5) it takes positive recombinant to be inoculated in BMGY fluid nutrient medium culture 18-24 h, then is transferred to BMMY(containing 1% methanol) It is primary that 96 h(of culture medium Fiber differentiation every 24 h adds methanol).Thalline were collected by centrifugation and is cracked with acidic glass pearl, utilizes Western Blot detects protein expression situation.
2, the preparation of yeast lysate, the recombinant yeast pichia pastoris for expressing VSV-G after mainly comprising the steps that induction Bacterium is resuspended in the PBS (20 mM, pH 7.4) that 1 ml contains 1 mM PMSF, will after isometric pickling glass pearl is added Mixture, which is acutely vortexed, shakes 1 min, is immediately placed on 1 min on ice.The step is repeated 10 times.Mixture at 4 DEG C with Supernatant is collected by centrifugation in 13000g.It is yeast lysate after 0.22 μm of membrane filtration of the supernatant.
3, Yeast expression protein product detects:
Expression product is detected using Western Blot technology, the specific steps are as follows:
Yeast lysate is mixed with PAGE Gel Loading Buffer, and 10 min are heated in boiling water bath, take 10-20 μ l mixed liquor is splined on PAGE gel (10% separation gel, 5% concentration glue).Albumen is turned in the way of half-dried turn after electrophoresis It moves on pvdf membrane.1 h is incubated for confining liquid at room temperature after film is rinsed；1:3000 rabbit-anti VSV-G primary antibody is transferred to after rinsing In solution, 4 DEG C of overnight incubations；1 h is incubated in the secondary antibody of room temperature and 1:50000 mouse anti-rabbit after rinsing.ECL developing solution is used after rinsing Exposure colour developing.
Embodiment 2
The detection and functional verification that albumen transmits between yeast lysate mediated cell.
Ad293 cell is transfected respectively with pCAG-CreER and pCMV-DsRed/loxP2-EGFP.24 h after transfection, will be thin Born of the same parents' digestion, and the ratio with pCAG-CreER:pCMV-DsRed/loxP2-EGFP for 2:1 is laid in 96 orifice plates.It is complete to cell After adherent, the yeast lysate containing VSV-G that above-described embodiment 1 is obtained is added in the middle of Ad293 with culture solution, 3 h After 80% lysate is sucked out and be added isometric sodium citrate buffer solution (pH5.4) be incubated at room temperature 1 min, be sucked out buffer, be added The 4- hydroxyl tamoxifen (4-HT) of fresh cell medium and 10 mM are placed on 37 DEG C of 24 h of culture, under fluorescence microscope Observation.CreER fusion protein can enter nucleus from cytoplasm, and DNA recombination is carried out between the site loxP, make recipient cell Born of the same parents switch to green from red.Apparent EGFP positive cell as seen from Figure 2 illustrates that yeast lysate can be delivered successfully CreER fusion protein, and after entering cell, it can successfully discharge red protein matter, it was demonstrated that VSV-G cell membrane particles can have Effect transferrin matter simultaneously plays biological function.
Embodiment 3
Yeast lysate promotes plasmid DNA transfection.
It requires to incubate with pGL3-control plasmid with 2000 transfection reagent by specification of PolyJet and Lipofectamine It educates, transfects Ad293 and HeLa cell respectively.Cell is with 1 × 104The density in every hole is laid in advance in 96 orifice plates.Transfection reagent with After cell is added in DNA compound, yeast lysate is added, liquid is changed after 37 DEG C of 12 h of culture, fluorescein is carried out when 48 h Enzyme detection.From Fig. 3 and Fig. 4 this it appears that being incubated for using recombination yeast bacterial lysate, when transfection time is 24 h, Ad293 Cell and HeLa cell relative light intensity unit (white column) have maximum value, about the 3 of blank control at 5 μ l additional amounts Times.Correspondingly, when being incubated for (black column) with wild type KM71 lysate, then without apparent effect.
It requires to incubate with pCMV-DsRed plasmid with 2000 transfection reagent by specification of PolyJet and Lipofectamine It educates, transfects Ad293 and HeLa cell respectively.Cell is with 1 × 104The density in every hole is laid in advance in 96 orifice plates.Transfection reagent with After cell is added in DNA compound, yeast lysate is added, changes liquid after 37 DEG C of 12 h of culture, a part of cell when 48 h Luminescent cell proportion is counted with flow cytometer after fluorescence microscopy microscopic observation, another part cell dissociation.From Fig. 5 with Fig. 7 can be seen that with after VSV-G incubation, and Ad293 and HeLa cell red quantity dramatically increases；It can be seen that from Fig. 6 and Fig. 8 The ratio that ratio increase about 10%, the HeLa red cell quantity that Ad293 red quantity accounts for total number of cells accounts for total number of cells is promoted about 1 times.It is above-mentioned the experimental results showed that, the recombinant yeast pichia pastoris lysate for expressing VSV-G can effectively improve DNA in mammalian cells Transfection efficiency.
Above disclosed is only presently preferred embodiments of the present invention, cannot limit the right of the present invention with this certainly Range, therefore equivalent changes made in accordance with the claims of the present invention, are still within the scope of the present invention.
<110>University Of Shantou
<120>a kind of recombinant yeast pichia pastoris bacterium that can express VSV-G and its preparation and application
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213>artificial sequence
<400> 1
ccatggcctg ttgctccacc agctt 25
<210> 2
<211> 26
<212> DNA
<213>artificial sequence
<400> 2
aagctttcag cagtggctca cagcag 26
<210> 3
<211> 1536
<212> DNA
<213>artificial sequence
<400> 3
atgaagtgcc ttttgtactt agccttttta ttcattgggg tgaattgcaa gttcaccata 60
gtttttccac acaaccaaaa aggaaactgg aaaaatgttc cttctaatta ccattattgc 120
ccgtcaagct cagatttaaa ttggcataat gacttaatag gcacagcctt acaagtcaaa 180
atgcccaaga gtcacaaggc tattcaagca gacggttgga tgtgtcatgc ttccaaatgg 240
gtcactactt gtgatttccg ctggtatgga ccgaagtata taacacattc catccgatcc 300
ttcactccat ctgtagaaca atgcaaggaa agcattgaac aaacgaaaca aggaacttgg 360
ctgaatccag gcttccctcc tcaaagttgt ggatatgcaa ctgtgacgga tgccgaagca 420
gtgattgtcc aggtgactcc tcaccatgtg ctggttgatg aatacacagg agaatgggtt 480
gattcacagt tcatcaacgg aaaatgcagc aattacatat gccccactgt ccataactct 540
acaacctggc attctgacta taaggtcaaa gggctatgtg attctaacct catttccatg 600
gacatcacct tcttctcaga ggacggagag ctatcatccc tgggaaagga gggcacaggg 660
ttcagaagta actactttgc ttatgaaact ggaggcaagg cctgcaaaat gcaatactgc 720
aagcattggg gagtcagact cccatcaggt gtctggttcg agatggctga taaggatctc 780
tttgctgcag ccagattccc tgaatgccca gaagggtcaa gtatctctgc tccatctcag 840
acctcagtgg atgtaagtct aattcaggac gttgagagga tcttggatta ttccctctgc 900
caagaaacct ggagcaaaat cagagcgggt cttccaatct ctccagtgga tctcagctat 960
cttgctccta aaaacccagg aaccggtcct gctttcacca taatcaatgg taccctaaaa 1020
tactttgaga ccagatacat cagagtcgat attgctgctc caatcctctc aagaatggtc 1080
ggaatgatca gtggaactac cacagaaagg gaactgtggg atgactgggc accatatgaa 1140
gacgtggaaa ttggacccaa tggagttctg aggaccagtt caggatataa gtttccttta 1200
tacatgattg gacatggtat gttggactcc gatcttcatc ttagctcaaa ggctcaggtg 1260
ttcgaacatc ctcacattca agacgctgct tcgcaacttc ctgatgatga gagtttattt 1320
tttggtgata ctgggctatc caaaaatcca atcgagcttg tagaaggttg gttcagtagt 1380
tggaaaagct ctattgcctc ttttttcttt atcatagggt taatcattgg actattcttg 1440
gttctccgag ttggtatcca tctttgcatt aaattaaagc acaccaagaa aagacagatt 1500
tatacagaca tagagatgaa ccgacttgga aagtaa 1536

Claims (5)

1. the lysate that one kind can express the recombinant yeast pichia pastoris bacterium of VSV-G, which is characterized in that mammalian cell can be promoted DNA transfection, wherein the preparation that can express the recombinant yeast pichia pastoris bacterium of VSV-G mainly comprises the steps that
(1) VSV-G genetic fragment is cloned from plasmid pLP/VSV-G using PCR reaction；
(2) VSV-G genetic fragment obtained by recycling step (1), and the VSV-G genetic fragment and pGM-T carrier are subjected to T-A company It connects, is transformed into Escherichia coli Top10, amplification cultivation, digestion is detected and is sequenced, and recombinant plasmid pGM-T-VSV-G is obtained, to obtain EcoRI restriction enzyme site；
(3) expression vector is connected into VSV-G segment is obtained after pGM-T-VSV-G digestion obtained by step (2) using EcoRI PPIC3.5K is transformed into Escherichia coli Top10, amplification cultivation, and digestion is detected and is sequenced, and obtains recombinant expression carrier pPIC3.5K-VSV-G；
(4) recombinant expression carrier pPIC3.5K-VSV-G obtained by step (3) is converted into Pichia pastoris KM71, benefit by electrotransformation It is screened with histidine defect plate, Geneticin plate and PCR, with positive recombinant；
(5) it takes positive recombinant obtained by step (4) to be inoculated in BMGY fluid nutrient medium culture 18-24h, then is transferred to BMMY culture medium Fiber differentiation 96h, thalline were collected by centrifugation and is cracked with acidic glass pearl, and Western Blot is recycled to detect protein expression feelings Condition confirms the expression of vesicular stomatitis virus-G protein；
The recombinant yeast pichia pastoris bacterium for expressing VSV-G after induction is resuspended in the PBS containing 1mM PMSF, is added isometric After pickling glass pearl, mixture is acutely vortexed concussion 1min, is immediately placed on 1min on ice；Mixture is at 4 DEG C after repeatedly Under be collected by centrifugation supernatant, then with 0.22 μm of membrane filtration；1min is incubated at room temperature with isometric sodium citrate buffer solution of pH5.4 again It saves and uses afterwards.
2. lysate according to claim 1, which is characterized in that the primer and reaction condition of the PCR reaction are as follows:
Primer 1 (F): 5 '-CCATGGCCTGTTGCTC CACCAGCTT-3 '
Primer 2 (R): 5 '-AAGCTTTCAGCAGTGGCTCACAGCAG-3 '
Reaction condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of unwindase 13s 0s, 59.5 DEG C of 1min, 60 DEG C of extension 30s, 30 recycle；72 DEG C heat preservation 10min.
3. lysate according to claim 1, which is characterized in that contain 1% methanol in step (5) the BMMY culture medium； It is primary every adding methanol for 24 hours when the Fiber differentiation process.
4. the preparation method of lysate according to claim 1, which is characterized in that specifically include that expressing after induction The recombinant yeast pichia pastoris bacterium of VSV-G is resuspended in the PBS containing 1mM PMSF, after isometric pickling glass pearl is added, will mix Object, which is acutely vortexed, shakes 1min, is immediately placed on 1min on ice；Supernatant is collected by centrifugation in mixture at 4 DEG C after repeatedly, then uses 0.22 μm of membrane filtration.
5. the diagnosing and treating application of the non-disease of lysate according to claim 1, which is characterized in that be used for mediated dna Transfection.
CN201610488661.2A 2016-06-29 2016-06-29 A kind of recombinant yeast pichia pastoris bacterium that expressing VSV-G and its preparation and application Active CN106047739B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610488661.2A CN106047739B (en) 2016-06-29 2016-06-29 A kind of recombinant yeast pichia pastoris bacterium that expressing VSV-G and its preparation and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610488661.2A CN106047739B (en) 2016-06-29 2016-06-29 A kind of recombinant yeast pichia pastoris bacterium that expressing VSV-G and its preparation and application

Publications (2)

Publication Number Publication Date
CN106047739A CN106047739A (en) 2016-10-26
CN106047739B true CN106047739B (en) 2019-06-18

Family

ID=57166986

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610488661.2A Active CN106047739B (en) 2016-06-29 2016-06-29 A kind of recombinant yeast pichia pastoris bacterium that expressing VSV-G and its preparation and application

Country Status (1)

Country Link
CN (1) CN106047739B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434638A (en) * 2016-11-11 2017-02-22 河南省农业科学院小麦研究所 Method of extracting bacterial plasmids in large amount
EP3670646A1 (en) * 2018-12-21 2020-06-24 Ohly GmbH Functional yeast protein concentrate
CN113832114A (en) * 2020-06-23 2021-12-24 南京大学 Novel oncolytic adenovirus EM/VSV-G Ad5-P and application thereof in preparation of antitumor drugs
CN113832111A (en) * 2020-06-23 2021-12-24 南京大学 Method for preparing novel oncolytic virus by exosome-like technology

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection;Xin Liu等;《Appl Biochem Biotechnol》;Springer Science+Business Media;20160924;第181卷;748–761
VSV-G Viral Envelope Glycoprotein Prepared from Pichia pastoris;Xin Liu等;《J. Microbiol. Biotechnol》;The Korean Society for Microbiology;20170315;第27卷(第6期);1098-1105
鲤春病毒血症病毒糖蛋白基因在毕赤酵母中的分泌表达;王树云等;《淡水渔业》;20150731;第45卷(第4期);31-35

Also Published As

Publication number Publication date
CN106047739A (en) 2016-10-26

Similar Documents

Publication Publication Date Title
CN106047739B (en) A kind of recombinant yeast pichia pastoris bacterium that expressing VSV-G and its preparation and application
CN107012121A (en) Carry the structure of the stable cell lines of orthogonal tRNA/ aminoacyl tRNA synthetases
CN109182368B (en) Genetic transformation method using aspergillus flavus hyphae as receptor and mediated by agrobacterium tumefaciens
CN105073995A (en) Enhanced transgene expression and processing
US10329594B1 (en) Cell lines for high level production of protein-based pharmaceuticals
CN102250952B (en) The carrier that mediate foreign gene is expressed at mammal cell with high efficient and application thereof
CN104818295A (en) Method for preparing and screening cell line expressing bispecific antibody
CN106399370B (en) It is a kind of to establish the flounder embryo cell strain method for stablizing transgenosis
CN103436557A (en) Visual tracking method for rapid expression of NS1 protein by bombyx mori bioreactor
CN107653266B (en) Application of recombinant expression vector containing lepidoptera virus enhancer in expressing protein in diptera insect cells
CN103352048A (en) Microalgae chloroplast carrier for efficient cloning and expression and application thereof
CN106755093A (en) The technique that drosophila cell is transiently transfected
CN102533722B (en) Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump
CN106222202A (en) A kind of method of high-efficiency transfection turbot muscle cell
JP6099075B2 (en) Method for promoting transport of target mRNA from nucleus to cytoplasm, protein expression method and production method, and kit used therefor
CN105779499A (en) Recombinant baculovirus, application thereof and vector for constructing same
CN108486115A (en) A kind of transfection method and its application for the expression of crayfish Cells Exogenous genetic
CN101372686B (en) Infected protein mediated method for producing recombinant baculovirus particle
CN104711288A (en) Recombinant vector, recombinant baculovirus prepared from the same and application of virus in preparation of malaria vaccines
CN107236732B (en) A kind of saccharomyces neoformans inducible promoter and its application
CN107937400A (en) The small ribonucleic acid molecules of silence NS1 gene expressions and screening technique and application
CN109536517B (en) Silkworm microparticle hypothetical protein NB29, recombinant expression vector and application thereof
CN116411022B (en) Vector and cell for indicating activation degree of porcine somatic clone embryo genome
US20240167006A1 (en) High production of recombinant protein by making cell hybrids and enriching for a preferred mitochondrial phenotype
CN116334070A (en) Application of fusion protein and cell fusion method

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant