CN103911371A - Saccharomyces cerevisiae integrated expression vector - Google Patents
Saccharomyces cerevisiae integrated expression vector Download PDFInfo
- Publication number
- CN103911371A CN103911371A CN201310003066.1A CN201310003066A CN103911371A CN 103911371 A CN103911371 A CN 103911371A CN 201310003066 A CN201310003066 A CN 201310003066A CN 103911371 A CN103911371 A CN 103911371A
- Authority
- CN
- China
- Prior art keywords
- saccharomyces cerevisiae
- yeast saccharomyces
- sequence
- yeast
- homology arm
- 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.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a saccharomyces cerevisiae integrated expression vector, and provides a double-stranded DNA fragment, which sequentially comprises elements such as a saccharomyces cerevisiae chromosome upstream homology arm, an eukaryotic promoter, a saccharomyces cerevisiae IRES sequence, an eukaryotic screening gene, an eukaryotic transcription termination sequence and a saccharomyces cerevisiae chromosome downstream homology arm from upstream to downstream. The invention further relates to plasmid containing the double-stranded DNA fragment, wherein the plasmid is a saccharomyces cerevisiae integrated expression vector, can be provided for making exogenous or endogenous genes express in saccharomyces cerevisiae without induction, can adopt saccharomyces cerevisiae to produce the target protein, or can be used for metabolic pathway construction of genetic engineering strains. The saccharomyces cerevisiae integrated expression vector provides a good tool for protein expression and metabolic pathway construction of saccharomyces cerevisiae.
Description
Technical field
The present invention relates to the integrated expression vector of a kind of yeast saccharomyces cerevisiae.
Background technology
Yeast saccharomyces cerevisiae (Saccharomycescerevisiae), claims again bread yeast or budding yeast.Yeast saccharomyces cerevisiae is a primary yeast the closest with mankind's relation.Yeast saccharomyces cerevisiae is traditional microorganism of making the food such as bread and steamed bun and wine brewing, is the microorganism of tool biological safety up to now.Meanwhile, in modern molecule and cytobiology, yeast saccharomyces cerevisiae is used as eucaryon model animals, and its effect is equivalent to the model animals intestinal bacteria of protokaryon.Yeast saccharomyces cerevisiae is the most frequently used biological species in fermentation.
The cell of yeast saccharomyces cerevisiae is spherical or avette, diameter 5 – 10 μ m.The method of its breeding is gemmation.
Yeast saccharomyces cerevisiae is conventional microorganism in modern industry biotechnology, health products trade and medical bio technology.Because yeast saccharomyces cerevisiae has good leavening property, as good robustness, tolerate the characteristics such as living contaminants in cell proliferation, production process fast, in industrial biotechnology, be often used as the biology that sets out of metabolic engineering approach transformation, be used to the production of bulk chemical and fine chemistry industry through the improved saccharomyces cerevisiae engineered yeast of various metabolic pathway approach, as alcohol fuel, ergosterol etc.In addition, because yeast saccharomyces cerevisiae belongs to eukaryote and has high biological safety, be also widely used in medical bio technical field and health products trade, as the production of Hepatitis B virus vaccine, the exploitation of functional health yeast etc.Saccharomyces cerevisiae expression is the indispensable instrument of above-mentioned application and research, and the construction work of all kinds expression vector is more and more subject to people's attention.
The expression vector of yeast saccharomyces cerevisiae is generally plasmid-type expression vector.Conventional plasmid-type expression vector belongs to Escherichia coli-Saccharomyces cerevisiae shuttle vectors, when operation, colibacillary replication origin and protokaryon selection markers (as ampicillin resistance gene) can be brought in brewing yeast cell, be had recessive Biosafety problem.If plasmid-type expression vector is proceeded to yeast saccharomyces cerevisiae by package technique in use body, although can remove procaryotic DNA original paper, increase the complexity of operation.In addition, the problem that exists plasmid easily to lose with plasmid-type expression vector establishment engineering bacteria, causes the unstable of engineering bacteria production performance.
Summary of the invention
The object of this invention is to provide the integrated expression vector of a kind of yeast saccharomyces cerevisiae.
The invention provides a kind of double chain DNA fragment (eucaryon element group), comprise successively following element from upstream to downstream: IRES sequence, eucaryon screening-gene, eukaryotic transcription terminator sequence and the yeast saccharomyces cerevisiae karyomit(e) downstream homology arm of yeast saccharomyces cerevisiae karyomit(e) upstream homology arm, eukaryotic promoter, yeast saccharomyces cerevisiae.
The IRES sequence of described yeast saccharomyces cerevisiae is in yeast saccharomyces cerevisiae, to have internal ribosome entry site and have the DNA sequence dna that starts interpretative function.The IRES sequence of described yeast saccharomyces cerevisiae specifically can be if the sequence of sequence table 1 be from as shown in the 2312nd to 2659 Nucleotide of 5 ' end.
Described eukaryotic promoter can be the promotor of yeast, specifically can be the promotor of yeast saccharomyces cerevisiae.The promotor of described yeast saccharomyces cerevisiae specifically can be ILV5 gene promoter, the TDH3 gene promoter of yeast saccharomyces cerevisiae or the ADH2 gene promoter of yeast saccharomyces cerevisiae of yeast saccharomyces cerevisiae.The ILV5 gene promoter of described yeast saccharomyces cerevisiae specifically can be if the sequence of sequence table 1 be from as shown in the 1014th to 1567 Nucleotide of 5 ' end.The TDH3 gene promoter of described yeast saccharomyces cerevisiae specifically can be as shown in the sequence of sequence table 2.The ADH2 gene promoter of described yeast saccharomyces cerevisiae specifically can be as shown in the sequence of sequence table 3.
Described eukaryotic transcription terminator sequence can be the transcription termination sequence of yeast, specifically can be the transcription termination sequence of yeast saccharomyces cerevisiae.The transcription termination sequence of described yeast saccharomyces cerevisiae can be the URA3 genetic transcription terminator sequence of yeast saccharomyces cerevisiae.The URA3 genetic transcription terminator sequence of described yeast saccharomyces cerevisiae specifically can be if the sequence of sequence table 1 be from as shown in the 3477th to 3551 Nucleotide of 5 ' end.
Described yeast saccharomyces cerevisiae karyomit(e) upstream homology arm, can be the upstream homology arm of the 18SrDNA on yeast saccharomyces cerevisiae karyomit(e), specifically can be if the sequence of sequence table 1 is from as shown in the 675th to 1007 Nucleotide of 5 ' end.Described yeast saccharomyces cerevisiae karyomit(e) downstream homology arm, can be the downstream homology arm of the 18SrDNA on yeast saccharomyces cerevisiae karyomit(e), specifically can be if the sequence of sequence table 1 is from as shown in the 3558th to 3921 Nucleotide of 5 ' end.
Described eucaryon screening-gene can be auxotroph selectable marker gene or microbiotic selectable marker gene, specifically can be the URA3 gene of yeast saccharomyces cerevisiae.The URA3 gene of described yeast saccharomyces cerevisiae specifically can be if the sequence of sequence table 1 be from as shown in the 2673rd to 3476 Nucleotide of 5 ' end.The URA3 gene of described yeast saccharomyces cerevisiae specifically can be if the sequence of sequence table 1 be from as shown in the 2673rd to 3473 Nucleotide of 5 ' end.
The present invention also protects a kind of plasmid (the integrated expression vector of yeast saccharomyces cerevisiae) with described eucaryon element group.
The integrated expression vector of described yeast saccharomyces cerevisiae also comprises protokaryon element group; Described protokaryon element group comprises following assembly successively to downstream from upstream: protokaryon replication orgin and protokaryon screening-gene.
Described protokaryon replication orgin specifically can be the replication origin of pMB1.
Described protokaryon screening-gene specifically can be ampicillin resistance gene.
The integrated expression vector of described yeast saccharomyces cerevisiae specifically can be the plasmid that the plasmid removing sequence 1 shown in the sequence of sequence table 1 is obtained after DNA fragmentation shown in the 1580th to 2296 Nucleotide of 5 ' end.
Described eucaryon element group or the integrated expression vector of described yeast saccharomyces cerevisiae can be used for preparing albumen.
When applying described eucaryon element group and preparing albumen, the encoding gene of target protein is inserted between the eukaryotic promoter and the IRES sequence of yeast saccharomyces cerevisiae of eucaryon element group, obtain fusion dna molecule, then fusion dna molecule is imported to yeast strain, by the genomic dna generation homologous recombination of described fusion dna molecule and yeast strain, the IRES sequence of the encoding gene of eukaryotic promoter, target protein, yeast saccharomyces cerevisiae, eucaryon screening-gene and eukaryotic transcription terminator sequence are integrated in the genomic dna of yeast strain, can obtain target protein by cultivating bacterial strain.Described eukaryotic promoter is used for the expression of the encoding gene that starts described target protein.Described eucaryon screening-gene can provide the selective pressure of screening recombinant bacterium, to avoid the following limitation in existing method: selective pressure is low, cannot improve the copy number of object fragment on host chromosome, makes goal gene cannot obtain satisfied expression amount.
Described yeast strain can be yeast saccharomyces cerevisiae, specifically can be yeast saccharomyces cerevisiae w303 bacterial strain.
Described target protein can be mCherry albumen.The encoding gene of described mCherry albumen specifically can be if the sequence of sequence table 1 be from as shown in the 1580th to 2296 Nucleotide of 5 ' end.
The present invention also protects a kind of method of preparing albumen; that the encoding gene of target protein is inserted between the described eukaryotic promoter of described eucaryon element group and the IRES sequence of described yeast saccharomyces cerevisiae; obtain fusion dna molecule; described fusion dna molecule is imported to yeast strain and cultivates described yeast strain, obtain described target protein.
Described yeast strain can be yeast saccharomyces cerevisiae, specifically can be yeast saccharomyces cerevisiae w303 bacterial strain.
Described target protein can be mCherry albumen.The encoding gene of described mCherry albumen specifically can be if the sequence of sequence table 1 be from as shown in the 1580th to 2296 Nucleotide of 5 ' end.
The present invention also protects a kind of method of preparing albumen, that the encoding gene of target protein is inserted between the described eukaryotic promoter of the integrated expression vector of described yeast saccharomyces cerevisiae and the IRES sequence of described yeast saccharomyces cerevisiae, obtain recombinant plasmid, (mode that can cut described recombinant plasmid by enzyme obtains fusion dna molecule from recombinant plasmid to obtain fusion dna molecule in described recombinant plasmid, also can be using described recombinant plasmid as template, obtain fusion dna molecule by the mode of pcr amplification), described fusion dna molecule is imported to yeast strain and cultivates described yeast strain, obtain described target protein, described fusion dna molecule is that the encoding gene of described target protein is inserted to the DNA molecular obtaining between the described eukaryotic promoter of described eucaryon element group and the IRES sequence of described yeast saccharomyces cerevisiae.
Described yeast strain can be yeast saccharomyces cerevisiae, specifically can be yeast saccharomyces cerevisiae w303 bacterial strain.
Described target protein can be mCherry albumen.The encoding gene of described mCherry albumen specifically can be if the sequence of sequence table 1 be from as shown in the 1580th to 2296 Nucleotide of 5 ' end.
When applying method of the present invention and preparing albumen, the DNA fragmentation that proceeds to yeast saccharomyces cerevisiae is the chromosomal DNA fragmentation of yeast saccharomyces cerevisiae self except goal gene, and other DNA fragmentations are cut in the time of carrier linearizing, containing any procaryotic DNA element.Saccharomyces cerevisiae expression of the present invention can make external source or native gene express in yeast saccharomyces cerevisiae, and without induction, utilizes yeast saccharomyces cerevisiae to produce target protein, or builds for the pathways metabolism of engineering strain.The good instrument that provides is provided for the protein expression that saccharomyces cerevisiae expression of the present invention is yeast saccharomyces cerevisiae and pathways metabolism.
Accompanying drawing explanation
Fig. 1 is the structural representation of recombinant plasmid pEXILV5-mCherry.
Fig. 2 is the result that characterizes mCherry expression level in embodiment 2 by fluorescence intensity.
Fig. 3 is the SDS-PAGE collection of illustrative plates in embodiment 2.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
The preparation method of plasmid pSK-mCherry is as follows: by the sequence of the sequence table of synthetic 1, from the DNA molecular shown in the 1568th to 2296 Nucleotide of 5 ' end, (wherein the 1568th to 1573 Nucleotide are ClaI restriction endonuclease recognition sequence " ATCGAT ", the 1574th to 1579 Nucleotide are NdeI restriction endonuclease recognition sequence " CATATG ", the 1580th to 2296 Nucleotide are the encoding gene of mCherry albumen, 2297-2302 position Nucleotide is EcoRI restriction endonuclease recognition sequence " GAATTC ") insert between the ClaI and EcoRI restriction enzyme site of pBlueScriptIISK (-) carrier, obtain plasmid pSK-mCherry.
PBlueScriptIISK (-) carrier: purchased from Agilent Technologies Genomics, Catalog No.212206.
Yeast saccharomyces cerevisiae w303 bacterial strain (bacterial strain attribute: MATa; Ura3-52; Trp1 Δ 2; Leu2-3,112; Hi s3-11; Ade2-1; Can1-100): purchased from EUROSCARF, numbering 20000A.
The preparation of embodiment 1, saccharomyces cerevisiae expression
One, the structure of recombinant plasmid pEXILV5-mCherry
1, (Latin is called Saccharomyces cerevisiae to extract yeast saccharomyces cerevisiae; Common micro-organisms culture presevation administrative center of China, strain number is 2.1882) genomic dna.
2, the genomic dna extracting take step 1 is template, with ILV5P-S(underscore mark SalI recognition sequence) and ILV5P-AS(underscore mark ClaI recognition sequence) primer pair (target sequence is 554bp) that forms carries out pcr amplification, obtains the pcr amplification product of the ILV5 gene promoter with yeast saccharomyces cerevisiae.
ILV5P-S：5’-ACGC
GTCGACCCCTATCTGTTCTTCCGCTCTACC-3’；
ILV5P-AS：5’-CC
ATCGATGTTTTATTTTTTACTTATATTGCTGGTAGG-3’。
3, with the pcr amplification product of restriction enzyme SalI and ClaI double digestion step 2, reclaim enzyme and cut product.
4,, with restriction enzyme SalI and ClaI double digestion plasmid pSK-mCherry, reclaim the carrier framework of about 3997bp.
5, the carrier framework of the enzyme of step 3 being cut to product and step 4 is connected, and obtains recombinant plasmid pILV5mCherry.
6, the genomic dna extracting take step 1 is template, with 18sUP-S(underscore mark XhoI recognition sequence) and 18sUP-AS(underscore mark SalI recognition sequence) primer pair (target sequence is 333bp) that forms carries out pcr amplification, obtains the pcr amplification product of the upstream homology arm of the 18SrDNA with yeast saccharomyces cerevisiae.
18sUP-S：5’-CCG
CTCGAGAAGGACTCAAGGTTAGCCAGAAG-3’；
18sUP-AS：5’-ACGC
GTCGACAGGGAGCCTGAGAAACGGCTAC-3’。
7, with the pcr amplification product of restriction enzyme XhoI and SalI double digestion step 6, reclaim enzyme and cut product.
8, the recombinant plasmid obtaining by restriction enzyme XhoI and SalI double digestion step 5, reclaims the carrier framework of about 4551bp.
9, the carrier framework of the enzyme of step 7 being cut to product and step 8 is connected, and obtains recombinant plasmid pRI5mCherry.
10, the genomic dna extracting take step 1 is template, with TIF4631-ires-S(underscore mark EcoRI, SmaI recognition sequence) and TIF4631-ires-AS(underscore mark BamHI recognition sequence) primer pair (target sequence is 348bp) that forms carries out pcr amplification, obtains the pcr amplification product of the IRES sequence (TIF4631) with yeast saccharomyces cerevisiae.
TIF4631-ires-S：5’-G
GAATTCTCC
CCCGGGGACTCGATAACGACGTGAGAAAC-3’；
TIF4631-ires-AS：5’-CG
GGATCCTATTGTAATAGGTAATTACAGTTGTCCTCTTAC-3’。
11, with the pcr amplification product of restriction enzyme EcoRI and BamHI double digestion step 10, reclaim enzyme and cut product.
12, the recombinant plasmid obtaining by restriction enzyme EcoRI and BamHI double digestion step 9, reclaims the carrier framework of about 4884bp.
13, the carrier framework of the enzyme of step 11 being cut to product and step 12 is connected, and obtains recombinant plasmid pRI5mIRES.
14, the genomic dna extracting take step 1 is template, with Ura3-s(underscore mark BamHI, SpeI recognition sequence) and Ura3-AS(underscore mark XbaI recognition sequence) primer pair (target sequence is 879bp) that forms carries out pcr amplification, obtains having the URA3 gene (eucaryon selection markers gene) of yeast saccharomyces cerevisiae and the pcr amplification product of URA3 genetic transcription terminator sequence.
Ura3-s：5’-CG
GGATCCG
ACTAGTATGTCGAAAGCTACATATAAGGAACGTGCT-3’；
Ura3-AS：5’-GC
TCTAGATAATAACTGATATAATTAAATTGAAGCTCTAAT-3’。
15, with the pcr amplification product of restriction enzyme BamHI and XbaI double digestion step 14, reclaim enzyme and cut product.
16, the recombinant plasmid obtaining by restriction enzyme BamHI and XbaI double digestion step 13, reclaims the carrier framework of about 5232bp.
17, the carrier framework of the enzyme of step 15 being cut to product and step 16 is connected, and obtains recombinant plasmid pRI5mIRURA3.
18, the genomic dna extracting take step 1 is template, with 18sDOWN-S(underscore mark XbaI recognition sequence) and 18sDOWN-AS(underscore mark BstXI recognition sequence) primer pair (target sequence is 364bp) that forms carries out pcr amplification, obtains the pcr amplification product of the downstream homology arm of the 18SrDNA with yeast saccharomyces cerevisiae.
18sDOWN-S：5-GC
TCTAGACGTAAGGTGCCGAGTGGGTC-3
18sDOWN-AS：5-CTGCAGAA
CCACCGCGGTGGGTGGCTCTTGGCGAACCAGGAC-3
19, with the pcr amplification product of restriction enzyme XbaI and BstXI double digestion step 18, reclaim enzyme and cut product.
20, the recombinant plasmid obtaining by restriction enzyme XbaI and BstXI double digestion step 17, reclaims the carrier framework of about 6111bp.
21, the carrier framework of the enzyme of step 19 being cut to product and step 20 is connected, and obtains recombinant plasmid pEXILV5-mCherry.The structural representation of recombinant plasmid pEXILV5-mCherry is shown in Fig. 1.
The sequencing result of recombinant plasmid pEXILV5-mCherry is as shown in the sequence 1 of sequence table.In the sequence 1 of sequence table, from the upstream homology arm of the 675th to 1007 18SrDNA that Nucleotide is yeast saccharomyces cerevisiae of 5 ' end, the 1014th to 1567 ILV5 gene promoters that Nucleotide is yeast saccharomyces cerevisiae, the 1580th to 2296 Nucleotide are the encoding gene of mCherry albumen, the 2312nd to 2659 IRES sequences that Nucleotide is yeast saccharomyces cerevisiae, the 2673rd to 3476 URA3 genes that Nucleotide is yeast saccharomyces cerevisiae, the 3477th to 3551 URA3 genetic transcription terminator sequences that Nucleotide is yeast saccharomyces cerevisiae, the downstream homology arm of the 3558th to 3921 18SrDNA that Nucleotide is yeast saccharomyces cerevisiae, 4336-5003 position Nucleotide is pMB1 replication orgin, 5154-6011 position Nucleotide is protokaryon selection markers gene (ampicillin resistance gene).
Two, the structure of recombinant plasmid pEXTDH3-mCherry
1, with 1 of step 1.
2, the genomic dna extracting take step 1 is template, with GPDp-S(underscore mark SalI recognition sequence) and GPDp-AS(underscore mark NdeI recognition sequence) primer pair (target sequence is 698bp) that forms carries out pcr amplification, obtains the pcr amplification product of the TDH3 gene promoter with yeast saccharomyces cerevisiae.
GPDp-S：5’-ACGC
GTCGACATAAAAAACACGCTTTTTCAGTTCG-3’；
GPDp-AS：5’-GAATTCC
CATATGTTTGTTTGTTTATGTGTGTTTATTCG-3’。
3, with the pcr amplification product of restriction enzyme SalI and NdeI double digestion step 2, reclaim enzyme and cut product.
Subsequent step is same step 1 all.
Obtain recombinant plasmid pEXTDH3-mCherry.
The difference of the sequencing result of recombinant plasmid pEXTDH3-mCherry and recombinant plasmid pEXILV5-mCherry is only the sequence of sequence table 1 to replace for the nucleotide sequence shown in the sequence 2 of sequence table from the 1014th to 1573 nucleotide sequences of 5 ' end.
Three, the structure of recombinant plasmid pEXADH2-mCherry
1, with 1 of step 1.
2, the genomic dna extracting take step 1 is template, with ADH2P-S(underscore mark SalI recognition sequence) and ADH2P-AS(underscore mark ClaI recognition sequence) primer pair (target sequence is 521bp) that forms carries out pcr amplification, obtains the pcr amplification product of the ADH2 gene promoter with yeast saccharomyces cerevisiae.
ADH2P-S：5’-ACGC
GTCGACGCCAAGAACTCTAACCAGTCTTATC-3’；
ADH2P-AS：5’-CC
ATCGATTGTGTATTACGATATAGTTAATAGTTGATAG-3’。
Subsequent step is same step 1 all.
Obtain recombinant plasmid pEXADH2-mCherry.
The difference of the sequencing result of recombinant plasmid pEXADH2-mCherry and recombinant plasmid pEXILV5-mCherry is only the sequence of sequence table 1 to replace for the nucleotide sequence shown in the sequence 3 of sequence table from the 1014th to 1567 nucleotide sequences of 5 ' end.
The expression level of embodiment 2, saccharomyces cerevisiae expression of the present invention detects
One, express the structure of the yeast saccharomyces cerevisiae recombinant bacterium of mCherry
1, with restriction enzyme XhoI and BstXI double digestion recombinant plasmid pEXILV5-mCherry, reclaiming enzyme cuts product and (has upstream homology arm, the ILV5 gene promoter of 18SrDNA, the encoding gene of mCherry albumen, IRES sequence, the downstream homology arm of URA3 gene, URA3 genetic transcription terminator sequence and 18SrDNA).Enzyme is cut to product by lithium acetate method (Ito, H., Fukuda, Y., Murata, K., and Kimura, A.1983.Transformation of intact yeast cells treated with alkali cations.J.Bacteriol.53:163 – 168.) transformed saccharomyces cerevisiae w303, obtain recombinant bacterium, by its called after recombinant bacterium I-rfp.
2, with restriction enzyme XhoI and BstXI double digestion recombinant plasmid pEXTDH3-mCherry, reclaiming enzyme cuts product and (has upstream homology arm, the TDH3 gene promoter of 18SrDNA, the encoding gene of mCherry albumen, IRES sequence, the downstream homology arm of URA3 gene, URA3 genetic transcription terminator sequence and 18SrDNA).Enzyme is cut to product by lithium acetate method transformed saccharomyces cerevisiae w303, obtain recombinant bacterium, by its called after recombinant bacterium T-rfp.
3, with restriction enzyme XhoI and BstXI double digestion recombinant plasmid pEXADH2-mCherry, reclaiming enzyme cuts product and (has upstream homology arm, the ADH2 gene promoter of 18SrDNA, the encoding gene of mCherry albumen, IRES sequence, the downstream homology arm of URA3 gene, URA3 genetic transcription terminator sequence and 18SrDNA).Enzyme is cut to product by lithium acetate method transformed saccharomyces cerevisiae w303, obtain recombinant bacterium, by its called after recombinant bacterium A-rfp.
Two, recombinant bacterium is expressed the ability detection of mCherry
YGM substratum: purchased from general Jino company, article No. is YGM003A-19 by 8gYGM() and 20g glucose is water-soluble and water is settled to 1L.
YPD substratum: water-soluble to 10g yeast powder, 20g glucose and 20g peptone and water are settled to 1L.
Each recombinant bacterium prepared by step 1 and yeast saccharomyces cerevisiae w303 carry out respectively following cultivation and the detection of mCherry expression level:
1, picking list bacterium colony is in 3ml YGM substratum, and 30 ℃, 220rpm are cultivated 16hr.
2, get 1ml seed liquor in 30ml liquid fermentation medium (YGM substratum or YPD substratum), 30 ℃, 220rpm are cultivated 16hr.
3, get the fermented liquid that 5ml step 2 obtains, 4 ℃, the centrifugal 2min of 10000rpm, harvested cell.
4, with 5ml sterilized water re-suspended cell, 4 ℃, the centrifugal 2min of 10000rpm, harvested cell blots residual liquid as far as possible.
5, the cell obtaining by aqueous suspension step 4, makes its OD
600nm=1.
The fluorescence intensity of the cell suspending liquid that 6, detecting step 5 obtains, makes blank with sterilized water, and excitation wavelength is 587nM, and wavelength of transmitted light is 610nM.
With emitted luminescence intensity sign mCherry expression level, the ordinate zou that the results are shown in Figure 2(Fig. 2 while adopting YGM substratum as liquid fermentation medium is emitted luminescence intensity denary logarithm).Each recombinant bacterium carries out the emitted luminescence intensity that above step detects and all carries out higher than yeast saccharomyces cerevisiae w303 the emitted luminescence intensity that above step detects.Each recombinant bacterium carries out the level difference of each expression of recombinant plasmid of emitted luminescence intensity Discrepancy Description mCherry that above step detects, has realized the object of this serial carrier differential expression target protein.
7, get the cell suspending liquid that 10ml step 5 obtains, 4 ℃, the centrifugal 2min of 10000rpm, harvested cell; With 1ml sterilized water re-suspended cell, bacteria suspension is gone in 1.5ml centrifuge tube, 4 ℃, the centrifugal 2min of 10000rpm, abandon supernatant liquor and blot residual liquid as far as possible; With the potassium phosphate buffer suspension cell of 200ul50mmol/L pH7.5, add 0.2g pickling glass pearl (Sigma, G8772-10G), vortex vibration smudge cells (condition is vibration 10sec, ice bath 20sec, and the global oscillation time is 10min); After cytoclasis, with 4 ℃, the centrifugal 40min of 13000rpm, collect supernatant liquor (total protein extract); Measure protein concentration by Bradford method, with the potassium phosphate buffer of 50mmol/L pH7.5, total protein extract is diluted to same concentrations and carries out SDS-PAGE detection, applied sample amount is that every hole is containing total protein 10ug.
The results are shown in Figure 3,1,2,4,6 results when adopting YGM substratum as liquid fermentation medium, 9,10,11,12 results when adopting YPD substratum as liquid fermentation medium, 1 and 9 is yeast saccharomyces cerevisiae w303,2 and 10 is recombinant bacterium T-rfp, and 4 and 12 is recombinant bacterium I-rfp, and 6 and 11 is recombinant bacterium A-rfp, 8 for standard molecular weight mark (from upper under be followed successively by: 170,130,95,72,55,43,34,26,17,10kDa), arrow mark object band.
In the total protein of recombinant bacterium T-rfp, recombinant bacterium I-rfp and recombinant bacterium A-rfp, there is obvious mCherry band, and in yeast saccharomyces cerevisiae w303 total protein, there is no the corresponding band of obvious mCherry.
Claims (10)
1. a double chain DNA fragment, comprises following element successively to downstream from upstream: IRES sequence, eucaryon screening-gene, eukaryotic transcription terminator sequence and the yeast saccharomyces cerevisiae karyomit(e) downstream homology arm of yeast saccharomyces cerevisiae karyomit(e) upstream homology arm, eukaryotic promoter, yeast saccharomyces cerevisiae.
2. double chain DNA fragment as claimed in claim 1, it is characterized in that: described yeast saccharomyces cerevisiae karyomit(e) upstream homology arm is the upstream homology arm of the 18SrDNA on yeast saccharomyces cerevisiae karyomit(e), described yeast saccharomyces cerevisiae karyomit(e) downstream homology arm is the downstream homology arm of the 18SrDNA on yeast saccharomyces cerevisiae karyomit(e).
3. double chain DNA fragment as claimed in claim 2, it is characterized in that: described " the upstream homology arm of the 18SrDNA on yeast saccharomyces cerevisiae karyomit(e) ", if the sequence 1 of sequence table is from as shown in the 675th to 1007 Nucleotide of 5 ' end, described " the downstream homology arm of the 18SrDNA on yeast saccharomyces cerevisiae karyomit(e) " is if the sequence 1 of sequence table is from as shown in the 3558th to 3921 Nucleotide of 5 ' end.
4. as the double chain DNA fragment as described in arbitrary in claims 1 to 3, it is characterized in that: the transcription termination sequence that the promotor that described eukaryotic promoter is yeast saccharomyces cerevisiae, described eukaryotic transcription terminator sequence are yeast saccharomyces cerevisiae.
5. double chain DNA fragment as claimed in claim 4, is characterized in that: the ADH2 gene promoter of the ILV5 gene promoter that the promotor of described yeast saccharomyces cerevisiae is yeast saccharomyces cerevisiae, the TDH3 gene promoter of yeast saccharomyces cerevisiae or yeast saccharomyces cerevisiae; The transcription termination sequence of described yeast saccharomyces cerevisiae is the URA3 genetic transcription terminator sequence of yeast saccharomyces cerevisiae.
6. a plasmid, comprises eucaryon element group; Described eucaryon element group is arbitrary described double chain DNA fragment in claim 1 to 5.
7. plasmid as claimed in claim 6, is characterized in that: described plasmid also comprises protokaryon element group; Described protokaryon element group comprises following assembly successively to downstream from upstream: protokaryon replication orgin and protokaryon screening-gene.
In claim 1 to 5 plasmid described in arbitrary described double chain DNA molecule or claim 6 or 7 in the application of preparing in albumen.
9. prepare the method for albumen for one kind, that the encoding gene of target protein is inserted in claim 1 to 5 between the described eukaryotic promoter of arbitrary described double chain DNA molecule and the IRES sequence of described yeast saccharomyces cerevisiae, obtain fusion dna molecule, described in inciting somebody to action, obtain fusion dna molecule and import yeast strain and cultivate described yeast strain, obtain described target protein.
10. prepare the method for albumen for one kind, by between the described eukaryotic promoter of recombinant plasmid described in the encoding gene insertion claim 6 or 7 of target protein and the IRES sequence of described yeast saccharomyces cerevisiae, obtain recombinant plasmid, obtain the fusion dna molecule in described recombinant plasmid, described fusion dna molecule is imported to yeast strain and cultivates described yeast strain, obtain described target protein; Described fusion dna molecule is that the encoding gene of described target protein is inserted to the DNA molecular obtaining between the described eukaryotic promoter of described eucaryon element group and the IRES sequence of described yeast saccharomyces cerevisiae.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310003066.1A CN103911371B (en) | 2013-01-05 | 2013-01-05 | The integrated expression vector of a kind of saccharomyces cerevisiae |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310003066.1A CN103911371B (en) | 2013-01-05 | 2013-01-05 | The integrated expression vector of a kind of saccharomyces cerevisiae |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103911371A true CN103911371A (en) | 2014-07-09 |
| CN103911371B CN103911371B (en) | 2016-05-04 |
Family
ID=51037377
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310003066.1A Expired - Fee Related CN103911371B (en) | 2013-01-05 | 2013-01-05 | The integrated expression vector of a kind of saccharomyces cerevisiae |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103911371B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561080A (en) * | 2015-01-04 | 2015-04-29 | 浙江大学 | Brewing yeast integrated expression vector with recyclable selective marker and construction method thereof |
| CN104630258A (en) * | 2015-01-06 | 2015-05-20 | 江南大学 | Saccharomyces cerevisiae gene expression system, and establishment and application thereof |
| CN107988202A (en) * | 2017-11-27 | 2018-05-04 | 天津大学 | A kind of method for knocking out S. cerevisiae chromosomal |
| CN109423497A (en) * | 2017-08-31 | 2019-03-05 | 康码（上海）生物科技有限公司 | Enhance the RNA element of protein synthesis efficiency |
| CN112410366A (en) * | 2020-12-10 | 2021-02-26 | 科稷达隆生物技术有限公司 | Method for introducing linear plasmid into saccharomyces cerevisiae and prepared saccharomyces cerevisiae |
| CN113736789A (en) * | 2021-09-26 | 2021-12-03 | 江南大学 | Application of N-terminal sequence element in regulation and control of saccharomyces cerevisiae protein expression |
| CN114574517A (en) * | 2022-03-04 | 2022-06-03 | 江南大学 | Plasmid kit for saccharomyces cerevisiae multicopy integration |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040241829A1 (en) * | 2001-08-06 | 2004-12-02 | Rossolini Gian Maria | Vector for site-specific integration of heterologous dna sequences into metilotrophic yeasts |
| CN1871359A (en) * | 2003-10-22 | 2006-11-29 | 凯克研究生院 | Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy |
| WO2008143684A1 (en) * | 2006-10-05 | 2008-11-27 | Fred Hutchinson Cancer Research Center | Methods of generation use of in vivo biotinylated recombinant antibodies |
| CN102234661A (en) * | 2010-04-23 | 2011-11-09 | 北京生物制品研究所 | Preparation method of human papilloma virus virions, exogenous protein expression cassette and expression system |
-
2013
- 2013-01-05 CN CN201310003066.1A patent/CN103911371B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040241829A1 (en) * | 2001-08-06 | 2004-12-02 | Rossolini Gian Maria | Vector for site-specific integration of heterologous dna sequences into metilotrophic yeasts |
| CN1871359A (en) * | 2003-10-22 | 2006-11-29 | 凯克研究生院 | Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy |
| WO2008143684A1 (en) * | 2006-10-05 | 2008-11-27 | Fred Hutchinson Cancer Research Center | Methods of generation use of in vivo biotinylated recombinant antibodies |
| CN102234661A (en) * | 2010-04-23 | 2011-11-09 | 北京生物制品研究所 | Preparation method of human papilloma virus virions, exogenous protein expression cassette and expression system |
Non-Patent Citations (1)
| Title |
|---|
| ERIC ALANI ET AL.: "Saccharomyces cerevisiae MSH2, a Mispaired Base Recognition Protein, Also Recognizes Holliday Junctions in DNA", 《J. MOL. BIOL.》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561080A (en) * | 2015-01-04 | 2015-04-29 | 浙江大学 | Brewing yeast integrated expression vector with recyclable selective marker and construction method thereof |
| CN104630258A (en) * | 2015-01-06 | 2015-05-20 | 江南大学 | Saccharomyces cerevisiae gene expression system, and establishment and application thereof |
| CN104630258B (en) * | 2015-01-06 | 2018-08-14 | 江南大学 | A kind of genes of brewing yeast expression system and its structure and application |
| CN109423497A (en) * | 2017-08-31 | 2019-03-05 | 康码（上海）生物科技有限公司 | Enhance the RNA element of protein synthesis efficiency |
| CN107988202A (en) * | 2017-11-27 | 2018-05-04 | 天津大学 | A kind of method for knocking out S. cerevisiae chromosomal |
| CN107988202B (en) * | 2017-11-27 | 2020-09-04 | 天津大学 | Method for knocking out saccharomyces cerevisiae chromosome |
| CN112410366A (en) * | 2020-12-10 | 2021-02-26 | 科稷达隆生物技术有限公司 | Method for introducing linear plasmid into saccharomyces cerevisiae and prepared saccharomyces cerevisiae |
| CN112410366B (en) * | 2020-12-10 | 2022-03-04 | 科稷达隆(北京)生物技术有限公司 | Method for introducing linear plasmid into saccharomyces cerevisiae and prepared saccharomyces cerevisiae |
| CN113736789A (en) * | 2021-09-26 | 2021-12-03 | 江南大学 | Application of N-terminal sequence element in regulation and control of saccharomyces cerevisiae protein expression |
| CN113736789B (en) * | 2021-09-26 | 2023-08-29 | 江南大学 | Application of N-terminal sequence element in regulation and control of saccharomyces cerevisiae protein expression |
| CN114574517A (en) * | 2022-03-04 | 2022-06-03 | 江南大学 | Plasmid kit for saccharomyces cerevisiae multicopy integration |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103911371B (en) | 2016-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103911371B (en) | The integrated expression vector of a kind of saccharomyces cerevisiae | |
| Guimarães et al. | Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant | |
| Maqueda et al. | Wine yeast molecular typing using a simplified method for simultaneously extracting mtDNA, nuclear DNA and virus dsRNA | |
| CN103517981B (en) | Filamentous fungi having an altered viscosity phenotype | |
| MXPA06014165A (en) | Non-recombinant saccharomyces. | |
| CN107815458A (en) | A kind of genetic engineering bacterium for expressing people source acetaldehyde dehydrogenase gene and its application | |
| Zhang et al. | CRISPR/Cas9-mediated efficient genome editing via protoplast-based transformation in yeast-like fungus Aureobasidium pullulans | |
| WO2009116286A1 (en) | Flocculent yeast and method for production thereof | |
| CN112094934A (en) | Method for absolute quantification of fungi in sample by combining multiple internal standards | |
| CN105838782A (en) | Method for fast and stable separation and identification of Ustilago esculenta sexual compatible haploid strain | |
| Liu et al. | Development of genetic tools in glucoamylase-hyperproducing industrial Aspergillus niger strains | |
| CN101550605A (en) | Method for establishing saccharomyces integrated gene mutation library based on in vivo homologous recombination | |
| Naumova et al. | Molecular genetic characteristics of Saccharomyces cerevisiae distillers’ yeasts | |
| CN105039386A (en) | Method for constructing monascus strain capable of achieving high yield of acid protease | |
| CN111440890A (en) | Characteristic nucleotide sequence, identification primer and identification method of wild ganoderma lucidum W141201 | |
| CN115948265A (en) | Kluyveromyces marxianus haploid yeast and construction method and application thereof | |
| AU2020204574B2 (en) | B.licheniformis engineering bacteria for inhibiting bacterial autolysis, construction method and application thereof | |
| CN112646923A (en) | Primer, kit and method for rapidly identifying and quantifying saccharomyces cerevisiae | |
| CN109929853B (en) | Application of thermophilic bacteria source heat shock protein gene | |
| CN112695120A (en) | Primer, kit and method for rapid identification and quantification of saccharomyces cerevisiae | |
| CN102892884A (en) | Method for producing kluyveromyces marxianus transformant | |
| CN112481265A (en) | Bacillus subtilis artificial terminator and application thereof | |
| CN112940956A (en) | Pichia pastoris for enhancing lactoferrin expression by adopting double promoters and application thereof | |
| Minhas et al. | Development of an efficient transformation system for halotolerant yeast Debaryomyces hansenii CBS767 | |
| CN106148483A (en) | The primer of detection Bacillus coli cells DNA and 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 | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160504 Termination date: 20190105 |