CN102827860B - Construction method of double-gene mutant escherichia coli for secretory expression of recombinant protein - Google Patents
Construction method of double-gene mutant escherichia coli for secretory expression of recombinant protein Download PDFInfo
- Publication number
- CN102827860B CN102827860B CN201210286769.5A CN201210286769A CN102827860B CN 102827860 B CN102827860 B CN 102827860B CN 201210286769 A CN201210286769 A CN 201210286769A CN 102827860 B CN102827860 B CN 102827860B
- Authority
- CN
- China
- Prior art keywords
- gene
- protein
- escherichia coli
- dual
- recombinant
- 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.)
- Expired - Fee Related
Links
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims abstract description 60
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 60
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 43
- 238000010276 construction Methods 0.000 title claims abstract description 28
- 230000003248 secreting effect Effects 0.000 title claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 112
- 238000000034 method Methods 0.000 claims abstract description 76
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 56
- 239000012634 fragment Substances 0.000 claims abstract description 48
- 230000008569 process Effects 0.000 claims abstract description 37
- 238000010363 gene targeting Methods 0.000 claims abstract description 23
- 239000003550 marker Substances 0.000 claims abstract description 3
- 230000001580 bacterial effect Effects 0.000 claims description 115
- 239000013612 plasmid Substances 0.000 claims description 73
- 230000008859 change Effects 0.000 claims description 45
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 22
- 230000028327 secretion Effects 0.000 claims description 22
- 238000013461 design Methods 0.000 claims description 11
- 235000011187 glycerol Nutrition 0.000 claims description 11
- 210000001322 periplasm Anatomy 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 229940041514 candida albicans extract Drugs 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- 108010046845 tryptones Proteins 0.000 claims description 9
- 239000012138 yeast extract Substances 0.000 claims description 9
- 230000006798 recombination Effects 0.000 claims description 8
- 238000005215 recombination Methods 0.000 claims description 8
- 230000005611 electricity Effects 0.000 claims description 4
- 108091026890 Coding region Proteins 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 claims description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 230000004260 plant-type cell wall biogenesis Effects 0.000 claims description 2
- 238000000746 purification Methods 0.000 abstract description 13
- 210000002421 cell wall Anatomy 0.000 abstract description 11
- 238000003752 polymerase chain reaction Methods 0.000 abstract description 9
- 206010064571 Gene mutation Diseases 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 210000003000 inclusion body Anatomy 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 6
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 239000002510 pyrogen Substances 0.000 abstract description 4
- 230000007154 intracellular accumulation Effects 0.000 abstract description 2
- 108090000364 Ligases Proteins 0.000 abstract 1
- 102000018697 Membrane Proteins Human genes 0.000 abstract 1
- 108010052285 Membrane Proteins Proteins 0.000 abstract 1
- 210000000349 chromosome Anatomy 0.000 abstract 1
- 230000010039 intracellular degradation Effects 0.000 abstract 1
- 230000009466 transformation Effects 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 88
- 239000007788 liquid Substances 0.000 description 62
- 210000004027 cell Anatomy 0.000 description 45
- 230000006698 induction Effects 0.000 description 36
- 239000002609 medium Substances 0.000 description 35
- 238000000855 fermentation Methods 0.000 description 34
- 230000004151 fermentation Effects 0.000 description 34
- 239000000047 product Substances 0.000 description 29
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 28
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 28
- 230000000968 intestinal effect Effects 0.000 description 26
- 238000012216 screening Methods 0.000 description 24
- 238000012360 testing method Methods 0.000 description 23
- 238000002744 homologous recombination Methods 0.000 description 21
- 230000006801 homologous recombination Effects 0.000 description 21
- 101150077062 pal gene Proteins 0.000 description 21
- 238000012163 sequencing technique Methods 0.000 description 21
- 235000015097 nutrients Nutrition 0.000 description 20
- 238000002360 preparation method Methods 0.000 description 20
- 230000009182 swimming Effects 0.000 description 17
- 238000006467 substitution reaction Methods 0.000 description 14
- 230000004913 activation Effects 0.000 description 13
- 238000003209 gene knockout Methods 0.000 description 12
- 101150074251 lpp gene Proteins 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 101150041450 palH gene Proteins 0.000 description 10
- 101001135770 Homo sapiens Parathyroid hormone Proteins 0.000 description 9
- 101001135995 Homo sapiens Probable peptidyl-tRNA hydrolase Proteins 0.000 description 9
- 102000058004 human PTH Human genes 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 7
- 239000011248 coating agent Substances 0.000 description 7
- 238000000576 coating method Methods 0.000 description 7
- 238000012790 confirmation Methods 0.000 description 7
- 238000005520 cutting process Methods 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 239000012467 final product Substances 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 7
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 7
- 238000004321 preservation Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 210000001185 bone marrow Anatomy 0.000 description 5
- 230000028023 exocytosis Effects 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 210000004698 lymphocyte Anatomy 0.000 description 5
- 101150116543 mrcB gene Proteins 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 230000004936 stimulating effect Effects 0.000 description 5
- 239000005515 coenzyme Substances 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 101150058012 mrcA gene Proteins 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 108010028006 B-Cell Activating Factor Proteins 0.000 description 3
- 230000035508 accumulation Effects 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000008676 import Effects 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 108050001049 Extracellular proteins Proteins 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000002045 lasting effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 101150064202 mrdA gene Proteins 0.000 description 2
- 101150001852 mrdB gene Proteins 0.000 description 2
- 101150034514 murC gene Proteins 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 108020001775 protein parts Proteins 0.000 description 2
- 101150042737 rodA gene Proteins 0.000 description 2
- 102000016605 B-Cell Activating Factor Human genes 0.000 description 1
- 101100203377 Bacillus subtilis (strain 168) slp gene Proteins 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 101100242529 Drosophila melanogaster Pal2 gene Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101100082540 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) pcp gene Proteins 0.000 description 1
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 101150012565 LRIT1 gene Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 101100477497 Mus musculus Shcbp1 gene Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 102100036893 Parathyroid hormone Human genes 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 101710195090 Protein kil Proteins 0.000 description 1
- XOCUXOWLYLLJLV-UHFFFAOYSA-N [O].[S] Chemical compound [O].[S] XOCUXOWLYLLJLV-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940116978 human epidermal growth factor Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000003161 proteinsynthetic effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a construction method of double-gene mutation escherichia coli for recombinant protein secretory expression, which comprises the steps of carrying out PCR (polymerase chain reaction) by using pre-designed primers of double knockout genes of cell membrane protein and cell wall synthetase genes to obtain gene targeting fragments of the double knockout genes, wherein the two ends of the gene targeting fragments are a specific target gene homologous sequence of 10-250 bp and a resistance marker sequence in the middle, and integrating the gene targeting fragments into a host escherichia coli chromosome through electric transformation to obtain a double-gene mutation secretory escherichia coli strain. The invention improves the expression of recombinant foreign protein and reduces the intracellular degradation of target protein; intracellular accumulation of recombinant foreign protein is reduced, and formation of inclusion bodies is eliminated; the cell wall breaking process is cancelled, the pyrogen is reduced, and the separation and purification are simplified; thereby achieving the purposes of reducing the production cost and improving the protein expression and the product quality.
Description
Technical field
The present invention relates to transform intestinal bacteria heredity group gene field, is exactly a kind of construction process of the dual-gene mutant Escherichia coli for recombinant protein secretion type expression.
Background technology
In recent years, recombinant protein medicine market development is swift and violent, has become an important new high-tech industry.Wherein important market recombinant protein medicine mainly contains: human parathyroid hormone, bone-marrow-derived lymphocyte stimulating factor, human epidermal growth factor, gamma-interferon, lysozyme element and various antibody etc.In the production of various recombinant proteins, intestinal bacteria remain most popular host system.In escherichia coli host system, the spatial positioning after synthesizing according to recombinant protein, can be divided into intracellular expression and secreting, expressing.The recombinant protein of intestinal bacteria intracellular expression often forms inclusion body, because of abnormal space folding structure non-activity precipitation.Secreting, expressing can be divided into again periplasmic space expresses and extracellular expression, and exocytosis express recombinant protein because its redox environment is suitable for forming normal albumen space structure, do not form inclusion body, under normal circumstances for there being active soluble proteins, and the albumen of secreting, expressing is seldom subject to proteasome degradation.Thereby improve the expression level of activated protein, reduced separation costs.Therefore, extracellular expression, for improving recombinant protein output and purity, is simplified downstream process significant.
The natural exocytosis mechanism of the normal shortage of intestinal bacteria, most of foreign proteins cannot be secreted in substratum.The outer leakage expression of born of the same parents refers to and adopts reason, change and genetic method to make host cell wall or epicyte that disappearance or damage occur, thereby target protein is leaked in the outer substratum of born of the same parents.The method that the outer leakage expression of born of the same parents adopts can be divided into: chemical leakage, apply fatsolvent and other chemical reagent control cell seepage; Physics seepage, applies the physical methods such as freeze thawing and makes cell seepage; Biological seepage, applies the biological reagents such as microbiotic and makes the biosynthesis block of cytolemma or wall and cause seepage; Heredity seepage, is used hereditary means to change the permeability of cell walls or film, and intracellular protein part is optionally leaked out in substratum.Heredity seepage has multiple advantage, as: optionally secrete target protein, without special seepage technique, without poisonous chemical reagent interpolation etc.Therefore hereditary seepage is often the first-selection of target protein leakage expression.
The method of the intestinal bacteria extracellular production recombinant protein of report is often the outer superior strain of born of the same parents by physics and chemistry mutagenesis screening genetic mutation at present.But the method screening process complexity, mechanism is not clear, poor repeatability.In theory, genetically engineered intestinal bacteria can cause its epicyte or Cell Wall Deficient, and its periplasmic space protein part is discharged in substratum.The production that the seepage bacterial strain of use genetically engineered mutant carries out extracellular protein rarely has report.As: colibacillary
lppmutant can leak out to periplasmic space albumen in substratum and the normal growth of not remarkably influenced bacterial strain, but the seepage ratio of target protein and output thereof still there is ample room for improvement.In addition, coexpression crack protein Kil or BRP(bacteriocin discharge albumen) also can change the permeability of Bacillus coli cells adventitia, thus make periplasmic space protein delivery in substratum.
Summary of the invention
The invention provides the construction process of the dual-gene mutant Escherichia coli for recombinant protein secretion type expression of the recombinant protein extracellular expression that the fermentation of a kind of intestinal bacteria foreign protein and exocytosis carry out simultaneously, the secretor type bacterial strain that the dual-gene permeability knocking out of the cell walls obtaining by genetically engineered method or film strengthens, and foreign protein synthetic in born of the same parents is imported to cell periplasmic space by signal peptide or fusion rotein, and then optionally leak out in substratum, improving protein expression level and reducing separation costs becomes object of the present invention.
For a construction process for the dual-gene mutant Escherichia coli of recombinant protein secretion type expression, it is characterized in that: the epicyte protein and the Cell wall synthesis enzyme gene that use design in advance
palwith
mrcB, or
mrcBwith
lpptwo two primers that knock out gene that knock out gene or other assortments of genes carry out PCR reaction, obtain two ends and be 10～250 bp specific objective DNA homolog sequences and middle concentration for resistance marker sequence is 1～1000 ng/ μ l's
palwith
mrcB, or
mrcBwith
lpptwo two gene targeting fragments that knock out gene that knock out gene or other assortment of genes, and transform and be incorporated in host e. coli karyomit(e) by electricity respectively, obtain the secretory coli bacterial strain of dual-gene sudden change; Other described assortments of genes refer to
mrcA,
mrdA,
rodA,
murC,
rodZin the combination of any two.
The construction process of described a kind of dual-gene mutant Escherichia coli for recombinant protein secretion type expression, is characterized in that: the gene that the secretory coli bacterial strain of described dual-gene sudden change contains
paland gene
mrcBthere is sudden change in coding region, or gene
paland gene
mrcBpromoter region there is sudden change, thereby make
palgene and gene
mrcBbe beyond expression out corresponding Pal and MrcB protein product, or give expression to the Pal and the MrcB protein product that have reduced function, and then normal cytolemma transportation function changes.
The construction process of described a kind of dual-gene mutant Escherichia coli for recombinant protein secretion type expression, is characterized in that:
The application of described dual-gene mutant Escherichia coli comprises: the secretor type recombinant escherichia coli strain of exogenous protein expression builds, the target protein of secretor type recombination bacillus coli is expressed process;
The secretor type recombinant escherichia coli strain construction process of described exogenous protein expression is: external source target protein plasmagene is first recombinated in secretor type plasmid pET32a (+), be cloned into subsequently in the secretory coli bacterial strain of described dual-gene sudden change, and then obtain the secretor type recombinant escherichia coli strain of exogenous protein expression.
The construction process of described a kind of dual-gene mutant Escherichia coli for recombinant protein secretion type expression, it is characterized in that: the recombination plasmid that the secretor type recombinant escherichia coli strain of described exogenous protein expression contains encoding exogenous albumen, in this recombinant plasmid, foreign protein genes is connected with the signal coding sequence that is positioned periplasmic space, thereby makes synthetic foreign protein in born of the same parents be directed in periplasmic space and then be leaked in substratum by signal peptide.
The construction process of described a kind of dual-gene mutant Escherichia coli for recombinant protein secretion type expression, is characterized in that: the target protein expression process of described secretor type recombination bacillus coli refers to and in fermention medium, uses the secretor type recombinant escherichia coli strain of exogenous protein expression to produce foreign protein.
The construction process of described a kind of dual-gene mutant Escherichia coli for recombinant protein secretion type expression, it is characterized in that: in described fermention medium, contain: 0.1～2% Tryptones, 0.2～10% yeast extract powder, 0.05～5% glycerine, 0.02～0.5% KH
2pO
4, 0.1～2% K
2hPO
4.
Principle of the present invention is:
The construction process of the said a kind of dual-gene mutant Escherichia coli for recombinant protein secretion type expression of the present invention, mainly comprises: the intestinal bacteria that build the recombinant protein extracellular expression of specific cytolemma and cell walls change produce bacterial strain and in fermention medium, use this production bacterial strain to produce foreign protein.It is characterized in that what use designed in advance
palgene and
mrcBgene knockout primer carries out PCR reaction, and obtaining two ends is 10～250 bp specific objective DNA homolog sequences and the middle high density (10～1000 ng/ml) for resistance screening flag sequence
palgene and
mrcBgene targeting fragment.And by its successively electricity transform import to through induction containing the treating in mutant Escherichia coli starting strain of pKD46, thereby realize
palwith
mrcBdual-gene sudden change.The secretor type recombination bacillus coli that wherein said cytolemma and cell walls change is produced bacterial strain and is referred to:
palwith
mrcB,or
mrcBwith
lpptwo knock out gene and other assortment of genes (
mrcA,
mrdA,
rodA,
murC,
rodZdeng) nucleotide deletion or the replacement of coding region; Or the nucleotide deletion of promoter region or the mutant strain of replacement of gene.Preferably
palwith
mrcBdual-gene sudden change.
Beneficial effect:
1, two secretor type bacterial strains that knock out that the present invention builds provide a kind of new intestinal bacteria seepage fermentation recombinant protein approach, and seepage ratio and the output of target protein have obtained further raising.
2, the present invention has realized recombinant protein fermentation and exocytosis is carried out simultaneously.During the fermentation, target recombinant albumen can be from the lasting growth of seepage in born of the same parents and not remarkably influenced thalline, has reduced the interior degraded of born of the same parents of target protein, improves expression of recombinant proteins; Reduce the interior excessively accumulation of born of the same parents of recombinant protein simultaneously, and reduced inclusion body formation; Eliminate cell wall breaking technique, reduced pyrogen contamination; Design substratum composition, is convenient to separation and purification, and then reaches and reduced production cost, the object of improving the quality of products.Seepage has not only been simplified the technique of purification of recombinant proteins, has also improved the expression level of target product.After testing, exceed 50% recombinant protein and leak in substratum, expression level is 10～30%.
3, the present invention adopts colibacillary
palwith
mrcBbe host and locate foreign protein in the carrier of periplasmic space etc. dual-gene mutant strain, the expression of foreign protein and the outer accumulation of born of the same parents are carried out simultaneously.Thereby improve the expression of recombinant exogenous protein, reduce the interior degraded of born of the same parents of target protein; Reduce the intracellular accumulation of recombinant exogenous protein, eliminated the formation of inclusion body; Cancel cell wall breaking technique, reduced pyrogen, simplified separation and purification; And then reach and reduce production costs, improve the object of protein expression and quality product.After testing, exceed 50% external source recombinant protein and can leak in substratum, expression level is 10～30%.
Accompanying drawing explanation
Fig. 1 is that dual-gene knockout mutant strain target protein is expressed SDS-PAGE result (target protein: also human parathyroid hormone fusion rotein of restructuring sulphur oxygen; Swimming lane 1-7 is extracellular protein, and swimming lane 8-14 is intracellular protein, and M is standard protein).Swimming lane 1 is
mrcAwith
paldual-gene mutant strain; Swimming lane 2 is
mrcBwith
paldual-gene mutant strain (embodiment 1); Swimming lane 3 is
lppwith
paldual-gene mutant strain; Swimming lane 4 is
mrcAwith
lppdual-gene mutant strain; Swimming lane 5 is
lpp and mrcBdual-gene mutant strain (embodiment 2); The positive contrast of swimming lane 6; The negative contrast of swimming lane 7; Swimming lane 8 is
mrcAwith
paldual-gene mutant strain; Swimming lane 9 is
mrcBwith
paldual-gene mutant strain (embodiment 1); Swimming lane 10 is
lppwith
paldual-gene mutant strain; Swimming lane 11 is
mrcAwith
lppdual-gene mutant strain; Swimming lane 12 is
lpp and mrcBdual-gene mutant strain (embodiment 2); The positive contrast of swimming lane 13; The negative contrast of swimming lane 14.(the dual-gene sudden change combination of other swimming lanes is contained among claim 1)
Embodiment
The intestinal bacteria construction process of the said recombinant protein secretion type expression of the present invention, comprises the following steps:
The secretory coli strain construction of A, dual-gene sudden change, build
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change:
A, structure intestinal bacteria
palthe secretor type bacterial strain of transgenation
(1) use the specific of design in advance
palgene knockout primer, take resistant maker gene plasmid pKD3 as template, carries out the synthetic specific PCR product in polymerase chain reaction, is called
palgene targeting fragment.Target practice fragment two ends are 10～250 bp's (preferably 30～100 bp)
paldNA homolog sequence, centre is chlorampenicol resistant flag sequence.Preferably 100～500 ng/ μ are l) to be used PCR product purification test kit to obtain high density 10～1000 ng/ μ l(
palgene targeting fragment;
(2) utilize preferably 5～20 mmol/L of 1～100 mmol/L() L-arabinose induction containing intestinal bacteria starting strain 0.2～10 h(that knocks out helper plasmid pKD46 preferably 1～3 h), then by the bacterium solution preparation Electroporation-competent cells after induction;
(3) by the high density after purifying
palgene targeting fragment electricity transforms among the intestinal bacteria starting strain Electroporation-competent cells after the induction that imports gained, SOC substratum at the L-arabinose containing 0.5～50 mmol/L (preferably 2～25 mmol/L) recovers 0.2～20 h, use the screening of resistance LB solid medium that the mutant strain of homologous recombination occurs, and use PCR method and sequencing to determine
palgene region occurs, after correct Substitution, to obtain final product
paltransgenation bacterial strain; Concrete steps please refer to document (Datsenko and Wanner 2000);
B,
palwith
mrcBthe secretor type strain construction of dual-gene sudden change
(1) with step a gained
palthe secretor type bacterial strain of transgenation is prepared chemoreception state cell, after imported plasmid pCP20, the resistant panel of coating again the Amp that contains 5～500 μ g/ml (preferably 10～200 μ g/ml) filters out the single bacterium colony recon containing plasmid pCP20 under the condition of 20～45 ℃ of temperature (preferably 25～35 ℃) left and right, the LB liquid nutrient medium of transferring is again cultivated 3～48 h(preferably 12～24 h) under 30～50 ℃ of temperature (preferably 35～45 ℃) condition, afterwards gained bacterium liquid is carried out to dilution spread, the single bacterium colony obtaining is transferred respectively and is carried out the test of mark resistance into LB resistant panel, and identify the rejecting that whether has completed resistant maker gene by PCR method and sequencing, unmarked through what confirm
paltransgenation bacterial strain adopts final concentration 4～36%(preferably 8～18 %) glycerine in temperature-5～-80 ℃ of (preferably-10～-40 ℃) preservations,
palwith
mrcBthe starting strain of dual-gene sudden change,
(2) by gained
palwith
mrcBthe dual-gene starting strain knocking out is drawn LB flat board and is activated at 20～45 ℃ (preferably 30～42 ℃), transfer and prepare chemoreception state cell into 20～45 ℃ of LB liquid nutrient mediums (preferably 30～42 ℃), rear importing knocks out helper plasmid pKD46, again the bacterium liquid obtaining is coated to the resistant panel containing the Amp of 5～500 μ g/ml (preferably 10～200 μ g/ml), under 30～50 ℃ of temperature (preferably 35～45 ℃) condition, screening is containing single bacterium colony recon of plasmid pKD46, use enzyme cutting method to identify that whether this recon is containing pKD46 plasmid, bacterial classification containing pKD46 plasmid is containing pKD46's
palthe secretor type bacterial strain of transgenation,
(3) gained is containing pKD46's
paltransgenation bacterial strain is prepared Electroporation-competent cells under the condition of 20～45 ℃ (preferably 30～42 ℃), passed through again preferably 5～20 mmol/L of 1～100 mmol/L() L-arabinose induction 0.2～10 h(preferably 1～3 h), after by high density after purifying
mrcBgene targeting fragment imports wherein, high density after purifying
mrcBthe preparation method of gene targeting fragment is with step a, recover 0.2～20 h at the SOC substratum containing the L-arabinose of 0.5～50 mmol/L (preferably 2～25 mmol/L), use resistant panel, in 30～50 ℃ of temperature (preferably 35～45 ℃) screenings, homologous recombination occurs
mrcBmutant strain, and use PCR method and sequencing to identify
mrcBthere is Substitution in gene region, the mutant bacteria speciogenesis after confirmation twice homologous recombination, be
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change;
The secretor type recombinant escherichia coli strain of B, exogenous protein expression builds (structure of the secretor type recombinant bacterial strain of human parathyroid hormone)
By synthetic human parathyroid hormone hPTH gene segment, after order-checking is correct, be cloned in secretive expression vector pET32a (+), obtain recombinant plasmid pET32a-
pthH, be transformed into
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change obtains secretor type recombinant bacterial strain, produces the secretor type recombinant bacterial strain of human parathyroid hormone.
The target protein of C, secretor type recombination bacillus coli is expressed process
First use the LB solid medium activation this project bacterial strain containing the Amp of 5～500 μ g/ml (preferably 25～100 μ g/ml), 3～72 h(preferably 12～36 choose single colony inoculation after h) in 250 ml shaking flasks of the LB seed culture medium of the Amp containing 5～500 μ g/ml (preferably 10～200 μ g/ml) with 3～300 ml (preferably 10～50 ml), at 20～45 ℃ of temperature (preferably 30～42 ℃), preferably 150～300 rpm of rotating speed 50～500 rpm() condition under cultivate 3～72 h(preferably 12～36 h), again by preferably 0.5～5 ml of 0.1～10 ml() this bacterium liquid be inoculated into 3～300 ml (preferably 10～50 ml) in the 250 ml shaking flasks containing the TB liquid fermentation medium of the Amp of 5～500 μ g/ml (preferably 10～200 μ g/ml), be 0.05～1.8(preferably 0.2～0.8 when cell concentration in substratum reaches at OD600) time to add final concentration be preferably 0.2～0.8 mmol of 0.05～5 mmol() IPTG, induction fermentation 1～24 h(is 2～12 h) rear termination fermentations preferably.Obtain fermented liquid centrifugation under normal temperature, collect supernatant liquor and obtain recombinant protein crude extract.
Wherein said LB seed culture medium is: 0.1～5% Tryptones, 0.05～5% yeast extract powder and 0.05～5%NaCl;
TB liquid fermentation medium is: 0.1～2% Tryptones, 0.2～10% yeast extract powder, 0.05～5% glycerine, 0.02～0.5% KH
2pO
4, 0.1～2% K
2hPO
4;
LB solid medium: for: 0.5～5% agar, 0.1～5% Tryptones, 0.05～5% yeast extract powder and 0.05～5%NaCl.
The present invention adopts
palwith
mrcBthe intestinal bacteria of dual-gene sudden change, as exogenous protein host strain, are positioned the carrier of periplasmic space as target recombinant protein expressing plasmid.The raw materials used commercially available product that is.
The present invention has realized recombinant protein fermentation and exocytosis is carried out simultaneously.During the fermentation, target recombinant albumen can be from the lasting growth of seepage in born of the same parents and not remarkably influenced thalline, has reduced the interior degraded of born of the same parents of target protein, improves expression of recombinant proteins; Reduce the interior excessively accumulation of born of the same parents of recombinant protein simultaneously, and reduced inclusion body formation; Eliminate cell wall breaking technique, reduced pyrogen contamination; Design substratum composition, is convenient to separation and purification, and then reaches and reduced production cost, the object of improving the quality of products.Seepage has not only been simplified the technique of purification of recombinant proteins, has also improved the expression level of target product.After testing, exceed 50% recombinant protein and leak in substratum, expression level is 10～30%.
Below in conjunction with embodiment, the present invention is further elaborated, its objective is the better content of the present invention of understanding.Therefore, the cited case does not limit the scope of the invention.In addition the following material of even employing if no special instructions in listed embodiment,
1) bacterial classification:
Host is
escherichia colijM109 (DE3), knocks out helper plasmid pKD46, resistant maker gene plasmid pKD3 and pKD4, and resistant maker gene is rejected plasmid pCP20.
palgene knockout primer palH+PF and palH+PR,
palgene identification primer palT1 and palT2.
mrcBgene knockout primer mrcBH+PF and mrcBH+PR,
mrcBgene identification primer mrcBT1 and mrcBT2.
2) substratum:
Seed culture medium: 1% Tryptones, 0.5% yeast extract powder and 0.5%NaCl;
Fermention medium: 1.2% Tryptones, 2.4% yeast extract powder, 0.5% glycerine, 0.231%KH
2pO
4, 1.254%K
2hPO
4;
Solid medium: 1.5% agar, 1% Tryptones, 0.5% yeast extract powder and 0.5%NaCl;
SOC substratum: 2% Tryptones, 0.5% yeast extract powder, 0.05%NaCl, 2.5 mM KCl 10 mM MgCl2,20 mM glucose.
Agents useful for same is commercially available product.
An intestinal bacteria construction process for recombinant protein secretion type expression, comprises the following steps:
A,
palwith
mrcBthe structure of the secretor type bacterial strain of dual-gene sudden change comprises the following steps:
A, structure
palthe secretor type bacterial strain of transgenation:
(1) use designs in advance
palgene knockout primer palH+PF and palH+PR, take resistant maker gene plasmid pKD3 as template, carry out the synthetic specific PCR product in polymerase chain reaction, obtains
palgene targeting fragment, the two ends of target practice fragment are 50bp
paldNA homolog sequence, centre is chlorampenicol resistant flag sequence, uses PCR product purification test kit to obtain the high density target practice fragment after the purifying of 20ng/ μ l the PCR product of gained;
(2) utilize the L-arabinose induction of 10mmol/L to contain to knock out set out bacterial classification JM109 (DE3) 2 hours of the intestinal bacteria of helper plasmid pKD46, then by the bacterium solution preparation Electroporation-competent cells after the induction of gained;
(3) by the high density after step (1) gained purifying
palafter among the Electroporation-competent cells of the bacterium liquid after the induction of gene targeting fragment importing step (2) gained, recover after 2 hours at the SOC substratum of the L-arabinose containing 5 mmol/L, use resistant panel screening that the mutant strain of homologous recombination occurs, and use primers designed palT1 and palT2 to carry out PCR method and sequencing is determined
palafter gene region generation Substitution, to obtain final product
palthe secretor type bacterial strain of transgenation;
B, structure
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change:
(1) use gained
paltransgenation bacterial strain is prepared chemoreception state cell, after imported plasmid pCP20, the resistant panel of coating again the Amp that contains 50 μ g/ml filters out the single bacterium colony recon containing plasmid pCP20 under 30 ℃ of conditions, the LB liquid nutrient medium of transferring is again cultivated 15 hours under 39 ℃ of conditions, afterwards gained bacterium liquid is carried out to dilution spread, the single bacterium colony obtaining proceeds to respectively LB resistant panel again and carries out the test of mark resistance, and identify by PCR method and sequencing the rejecting that whether has completed resistant maker gene, it is unmarked that process is confirmed
paltransgenation bacterial strain adopts final concentration 13% glycerine in-20 ℃ of preservations,
palwith
mrcBthe dual-gene starting strain knocking out;
(2) by gained
palwith
mrcBthe dual-gene starting strain knocking out is drawn dull and stereotyped about the 37 ℃ activation of LB, transfer into about 37 ℃ preparation chemoreception state cells of LB liquid nutrient medium, rear importing knocks out helper plasmid pKD46, again the bacterium liquid obtaining is coated to the resistant panel containing the Amp of 50 μ g/ml, under 30 ℃ of conditions, screening is containing single bacterium colony recon of plasmid pKD46, use enzyme cutting method to identify that this recon, whether containing pKD46 plasmid, is containing pKD46's containing the bacterial classification of pKD46 plasmid
palthe secretor type bacterial strain of transgenation;
(3) gained is prepared Electroporation-competent cells containing the single gene mutation bacterial strain of pKD46 under 30 ℃ of conditions, passed through again 10mmol L-arabinose induction 2 hours, after the specific target practice fragment of high density after purifying is imported wherein, after purifying, the preparation method of the specific target practice fragment of high density is with embodiment 1, again it is recovered after about 2 hours through the SOC substratum containing 5 mmol L-arabinose, 37 ℃ of screening generation homologous recombination of use resistant panel
mrcBmutant strain, and use PCR method and sequencing to identify
mrcBthere is Substitution in gene region, the mutant bacteria speciogenesis after confirmation twice homologous recombination, be
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change;
B, recombinant protein fermentation:
By synthetic human parathyroid hormone hPTH gene segment, after order-checking is correct, be cloned in secretive expression vector pET32a (+), obtain recombinant plasmid pET32a-
pthH, be transformed into
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change obtains secretor type recombinant bacterial strain, produces the secretor type recombinant bacterial strain of human parathyroid hormone.First use the dull and stereotyped activation of the LB this project bacterial strain containing the Amp of 50 μ g/ml, after 22 hours, choose single colony inoculation in the 250ml shaking flask of the LB liquid nutrient medium of the Amp containing 50 μ g/ml with 30ml, turning shaking table for 37 ℃ 200 cultivates 18 hours, again this bacterium liquid is inoculated into 30ml in the 250ml shaking flask containing the TB liquid fermentation medium of the Amp of 50 μ g/ml, making substratum cell concentration remain on OD600 is 0.1 left and right, 200 turn shaking table cultivates that to add final concentration after 8 hours be the IPTG of 0.5mmol, induction fermentation stops fermentation after 4 hours, obtain, get 1ml fermented liquid as sample, for subsequent analysis.After testing, exceeding 50% recombinant protein leaks in substratum.
An intestinal bacteria construction process for recombinant protein secretion type expression, comprises the following steps:
A,
mrcBwith
lppthe structure of the secretor type bacterial strain of dual-gene sudden change comprises the following steps:
A, structure
lppthe secretor type bacterial strain of transgenation:
(1) use designs in advance
lppgene knockout primer lppH+PF and lppH+PR, take resistant maker gene plasmid pKD3 as template, carry out the synthetic specific PCR product in polymerase chain reaction, obtains
lppgene targeting fragment, the two ends of target practice fragment are 50bp
lppdNA homolog sequence, centre is chlorampenicol resistant flag sequence, uses PCR product purification test kit to obtain the high density target practice fragment after the purifying of 20ng/ μ l the PCR product of gained;
(2) utilize the L-arabinose induction of 10mmol/L to contain to knock out set out bacterial classification JM109 (DE3) 2 hours of the intestinal bacteria of helper plasmid pKD46, then by the bacterium solution preparation Electroporation-competent cells after the induction of gained;
(3) by the high density after step (1) gained purifying
lppafter among the Electroporation-competent cells of the bacterium liquid after the induction of gene targeting fragment importing step (2) gained, recover after 2 hours at the SOC substratum of the L-arabinose containing 5 mmol/L, use resistant panel screening that the mutant strain of homologous recombination occurs, and use primers designed lppT1 and lppT2 to carry out PCR method and sequencing is determined
lppafter gene region generation Substitution, to obtain final product
lppthe secretor type bacterial strain of transgenation;
B, structure
mrcBwith
lppthe secretor type bacterial strain of dual-gene sudden change:
(1) use gained
lpptransgenation bacterial strain is prepared chemoreception state cell, after imported plasmid pCP20, the resistant panel of coating again the Amp that contains 50 μ g/ml filters out the single bacterium colony recon containing plasmid pCP20 under 30 ℃ of conditions, the LB liquid nutrient medium of transferring is again cultivated 15 hours under 39 ℃ of conditions, afterwards gained bacterium liquid is carried out to dilution spread, the single bacterium colony obtaining proceeds to respectively LB resistant panel again and carries out the test of mark resistance, and identify by PCR method and sequencing the rejecting that whether has completed resistant maker gene, it is unmarked that process is confirmed
lpptransgenation bacterial strain adopts final concentration 13% glycerine in-20 ℃ of preservations,
mrcBwith
lppthe dual-gene starting strain knocking out;
(2) by gained
mrcBwith
lppthe dual-gene starting strain knocking out is drawn dull and stereotyped about the 37 ℃ activation of LB, transfer into about 37 ℃ preparation chemoreception state cells of LB liquid nutrient medium, rear importing knocks out helper plasmid pKD46, again the bacterium liquid obtaining is coated to the resistant panel containing the Amp of 50 μ g/ml, under 30 ℃ of conditions, screening is containing single bacterium colony recon of plasmid pKD46, use enzyme cutting method to identify that this recon, whether containing pKD46 plasmid, is containing pKD46's containing the bacterial classification of pKD46 plasmid
lppthe secretor type bacterial strain of transgenation;
(3) gained is prepared Electroporation-competent cells containing the single gene mutation bacterial strain of pKD46 under 30 ℃ of conditions, passed through again 10mmol L-arabinose induction 2 hours, after the specific target practice fragment of high density after purifying is imported wherein, after purifying, the preparation method of the specific target practice fragment of high density is with embodiment 1, again it is recovered after about 2 hours through the SOC substratum containing 5 mmol L-arabinose, 37 ℃ of screening generation homologous recombination of use resistant panel
mrcBmutant strain, and use PCR method and sequencing to identify
mrcBthere is Substitution in gene region, the mutant bacteria speciogenesis after confirmation twice homologous recombination, be
mrcBwith
lppthe secretor type bacterial strain of dual-gene sudden change;
B, recombinant protein fermentation:
By synthetic human parathyroid hormone hPTH gene segment, after order-checking is correct, be cloned in secretive expression vector pET32a (+), obtain recombinant plasmid pET32a-
pthH, be transformed into
mrcBwith
lppthe secretor type bacterial strain of dual-gene sudden change obtains secretor type recombinant bacterial strain, produces the secretor type recombinant bacterial strain of human parathyroid hormone.First use the dull and stereotyped activation of the LB this project bacterial strain containing the Amp of 50 μ g/ml, after 22 hours, choose single colony inoculation in the 250ml shaking flask of the LB liquid nutrient medium of the Amp containing 50 μ g/ml with 30ml, turning shaking table for 37 ℃ 200 cultivates 18 hours, again this bacterium liquid is inoculated into 30ml in the 250ml shaking flask containing the TB liquid fermentation medium of the Amp of 50 μ g/ml, making substratum cell concentration remain on OD600 is 0.1 left and right, 200 turn shaking table cultivates that to add final concentration after 8 hours be the IPTG of 0.5mmol, induction fermentation stops fermentation after 4 hours, obtain, get 1ml fermented liquid as sample, for subsequent analysis.After testing, exceeding 50% recombinant protein leaks in substratum.
An intestinal bacteria construction process for recombinant protein secretion type expression, comprises the following steps:
A,
palwith
mrcBthe structure of the secretor type bacterial strain of dual-gene sudden change comprises the following steps:
A, structure
palthe secretor type bacterial strain of transgenation:
(1) use designs in advance
palgene knockout primer palH+PF and palH+PR, take resistant maker gene plasmid pKD3 as template, carry out the synthetic specific PCR product in polymerase chain reaction, obtains
palgene targeting fragment, the two ends of target practice fragment are 50bp
paldNA homolog sequence, centre is chlorampenicol resistant flag sequence, uses PCR product purification test kit to obtain the high density target practice fragment after the purifying of 20ng/ μ l the PCR product of gained;
(2) utilize the L-arabinose induction of 10mmol/L to contain to knock out set out bacterial classification JM109 (DE3) 2 hours of the intestinal bacteria of helper plasmid pKD46, then by the bacterium solution preparation Electroporation-competent cells after the induction of gained;
(3) by the high density after step (1) gained purifying
palafter among the Electroporation-competent cells of the bacterium liquid after the induction of gene targeting fragment importing step (2) gained, recover after 2 hours at the SOC substratum of the L-arabinose containing 5 mmol/L, use resistant panel screening that the mutant strain of homologous recombination occurs, and use primers designed palT1 and palT2 to carry out PCR method and sequencing is determined
palafter gene region generation Substitution, to obtain final product
palthe secretor type bacterial strain of transgenation;
B, structure
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change:
(1) use gained
paltransgenation bacterial strain is prepared chemoreception state cell, after imported plasmid pCP20, the resistant panel of coating again the Amp that contains 50 μ g/ml filters out the single bacterium colony recon containing plasmid pCP20 under 30 ℃ of conditions, the LB liquid nutrient medium of transferring is again cultivated 15 hours under 39 ℃ of conditions, afterwards gained bacterium liquid is carried out to dilution spread, the single bacterium colony obtaining proceeds to respectively LB resistant panel again and carries out the test of mark resistance, and identify by PCR method and sequencing the rejecting that whether has completed resistant maker gene, it is unmarked that process is confirmed
paltransgenation bacterial strain adopts final concentration 13% glycerine in-20 ℃ of preservations,
palwith
mrcBthe dual-gene starting strain knocking out;
(2) by gained
palwith
mrcBthe dual-gene starting strain knocking out is drawn dull and stereotyped about the 37 ℃ activation of LB, transfer into about 37 ℃ preparation chemoreception state cells of LB liquid nutrient medium, rear importing knocks out helper plasmid pKD46, again the bacterium liquid obtaining is coated to the resistant panel containing the Amp of 50 μ g/ml, under 30 ℃ of conditions, screening is containing single bacterium colony recon of plasmid pKD46, use enzyme cutting method to identify that this recon, whether containing pKD46 plasmid, is containing pKD46's containing the bacterial classification of pKD46 plasmid
palthe secretor type bacterial strain of transgenation;
(3) gained is prepared Electroporation-competent cells containing the single gene mutation bacterial strain of pKD46 under 30 ℃ of conditions, passed through again 10mmol L-arabinose induction 2 hours, after the specific target practice fragment of high density after purifying is imported wherein, after purifying, the preparation method of the specific target practice fragment of high density is with embodiment 1, again it is recovered after about 2 hours through the SOC substratum containing 5 mmol L-arabinose, 37 ℃ of screening generation homologous recombination of use resistant panel
mrcBmutant strain, and use PCR method and sequencing to identify
mrcBthere is Substitution in gene region, the mutant bacteria speciogenesis after confirmation twice homologous recombination, be
palwith
mrcBthe secretor type bacterial strain of transgenation;
B, recombinant protein fermentation:
By synthetic bone-marrow-derived lymphocyte stimulating factor BLyS gene segment, after order-checking is correct, be cloned in secretive expression vector pET32a (+), obtain recombinant plasmid pET32a-
blys, be transformed into
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change obtains secretor type recombinant bacterial strain, produces the secretor type recombinant bacterial strain of bone-marrow-derived lymphocyte stimulating factor.First use the dull and stereotyped activation of the LB this project bacterial strain containing the Amp of 50 μ g/ml, after 22 hours, choose single colony inoculation in the 250ml shaking flask of the LB liquid nutrient medium of the Amp containing 50 μ g/ml with 30ml, turning shaking table for 37 ℃ 200 cultivates 18 hours, again this bacterium liquid is inoculated into 30ml in the 250ml shaking flask containing the TB liquid fermentation medium of the Amp of 50 μ g/ml, making substratum cell concentration remain on OD600 is 0.1 left and right, 200 turn shaking table cultivates that to add final concentration after 8 hours be the IPTG of 0.5mmol, induction fermentation stops fermentation after 4 hours, obtain, get 1ml fermented liquid as sample, for subsequent analysis.After testing, exceeding 50% recombinant protein leaks in substratum.
An intestinal bacteria construction process for recombinant protein secretion type expression, comprises the following steps:
A,
mrcBwith
lppthe structure of the secretor type bacterial strain of dual-gene sudden change comprises the following steps:
A, structure
lppthe secretor type bacterial strain of transgenation:
(1) use designs in advance
lppgene knockout primer lppH+PF and lppH+PR, take resistant maker gene plasmid pKD3 as template, carry out the synthetic specific PCR product in polymerase chain reaction, obtains
lppgene targeting fragment, the two ends of target practice fragment are 50bp
lppdNA homolog sequence, centre is chlorampenicol resistant flag sequence, uses PCR product purification test kit to obtain the high density target practice fragment after the purifying of 20ng/ μ l the PCR product of gained;
(2) utilize the L-arabinose induction of 10mmol/L to contain to knock out set out bacterial classification JM109 (DE3) 2 hours of the intestinal bacteria of helper plasmid pKD46, then by the bacterium solution preparation Electroporation-competent cells after the induction of gained;
(3) by the high density after step (1) gained purifying
lppafter among the Electroporation-competent cells of the bacterium liquid after the induction of gene targeting fragment importing step (2) gained, recover after 2 hours at the SOC substratum of the L-arabinose containing 5 mmol/L, use resistant panel screening that the mutant strain of homologous recombination occurs, and use primers designed lppT1 and lppT2 to carry out PCR method and sequencing is determined
lppafter gene region generation Substitution, to obtain final product
lppthe secretor type bacterial strain of transgenation;
B, structure
mrcBwith
lppthe secretor type bacterial strain of dual-gene sudden change:
(1) use gained
lpptransgenation bacterial strain is prepared chemoreception state cell, after imported plasmid pCP20, the resistant panel of coating again the Amp that contains 50 μ g/ml filters out the single bacterium colony recon containing plasmid pCP20 under 30 ℃ of conditions, the LB liquid nutrient medium of transferring is again cultivated 15 hours under 39 ℃ of conditions, afterwards gained bacterium liquid is carried out to dilution spread, the single bacterium colony obtaining proceeds to respectively LB resistant panel again and carries out the test of mark resistance, and identify by PCR method and sequencing the rejecting that whether has completed resistant maker gene, it is unmarked that process is confirmed
lpptransgenation bacterial strain adopts final concentration 13% glycerine in-20 ℃ of preservations,
mrcBwith
lppthe dual-gene starting strain knocking out;
(2) by gained
mrcBwith
lppthe dual-gene starting strain knocking out is drawn dull and stereotyped about the 37 ℃ activation of LB, transfer into about 37 ℃ preparation chemoreception state cells of LB liquid nutrient medium, rear importing knocks out helper plasmid pKD46, again the bacterium liquid obtaining is coated to the resistant panel containing the Amp of 50 μ g/ml, under 30 ℃ of conditions, screening is containing single bacterium colony recon of plasmid pKD46, use enzyme cutting method to identify that this recon, whether containing pKD46 plasmid, is containing pKD46's containing the bacterial classification of pKD46 plasmid
lppthe secretor type bacterial strain of transgenation;
(3) gained is prepared Electroporation-competent cells containing the single gene mutation bacterial strain of pKD46 under 30 ℃ of conditions, passed through again 10mmol L-arabinose induction 2 hours, after the specific target practice fragment of high density after purifying is imported wherein, after purifying, the preparation method of the specific target practice fragment of high density is with embodiment 1, again it is recovered after about 2 hours through the SOC substratum containing 5 mmol L-arabinose, 37 ℃ of screening generation homologous recombination of use resistant panel
mrcBmutant strain, and use PCR method and sequencing to identify
mrcBthere is Substitution in gene region, the mutant bacteria speciogenesis after confirmation twice homologous recombination, be
mrcBwith
lppthe secretor type bacterial strain of dual-gene sudden change;
B, recombinant protein fermentation:
By synthetic bone-marrow-derived lymphocyte stimulating factor BLyS gene segment, after order-checking is correct, be cloned in secretive expression vector pET32a (+), obtain recombinant plasmid pET32a-
blys, be transformed into
mrcBwith
lppthe secretor type bacterial strain of dual-gene sudden change obtains secretor type recombinant bacterial strain, produces the secretor type recombinant bacterial strain of bone-marrow-derived lymphocyte stimulating factor.First use the dull and stereotyped activation of the LB this project bacterial strain containing the Amp of 50 μ g/ml, after 22 hours, choose single colony inoculation in the 250ml shaking flask of the LB liquid nutrient medium of the Amp containing 50 μ g/ml with 30ml, turning shaking table for 37 ℃ 200 cultivates 18 hours, again this bacterium liquid is inoculated into 30ml in the 250ml shaking flask containing the TB liquid fermentation medium of the Amp of 50 μ g/ml, making substratum cell concentration remain on OD600 is 0.1 left and right, 200 turn shaking table cultivates that to add final concentration after 8 hours be the IPTG of 0.5mmol, induction fermentation stops fermentation after 4 hours, obtain, get 1ml fermented liquid as sample, for subsequent analysis.After testing, exceeding 50% recombinant protein leaks in substratum.
An intestinal bacteria construction process for recombinant protein secretion type expression, comprises the following steps:
A,
palwith
mrcBthe structure of the secretor type bacterial strain of dual-gene sudden change comprises the following steps:
A, structure
palthe secretor type bacterial strain of transgenation:
(1) use designs in advance
palgene knockout primer palH+PF and palH+PR, take resistant maker gene plasmid pKD3 as template, carry out the synthetic specific PCR product in polymerase chain reaction, obtains
palgene targeting fragment, the two ends of target practice fragment are 50bp
paldNA homolog sequence, centre is chlorampenicol resistant flag sequence, uses PCR product purification test kit to obtain the high density target practice fragment after the purifying of 20ng/ μ l the PCR product of gained;
(2) utilize the L-arabinose induction of 10mmol/L to contain to knock out set out bacterial classification JM109 (DE3) 2 hours of the intestinal bacteria of helper plasmid pKD46, then by the bacterium solution preparation Electroporation-competent cells after the induction of gained;
(3) by the high density after step (1) gained purifying
palafter among the Electroporation-competent cells of the bacterium liquid after the induction of gene targeting fragment importing step (2) gained, recover after 2 hours at the SOC substratum of the L-arabinose containing 5 mmol/L, use resistant panel screening that the mutant strain of homologous recombination occurs, and use primers designed palT1 and palT2 to carry out PCR method and sequencing is determined
palafter gene region generation Substitution, to obtain final product
palthe secretor type bacterial strain of transgenation;
B, structure
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change:
(1) use gained
paltransgenation bacterial strain is prepared chemoreception state cell, after imported plasmid pCP20, the resistant panel of coating again the Amp that contains 50 μ g/ml filters out the single bacterium colony recon containing plasmid pCP20 under 30 ℃ of conditions, the LB liquid nutrient medium of transferring is again cultivated 15 hours under 39 ℃ of conditions, afterwards gained bacterium liquid is carried out to dilution spread, the single bacterium colony obtaining proceeds to respectively LB resistant panel again and carries out the test of mark resistance, and identify by PCR method and sequencing the rejecting that whether has completed resistant maker gene, it is unmarked that process is confirmed
paltransgenation bacterial strain adopts final concentration 13% glycerine in-20 ℃ of preservations,
palwith
mrcBthe dual-gene starting strain knocking out;
(2) by gained
palwith
mrcBthe dual-gene starting strain knocking out is drawn dull and stereotyped about the 37 ℃ activation of LB, transfer into about 37 ℃ preparation chemoreception state cells of LB liquid nutrient medium, rear importing knocks out helper plasmid pKD46, again the bacterium liquid obtaining is coated to the resistant panel containing the Amp of 50 μ g/ml, under 30 ℃ of conditions, screening is containing single bacterium colony recon of plasmid pKD46, use enzyme cutting method to identify that this recon, whether containing pKD46 plasmid, is containing pKD46's containing the bacterial classification of pKD46 plasmid
palthe secretor type bacterial strain of transgenation;
(3) gained is prepared Electroporation-competent cells containing the single gene mutation bacterial strain of pKD46 under 30 ℃ of conditions, passed through again 10mmol L-arabinose induction 2 hours, after the specific target practice fragment of high density after purifying is imported wherein, after purifying, the preparation method of the specific target practice fragment of high density is with embodiment 1, again it is recovered after about 2 hours through the SOC substratum containing 5 mmol L-arabinose, 37 ℃ of screening generation homologous recombination of use resistant panel
mrcBmutant strain, and use PCR method and sequencing to identify
mrcBthere is Substitution in gene region, the mutant bacteria speciogenesis after confirmation twice homologous recombination, be
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change;
B, recombinant protein fermentation:
By synthetic caffeoyl coenzyme A-O-methyl transferase gene SvOM segment, after checking order correctly, be cloned in expression plasmid pET32a (+), obtain secretor type plasmid pET32a-
svom, transform Host Strains
palwith
mrcBthe secretor type bacterial strain of dual-gene sudden change.Must produce the secretor type recombinant bacterial strain of caffeoyl coenzyme A-O-methyltransgerase.First use the dull and stereotyped activation of the LB this project bacterial strain containing the Amp of 50 μ g/ml, after 22 hours, choose single colony inoculation in the 250ml shaking flask of the LB liquid nutrient medium of the Amp containing 50 μ g/ml with 30ml, turning shaking table for 37 ℃ 200 cultivates 18 hours, again this bacterium liquid is inoculated into 30ml in the 250ml shaking flask containing the TB liquid fermentation medium of the Amp of 50 μ g/ml, making substratum cell concentration remain on OD600 is 0.1 left and right, 200 turn shaking table cultivates that to add final concentration after 8 hours be the IPTG of 0.5mmol, induction fermentation stops fermentation after 4 hours, obtain, get 1ml fermented liquid as sample, for subsequent analysis.After testing, exceeding 50% recombinant protein leaks in substratum.
An intestinal bacteria construction process for recombinant protein secretion type expression, comprises the following steps:
A,
mrcBwith
lppthe structure of the secretor type bacterial strain of dual-gene sudden change comprises the following steps:
A, structure
lppthe secretor type bacterial strain of transgenation:
(1) use designs in advance
lppgene knockout primer lppH+PF and lppH+PR, take resistant maker gene plasmid pKD3 as template, carry out the synthetic specific PCR product in polymerase chain reaction, obtains
lppgene targeting fragment, the two ends of target practice fragment are 50bp
lppdNA homolog sequence, centre is chlorampenicol resistant flag sequence, uses PCR product purification test kit to obtain the high density target practice fragment after the purifying of 20ng/ μ l the PCR product of gained;
(2) utilize the L-arabinose induction of 10mmol/L to contain to knock out set out bacterial classification JM109 (DE3) 2 hours of the intestinal bacteria of helper plasmid pKD46, then by the bacterium solution preparation Electroporation-competent cells after the induction of gained;
(3) by the high density after step (1) gained purifying
lppafter among the Electroporation-competent cells of the bacterium liquid after the induction of gene targeting fragment importing step (2) gained, recover after 2 hours at the SOC substratum of the L-arabinose containing 5 mmol/L, use resistant panel screening that the mutant strain of homologous recombination occurs, and use primers designed lppT1 and lppT2 to carry out PCR method and sequencing is determined
lppafter gene region generation Substitution, to obtain final product
lppthe secretor type bacterial strain of transgenation;
B, structure
mrcBwith
lppthe secretor type bacterial strain of dual-gene sudden change:
(1) use gained
lpptransgenation bacterial strain is prepared chemoreception state cell, after imported plasmid pCP20, the resistant panel of coating again the Amp that contains 50 μ g/ml filters out the single bacterium colony recon containing plasmid pCP20 under 30 ℃ of conditions, the LB liquid nutrient medium of transferring is again cultivated 15 hours under 39 ℃ of conditions, afterwards gained bacterium liquid is carried out to dilution spread, the single bacterium colony obtaining proceeds to respectively LB resistant panel again and carries out the test of mark resistance, and identify by PCR method and sequencing the rejecting that whether has completed resistant maker gene, it is unmarked that process is confirmed
lpptransgenation bacterial strain adopts final concentration 13% glycerine in-20 ℃ of preservations,
mrcBwith
lppthe dual-gene starting strain knocking out;
(2) by gained
mrcBwith
lppthe dual-gene starting strain knocking out is drawn dull and stereotyped about the 37 ℃ activation of LB, transfer into about 37 ℃ preparation chemoreception state cells of LB liquid nutrient medium, rear importing knocks out helper plasmid pKD46, again the bacterium liquid obtaining is coated to the resistant panel containing the Amp of 50 μ g/ml, under 30 ℃ of conditions, screening is containing single bacterium colony recon of plasmid pKD46, use enzyme cutting method to identify that this recon, whether containing pKD46 plasmid, is containing pKD46's containing the bacterial classification of pKD46 plasmid
lppthe secretor type bacterial strain of transgenation;
(3) gained is prepared Electroporation-competent cells containing the single gene mutation bacterial strain of pKD46 under 30 ℃ of conditions, passed through again 10mmol L-arabinose induction 2 hours, after the specific target practice fragment of high density after purifying is imported wherein, after purifying, the preparation method of the specific target practice fragment of high density is with embodiment 1, again it is recovered after about 2 hours through the SOC substratum containing 5 mmol L-arabinose, 37 ℃ of screening generation homologous recombination of use resistant panel
mrcBmutant strain, and use PCR method and sequencing to identify
mrcBthere is Substitution in gene region, the mutant bacteria speciogenesis after confirmation twice homologous recombination, be
mrcBwith
lppthe secretor type bacterial strain of dual-gene sudden change;
B, recombinant protein fermentation:
By synthetic caffeoyl coenzyme A-O-methyl transferase gene SvOM segment, after checking order correctly, be cloned in expression plasmid pET32a (+), obtain secretor type plasmid pET32a-
svom, transform Host Strains
mrcBwith
lppthe secretor type bacterial strain of dual-gene sudden change.Must produce the secretor type recombinant bacterial strain of caffeoyl coenzyme A-O-methyltransgerase.First use the dull and stereotyped activation of the LB this project bacterial strain containing the Amp of 50 μ g/ml, after 22 hours, choose single colony inoculation in the 250ml shaking flask of the LB liquid nutrient medium of the Amp containing 50 μ g/ml with 30ml, turning shaking table for 37 ℃ 200 cultivates 18 hours, again this bacterium liquid is inoculated into 30ml in the 250ml shaking flask containing the TB liquid fermentation medium of the Amp of 50 μ g/ml, making substratum cell concentration remain on OD600 is 0.1 left and right, 200 turn shaking table cultivates that to add final concentration after 8 hours be the IPTG of 0.5mmol, induction fermentation stops fermentation after 4 hours, obtain, get 1ml fermented liquid as sample, for subsequent analysis.After testing, exceeding 50% recombinant protein leaks in substratum.
sequence table
mrcB, mrcA, lpp, palgene knockout primer:
| mrcBH+PF | agaacagaaaatcgggcttttgcgcctgaatattgcggagaaaaagcatgGTGTAGGCTGGAGCTGCTTC |
| mrcBH+PF | ctcgccatccggtatttcacgcttagatgttaattactaccaaacatatcATGGGAATTAGCCATGGTCC |
| lppH+PF | acttgtaacgctacatggagattaactcaatctagagggtattaataatgGTGTAGGCTGGAGCTGCTTC |
| lppH+PR | gtgcgccatttttcacttcacaggtactattacttgcggtatttagtagcATGGGAATTAGCCATGGTCC |
| palH+PF | gccgtatctgtgataataattaattgaatagtaaaggaatcattgaaatgGTGTAGGCTGGAGCTGCTTC |
| palH+PR | tgtctgaagttactgctcatgcaattctcttagtaaaccagtaccgcacgATGGGAATTAGCCATGGTCC |
| rodZH+PF | gatggttcaccggcatctcaattctcatttaaacgtacctgcagcgaatgGTGTAGGCTGGAGCTGCTTC |
| rodZH+PR | catgaaaaatctcccgcgttacccgtctgttactgcgccggtgattgttcATGGGAATTAGCCATGGTCC |
| mrdAH+PF | cttatcaccgtgagtgataagggagctttgagtagaaaacgcagcggatgGTGTAGGCTGGAGCTGCTTC |
| mrdAH+PR | tgtttttttattcggattatccgtcatgattaatggtcctccgctgcggcATGGGAATTAGCCATGGTCC |
| mrcAH+PF | gcgtttgtttataaactgcccaaatgaaactaaatgggaaatttccagtgGTGTAGGCTGGAGCTGCTTC |
| mrcAH+PR | actgaaaaggcgccgaagcgcctttttaatcagaacaattcctgtgcctcATGGGAATTAGCCATGGTCC |
| rodAH+PF | ctgcctgcggaaaatccagcggttgccgcagcggaggaccattaatcatgGTGTAGGCTGGAGCTGCTTC |
| rodAH+PR | cgagccactgcttacgcattgcgcacctcttacacgcttttcgacaacatATGGGAATTAGCCATGGTCC |
| murCH+PF | cgatgccttttgcatcgtatgaatttaagaagttaatggcgtaaagaatgGTGTAGGCTGGAGCTGCTTC |
| murCH+PR | aggtcccacccaacaggaccgcgattttatcagtcatgttgttcttcctcATGGGAATTAGCCATGGTCC |
(note: lowercase represents the homologous sequence of specific gene)
mrcA, mrcB, pal, lppgene identification primer:
| mrcBT1 | TCGAGCACAAATTTTGAGAG |
| mrcBT2 | CAACCAGATGAAAAGAAAGG |
| lppT1 | CAGCGTTCGATGCTTCTTT |
| lppT2 | GTTTCGGTGAGCAGTGGAG |
| palT1 | GTGCGCCACAACGTATTAC |
| palT2 | AAGTTGTTGCTGGAGTTGG |
| rodZT1 | TAAAGCGGTCTGATAGCCA |
| rodZT2 | GCCTTGATTTGATTGACCG |
| mrdAT1 | CAAGAATGCGGACATCAAG |
| mrdAT2 | CCATCAGTGGTACGGCTATT |
| mrcAT1 | CTGCCCAAATGAAACTAAATG |
| mrcAT2 | TTGCTGGAAGACCCTGACT |
| rodAT1 | GTGTTGCTAACCGCCCTAA |
| rodAT2 | GCCTGGCTAAATCGAGACG |
| murCT1 | AAATTATCGAGCAGCCACAG |
| murCT2 | GCAGCGTACCATCTTCACC |
palgene order:
atgcaactgaacaaagtgctgaaagggctgatgattgctctgcctgttatggcaattgcggcatgttcttccaacaagaacgccagcaatgacggcagcgaaggcatgctgggtgccggcactggtatggatgcgaacggcggcaacggcaacatgtcttccgaagagcaggctcgtctgcaaatgcaacagctgcagcagaacaacatcgtttacttcgatctggacaagtacgatatccgttctgacttcgctcaaatgctggatgcacatgcaaacttcctgcgtagcaacccgtcttacaaagtcaccgtagaaggtcacgcggacgaacgtggtactccggaatacaacatctccctgggtgaacgtcgtgcgaacgccgttaagatgtacctgcagggtaaaggcgtttctgcagaccagatctccatcgtttcttacggtaaagaaaaacctgcagtactgggtcatgacgaagcggcatactccaaaaaccgtcgtgcggtactggtttactaa
mrcBgene order:
atggccgggaatgaccgcgagccaattggacgcaaagggaaaccgacgcgtccggtcaaacaaaaggtaagccgtcgtcgttacgaagatgacgatgattacgacgattatgatgactatgaggatgaagaaccgatgccgcgcaaaggtaagggcaaaggcaaagggcgtaagcctcgtggcaaacgcggctggctatggctactgctaaaactggctatcgtttttgccgtgctgatcgccatttacggcgtttatctcgatcaaaaaattcgtagccgtattgatggcaaggtctggcaactgcctgcggcagtttatggccgaatggtcaatcttgagccagacatgaccatcagcaagaacgagatggtgaagctgctggaggcgacccagtatcgtcaggtgtcgaaaatgacccgtcctggcgaatttaccgtgcaggccaacagcattgagatgattcgccgtccgtttgatttcccggacagtaaagaaggacaggtgcgcgcgcgtctgacctttgatggcgatcatctggcgacgatcgtcaatatggagaacaaccgtcagttcggtttcttccgtcttgatccgcgtctgatcaccatgatctcttcgccaaacggtgagcagcgtctgtttgtgccgcgcagtggtttcccggatttgctggtggatactttgctggcgacagaagaccgtcatttttacgagcatgatggaatcagtctctactcaatcggacgtgcggtgctggcaaacctgaccgccggacgcacggtacagggtgcgagtacgctgacgcaacagctggtgaaaaacctgttcctctccagcgagcgttcttactggcgtaaagcgaacgaagcttacatggcgctgatcatggacgcgcgttacagcaaagaccgtattcttgagctgtatatgaacgaggtgtatctcggtcagagcggcgacaacgaaatccgcggcttcccgctggcaagcttgtattactttggtcgcccggtagaagagctaagcctcgaccagcaggcgctgttagtcggtatggtgaaaggggcgtccatctacaacccgtggcgtaacccaaaactggcgctggagcgacgtaatctggtgctgcgtctgctgcaacagcaacagattattgatcaagaactctatgacatgttgagtgcccgtccgctgggggttcagccgcgcggtggggtgatctctcctcagccagcctttatgcaactggtgcgtcaggagctgcaggcaaaactgggcgataaggtaaaagatctctccggcgtgaagatcttcactacctttgactcggtggcccaggacgcggcagaaaaagccgccgtggaaggcattccggcactgaagaaacagcgtaagttgagcgatcttgaaactgcgattgtggtcgtcgaccgctttagtggtgaagttcgtgcgatggtcggaggttctgagccgcagtttgcgggctacaaccgtgcgatgcaggcgcgtcgttcgattggttcccttgcaaaaccagcgacttatctgacggccttaagccagccgaaaatctatcgtctgaatacgtggattgcggatgcgccaattgcgctgcgtcagccgaatggccaggtctggtcaccgcagaatgatgaccgtcgttatagcgaaagcggcagagtgatgctggtggatgcgttgacccgttcgatgaacgtgccgacggtaaatctggggatggcgctggggctgcctgcggttacggagacctggattaaactgggcgtaccgaaagatcagttgcatccggttccggcaatgctgctgggggcgttgaacttaacgccaatcgaagtggcgcaggcattccagaccatcgccagcggtggtaaccgtgcaccgctttctgcgctgcgttcggtaatcgcggaagatggcaaagtgctgtatcagagcttcccgcaggcggaacgcgctgttccggcgcaggcggcgtatctgacactatggaccatgcagcaggtggtacaacgcggtacgggtcgtcagcttggggcgaaatacccgaacctgcatctggcagggaaaacagggactaccaacaataacgtagatacctggtttgcgggcattgacggcagcacggtgaccatcacctgggtcggccgtgataacaaccagccgaccaaactgtatggtgccagcggggcaatgtcgatttatcagcgttatctggctaaccagacgccaacgccgctgaatcttgttccgccagaagatattgcagatatgggcgtggactacgacggcaactttgtttgcagcggtggcatgcgtatcttgccggtctggaccagcgatccgcaatcgctgtgccagcagagcgagatgcagcagcagccgtcaggcaatccgtttgatcagtcttctcagccgcagcaacagccgcaacagcaacctgctcagcaagagcagaaagacagcgacggtgtagccggttggatcaaggatatgtttggtagtaattaa
mrcAgene order:
gtgaagttcgtaaagtattttttgatccttgcagtctgttgcattctgctgggagcaggctcgatttatggcctataccgctacatcgagccacaactgccggatgtggcgacattaaaagatgttcgcctgcaaattccgatgcagatttacagcgccgatggcgagctgattgctcaatacggtgagaaacgtcgtattccggttacgttggatcaaatcccaccggagatggtgaaagcctttatcgcgacagaagacagccgcttctacgagcatcacggcgttgacccggtggggatcttccgtgcagcaagcgtggcgctgttctccggtcacgcgtcacaaggggcaagtaccattacccagcagctggcgagaaacttcttcctcagtccagaacgcacgctgatgcgtaagattaaggaagtcttcctcgcgattcgcattgaacagctgctgacgaaagacgagatcctcgagctttatctgaacaagatttaccttggttaccgcgcctatggtgtcggtgctgcggcacaagtctatttcggaaaaacggtcgaccaactgacgctgaacgaaatggcggtgatagccgggctgccgaaagcgccttccaccttcaacccgctctactcgatggatcgtgccgtcgcgcggcgtaacgtcgtgctgtcgcggatgctggatgaagggtatatcacccaacaacagttcgatcagacacgcactgaggcgattaacgctaactatcacgcgccggagattgctttctctgcgccgtacctgagcgaaatggtgcgccaggagatgtataaccgttatggcgaaagtgcctatgaagacggttatcgcatttacaccaccatcacccgcaaagtgcagcaggccgcgcagcaggcggtacgtaataacgtgctggactacgacatgcgccacggctatcgcggcccggcaaatgtgctgtggaaagtgggcgagtcggcgtgggataacaacaagattaccgatacgctgaaggcgctgccaacctatggtccgctgctgcctgccgcagtcaccagcgccaatcctcagcaagcgacggcgatgctggcggacgggtcgaccgtcgcattgagtatggaaggcgttcgctgggcgcgtccttaccgttcggatactcagcaaggaccgacgccgcgtaaagtgaccgatgttctgcaaacgggtcagcaaatctgggttcgtcaggttggcgatgcatggtggctggcacaagtgccggaagtgaactcggcgctggtgtcgatcaatccgcaaaacggtgccgttatggcgctggtcggtggctttgatttcaatcagagcaagtttaaccgcgccacccaggcactgcgtcaggtgggttccaacatcaaaccgttcctctacaccgcggcgatggataaaggtctgacgctggcaagtatgttgaacgatgtgccaatttctcgctgggatgcaagtgccggttctgactggcagccgaagaactcaccaccgcagtatgctggtccaattcgcttacgtcaggggctgggtcagtcgaaaaacgtggtgatggtacgcgcaatgcgggcgatgggcgtcgactacgctgcagaatatctgcaacgcttcggcttcccggcacaaaacattgtccacaccgaatcgctggcgctgggttcagcgtccttcaccccaatgcaggtggcgcgcggctacgcggtcatggcgaacggcggcttcctggtggacccgtggtttatcagcaaaattgaaaacgatcagggcggcgtgattttcgaagcgaaaccgaaagtagcctgcccggaatgcgatattccggtgatttacggtgatacgcagaaatcgaacgtgctggaaaataacgatgttgaagatgtcgctatctcccgcgagcagcagaatgtttctgtaccaatgccgcagctggagcaggcaaatcaggcgttagtggcgaagactggcgcgcaggagtacgcaccgcacgtcatcaacactccgctggcattcctgattaagagtgctttgaacaccaatatctttggtgagccaggctggcagggtactggctggcgtgcaggtcgtgatttgcagcgtcgcgatatcggcgggaaaaccgggaccactaacagttcgaaagatgcgtggttctcgggttacggtccgggcgttgtgacctcggtctggattggctttgatgatcaccgtcgtaatctcggtcatacaacggcttccggagcgattaaagatcagatctcaggttacgaaggcggtgccaagagtgcccagcctgcatgggacgcttatatgaaagccgttcttgaaggtgtgccggagcagccgctgacgccgccaccgggtattgtgacggtgaatatcgatcgcagcaccgggcagttagctaatggtggcaacagccgcgaagagtatttcatcgaaggtacgcagccgacacaacaggcagtgcacgaggtgggaacgaccattatcgataatggcgaggcacaggaattgttctga
mrdAgene order:
atgaaactacagaactcttttcgcgactatacggcagagtccgcgctgtttgtgcgccgggcgctggtcgcctttttggggattttgctgctgaccggcgtgcttatcgccaacctgtataatctgcaaattgttcgctttaccgactaccagacccgctctaatgaaaaccgcattaagctggtgcctatcgcgcccagccgcggcattatctacgatcgtaacggtatccctctggccctcaaccgcactatctaccagatagaaatgatgccggagaaagtcgataacgtgcagcaaacgctggacgctttgcgcagcgtggtagatctgaccgatgacgatattgctgcattccgaaaagagcgcgcacgttcacaccgtttcacctctattccggtgaaaactaacctgaccgaagtacaagtagctcgctttgccgtcaatcagtaccgttttccgggtgtcgaagttaaaggctataaacgtcgttactatccttacggttcggcgttgacccacgtcatcggctatgtgtcgaaaatcaacgataaagacgtcgaacgcctgaataatgacggcaaactggccaactatgcggcaacgcatgatatcggtaagctgggcattgagcgttactatgaagatgtgctgcacggtcagaccggttatgaagaggttgaagttaacaaccgtgggcgtgttattcgccagttaaaagaagtaccaccgcaagccggacacgatatttacctgacgctggatctcaaactccagcaatatattgaaacgctgctggcgggtagccgcgcagctgtggtagtcaccgatccgcgtacaggtggggtgctggcgctggtttccacgcctagttatgacccaaacttgtttgttgacggtatctccagcaaagattattccgccttgttgaacgatccgaatacaccgctggtgaaccgcgccacacagggggtttatcctcccgcgtctacagttaaaccctatgtggcggtttcggcattgagcgccggggtgatcacgcgcaatacgacgctgtttgacccaggctggtggcaactgccaggttcggaaaaacgttatcgtgactggaaaaaatggggccacgggcgtctgaatgtcacaagatcgctggaagaatctgcggataccttcttctatcaggtggcctacgatatggggatcgatcgcctctccgaatggatgggtaaattcggttatggtcattacaccggtatcgacctggcggaagaacgttccggcaacatgcctacccgcgaatggaaacagaaacgctttaaaaaaccgtggtatcagggtgacaccattccggttggtatcggtcagggttactggacagcgaccccaatccagatgagtaaggcactgatgatcctgattaatgacggtatcgtgaaggttcctcatttgctgatgagcaccgccgaagacggcaaacaggtgccatgggtacagccgcatgaaccgcccgtcggcgatattcattccggttactgggagctggcgaaagacggtatgtacggtgttgctaaccgccctaacggtacggcgcataaatactttgctagcgcaccgtacaaaattgcggcgaaatccggtaccgctcaggtcttcggtctgaaagcgaacgaaacctataatgcgcacaaaattgccgagcgtttacgtgaccacaaactgatgaccgcctttgcgccatacaacaatccgcaagtggctgtcgccatgattctggagaacggtggtgcgggtccggcggttggtacactgatgcgccagatcctcgaccacattatgctgggtgataacaacaccgatctgcctgcggaaaatccagcggttgccgcagcggaggaccattaa
rodZgene order:
atgaatactgaagccacgcacgaccaaaatgaagcacttactaccggcgctcgcctgcgtaatgctcgcgaacaactaggacttagtcagcaggccgttgccgagcgactttgcctgaaggtttccacggtacgcgacattgaagaagataaggcacccgccgatcttgcttcaacattcctgcgcggatatatccgctcttatgcgcgtctggtacatattccagaagaagaactgctgccagggctggaaaagcaggctccacttcgggctgcaaaagttgcgccgatgcagagtttttccctcggtaaacgccgcaaaaaacgcgacggctggctgatgaccttcacttggctggtgttgtttgtggttatcggcctgagcggtgcctggtggtggcaagaccgcaaagctcagcaggaagagatcaccactatggccgatcaatcttcggcggaactgagcagtaatagcgagcaggggcagagtgttccattaaatacgtcgacaactacagacccggctacaacgtccacgccgccagcgtctgtggatactaccgcaaccaacacgcaaacacctgccgtaactgcgccagcaccagctgttgatccgcaacagaatgcggttgtttcgccttcgcaggcaaatgttgataccgccgcgaccccggcaccaacggcagcaacaacgccagatggtgctgcgccgttgccaaccgatcaggctggcgtgaccacgccggtggctgatccgaatgcgctggtgatgaactttactgccgattgctggctggaggtcactgatgctaccggtaaaaaattgtttagcggtatgcagcgtaaagacggtaatttgaacttaaccggccaggcaccgtacaaactgaaaattggtgcgccagccgcagtacagatccagtatcaagggaaacctgtcgatctgagtcgttttatcagaactaaccaggttgcgcgtctgaccctcaatgccgaacaatcaccggcgcagtaa
lppgene order:
atgaaagctactaaactggtactgggcgcggtaatcctgggttctactctgctggcaggttgctccagcaacgctaaaatcgatcagctgtcttctgacgttcagactctgaacgctaaagttgaccagctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaa
rodAgene order:
atgacggataatccgaataaaaaaacattctgggataaagtccatctcgatcccacaatgctgctgatcttactggcattgctggtttacagcgccctggttatctggagcgccagcggtcaggatattggcatgatggagcgtaaaatcggccaaatcgcgatgggtctggtcatcatggtggtgatggcgcaaattcctccacgcgtttatgaaggctgggccccctatctctatatcatctgtattattttgctggtggcggtagatgctttcggtgccatctctaaaggtgctcaacgctggctggacctcggtattgttcgttttcagccgtcggaaattgccaaaatagccgtaccactgatggttgcgcgctttatcaaccgcgacgtttgcccgccatcgttgaagaacactggcatcgcgctggtgctgatatttatgcccacgctgctggtggctgcacagcctgacctgggaacatcaatcctcgttgcgctttccggtctgtttgtactgttcctctctggccttagctggcgtctgattggcgtcgcagtagtgctggtagcggcgttcattccgattctgtggttcttcctgatgcatgattaccagcgccagcgcgtaatgatgctcctggacccggaatcagacccactcggcgcgggctatcacattattcagtctaaaattgctattggctccggcggattacgcggcaaaggctggctgcacggcactcagtcacagcttgaatttctccccgaacgccatactgactttatcttcgcggtactggcggaagagctgggattagtgggcattctgattctgctcgctctctacattctgctgatcatgcgcgggctgtggatagccgccagagcgcaaaccacctttggtcgcgtcatggctggcggcttaatgctgatattattcgtttatgtcttcgtaaatattggtatggtaagcggtattctgccggttgtaggggttccgctcccactggtcagttatggaggatcggcgctaattgtgctgatggctgggttcgggattgtaatgtcaatccacacccacaggaaaatgttgtcgaaaagcgtgtaa
murCgene order:
atgaatacacaacaattggcaaaactgcgttccatcgtgcccgaaatgcgtcgcgttcggcacatacattttgtcggcattggtggtgccggtatgggcggtattgccgaagttctggccaatgaaggttatcagatcagtggttccgatttagcgccaaatccggtcacgcagcagttaatgaatctgggtgcgacgatttatttcaaccatcgcccggaaaacgtacgtgatgccagcgtggtcgttgtttccagcgcgatttctgccgataacccggaaattgtcgccgctcatgaagcgcgtattccggtgatccgtcgtgccgaaatgctggctgagttaatgcgttttcgtcatggcatcgccattgccggaacgcacggcaaaacgacaaccaccgcgatggtttccagcatctacgcagaagcggggctcgacccaaccttcgttaacggcgggctggtaaaagcggcgggggttcatgcgcgtttggggcatggtcggtacctgattgccgaagcagatgagagtgatgcatcgttcctgcatctgcaaccgatggtggcgattgtcaccaatatcgaagccgaccacatggatacctaccagggcgactttgagaatttaaaacagacttttattaattttctgcacaacctgccgttttacggtcgtgcggtgatgtgtgttgatgatccggtgatccgcgaattgttaccgcgagtggggcgtcagaccacgacttacggcttcagcgaagatgccgacgtgcgtgtagaagattatcagcagattggcccgcaggggcactttacgctgctgcgccaggacaaagagccgatgcgcgtcaccctgaatgcgccaggtcgtcataacgcgctgaacgccgcagctgcggttgcggttgctacggaagagggcattgacgacgaggctattttgcgggcgcttgaaagcttccaggggactggtcgccgttttgatttcctcggtgaattcccgctggagccagtgaatggtaaaagcggtacggcaatgctggtcgatgactacggccaccacccgacggaagtggacgccaccattaaagcggcgcgcgcaggctggccggataaaaacctggtaatgctgtttcagccgcaccgttttacccgtacgcgcgacctgtatgatgatttcgccaatgtgctgacgcaggttgataccctgttgatgctggaagtgtatccggctggcgaagcgccaattccgggagcggacagccgttcgctgtgtcgcacaattcgtggacgtgggaaaattgatcccattctggtgccggatccggcgcgggtagccgagatgctggcaccggtattaaccggtaacgacctgattctcgttcagggggctggtaatattggaaaaattgcccgttctttagctgaaatcaaactgaagccgcaaactccggaggaagaacaacatgactga
PTH protein sequence:
SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVLTKSQ
BLyS protein sequence:
AVQGPETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRCSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFCALKLL
SVOM protein sequence:
MTKGNSTKITDELYRYVLAHNPPLDEFQRGLVALTWEKFPDSAGMQSAEEQGPLLAFLVRLTGARHIVEVGTFTGFSALSMAQALPADGRLIACDVSEEWTAYGRAAWEAAGVADRIDLRIAPALDTLRAMPQEPHVDLAYVDADKQNQILYWEELVPRMRPGGLIVTDNTLFHGTVLDESPESRGAAVRAFNDHVMADTRMDSVLLAVSDGLSLSRKR
Claims (5)
1. for a construction process for the dual-gene mutant Escherichia coli of recombinant protein secretion type expression, it is characterized in that: the epicyte protein and the Cell wall synthesis enzyme gene that use design in advance
palwith
mrcBtwo primers that knock out gene carry out PCR reaction, obtain two ends and be 10～250 bp specific objective DNA homolog sequences and middle concentration for resistance marker sequence is 1～1000 ng/ μ l's
palwith
mrcBtwo gene targeting fragments that knock out gene, and transform and be incorporated in host e. coli karyomit(e) by electricity respectively, obtain the secretory coli bacterial strain of dual-gene sudden change.
2. the construction process of a kind of dual-gene mutant Escherichia coli for recombinant protein secretion type expression according to claim 1, is characterized in that:
The application of described dual-gene mutant Escherichia coli comprises: the secretor type recombinant escherichia coli strain of exogenous protein expression builds, the target protein of secretor type recombination bacillus coli is expressed process;
The secretor type recombinant escherichia coli strain construction process of described exogenous protein expression is: external source target protein plasmagene is first recombinated in secretor type plasmid pET32a (+), be cloned into subsequently in the secretory coli bacterial strain of described dual-gene sudden change, and then obtain the secretor type recombinant escherichia coli strain of exogenous protein expression.
3. the construction process of a kind of dual-gene mutant Escherichia coli for recombinant protein secretion type expression according to claim 2, it is characterized in that: the recombination plasmid that the secretor type recombinant escherichia coli strain of described exogenous protein expression contains encoding exogenous albumen, in this recombinant plasmid, foreign protein genes is connected with the signal coding sequence that is positioned periplasmic space, thereby makes synthetic foreign protein in born of the same parents be directed in periplasmic space and then be leaked in substratum by signal peptide.
4. the construction process of a kind of dual-gene mutant Escherichia coli for recombinant protein secretion type expression according to claim 2, is characterized in that: the target protein expression process of described secretor type recombination bacillus coli refers to and in fermention medium, uses the secretor type recombinant escherichia coli strain of exogenous protein expression to produce foreign protein.
5. the construction process of a kind of dual-gene mutant Escherichia coli for recombinant protein secretion type expression according to claim 4, it is characterized in that: in described fermention medium, contain: 0.1～2% Tryptones, 0.2～10% yeast extract powder, 0.05～5% glycerine, 0.02～0.5% KH
2pO
4, 0.1～2% K
2hPO
4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210286769.5A CN102827860B (en) | 2012-08-13 | 2012-08-13 | Construction method of double-gene mutant escherichia coli for secretory expression of recombinant protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210286769.5A CN102827860B (en) | 2012-08-13 | 2012-08-13 | Construction method of double-gene mutant escherichia coli for secretory expression of recombinant protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102827860A CN102827860A (en) | 2012-12-19 |
| CN102827860B true CN102827860B (en) | 2014-06-04 |
Family
ID=47331171
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210286769.5A Expired - Fee Related CN102827860B (en) | 2012-08-13 | 2012-08-13 | Construction method of double-gene mutant escherichia coli for secretory expression of recombinant protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102827860B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287225A (en) * | 2017-06-27 | 2017-10-24 | 深圳劲宇生物科技有限公司 | Gold ion is detected and adsorption system and its Host Strains, gold ion recovery method |
| JP7185006B2 (en) * | 2018-07-06 | 2022-12-06 | ワッカー ケミー アクチエンゲゼルシャフト | Bacterial strains that release recombinant proteins by fermentation |
| EP3827085A1 (en) * | 2018-07-24 | 2021-06-02 | Wacker Chemie AG | Novel bacterial lpp mutants and the use thereof for the secretory production of recombinant proteins |
-
2012
- 2012-08-13 CN CN201210286769.5A patent/CN102827860B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| Eric Cascales等.Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity.《J. Bacteriol.》.2002,第184卷(第3期),754-759. |
| Lipoprotein Cofactors Located in the Outer Membrane Activate Bacterial Cell Wall Polymerases;Paradis-Bleau等;《Cell》;20101123;第143卷;1110–1120 * |
| Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity;Eric Cascales等;《J. Bacteriol.》;20020228;第184卷(第3期);754-759 * |
| Paradis-Bleau等.Lipoprotein Cofactors Located in the Outer Membrane Activate Bacterial Cell Wall Polymerases.《Cell》.2010,第143卷1110–1120. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102827860A (en) | 2012-12-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101098353B1 (en) | A method of secretion expression of lysostaphin in escherichia coli at high level | |
| CN113388633B (en) | Preparation of human basic fibroblast growth factor by using bacillus subtilis and endonuclease | |
| CN112522173A (en) | Engineering bacterium for producing heterologous alkaline protease and construction method thereof | |
| CN114746553B (en) | Modified cyanobacteria, method for producing modified cyanobacteria, and method for producing protein | |
| Oudega et al. | Protein H encoded by plasmid CloDF13 is involved in excretion of cloacin DF13 | |
| CN102827860B (en) | Construction method of double-gene mutant escherichia coli for secretory expression of recombinant protein | |
| CN105247036B (en) | The novel method of protein purification | |
| CN104232611A (en) | Recombinant beauveria brongniartii proteinase K as well as industrial production and purification method thereof | |
| CN102827904B (en) | Integrated method for extracellular production of foreign protein by escherichia coli | |
| CN107723287B (en) | Expression system for enhancing production and preparation of silk protein | |
| JP2022134906A (en) | Methods for producing biofilms, biofilms and methods for producing organic materials | |
| CN114672525A (en) | Biosynthesis method and application of N-acetyl-5-methoxytryptamine | |
| KR102473375B1 (en) | Recombinant microorganisms, their preparation methods and their use in the production of coenzyme Q10 | |
| CN116555319A (en) | Method for synthesizing alligator antimicrobial peptide AM-CATH by multi-copy integration and high-density fermentation | |
| TW201738263A (en) | A preparation method of recombinant human granulocyte colony stimulating factor | |
| CN111349575B (en) | Pichia pastoris engineering bacteria for constitutive expression of porcine pepsinogen C and application thereof | |
| KR20160078068A (en) | Polypeptide for purification of target protein and use thereof | |
| CN107988290A (en) | A kind of biological method for improving glutathione cumulant | |
| CN108220317B (en) | Recombinant expression plasmid and preparation method and application thereof | |
| US10155964B2 (en) | Transformed Synechococcus elongatus strain having capability of producing squalene from carbon dioxide and method for producing squalene using the same | |
| CN117660511B (en) | Rhodobacter sphaeroides gene editing method based on RecET recombination system and application thereof | |
| CN111349576B (en) | Pichia pastoris engineering bacteria for constitutive expression of porcine pepsinogen A and application thereof | |
| CN116731144B (en) | Recombinant rice antioxidant peptide and preparation method and application thereof | |
| CN117701487B (en) | Bacillus licheniformis engineering bacteria for efficiently expressing amylase genes and application thereof | |
| CN116200420A (en) | Tandem multi-copy recombinant expression vector for efficiently expressing king cobra antibacterial peptide OH-CATH30 and application thereof |
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 |
Granted publication date: 20140604 Termination date: 20140813 |
|
| EXPY | Termination of patent right or utility model |