CN103757012A - Expression equipment for secretory expression of foreign protein by penicillium expansum and genetically engineered bacterium thereof - Google Patents
Expression equipment for secretory expression of foreign protein by penicillium expansum and genetically engineered bacterium thereof Download PDFInfo
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- CN103757012A CN103757012A CN201410005275.4A CN201410005275A CN103757012A CN 103757012 A CN103757012 A CN 103757012A CN 201410005275 A CN201410005275 A CN 201410005275A CN 103757012 A CN103757012 A CN 103757012A
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Abstract
The invention discloses expression equipment for secretory expression of a foreign protein by penicillium expansum and a genetically engineered bacterium thereof. The expression equipment sequentially comprises the following members from 5' to 3': (1) a lipase gene promoter of penicillium expansum; (2) a signal peptide of secretory expression; (3) multiple cloning sites; (4) a terminator of a gene C of aspergillus nidulans tryptophan synthetase. By inserting the foreign gene into the expression equipment, converting into agrobacterium tumefaciens by T-DNA (Transferred-Deoxyribonucleic Acid) and combining with penicillium expansum, the genetically engineered bacterium of penicillium expansum obtained can efficiently secretory-express heterologous genes originated from animals, plants and microorganisms, so that the foreign protein obtained can be produced on a large scale. The penicillium expansum is exuberant to grow, the condition of culture is extensive and cheap, both solid culture and liquid submerged fermentation are feasible, and the extracelluar protein produced is easy to separate and purify, so that the expression equipment is suitable for producing proteins with great industrial demands.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of expression equipment and genetic engineering bacterium thereof of Penicilllum expansum secreting, expressing foreign protein.
Background technology
Protein recombinant expressed aspect, filamentous fungus expression system has the unique advantage that is superior to prokaryotic expression system and yeast expression system.Prokaryotic expression system take intestinal bacteria as representative, lacks translation post-treatment and folding mechanism; Therefore, when high efficient expression foreign protein, easily produce inclusion body, and renaturing inclusion bodies is an extremely complicated and difficult process.Although the yeast expression system take yeast saccharomyces cerevisiae as representative can correctly be translated and fold when expressing foreign protein, easily produce excessive glycosylation, while therefore expressing higher eucaryote gene, produce activated protein ratio more difficult.Filamentous fungus has more senior translation, folding and modification system, can make up above-mentioned deficiency completely.
Penicilllum expansum is a kind of filamentous fungus that is separated to from soil, this filamentous fungus has powerful lipase throughput, through mutagenesis and seed selection, the level of its yielding lipase reached industrial requirement (Li Qiang. the expression of Penicilllum expansum FS1884 lipase gene in Pichia pastoris GS115 and orthogenesis research. Fujian Normal University master thesis .2009, pp8.); This promotor vigor of lipase gene that shows Penicilllum expansum is very strong, and the translation of intracellular protein, folding and modification system is very flourishing.In addition, Penicilllum expansum is very easily cultivated, and between pH3-10, all can well grow, and fermention medium only needs soybean cake powder, starch and a small amount of inorganic microelement, and zymotechnique maturation, has good industrial foundation.Therefore, Penicilllum expansum is applicable to being developed to the expression system of recombinant protein very much, for expressing the heterologous gene that derives from animal, plant and microorganism etc.
Summary of the invention
The object of the invention is to for exogenous protein expression amount lowly, the deficiency of separation and purification difficulty, provides expression equipment and the genetic engineering bacterium thereof of a kind of Penicilllum expansum simple and efficient to handle (Penicillium expansum) secreting, expressing foreign protein.The Penicilllum expansum genetic engineering bacterium that contains this expression equipment, can efficient secretory expression derives from the heterologous genes of animal, plant and microorganism etc.
The object of the invention is to be achieved through the following technical solutions:
First aspect, the present invention relates to the expression equipment of a kind of Penicilllum expansum (Penicillium expansum) secreting, expressing foreign protein, wherein, described expression equipment from 5 ' to 3 ' comprises following element successively: (1) controls the penicillium expansum lipase gene promoter P of foreign gene at Penicilllum expansum transcription
lip; (2) encoding sequence of control foreign protein signal peptide of secreting, expressing in Penicilllum expansum; (3) multiple clone site; (4) control foreign gene and in Penicilllum expansum, stop the Aspergillus nidulans tryptophan synthetase terminator T transcribing
trpC.
In the present invention, described promotor is to control the promotor of heterologous gene at Penicilllum expansum transcription, is preferably penicillium expansum lipase gene promoter P
lip, as shown in SEQ ID NO:7 the 1st to 1859 of better promoter sequence.
In the present invention, described signal peptide can be any signal peptide (secretion peptide) that can control foreign protein secreting, expressing in Penicilllum expansum in this area, preferably as shown in SEQ ID NO:7 the 1860th to 1922 of the encoding sequence of signal peptide.
In the present invention, described multiple clone site can be the multiple clone site of the various routines in this area, and preferably multiple clone site sequence is as shown in SEQ ID NO:8.
In the present invention, described terminator is can control heterologous gene in Penicilllum expansum, to stop the terminator of transcribing, the preferably terminator T of Aspergillus nidulans tryptophan synthetase C
trpC, better terminator sequence the 1st to the 776th of sequence as shown in SEQ ID NO:9.
Preferably described expression equipment also comprises following element: the expression cassette of (5) selection markers gene.It is positioned at Expression element (1) promotor P
lipupstream or element (4) terminator T
trpCdownstream.
In the present invention, the expression cassette of described selection markers gene can be expressed the conventional selection markers of this area in Penicilllum expansum, preferably selection markers is that neomycin phosphotransferase or hygromix phosphotransferase, better selection markers are hygromix phosphotransferases, and its better gene order is as shown in SEQ ID NO:10.
Preferably, the expression equipment of described Penicilllum expansum secreting, expressing foreign protein also further comprises the encoding sequence of foreign protein, it is positioned among described (3) element multiple clone site, before or after.Described foreign protein genes derives from plant, animal, microorganism and synthetic etc.
Second aspect, the present invention relates to a kind of carrier that contains above-mentioned any expression equipment.Preferably agrobacterium tumefaciens Second Academy carrier, as T-DNA expression vector pPZP100, pPZP201, pPZP201B, pPZP201BK, pBI121, pCAMBIA or pPK2, but is not limited to these carriers, the better pCAMBIA that is selected from.
The third aspect, the present invention relates to a kind of transformant that contains above-mentioned any carrier (host cell).Preferably agrobacterium tumefaciens LBS4404, GV2260, C58C1, GV3100, A136, GV3101, AGL-1, EHA101, EHA105, more preferably agrobacterium tumefaciens EHA105.
Fourth aspect, the present invention relates to a kind of Penicilllum expansum genetic engineering bacterium of secreting, expressing foreign protein, contains the aforesaid expression equipment of the encoding sequence that further comprises foreign protein in the genome of described genetic engineering bacterium.
The 5th aspect, the present invention relates to a kind of preparation method of Penicilllum expansum genetic engineering bacterium of secreting, expressing foreign protein, comprise the agrobacterium tumefaciens binary vector of the aforementioned expression equipment that contains foreign gene is transformed to agrobacterium tumefaciens, agrobacterium tumefaciens transformant and the Penicilllum expansum of gained are cultivated altogether, select the zygote that is integrated with foreign protein genes expression equipment in genome, be the Penicilllum expansum genetic engineering bacterium of expressing foreign protein.This Penicilllum expansum genetic engineering bacterium can high efficient expression derives from the heterologous gene of animal, plant and microorganism etc.
The 6th aspect, the present invention relates to a kind of raw albuminiferous method, comprises the Penicilllum expansum genetic engineering bacterium of cultivating described expression foreign protein, obtains this foreign protein from culture system.
The 7th aspect, the present invention relates to a kind of Penicilllum expansum genetic engineering bacterium of above-mentioned secreting, expressing foreign protein in the purposes of preparing in industrial enzyme preparation, fodder additives or pharmaceutical grade protein.Described zymin is lipase, polygalacturonase, cellulase or tannic acid enzyme.
Compared with prior art, the present invention has following beneficial effect:
1, Penicilllum expansum of the present invention is expressed equipment on cyclisation plasmid, is easy to preserve and DNA operates.
2, Penicilllum expansum of the present invention is expressed equipment utilization Agrobacterium in conjunction with transfer techniques, can improve transformation efficiency, shortens operating time and workload.
3, the promotor (P that Penicilllum expansum expression equipment of the present invention contains Penicilllum expansum source
lip), this promotor and bacterial strain have been evolved a lot of years jointly, and the matching degree of promotor and bacterial strain is high, active strong; Can drive the high efficient expression of most heterologous genes.
4, Penicilllum expansum expression equipment of the present invention has the secreting, expressing ability of optimization, is applicable to the secreting, expressing of most of heterologous genes.
5, the multiple clone site of Penicilllum expansum expression equipment of the present invention contains rare interface, and the connection of compatible most heterologous genes is saved the operating time and increased work efficiency.
6, genetic engineering bacterium of the present invention can be dredged cotton shape thermophilic silk born of the same parents' bacterium lipase (TLL) by high efficient expression, in washing and process hides field, has important application.
7, expression equipment provided by the invention, can realize the high efficient expression of the heterologous gene that derives from animal, plant and microorganism in Penicilllum expansum.Because Penicilllum expansum culture condition is extensive, be suitable for solid culture and liquid submerged fermentation, with low cost; Therefore the present invention can produce bacterial strain for the zymin industries such as washing, process hides, weaving, papermaking provide genetic engineering bacterium, and the production efficiency that improves zymin reduces production costs.
Accompanying drawing explanation
By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:
Fig. 1 is the physical map of the expression equipment of Penicilllum expansum secreting, expressing foreign protein; Wherein, P
lip: penicillium expansum lipase gene promoter; Sig: secreting signal peptide; MSC: multiple clone site; T
trpC: Aspergillus nidulans tryptophan synthetase C terminator; Hyg Box: hygromycin phosphotransferase gene selection markers box; Sal I, Pme I: be corresponding restriction enzyme site;
Fig. 2 is Penicilllum expansum expression vector pCHAMBIA2300::P
lip-Sig-T
trpCdesign of graphics;
Fig. 3 is the recombinant expression vector pCHAMBIA2300::P that contains beta-glucosiduronatase gene GUS
lip-Sig-GUS-T
trpCdesign of graphics;
Fig. 4 is for containing the recombinant expression vector pCHAMBIA2300::P that dredges cotton shape thermophilic silk born of the same parents' bacterium lipase (TLL)
lip-Sig-TLL-T
trpCdesign of graphics;
Fig. 5 is the secreting, expressing of gus gene in Penicilllum expansum, and wherein, A is the result of common Penicilllum expansum after GUS dyeing; B is the mould genetic engineering bacterium that the contains described secreting, expressing equipment result after GUS dyeing.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.Following examples will contribute to those skilled in the art further to understand the present invention, but not limit in any form the present invention.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make certain adjustments and improvements.These all belong to protection scope of the present invention.
In the present invention, penicillium expansum lipase gene promoter, at 5 ' end of this expression equipment, is approximately comprised of 1859 Nucleotide.Terminator, at 3 ' end of this expression equipment, is approximately comprised of 776 Nucleotide.In this expression equipment, after promotor, also connecting one section of secretion peptide sequence, the secretion peptide of 21 aminoacid sequences of coding, and added restriction endonuclease sites (multiple clone site after secretion peptide, MCS), thereby be convenient to foreign gene, smoothly embed and expressed, thus the present invention to have obtained structure be the expression equipment of promotor-secretion peptide-MCS-terminator.This expression equipment can be in Penicilllum expansum the various foreign proteins of secreting, expressing, expression amount is high, be easy to separation and purification, with low cost.
One, Penicilllum expansum is expressed the structure of equipment
The invention provides a kind of expression equipment of Penicilllum expansum secreting, expressing foreign protein.Described expression equipment is transferred to Penicilllum expansum cell by Agrobacterium combination.By cultivating Penicilllum expansum genetic engineering bacterium, foreign protein is able to efficient secretory expression.
As shown in Figure 1, its preparation method is as follows for the physical map of Penicilllum expansum expression equipment of the present invention:
(1), by Restriction Enzyme incision technology, obtain hygromycin phosphotransferase gene expression cassette, as selection markers.
(2) by pcr amplification, obtain respectively comprising the penicillium expansum lipase gene promoter (P that secretes peptide sequence
lip) and Aspergillus nidulans tryptophan synthetase C terminator (T
trpC).
(3) by hygromycin B selection markers box, promotor (P
lip), multiple clone site, terminator (T
trpC) be connected in expression vector, build and obtain recombinant expression vector 1, be pCHAMBIA2300::P
lips-Sig-T
trpC, corresponding design of graphics as shown in Figure 1.The expression vector here mainly plays a large amount of this table of copy equipment and provides Agrobacterium in conjunction with shifting site, so can select any Agrobacterium binary vector, as pPZP100, pPZP201, pPZP201B, pPZP201BK, pBI121, pCAMBIA or pPK2, but be not limited to these carriers.
(4) heterologous gene is inserted in above-mentioned expression vector 1, foreign gene is positioned at Sig and T
trpCbetween, obtain recombinant expression plasmid 2, be pCHAMBIA2300::P
lip-Sig-GUS-T
trpCand pCHAMBIA2300::P
lip-Sig-TLL-T
trpC; Corresponding design of graphics respectively as shown in Figure 3,4.
In above-mentioned steps (3), selection markers, promotor, multiple clone site and terminator are connected in destination carrier, the method that can be connected with ligase enzyme with digestion with restriction enzyme is connected into expression vector by each element, and it is all the conventional way in this area that described digestion with restriction enzyme is connected with ligase enzyme.
In above-mentioned steps (4), heterologous gene is inserted in above-mentioned expression vector 1, the method that can be connected with ligase enzyme with digestion with restriction enzyme is connected into target gene in expression vector 1, and it is all the conventional way in this area that described digestion with restriction enzyme is connected with ligase enzyme.
Two, Penicilllum expansum genetic engineering bacterium
The invention provides a kind of Penicilllum expansum genetic engineering bacterium, this genetic engineering bacterium is that expression equipment of the present invention is transferred to Penicilllum expansum cell by Agrobacterium combination, cultivates the preparation of screening transformant and obtains, and concrete preparation process is as follows:
(1) carrier that contains expression equipment of the present invention is transformed to agrobacterium tumefaciens; Recycling transforms Penicilllum expansum in conjunction with metastasis.
(2) cell of above-mentioned conversion is screened, identified, obtain expressing the restructuring Penicilllum expansum bacterial classification of foreign protein.
Below in conjunction with specific embodiment, further illustrate the present invention, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturers.Experiment material used in the following example, if no special instructions, is and purchases available from routine biochemistry reagent shop.
Penicilllum expansum bacterial strain therefrom traditional Chinese medicines is bought (Center for culture collection of pharmaceutical microorganisms, CPCC) with microbial strains preservation administrative center, and deposit number is CPCC460013.After purchase, be seeded on PDA slant medium, under 26 ℃ of conditions, cultivate 20 days, be then stored in 4 ℃ of refrigerators.
E.colistraindh5α, in < < Liu Jing China, wraps quintessence, Chen Yanfei; The improvement of bacillus coli DH 5 alpha competent cell transformation efficiency, Shaoguan College's journal, 2008,29 (03): open in 87-90 > > document.The competent cell of e.colistraindh5α can be bought by supplier TIANGEN Biotech (Beijing) Co., Ltd., article No. CB101-03.
Agrobacterium tumefaciens EHA105 is yellow sub-beautiful at < <, Jiang Xiliang, Yunlong, field, Guo Ping, Zhu Changxiong; The research of Agrobacterium tumefaciens mediated trichoderma harziarum genetic transformation, Chinese biological engineering magazine, 2008,28 (3): open in 38-43 > > document.Agrobacterium tumefaciens EHA105 can obtain by openly commercially available commercial channel, and as buied from Chinese plasmid vector strain cell pnca gene preservation center BiovectorScience Lab, article No. is Biovector-375.
In following embodiment of the present invention, substratum used and solution formula are as follows:
CTAB extracting solution: 2%CTAB; 2%PVP; 100mM Tris-HCl pH8.0; 20mM EDTA; 1.4M NaCl; During use, add the mercaptoethanol of 2% (v/v).
PDA substratum (g/L): potato, 200; Sucrose, 20; Agar, 15; PH nature.
MM substratum: 1M K
2hPO
4-KH
2pO
4(pH7.0) 10mL, M-N (MgSO
47H
2o30g/L, NaCl15g/L) 20mL, 1%CaCl
22H
2o1mL, 20% glucose 10mL, 0.01%FeSO
410mL, Spore element (ZnSO
47H
2o100mg/L, CuSO
45H
2o100mg/L, H
3bO
3100mg/L, MnSO
4h
2o100mg/L, Na
2moO
42H
2o100mg/L)) 5mL, 20%NH
4nO
32.5mL, sterilized water 941.5mL.
IM substratum: 1M K
2hPO
4-KH
2pO
4(pH7.0) 10mL, M-N (MgSO
47H
2o30g/L, NaCl15g/L) 20mL, 1%CaCl
22H
2o1mL, 20% glucose 10mL, 0.01%FeSO
410mL, Spore element (ZnSO
47H
2o100mg/L, CuSO
45H
2o100mg/L, H
3bO
3100mg/L, MnSO
4h
2o100mg/L, Na
2moO
42H
2o100mg/L) 5mL, 20%NH
4nO
32.5mL, 50% glycerine 10mL, 1M MES (pH5.5) 40mL, 100mM AS (Syringylethanone) 2mL, sterilized water 898.7mL.
CM substratum: add the glucose amount of half IM substratum, additional 1.5% agar, other composition is with IM substratum.
GM substratum (%): soybean cake powder 4, W-Gum 0.8, NaNO
30.3, Na
2hPO
40.2, K
2sO
40.25, MgSO
40.03, FeSO
40.003.
FM substratum (%): soybean cake powder 6, W-Gum 1, NaNO
30.4, Na
2hPO
40.2, K
2sO
40.3, MgSO
40.035, FeSO
40.02, CaCO
30.5, Na
2cO
30.02.
GUS formula for dye liquor: 100mM Na
3pO
4pH7.0,2mM X-Gluc, 0.5%Triton X-100,2mM K
3[Fe (CN)
6], 2mM K
4[Fe (CN)
6]
Czapek's solution formula (g/L): NaNO
33; K
2hPO
41; M
gsO
40.5; KCl0.5; FeSO
40.01; Sucrose 30;
Producing of sweet oil emulsion: 4%PVA solution and sweet oil are 2: 1 (v/v), is put into (outsourcing ice cube) in little triangular flask, and being adjusted into rotating speed is 10000 revs/min, an emulsification 3 minutes, 3 minutes, interval, altogether emulsification 3 times.
the preparation of embodiment 1, Penicilllum expansum heterogenous expression equipment
(1) utilize restriction enzyme Sac I, Sal I by hygromycin resistance expression cassette from plasmid pV2 (Wang Y, Guo B, Miao Z, Tang K.Transformation of taxol-producing endophytic fungi by restriction enzyme-mediated integration (REMI) .FEMS Microbiology letter, 2007,253-259) upper enzyme cuts out, fragment reclaims through gel, be cloned on the corresponding restriction enzyme site of pCAMBIA2300 plasmid, obtain the recombinant plasmid pCHAMBIA2300 with hygromycin resistance; Any DNA that Totomycin expression cassette also can contain this sequence from other, wherein the nucleotide sequence of hygromycin phosphotransferase gene is as shown in SEQ ID NO:10.
(2) get Penicilllum expansum mycelium 0.2g through liquid nitrogen grinding, add 1mL CTAB extracting solution, 65 ℃ of water-bath 30min, the centrifugal 10min of 12000rpm/min, gets supernatant liquor; The KAC (2.5M, pH4.8) that enters 0.7mL, 12000rpm/min is centrifugal, and 10min is centrifugal, gets supernatant liquor; Then toward the Virahol that adds 0.6mL in supernatant liquor, precipitation DNA; Genomic dna, through 75% washing with alcohol twice, is dissolved in distilled water standby.
(3) according to the sequence (GenBank Accession:AF288685) of the sequence of the flank of penicillium expansum lipase gene 5 ' end (GenBank Accession:DQ677520) and penicillium expansum lipase gene, design:
Forward primer P1, its nucleotide sequence, as shown in SEQ ID NO:1, wherein contains Restriction Enzyme Sal I point of contact;
Reverse primer P2, its nucleotide sequence, as shown in SEQ ID NO:2, wherein contains Restriction Enzyme Spe I point of contact;
Take Penicilllum expansum genomic dna as template, utilize round pcr amplification complete genome sequence.PCR reaction conditions is: 95 ℃ of 5min; 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 120s, 35 circulations; 72 ℃ of 10min; Then target fragment is reclaimed, obtained the nucleotide sequence that comprises promotor and secreting signal peptide as shown in SEQ ID NO:7.
(4) according to the sequences Design of plasmid pPAN7-1 (GenBank Accession:Z32698):
Forward primer P3, its nucleotide sequence is as shown in SEQ ID NO:3, and this primer comprises multiple clone site sequence as shown in SEQ ID NO:8, wherein contains Restriction Enzyme SpeI and Swa I point of contact;
Reverse primer P4, its nucleotide sequence, as shown in SEQ ID NO:4, wherein contains Restriction Enzyme Pme I point of contact;
Utilize round pcr to amplify tryptophan synthetase C terminator T
trpC, its nucleotide sequence is as shown in SEQ ID NO:9.PCR reaction conditions is: 95 ℃ of 5min; 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 60s, 35 circulations; 72 ℃ of 10min.
(5) get the each 1 μ L of reaction solution of (3) and (4) as template, using P1 as forward primer; Using P4 as reverse primer, carry out pcr amplification, the reaction conditions of PCR is: 95 ℃ of 5min; 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 180s, 35 circulations; 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out to gel column recovery, and with restriction enzyme Sal I and Pme I, carry out enzyme and cut, be then cloned into the corresponding site of pCHAMBIA2300, obtain recombinant expression vector 1, be pCHAMBIA2300::P
lip-Sig-T
trpC.
(6) according to the sequences Design of gus gene (GenBank Accession:S69414):
Forward primer P5, its nucleotide sequence, as shown in SEQ ID NO:5, wherein contains Restriction Enzyme Spe I point of contact;
Reverse primer P6, its nucleotide sequence, as shown in SEQ ID NO:6, wherein contains Restriction Enzyme Swa I point of contact;
Utilize round pcr amplification complete genome sequence.PCR reaction conditions is: 95 ℃ of 5min; 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 120s, 35 circulations; 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out to gel column recovery, and with restriction enzyme Spe I and Swa I, carry out enzyme and cut, be then cloned into pCHAMBIA2300::P
lip-Sig-T
trpCcorresponding site obtains final carrier pCHAMBIA2300::P
lip-Sig-GUS-T
trpC.
(7) to containing final carrier pCHAMBIA2300::P
lip-Sig-GUS-T
trpCbacterium carry out enlarged culturing, and extracting plasmid, utilizes freeze-thaw method Transformation Engineering Agrobacterium EHA105.Using P5 as forward primer; Using P6 as reverse primer, selected strain bacterium is carried out to PCR evaluation, be accredited as positive bacterial strain and be correct restructuring Agrobacterium EHA105-pCHAMBIA2300::P
lip-Sig-GUS-T
trpC, can further carry out follow-up experiment.
the acquisition of embodiment 2, Penicilllum expansum genetic engineering bacterium
Detailed step is as follows:
(1) the wild-type Penicilllum expansum after picking separation and purification is inoculated on PDA flat board, in 28 ℃ of cultivations about 20 days, with ripe spore under aseptic washing.
(2) whole carrier pCHAMBIA2300::P will be contained in example 1
lip-Sig-GUS-T
trpCengineering Agrobacterium EHA105 be inoculated in the LB liquid nutrient medium that contains 100 μ g/mL Streptomycin sulphates, 100 μ g/mL kantlex 28 ℃, 200rpm incubated overnight, reactivate with the MM substratum that contains 100 μ g/mL Streptomycin sulphates and 100 μ g/mL kantlex, 28 ℃, 220rpm cultivates 48 hours.Draw the centrifugal supernatant that goes of appropriate culture 5000rpm, and with the washing of IM liquid nutrient medium, finally with IM liquid nutrient medium, be diluted to OD
600=0.15, then at 28 ℃, under the condition of 220rpm, cultivate 6～8 hours, to OD
600=0.5～0.6.
(3) the fresh spore (1) step being obtained is mixed with 1 × 10
7the suspension of individual/mL concentration, getting subsequently above-mentioned spore suspension mixes with the engineering Agrobacterium equal-volume in step (2), getting 200 μ L is evenly applied on the CM substratum (containing AS200 μ g/mL) that is covered with glassine paper, cultivate altogether 60h for 28 ℃, then anti-glassine paper being taped against contained to Totomycin (100 μ g/mL transform and select microbiotic), cynnematin (500 μ g/mL, suppress Agrobacterium growth microbiotic) PDA substratum on 28 ℃ cultivate 2 days, take glassine paper off, under 28 ℃ of conditions, cultivate 1～3 day, the transformant of moisture resistance mycin is chosen and is inoculated on two sieve substratum, as stable, go down to posterity, be described Penicilllum expansum genetic engineering bacterium.
embodiment 3, utilize Penicilllum expansum to express device fabrication gus protein
By obtaining Penicilllum expansum genetic engineering bacterium and wild-type penicillium expansum in embodiment 2, be inoculated into respectively in GM substratum, in 220rpm, 26 ℃ of concussions, cultivate 2 days then filtered and recycled mycelium.Mycelium, through distillation washing three times, then, respectively from putting into an eppendorf pipe, adds GUS dye liquor, hatches 3-5h for 37 ℃, observes dyeing situation.Refer to Fig. 4, Fig. 4 demonstration has proceeded to pCHAMBIA2300::P
lip-Sig-GUS-T
trpCthe dye liquor of Penicilllum expansum genetic engineering bacterium presented significant blueness (Fig. 5 B), and the dye liquor color at the Penicilllum expansum bacterial strain place of wild-type does not change (Fig. 5 A); Result shows, using after expression equipment of the present invention, foreign protein genes GUS in Penicilllum expansum genetic engineering bacterium with high efficient expression, and secretion peptide guiding under be secreted into outside born of the same parents.
embodiment 4, utilize the present invention to express equipment secreting, expressing to dredge the thermophilic silk of cotton shape born of the same parents' bacterium lipase (TLL)
4.1, express the structure of equipment
(1) according to the sequence of TLL (GenBank Accession:AF054513), design:
Forward primer P7, its nucleotide sequence, as shown in SEQ ID NO:11, wherein contains Restriction Enzyme Spe I point of contact;
Reverse primer P8, its nucleotide sequence, as shown in SEQ ID NO:12, wherein contains Restriction Enzyme Swa I point of contact;
Take the TLL gene order (GenBank Accession:AF054513) of synthetic as template, utilize round pcr its mature peptide sequence (containing ttl signal peptide sequence) that increases.PCR reaction conditions is: 95 ℃ of 5min; 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 120s, 35 circulations; 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out to gel column recovery, and with restriction enzyme Spe I and Swa I, carry out enzyme and cut, be then cloned into pCHAMBIA2300::P
lip-Sig-T
trpCcorresponding site obtains final carrier pCHAMBIA2300::P
lip-Sig-TLL-T
trpC.
(2) to containing final carrier pCHAMBIA2300::P
lip-Sig-TLL-T
trpCbacterium carry out enlarged culturing, and extracting plasmid, utilizes freeze-thaw method Transformation Engineering Agrobacterium EHA105.Using P7 as forward primer; Using P8 as reverse primer, selected strain bacterium is carried out to PCR evaluation, be accredited as positive bacterial strain and be correct restructuring Agrobacterium EHA105-pCHAMBIA2300::P
lip-Sig-TLL-T
trpC, can further carry out follow-up experiment.
4.2, the acquisition of Penicilllum expansum genetic engineering bacterium
(1) the wild-type Penicilllum expansum after picking separation and purification is inoculated on PDA flat board, in 28 ℃ of cultivations about 20 days, with ripe spore under aseptic washing.
(2) whole carrier pCHAMBIA2300::P will be contained in example 4.1
lip-Sig-TLL-T
trpCengineering Agrobacterium EHA105 be inoculated in the LB liquid nutrient medium that contains 100 μ g/mL Streptomycin sulphates, 100 μ g/mL kantlex 28 ℃, 200rpm incubated overnight, reactivate with the MM substratum that contains 100 μ g/mL Streptomycin sulphates and 100 μ g/mL kantlex, 28 ℃, 220rpm cultivates 48 hours.Draw the centrifugal supernatant that goes of appropriate culture 5000rpm, and with the washing of IM liquid nutrient medium, finally with IM liquid nutrient medium, be diluted to OD
600=0.15, then at 28 ℃, under the condition of 220rpm, cultivate 6～8 hours, to OD
600=0.5～0.6.
(3) the fresh spore (1) step being obtained is mixed with 1 × 10
7the suspension of individual/mL concentration, getting subsequently above-mentioned spore suspension mixes with the engineering Agrobacterium equal-volume in step (2), getting 200 μ L is evenly applied on the CM substratum (containing AS200 μ g/mL) that is covered with glassine paper, cultivate altogether 60h for 28 ℃, then anti-glassine paper being taped against contained to Totomycin (100 μ g/mL transform and select microbiotic), cynnematin (500 μ g/mL, suppress Agrobacterium growth microbiotic) PDA substratum on 28 ℃ cultivate 2 days, take glassine paper off, under 28 ℃ of conditions, cultivate 1～3 day, the transformant of moisture resistance mycin is chosen and is inoculated on two sieve substratum, as stable, go down to posterity, be described Penicilllum expansum genetic engineering bacterium.
4.3, the secreting, expressing of allos lipase gene TLL in Penicilllum expansum genetically engineered
Because natural Penicilllum expansum itself just produces lipase, but the thermostability of the penicillium expansum lipase that it produces is very poor, under 55 ℃ of conditions, is incubated after 30min, and its lipase albumen is all sex change just, is difficult to detect lipase activity again.And TLL lipase is a kind of heat stable lipase, it is incubated after 30min under 50～60 ℃ of conditions, still retains the enzyme activity of 90% left and right, so the present embodiment just utilizes this characteristic to detect the secreting, expressing of TLL.
In random choose example 4.2, the mould genetic engineering bacterium of gained and unconverted penicillium expansum (in contrast) are inoculated on PDA substratum, cultivate after 15 days and access respectively in GM substratum 50mL, at 28 ℃, 210rpm shaking table is cultivated 24h, then with 10% inoculum size, proceed to respectively FM substratum (the bottled 30mL fermention medium of 250mL triangle), at 28 ℃, 210rpm condition bottom fermentation 48h; Then fermented liquid is centrifugal, supernatant liquor is placed under 55 ℃ of conditions and is incubated 30min, finally carries out lipase activity detection.
Utilize acid base titration to detect lipase activity, detecting step is: get 20 of 100mL triangular flasks, add respectively Gly-NaOH damping fluid and the 5.0mL sweet oil emulsion of 4.0mL pH9.4; Put into 36 ℃ of water-bath preheatings of concussion thermostat water bath 5 minutes.After enzyme liquid filters, with the Gly-NaOH damping fluid of 0.05mol/L pH9.4, dilute that to make enzyme work be to measure after 4-5u/mL.Toward wherein two enzyme liquid that respectively add 1mL to dilute, slowly vibration (60 times/min) 10 minutes (accurately timing).Other two bottles are done blank. and add immediately 95% alcohol 20mL (the enzyme liquid that blank adds 1mL to dilute), shake up, add the sodium chloride solution of 10mL30%, shake up; Make it identical with the pH of blank with 0.01mol/L NaOH solution titration sample, write down 0.01mol/L NaOH consumption.
Calculate
X=A×B×1/T×n
In formula:
The enzyme activity (U/g or U/mL) of X---sample;
A---the volume (mL) of quota of expenditure 0.01mol/L NaOH during titration sample;
The concentration (μ mol/mL) of NaOH for B--titration
T---time of enzymatic reacting (min);
N---extension rate;
Do simultaneously two parts parallel, results averaged, acquired results represents to integer.Parallel test relative error must not exceed 5.0%.
The enzyme activity determination of Penicilllum expansum genetic engineering bacterium and non-transgenic mould the results are shown in Table 1.
Table 1 lipase activity measurement result (enzyme activity unit: U/mL)
| Strain number | C | T 1 | T 2 | T 3 | T 4 | T 5 |
| Enzyme activity | 0 | 450±20 | 480±30 | 330±40 | 260±15 | 370±50 |
Wherein C is non-transgenic Penicilllum expansum bacterial strain (contrast), T
1, T
2, T
3, T
4and T
5for the independently Penicilllum expansum genetic engineering bacterium obtaining.As shown in Table 1, use expression equipment of the present invention, the TLL lipase of allos is able to efficient secretory expression in Penicilllum expansum, only with machine testing 5 strain Penicilllum expansum genetic engineering bacteriums, just detect that output is up to 480U/mL, therefore expression equipment of the present invention has important actual application value.
Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or modification within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (12)
1. an expression equipment for Penicilllum expansum secreting, expressing foreign protein, is characterized in that, described expression equipment from 5 ' to 3 ' comprises following element successively: (a) control the penicillium expansum lipase gene promoter P of foreign gene at Penicilllum expansum transcription
lip; (b) encoding sequence of control foreign protein signal peptide of secreting, expressing in Penicilllum expansum; (c) multiple clone site; (d) control foreign gene and in Penicilllum expansum, stop the Aspergillus nidulans tryptophan synthetase terminator T transcribing
trpC.
2. the expression equipment of Penicilllum expansum secreting, expressing foreign protein as claimed in claim 1, is characterized in that, described penicillium expansum lipase gene promoter P
lipsequence the 1st to the 1859th of sequence as shown in SEQ ID NO:7; Described Aspergillus nidulans tryptophan synthetase terminator T
trpCsequence the 1st to the 776th of sequence as shown in SEQ ID NO:9.
3. the expression equipment of Penicilllum expansum secreting, expressing foreign protein as claimed in claim 1, it is characterized in that the encoding sequence of described control foreign protein signal peptide of secreting, expressing in Penicilllum expansum the 1860th to the 1922nd of sequence as shown in SEQ ID NO:7.
4. the expression equipment of Penicilllum expansum secreting, expressing foreign protein as claimed in claim 1, is characterized in that, the sequence of described multiple clone site is as shown in SEQ ID NO:8.
5. the expression equipment of Penicilllum expansum secreting, expressing foreign protein as claimed in claim 1, is characterized in that, described expression equipment also comprises following element: (e) expression cassette of selection markers gene.
6. the expression equipment of the Penicilllum expansum secreting, expressing foreign protein as described in any one in claim 1～5, is characterized in that, described expression equipment also comprises the encoding sequence of foreign protein, and it is positioned among described multiple clone site, before or afterwards.
7. the carrier containing the expression equipment just like the Penicilllum expansum secreting, expressing foreign protein described in any one in claim 1～6.
8. one kind contains the transformant of carrier as claimed in claim 7.
9. a Penicilllum expansum genetic engineering bacterium for secreting, expressing foreign protein, is characterized in that, contains expression equipment as claimed in claim 6 in the genome of described genetic engineering bacterium.
10. the preparation method of the Penicilllum expansum genetic engineering bacterium of a secreting, expressing foreign protein as claimed in claim 9, it is characterized in that, comprise the agrobacterium tumefaciens binary vector that contains the equipment of expressing is as claimed in claim 7 transformed to agrobacterium tumefaciens, by the agrobacterium tumefaciens transformant of gained and Penicilllum expansum combination, select the zygote that is integrated with foreign protein genes expression equipment in genome, be the Penicilllum expansum genetic engineering bacterium of described secreting, expressing foreign protein.
11. 1 kinds of albuminiferous methods of life, is characterized in that, comprise the Penicilllum expansum genetic engineering bacterium of cultivating secreting, expressing foreign protein as claimed in claim 9, obtain foreign protein from culture system.
The Penicilllum expansum genetic engineering bacterium of 12. 1 kinds of secreting, expressing foreign proteins as claimed in claim 9 is in the purposes of preparing in industrial enzyme preparation, fodder additives or pharmaceutical grade protein.
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|---|---|---|---|---|
| CN110938648A (en) * | 2019-12-09 | 2020-03-31 | 中国热带农业科学院热带生物技术研究所 | Fungus secretion expression vector, construction method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154358A (en) * | 2011-01-04 | 2011-08-17 | 深圳市绿微康生物工程有限公司 | Construction method of efficiently-expressed plasmid for producing lipase gene |
| CN102154340A (en) * | 2011-01-04 | 2011-08-17 | 深圳市绿微康生物工程有限公司 | Method for obtaining strain capable of expressing lipase effectively |
-
2014
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154358A (en) * | 2011-01-04 | 2011-08-17 | 深圳市绿微康生物工程有限公司 | Construction method of efficiently-expressed plasmid for producing lipase gene |
| CN102154340A (en) * | 2011-01-04 | 2011-08-17 | 深圳市绿微康生物工程有限公司 | Method for obtaining strain capable of expressing lipase effectively |
Non-Patent Citations (2)
| Title |
|---|
| 杨月梅 等: "扩展青霉碱性脂肪酶基因5′端侧翼区域的克隆与鉴定", 《工业微生物》 * |
| 林琳 等，: "扩展青霉PF898 碱性脂肪酶cDNA 的克隆及序列分析", 《中国生物化学与分子生物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110938648A (en) * | 2019-12-09 | 2020-03-31 | 中国热带农业科学院热带生物技术研究所 | Fungus secretion expression vector, construction method and application thereof |
| CN110938648B (en) * | 2019-12-09 | 2021-11-05 | 中国热带农业科学院热带生物技术研究所 | Fungus secretion expression vector, construction method and application thereof |
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