CN101831452A - Method for efficiently expressing and producing T4 lysozyme through recombinant trichoderma reesei in inductive mode - Google Patents

Abstract

The invention discloses a method for expressing and producing T4 lysozyme recombinant protein in an inducible manner in Trichoderma reesei, which includes: 1) using the codon-optimized and artificially synthesized T4 lysozyme gene to improve its expression in Switzerland; 2) using the cellobiohydrolase gene promoter or terminator sequence derived from Trichoderma reesei as a homologous sequence integrated into the Trichoderma reesei genome as an exogenous plasmid vector; 3 ) using the T4 lysozyme exogenous gene promoter to regulate the T4 lysozyme exogenous gene in Trichoderma reesei to be highly expressed in an inducible manner; 4) Specific Trichoderma reesei engineering bacteria fermentation culture growth conditions to improve The biological yield of T4 lysozyme recombinant protein and the method for rapidly purifying the recombinant exogenous protein, and the pure T4 lysozyme recombinant protein prepared by this method has biological activity and can be widely used in the fields of medical treatment, food, feed, scientific research and the like.

Description

By the mould method that efficiently expresses and produce the T4 N,O-Diacetylmuramidase in the induction type mode of reorganization Rui Shi wood

Technical field

The present invention relates to the genetically engineered field, particularly relate to a kind of method of in Rui Shi wood is mould, expressing and producing T4 N,O-Diacetylmuramidase recombinant protein in the induction type mode.

Background technology

N,O-Diacetylmuramidase (Lysozyme, EC3.2.17) be meant that a class extensively is present in occurring in nature and multiple Gram-positive and negative pathogenetic bacteria, fungi and some virus had kill or inhibiting hydrolase that nineteen twenty-two is found first by Britain bacteriologist Fleming.The main sterilization mode of this lytic enzyme is to destroy-acetylmuramic acid in the bacteria cell wall and the β-1 between the N-acetylglucosamine, 4 glycosidic links cause cell wall to break and Dissolve things inside oozes out, and cause bacterium collapse and dead thus, therefore, the N,O-Diacetylmuramidase-acetylmuramic acid enzyme that is otherwise known as.At occurring in nature, the source of N,O-Diacetylmuramidase is very widely, is broadly divided into animal, plant and microbe-derived several classes such as N,O-Diacetylmuramidase.The N,O-Diacetylmuramidase of animal-origin generally is present in people and mammiferous multiple tissue and the secretory product, as it is medium to be present in tears, saliva, liver, kidney, Lymphoid tissue and egg white.Also can be separated to N,O-Diacetylmuramidase in the plants such as pawpaw, turnip and radish, they are considered to play an important role in the first road defensive barrier of biological human body.

In multiple microbe, find the existence of N,O-Diacetylmuramidase at present, as styreptomyces globispotus strain, acetone clostridium butylicum and multiple phage etc.The T4 N,O-Diacetylmuramidase derives from and infects colibacillary T4 phage, by T4 phage gpe coded by said gene (Arisaka F, et al., 2003), and 495 bases of full length gene, 164 amino acid of encoding, molecular weight 18700Da.When phage particle after the host cell internal packing is finished, under the effect of T4 N,O-Diacetylmuramidase, cause host-Bacillus coli cells cracking.Up to now, the T4 N,O-Diacetylmuramidase has had 30 years of researches history, and the crystalline structure of this enzyme and various mutations body thereof has obtained resolving.The T4 N,O-Diacetylmuramidase is made up of two structural domains: be positioned at the α/beta structure territory of N end and be positioned at the αLuo Xuanjiegou territory that C holds.Discover, the germicidal action of T4 N,O-Diacetylmuramidase mainly shows two aspects: the one, and this enzyme has the-acetylmuramic acid enzymic activity, the 2nd, being positioned at the positively charged α helical domain of this enzyme C end can combine with the electronegative cytolemma of bacterium or fungi, thereby disturbs the normal physiological metabolism of host cell.The αLuo Xuanjiegou territory that is positioned at T4 lysozyme C end mainly contains 4, is respectively α 1 (115-122), α 2 (126-134), α 3 (137-141) and α 4 (143-155).The α 2-α 3 and α 4 small peptides of synthetic do not have the-acetylmuramic acid enzymic activity, but still the biological activity of performance antibacterium or fungi.With after the T4 N,O-Diacetylmuramidase heat denatured, lose equally, but still keep stronger sterilizing ability though find its-acetylmuramic acid enzyme bioactivity.Studies show that, the T4 N,O-Diacetylmuramidase is carried out structure of modification and modify improving its fungicidal activity and thermostability that for example to have the fungicidal activity of the T4 N,O-Diacetylmuramidase of 6 histidine-tagged structures (6xHis-Tag) be 2 times of natural T4 N,O-Diacetylmuramidase to albumen n end; In addition, after holding the 6th amino acids to become the Methionin K of positively charged N by hydrophobic methionine(Met) M, anti-microbial activity can improve 4 times, but the stability decreases of protein molecular, infer that the reason that its fungicidal activity improves may be because C end degree of freedom strengthens, thereby the αLuo Xuanjiegou territory can more effectively be combined with the cytolemma target.

The T4 N,O-Diacetylmuramidase is except can killing multiple pathogenetic bacteria, and is also inhibited to some pathogenic fungi.Though the T4 N,O-Diacetylmuramidase can not pass the cytolemma of fungus conidium, the fungus conidium after this enzyme is handled no longer expands and germinates.The T4 N,O-Diacetylmuramidase also has certain destruction to potato protoplastis adventitia, but studies confirm that people and mammiferous cell toxicological harmless effect, and therefore, the T4 N,O-Diacetylmuramidase all has great application potential at industrial circles such as medicine, food and feeds.

Extract the T4 N,O-Diacetylmuramidase from the Bacillus coli cells of T4 phage-infect, production technique is not only loaded down with trivial details, yield poorly, and is difficult to industrialization.In recent years, fast development along with molecular biology and gene recombination technology, the method that makes up expression such as engineering strain such as yeast and intestinal bacteria and production external source recombinant protein more and more is subjected to scientific worker and industrial circle expert's favor, filamentous fungus then has many advantages equally as the new host of exogenous protein expression, as: 1) foreign protein genes also can be incorporated in the filamentous fungus genome and can obtain the transformant of genetic stability; 2) strong promoter that closely regulated and control by inductor can start efficiently expressing of downstream foreign protein genes, and recombinant exogenous protein can be secreted into outside the born of the same parents, thereby helps the purifying work in later stage; 3) can translate post-treatment and modification to the external source recombinant protein as eukaryotic expression system, comprise the generation of disulfide linkage and glycosylation etc.; 4) thalline and its meta-bolites have no side effect to human body and Mammals etc.; 5) industrial fermentation technology maturation makes things convenient for the suitability for industrialized production of recombinant exogenous protein etc.

Employed Rui Shi wood mould (Trichoderma reesei) is a kind of harmless saprophytic sex pilus shape fungi among the present invention, the industrial production that often is applied to cellobiohydrolase, and its output reaches as high as 40g/L (Jiang Tian first-class, 2007).In the cellulase system 1,4-beta fibers disaccharide-hydrolysing enzymes 1 (1,4-beta-D-glucan cellobiohydrolase) promotor of gene C BH1 is an extremely strong inducible promoter, this promotor can be by substrate for induction such as lactose, wheat straw powder and Mierocrystalline celluloses, but can be checked by glucose.Simultaneously, it also can be used as the homologous nucleotide sequence of exogenous origin gene integrator to the mould genome of wood.The mould method for transformation of Rui Shi wood mainly contains electro fusion method and protoplast transformation method etc., and the screening of recon mainly is by adopting antibiotics resistance as selected marker gene, and the most frequently used is Totomycin and G418 resistant gene.

Rui Shi wood is mould ripe not enough as a kind of new its operative technique of foreign protein genes expression system, and fragmentary successful report (seeing Table 1) is only arranged at present.

The external source recombinant protein of the mould expression of table 1 Rui Shi wood

Title	The source	Molecular weight (kDa)	Promotor	The host	Expression amount	Reference
							Ethylene synthetase	??Pseudomonas?syringae??pv.glycinea	??40	??Cbh1	??T.viride	?	??Li?Tao?et??al.，2008
Zytase	??Acrophialophora??nainiana	??19	??Cbh1	??T.reesei	??172mg/L	??Bruno?C，et??al.，2007
							Esterase	??Melanocarpus?albomyces	??74	??Cbh1	??T.reesei	?	??Hanna?K.，et??al.，2005
Antifungal protein	??Aspergillus?giganteus??MDH18894	??6	??Cbh1	??T.viride	?	Xu Jun etc., 2003
							Barley endopeptidase B	Barley	??30	??Cbh1	??T.reesei	??500mg/L	??Ritva?S.，et??al.，1997

Before the present invention, also do not adopt the report that codon is optimized, the T4 lysozyme gene of synthetic is expressed this foreign protein in the induction type mode in Rui Shi wood is mould, also nobody's promotor of using the mould cellobiohydrolase 1 gene C BH1 of Rui Shi wood is incorporated into report in the mould genome of Rui Shi wood as homologous sequence with the T4 lysozyme gene, more has no talent and carry out the high density fermentation production of T4 N,O-Diacetylmuramidase recombinant protein in Rui Shi wood is mould.

Summary of the invention

First purpose of the present invention is to provide that a kind of codon is optimized, the T4 lysozyme gene of synthetic, and it can obtain stable in Rui Shi wood is mould and efficiently express.

Second purpose of the present invention is to use one with the exogenous nucleotide integration sequence, the plasmid vector of important Expression elements such as carrying T4 N,O-Diacetylmuramidase foreign gene and selection markers can be integrated in the mould genome of Rui Shi wood, and obtain the mould engineering strain of reorganization Rui Shi wood of genetic stability.

The 3rd purpose of the present invention is that a kind of mould reorganization bacterium of Rui Shi wood that stably efficiently expresses the T4 lysozyme gene under mould cellobiohydrolase 1 gene promoter of Rui Shi wood starts will be provided.

The 4th purpose of the present invention provides the suitableeest reorganization Rui Shi mould growth of wood and expression condition and target protein purification process fast.

For achieving the above object, the present invention at first provides a kind of gene of the T4 of coding antalzyme protein, its be according to the codon optimized mistake of the mould preference of Rui Shi wood, coding or to the gene of small part coding T4 antalzyme protein.Wherein said T4 antalzyme protein primary structure is the aminoacid sequence shown in the SEQ IDNO.1.According to an embodiment preferred, described gene has or has the nucleotide sequence shown in the SEQ ID NO.2 to small part.

Described then gene is placed under the mould cellobiohydrolase 1 gene promoter manipulation of Rui Shi wood, is incorporated in the mould genome of Rui Shi wood, and along with the growth of reorganization Trichoderma body, the T4 lysozyme gene can stably be efficiently expressed in the induction type mode.

According to an embodiment preferred, the promotor of cellobiohydrolase 1 gene that Rui Shi wood is mould and terminator dna sequence dna are successively cloned out and are inserted in the new mould expression plasmid carrier of T4 N,O-Diacetylmuramidase wood, by the homologous recombination that takes place between this promotor or terminator dna sequence dna and the mould genome of Rui Shi wood, can will be incorporated into simultaneously in the mould genome of Rui Shi wood by T4 lysozyme gene and other important Expression element of the mould cellobiohydrolase 1 gene promoter regulation and control of Rui Shi wood, and the mould engineering strain of Rui Shi wood that obtains recombinating, it can be stablized under cellulosic inducing and express T4 N,O-Diacetylmuramidase recombinant protein efficiently, and it is secreted into outside the born of the same parents the most at last.

Concrete, the gene that at first is coding T4 N,O-Diacetylmuramidase is come out according to the complete synthetic of the Rui Shi mould codon of having a preference for of wood, then by the pcr amplification technology, add the promotor of the nucleotide coding sequence and mould cellobiohydrolase 1 gene of Rui Shi wood of mould cellobiohydrolase 1 secreting signal peptide of Rui Shi wood at its 5 ' end upstream region, terminator sequence in that its 3 ' end downstream area adds mould cellobiohydrolase 1 gene of Rui Shi wood forms a new foreign gene reading frame thus.According to an embodiment preferred, the T4 lysozyme gene at first is placed under the inducible promoter regulation and control, is inserted into then on the mould expression plasmid carrier of Rui Shi wood.On this expression plasmid carrier, this inducible promoter is positioned at the upstream of cellobiohydrolase 1 secreting signal peptide nucleotide coding sequence and the formed fusion gene of T4 N,O-Diacetylmuramidase nucleotide coding sequence, and it handles the stable and efficiently expression of so-called antigen-4 fusion protein gene in Rui Shi wood is mould.Simultaneously, this promotor nucleotides sequence is listed in the effect that the expression plasmid vector integration has also played the homologous recombination sequence in the mould genomic process of Rui Shi wood.At this expression plasmid carrier after complete linearization process, can be directed in the mould cell of Rui Shi wood by methods such as electro fusion method or protoplastiss, and then by the homologous recombination stable integration on the mould chromogene group of Rui Shi wood, the mould T4 N,O-Diacetylmuramidase recombinant protein of expressing in the fermentation culture process of this reorganization Rui Shi wood is secreted under the guiding of cellobiohydrolase 1 secreting signal peptide in the substratum outside the born of the same parents.

The recombinant protein of indication of the present invention can be the whole of T4 antalzyme protein, yet in some specific embodiments, expressed foreign gene also may be the part of this native protein.Sometimes, the protein gene that T4 N,O-Diacetylmuramidase recombinant protein can have a different biological function with another one, its objective is and purifies for convenience or improve its biological activity simultaneously at a mould cell inner expression of wood with independence or amalgamation mode.Proteic fusion can be passed through protein translation post-treatment, covalently bound mode, perhaps by the DNA recombinant technology gene just is stitched together mutually before accurate translation, and these two kinds of technology is technology that those skilled in the art know already.

Like this, the present invention more preferably embodiment is: 1) according to the mould codon of having a preference for of Rui Shi wood, carry out the synthetic of T4 lysozyme gene; 2) clone of Rui Shi mould cellobiohydrolase 1 gene promoter of wood and cellobiohydrolase 1 secreting signal peptide nucleotide coding sequence; 3) clone of cellobiohydrolase 1 gene terminator DNA; 4) make up a plasmid expression vector, the gene that wherein contains the gene or derivatives thereof of coding T4 N,O-Diacetylmuramidase recombinant protein (comprises that gene codon is optimised, change or merge mutually etc.) with other gene, this gene at first is spliced to form fusion gene mutually with the mould cellobiohydrolase 1 secreting signal peptide nucleotide coding sequence of Rui Shi wood, be placed in then under the mould cellobiohydrolase 1 gene promoter control of Rui Shi wood, be added with cellobiohydrolase 1 terminator dna sequence dna in this fusion gene downstream, also include in the plasmid expression vector simultaneously and be used for exogenous origin gene integrator to the mould genomic homologous recombination sequence of Rui Shi wood, this sequence can be Rui Shi mould cellobiohydrolase 1 gene promoter of wood or other homologous sequences; 5) by electro fusion method or additive method, plasmid expression vector DNA after the above-mentioned linearizing is transformed in the mould protoplastis of Rui Shi wood, and the mould protoplastis of the Rui Shi after will transforming wood coats on the HM solid plate substratum that contains 0.5mg/mL G418 and grows, and the wooden mould single bacterium colony that can grow on this substratum is exactly the wooden mould recon that contains foreign gene.

Further, the invention provides about reorganization reorganization Rui Shi wood the suitableeest mould grown cultures condition and exogenous protein expression condition, composition, inoculum size, pH value, dissolved oxygen amount and incubation time etc. as substratum make the increment of this wooden mould engineering bacteria of recombinating and the secretory volume of recombinant protein all reach maximization as much as possible thus.From fermented liquid, can obtain highly purified T4 N,O-Diacetylmuramidase recombinant protein by operations such as bactofugation, tubular fibre filtration, ion exchange chromatographies, for suitability for industrialized production, low-cost fermentation and the fast purifying of this enzyme lay the foundation later on.Specifically comprise following three phases: in 1) the yeast culture stage, behind 10% the mould engineering bacteria of inoculum size inoculation Rui Shi wood, through 72 hours cultivation, Trichoderma body weight in wet base will reach about 35～40g/L; 2) the abduction delivering stage adds Mierocrystalline cellulose, induces wooden mould cell high-efficient to express T4 N,O-Diacetylmuramidase recombinant protein and it is secreted in the substratum, keeps certain pH value and dissolved oxygen amount; 3) protein purification, fermented liquid is handled through the membrane filtration of centrifugal and three different sizes, and at process primary ions displacement chromatography, the purity of T4 antalzyme protein can reach more than 99%.

Most preferred, adopt the method for engineering strain fermentative production recombinant protein of the present invention to comprise: (1) seed liquor preparation: at first with mycelium inoculation on the PDA substratum, cultivated 6～7 days for 27～28 ℃, again spore is dissolved in the sterilized water, be inoculated in the liquid MM substratum 27～28 ℃ of shaking culture (375～380 rev/mins) 72～96 hours with 1～2% volume ratio; With its seed liquor as high density fermentation;

(2) yeast culture: in fermentor tank, hold an amount of MM basis fermention medium, the wooden mould seed liquor of volume ratio inoculation step (1) by 8～10%, 27～28 ℃ of aeration-agitations (rotating speed maintains 375～380 rev/mins from start to finish) were cultivated about 72～96 hours, and pH maintains about 5.5～6;

(3) protein induced expression: at postvaccinal the 4th～6 day, stream adds the Mierocrystalline cellulose that is dissolved in 50wt% in the MM substratum in the above-mentioned substratum, the substratum cellulose concentration is maintained about 2～3wt%, dissolved oxygen amount is greater than 20%, temperature maintenance is at 27～28 ℃, pH maintains about 5.5～6, continues vibration (rotating speed maintains 375～380 rev/mins from start to finish) inducing culture 3～4 days under this condition.

The MM culture medium prescription of using in said process is: every liter contains: glucose 20g, (NH ₄) ₂SO ₄5g, KH ₂PO ₄15g, MgSO ₄7H ₂O 0.6g, CaCl ₂0.6g, FeSO ₄7H ₂O 0.005g, MnSO ₄H ₂O 0.0016g, ZnSO ₄7H ₂O 0.0014g, CoCl ₂0.002g pH 5.5.

The purity of T4 N,O-Diacetylmuramidase recombinant protein is directly connected to the height of this Application of Recombinant scope and the related products cost of producing in the leavened prod, according to a preferential embodiment, for a large amount of T4 N,O-Diacetylmuramidase recombinant protein of fast purifying, can be at first use the filtration unit of the different molecular weight that dam and ion exchange chromatography etc. to carry out pre-treatment the mould fermented liquid supernatant liquid of Rui Shi wood, each link in purge process, all can use polyacrylamide gel electrophoresis to detect, can obtain purity by aforesaid method and reach 99% recombinant protein.

The present invention has only mentioned Rui Shi trichoderma strain QM9414 when introducing the mould expression system of Rui Shi wood in detail, yet, as the wooden mould expression system that those skilled in the art knew already, the wooden mould expression system of many kinds can utilize method provided by the present invention to carry out genetic transformation, expression and production.Therefore, all these wooden mould expression systems all should be included within the claim scope of the present invention.

Just as what will describe in detail in the following example, used plasmid vector is an integrated plasmid expression system in the mould method for transformation of Rui Shi wood that the present invention describes, according to a preferential embodiment, plasmid expression vector used in the present invention is one and obtains improved pPIC9K, its original AOX1 inducible promoter is replaced by the mould cellobiohydrolase 1 gene induced type promotor of Rui Shi wood, also include in the plasmid expression vector simultaneously and be used for the homologous recombination sequence of exogenous origin gene integrator to the yeast genes group, this sequence can be the mould cellobiohydrolase 1 gene induced type promotor of Rui Shi wood, terminator sequence or other homologous sequence.

The present invention has greatly improved the method for T4 N,O-Diacetylmuramidase recombinant protein biological yield in the mould cell of Rui Shi wood, and this recombinant protein is known to have very strong antibacterial or germicidal action under native state; Or or rather, the T4 N,O-Diacetylmuramidase recombinant protein of indication of the present invention is similar with this recombinant protein that the albumen that is derived from T4 phage gp e coded by said gene or other conventional expression system are produced, and can be widely applied to fields such as medical treatment, food, feed and scientific research as antibacterial or sterilant.Adopt method of the present invention; can be effectively codon optimized T4 lysozyme gene be incorporated in the mould genome of Rui Shi wood by Rui Shi mould cellobiohydrolase 1 gene promoter of wood or the terminator sequence mode with homologous recombination; the mould engineering strain of Rui Shi wood that obtains is can be with induction type mode formula stable and express T4 N,O-Diacetylmuramidase recombinant protein efficiently; the fermentation and the purifying process of this recombinant protein are provided simultaneously, have been applicable to large-scale production T4 N,O-Diacetylmuramidase recombinant protein.

Description of drawings

Fig. 1 is the codon optimized and transformation front and back contrast of T4 lysozyme gene, the gene order that the N representative is codon optimized and transformation is preceding; M represents codon optimized and improved gene order, has the expression nucleotide base of ordinate not change between two sequences;

Fig. 2 is the building process of the mould efficient expression vector T4-CBH9K of induction type wood;

Fig. 3 is that the pcr amplification of wooden mould conversion recon is identified: 1 is the DNA standard molecular weight; 2～9 is the T4 lysozyme gene amplification of 9 mould transformants of wood; CK+ is a T4 lysozyme gene positive control; CK-is a T4 lysozyme gene negative control;

Fig. 4 is standard molecular weight albumen (Blue Plus Protein marker 12～94KDa, Beijing TransGenBiotech company product) for SDS-PAGE electrophoresis detection T4 N,O-Diacetylmuramidase expression of recombinant proteins situation: M; The negative contrast of CK-, 120 hours fermented liquid supernatant of (not containing the T4 lysozyme gene) reorganization trichoderma strain inducing culture that CBH9K transforms; 1 cultivates 72 hours fermented supernatant fluid before inducing for TR-T4; 2～4 are respectively the fermented supernatant fluid of TR-T4 inducing culture after 24,48 and 72 hours;

Fig. 5 measures for reorganization T4 lysozyme activity: X-coordinate is the pure product of reorganization T4 N,O-Diacetylmuramidase (unit is mg/mL) of different weaker concns, ordinate zou is the OD350nm absorbance value, T4lysozyme is the determination of activity result of the pure product of T4 N,O-Diacetylmuramidase that prepare in wooden mould engineering bacteria TR-T4 fermented liquid, CK-is the determination of activity result that CBK9K (empty carrier) transforms the different weaker concns of Trichoderma fermented supernatant fluid extract.

Embodiment

Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

Embodiment 1: the synthetic of the T4 lysozyme gene after codon optimized

The T4 lysozyme gene derives from the T4 phage, its codon and prokaryotic organism are more approaching, and the Rui Shi Trichoderma is in eukaryote, therefore they are existing certain difference aspect the gene codon preference, and this species diversity has influence on T4 lysozyme gene and the stability and the expression efficiency of transcription product in the mould cell of Rui Shi wood thereof possibly.For improving the biological yield of T4 N,O-Diacetylmuramidase recombinant protein, dna nucleotide sequence and aminoacid sequence (GenBank:NY000866) according to the T4 lysozyme gene of having announced already, under the prerequisite that does not change its aminoacid sequence, (see SEQ ID NO.1), according to the Rui Shi wood mould codon of having a preference for (seeing CodonUsageDatabase:Hypocrea jecorina), the dna encoding sequence that manually designs and synthesized new T4 N,O-Diacetylmuramidase maturation protein.T4 N,O-Diacetylmuramidase mature protein gene after codon optimized with transform before compare, changed 159 nucleotide bases wherein, relate to 53 codons altogether, and the content of G+C becomes 58.8% (seeing SEQID NO.2) by original 36.6%.Accompanying drawing 1 (said gene splicing and synthetic work by Shanghai Bo Ya biotech company on behalf of finishing) is seen in contrast before and after the genetic modification.

Embodiment 2: the clone of mould cellobiohydrolase 1 gene promoter of Rui Shi wood-secreting signal peptide and terminator nucleotides sequence

1, the mould genomic extraction of Rui Shi wood

Rui Shi trichoderma strain QM9414 (this bacterial strain is from China Agricultural University) is seeded in every liter of MM[contains glucose 20g, (NH ₄) ₂SO ₄5g, KH ₂PO ₄15g, MgSO ₄7H ₂O0.6g, CaCl ₂0.6g, FeSO ₄7H ₂O 0.005g, MnSO ₄H ₂O 0.0016g, ZnSO ₄7H ₂O 0.0014g, CoCl ₂0.002g, 2% (w/v) agar powder, pH 5.5] on the solid medium, cultivated 4～5 days for 28 ℃, a small amount of mycelium of picking is transferred to the centrifuge tube from flat board, wash 2 times with physiological saline, add a small amount of granulated glass sphere and 500 μ lL STES damping fluid [200mM TrisHCl (pH8.5), 250mM NaCl, 25mM EDTA again, 2%SDS], behind the 3 * 40s that vibrates on the vortex vibrator, 65 ℃ of reaction 10min, ice bath 5min.After adding 200 μ L 10M ammonium acetate ice bath 5min again, the centrifugal 10min of 12000rpm.Get supernatant, add 200 μ L 10M ammonium acetates ice bath 5min once more, the centrifugal 10min of 12000rpm.With supernatant liquor Virahol-20 ℃ precipitation 20min.The centrifugal 10min of 12000rpm, precipitation is dried with 70% washing with alcohol twice, with 10 μ L water dissolution, adds an amount of RNase A, and behind 37 ℃ of reaction 30min ,-20 ℃ are standby.

2, the clone of mould cellobiohydrolase 1 gene promoter of Rui Shi wood-signal peptide and terminator nucleotides sequence

According to known Rui Shi wood mould cellobiose hydrolase gene promotor-secreting signal peptide (GenBank:D86235 and E00389) and terminator sequence (GenBank:E00389), designed primer1, primer2, primer3 and 4 primers of primer4 respectively, wherein primer Primer1 sequence is (seeing SEQ ID NO.3): 5 '-AC

AATTCTGGAGACGGCTTGTTGA ATC-3 ', Primer2 are (seeing SEQ ID NO.4): 5 '- CTCGAAGATGTTCATAGCACGAGCTGTGGCCAAGAAGGC-3 ', Primer3 are (seeing SEQ ID NO.5): 5 '- TACAAGAACTTGTAAAGGTCACCTTCTCCAACATCAAGTTCG-3 ', Primer4 are (seeing SEQ ID NO.6): 5 '-AC

CACGAAGAGCGGCGATTCTACGGG-3 '.Following stroke wavy line is partly represented restriction enzyme digestion sites in the primer, and following stroke horizontal line partly is the part that is base complementrity with T4 lysozyme gene two terminal preceding 15 nucleotide base sequences of synthetic respectively.With Primer1 and Primer2 is primer, with Rui Shi trichoderma strain QM9414 genomic dna is template, through pcr amplification, amplification obtains mould cellobiohydrolase 1 gene promoter of Rui Shi wood-secreting signal peptide nucleotide coding sequence (Pcbh1-sig), and (annotate: cellobiohydrolase 1 secreting signal peptide length is 17 amino-acid residues, coded by 51 nucleotide bases), structure for convenient follow-up expression vector, in Primer1, added Bgl II restriction enzyme digestion sites (seeing following stroke of wavy line part among the Primer1); In Primer2, added the dna sequence dna (seeing the part that following stroke horizontal line is arranged among the Primer2) that is base complementrity with preceding 15 nucleotide bases of T4 N,O-Diacetylmuramidase nucleotide coding sequence 5 ' end; The pcr amplification condition is: 94 ℃ of pre-sex change 5 minutes, and 94 ℃ of sex change 40 seconds, 55 ℃ of annealing 40 seconds, 72 ℃ were extended 1.5 minutes, 30 circulations, last 72 ℃ were extended 10 minutes.Similarly, be primer with Primer3 and Primer4, be template with Rui Shi trichoderma strain QM9414 genomic dna, through pcr amplification, obtained the mould cellobiohydrolase 1 gene terminator sequence (Tcbh1) of Rui Shi wood; Structure for convenient follow-up expression vector in Primer3, has added the dna sequence dna (seeing the part that following stroke horizontal line is arranged among the Primer3) that is base complementrity with preceding 15 nucleotide bases of T4 N,O-Diacetylmuramidase nucleotide coding sequence 3 ' end; In Primer4, added Sal I restriction enzyme digestion sites (seeing following stroke of wavy line part among the Primer4).The pcr amplification condition is: 94 ℃ of pre-sex change 5 minutes, and 94 ℃ of sex change 40 seconds, 55 ℃ of annealing 40 seconds, 72 ℃ were extended 45 seconds, 30 circulations, last 72 ℃ were extended 10 minutes.

The splicing of the mould gene reading frame of embodiment 3:T4 N,O-Diacetylmuramidase Rui Shi wood

With above-mentioned codon optimized T4 lysozyme gene (artificial sequence), the PCR product of Pcbh1-sig and Tcbh1 carries out agarose gel electrophoresis, use gel to reclaim test kit (Beijing hundred Tyke Bioisystech Co., Ltd, method is seen this description of product) the recovery target DNA fragment, ratio according to 1: 1: 1, with Pcbh1-sig, three kinds of dna fragmentations of T4 lysozyme gene and Tcbh1 (gel recovery product) mix, as template, carrying out PCR increases again, used two ends primer is respectively above-mentioned primer1 and primer4, the pcr amplification condition is: 94 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change 40 seconds, annealed 40 seconds for 55 ℃, 72 ℃ were extended 2 minutes, 30 circulations, and last 72 ℃ were extended 10 minutes.

The PCR product of above-mentioned total length directly is inserted into pEASY-T1 (available from the Beijing Quanshijin Biotechnology Co., Ltd, working method is seen this product description) in the T site in the plasmid, according to the method that the said firm provided, the middle plasmid vector T4-T that obtains containing the exogenous dna fragment insertion (contains the mould gene reading frame of T4 N,O-Diacetylmuramidase Rui Shi wood, it is in proper order: the bacterial clone T4 lysozyme gene-cellobiohydrolase 1 gene terminator sequence behind cellobiohydrolase 1 gene promoter-cellobiohydrolase 1 secretion signal peptide-coding sequence-codon optimized), then, by the nucleotide sequencing analysis, determine that the dna sequence dna in the mould gene reading frame of T4 N,O-Diacetylmuramidase Rui Shi wood is correct and complete (dna sequencing is finished by Beijing Biokit, Inc.).

Embodiment 4: the structure of the mould expression vector T4-CBH9K of Rui Shi wood

Carry out double digestion with restriction enzyme Bgl II and Sal I, the mould gene reading frame of the wood of the T4 N,O-Diacetylmuramidase Rui Shi in the plasmid vector T4-T in the middle of being connected is scaled off, utilize agarose gel electrophoresis to separate the purpose fragment.Handle plasmid pPIC9K with identical restriction enzyme, utilize agarose gel electrophoresis to separate the purpose fragment.Just obtain Rui Shi wood mould expression vector T4-CBH9K (seeing accompanying drawing 2) with linking together after the dna fragmentation mixing of above-mentioned two recovery and with ligase enzyme, use above-mentioned plasmid vector transformed into escherichia coli cell DH5 α (available from U.S. GIBCO company) then conveniently to carry out duplicating and preserving of this plasmid.

Plasmid vector pPIC9K is available from American I nvitrogen company, it is a yeast inducible expression plasmid vector, it contains an inducible promoter-alcohol oxidase promotor (AOX1) efficiently, and under the inducing of methyl alcohol, efficiently expressing of foreign gene inserted in adjustable downstream.

Embodiment 5: the preparation of linearizing T4-CBH9K plasmid DNA

At first use alkaline lysis (referring to the molecular cloning test guide) from Bacillus coli cells DH5 α, to extract the T4-CBH9K plasmid DNA, cut with 1～2 times of excessive restriction enzyme Nde I enzyme then, make it complete linearizing, utilize agarose gel electrophoresis to detect enzyme and cut whether fully.Then with phenol and chloroform respectively the above-mentioned enzyme of extracting cut product, ethanol sedimentation is abandoned supernatant, collecting precipitation after lyophilize, is dissolved in precipitation in the aseptic deionized water again ,-20 ℃ of preservations are standby.

Embodiment 6: the genetic transformation of the mould protoplastis of Rui Shi wood

The conidium that to cultivate about 7 days the mould QM9414 of Rui Shi wood on PDA substratum (agar 15g adds water to 1L for peeling potato 200g, glucose 20g) inclined-plane washes with physiological saline.On the PDA plate culture medium, place an aseptic glassine paper, with Rui Shi trichoderma conidium suspension (concentration be about 107/mL) coat on the substratum, cultivate about 18h for 28 ℃.20mg/mL lywallzyme (available from the Guangdong Microbes Inst), 20mg/mL helicase and 10mg/mL cellulase are mixed, be dissolved in fully in 1.2M sorbyl alcohol～100mM potassium phosphate buffer (pH5.6), again the young tender mycelia on the glassine paper in the flat board is transferred to wherein 28 ℃ of water-bath 1.5h～2h.Microscopy frequently in this process, the generation situation of observing protoplastis.Filter by 6 layers of lens wiping paper, protoplastis and the mycelium that is not digested are separated, and washed 2 times with 1.2M sorbyl alcohol～10mMTrisHCl (pH7.5).6000 rev/mins centrifugal 10 minutes, collect protoplastis, and wash 2 times with 1M sorb～10mMTrisHCI (pH7.5), be resuspended at last among 1M sorbyl alcohol～10mMCaCl2-10mM TrisHCl (pH7.5), making protoplastis concentration is 5x10 ⁷-5x10 ⁸/ mL.Linear recombinant plasmid (2～10 μ g) is mixed with the Rui Shi mould protoplastis of wood (about 200 μ L) for preparing, add 200 μ L 60%PEG4000～50mMCaCl ₂～10mMTrisHCl (pH7.5), ice bath 30min, the room temperature temperature is bathed 20min then, adds 2mL 60%PEG4000～50mM CaCl again ₂～10mmol/LTrisHCl (pH7.5) continues at room temperature and places 5min, adds 4mL 1M sorbyl alcohol～10mmol/LCaCl at last ₂～10mmol/LTrisHCl (pH7.5), mixing is coated on above-mentioned conversion fluid that to contain G418 concentration be that the MH[of 0.5mg/mL contains dextrose 20g, (NH for every liter ₄) ₂SO ₄5g, KH ₂PO ₄15g, MgSO ₄7H ₂O 0.6g, CaCl ₂0.6g, FeSO ₄7H ₂O 0.005g, MnSO ₄H ₂O0.0016g, ZnSO ₄7H ₂O 0.0014g, CoCl ₂0.002g, sorbitol 182.18g, pH5.5] on the flat board, cultivate for 28 ℃ and grew to wooden mould transformant in 4～5 days.

Embodiment 7: the screening of reorganization Rui Shi trichoderma strain

The G418 resistance transformant of growing on the MH substratum is seeded in respectively on the MM culture medium flat plate, cultivate after 4～5 days for 28 ℃, the a small amount of mycelium of picking is transferred to the different centrifuge tubes respectively from flat board, washes 2 times with physiological saline, adds a small amount of granulated glass sphere and 500 μ LSTES damping fluid [200mM TrisHCl (pH8.5) again, 250mM NaCl, 25mM EDTA, 2%SDS], behind the 3 * 40s that vibrates on the vibrator, 65 ℃ were reacted ice bath 5 minutes 10 minutes.Add 200 μ L 10M ammonium acetate solutions again, ice bath is after 5 minutes, 12000 rev/mins centrifugal 10 minutes, get supernatant, add 200 μ L 10M ammonium acetate solutions, ice bath is 5 minutes once more, 12000 rev/mins centrifugal 10 minutes.Get supernatant liquor and with Virahol-20 ℃ precipitation 20 minutes, centrifugal then, precipitation is with 70% washing with alcohol twice, air-dry after, with 10 μ L water dissolution, add an amount of RNase A, 37 ℃ were reacted after 30 minutes, standby.

The mould genomic dna of Rui Shi wood with said extracted is a template, with P1 (seeing SEQ IDNO.7) (5 '-AAATGA ACATCTTCGAGAAGT TGAGG-3 ') and P2 (seeing SEQ ID NO.8) (5 '-AATTACAAGTTCTTGTAGGCGTCCC-3 ') is primer, by PCR, the T4 lysozyme gene of amplification 495bp.Reaction conditions is: 95 ℃ of pre-sex change 5min, and 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 40s, totally 30 circulations, 72 ℃ are extended 10min, select clone's (seeing the swimming lane 7 in the accompanying drawing 3) of positive band.

To there be the Trichoderma silk of positive band to be seeded in once more on the PDA inclined-plane, cultivate about a week for 28 ℃, make its a large amount of generation conidiums.With physiological saline conidium is washed earlier, filter with 12 layers of lens wiping paper then, to remove unnecessary mycelia.Trichoderma conidium is coated on the thin PDA culture medium flat plate uniformly according to 100 spore/wares, cultivate about 10h for 28 ℃, in super clean bench, observe spore germination situation on the plate culture medium with 10 times of object lens of microscope, with sterile razor blade with in the visual field independently and the conidium that sprouts together downcut together with substratum.Change on the PDA inclined-plane that contains 0.4mg/mL G418, cultivate about a week for 28 ℃, when in vitro having a large amount of mycelia to produce, it is chosen a part, the rapid extraction genome carries out the pcr amplification checking and whether still contains the T4 lysozyme gene, and amplification condition is the same.Be confirmed to be the wooden mould transformant of success through the still positive clone after above-mentioned monospore separation and the PCR detection, and be named as TR-T4.

Embodiment 8: the high density fermentation of the wooden mould engineering bacteria of recombinating

1, the preparation of seed liquor

With the mycelium inoculation of engineering strain TR-T4 in a plurality of PDA solid slant culture bases, cultivated 6～7 days for 27～28 ℃, after treating that conidium grows up to green, scrape spore and be suspended in the sterilized water, be inoculated in the 500mL MM liquid nutrient medium with 1～2% inoculum size, 48～72h is cultivated in 27～28 ℃ of lucifuge concussions (375～380 rev/mins), with its seed liquor as high density fermentation.

2, the mould high density fermentation in the 5L fermentor tank of reorganization wood

This fermenting process can be divided into following two stages: 1) the yeast culture stage: held 3.5L MM basis fermention medium in 5L fermentor tank (Zhenjiang Oriental Bio-engineering Technology Co., Ltd), the wooden mould seed liquor that ratio inoculation in 10% prepares before this, 27～28 ℃ of aeration-agitations (rotating speed maintains 375～380 rev/mins from start to finish) were cultivated about 72～96 hours, and pH maintains about 5.5～6.In this culturing process, along with the growth of Trichoderma body, the dissolved oxygen amount in the substratum will reduce gradually by 100%, and after the carbon source in the substratum runs out of substantially, dissolved oxygen amount will be increased to more than 80% once again, and the weight in wet base of thalline will reach 35～40g/L this moment.2) the abduction delivering stage (72～144h): after inoculation the 4th day, add 50% cellulose suspension (with the preparation of MM substratum) by peristaltic pump stream, make its working concentration maintain about 2～3% all the time, dissolved oxygen amount is all the time greater than 20%, 27～28 ℃ of aeration-agitations are cultured to inoculation back 144～166 hours, and pH maintains about 5.5～6.Descended centrifugal 10 minutes for 4 ℃ through 5000rpm every 24 hours peek milliliter fermented liquids, get 30uL fermented liquid supernatant liquid and carry out the SDS-PAGE detection, discovery has the observable protein band of naked eyes, and molecular weight is about 19KDa, matches with the molecular weight of T4 N,O-Diacetylmuramidase recombinant protein in inferring.In addition, from the PAGE electrophorogram, adding the Mierocrystalline cellulose inducing culture after 72 hours, the Recombinant Protein Expression amount reaches climax (seeing accompanying drawing 4).

Embodiment 9: the purifying of recombinant protein

After treating that a fermentation period is all over, leave and take the 400mL fermented liquid directly carries out next round as seed liquor (inoculum size is 10%) fermenting process.Similar operation adds up to carry out 3 takes turns, and takes turns in the fermenting process every, all the increment and the T4 N,O-Diacetylmuramidase Recombinant Protein Expression amount of thalline is measured.In addition, take turns after fermenting process finishes fully every, also getting a little bacterium liquid coats on the PDA solid plate, and therefrom any mould single bacterium colony of 10 wood of picking, its genomic dna of rapid extraction carries out PCR and detects, found that, the biomass of thalline, the speed of growth and Recombinant Protein Expression amount are taken turns kept stable in the fermenting process at each, and in addition, the detected result of PCR also confirms the wooden mould good genetic stability (seeing Table 2) that has of reorganization.

The genetic stability of table 2TR-T4 bacterial strain and the mensuration of exogenous protein expression stability

The PCR positive	The thalline weight in wet base (g/L) of growing 72 hours	Induce 72 hours T4 N,O-Diacetylmuramidase expression amounts (mg/L)
			?100％	??39	??550
?100％	??37	??540
			?100％	??40	??590

Except that the seed liquor of retaining, remaining fermented liquid is used for the purifying of T4 N,O-Diacetylmuramidase recombinant protein.Fermented liquid is left and taken supernatant through centrifugal 10 minutes of 4 ℃ of 5000rpm.Fermented liquid supernatant at first is the tubular fibre filter post (Tianjin MoTian Membrane Engineering Technology Co., Ltd of 50KDa with the molecular weight that dams, product type: MOF-503, using method is seen the said firm's explanation) filter, collect and see through liquid, the clarifying liquid that sees through is that the nanofiltration membrane (the bright utmost point in Shanghai Chemical Industry Science Co., Ltd, production code member: 2426538, using method is seen the said firm's explanation) of 10KDa is handled with the molecular weight that dams again, keep phegma, final volume is about 700mL.This phegma is carried out desalting treatment, in the 700mL phegma, add 6.3L distilled water, be about to 10 times of phegma dilutions, the nanofiltration membrane treatment by above-mentioned 10KDa once more with diluent then, the also corresponding dilution of salt ionic concentration is 10 times in the phegma that obtain this moment, so repetitive operation is 5 times, i.e. salt ionic concentration dilution 10 in the phegma ⁵Doubly, finally obtain the 700mL phegma, after this phegma lyophilize, can obtain the recombinant protein lyophilized powder of preliminary purification (purity is big by about 60%).

This albumen lyophilized powder sample can obtain further purifying by ion exchange chromatography, to satisfy different production demands.Employed cation ion exchange resin is CM-Sephadex-C25 (U.S. GE company product).According to the said firm's description of product, resin is carried out pre-treatment, adorn post (the dress column method is seen the description of product, and chromatograph is Shanghai Hu Xi analytical instrument Co., Ltd., Factory product-model MA99-3) then, concrete operation method is as follows

1) distilled water of 5 times of volumes of albumen lyophilized powder adding dilutes, and with third acetic acid this diluent pH value is transferred to 5.5～5.8 then.

2) sample upper prop (annotate: when being 2g+500mL distilled water as if the lyophilized powder sample, use the chromatography column of 1.5x50cm, the resin bed volume height is 40cm), flow velocity 3mL/ minute.Go out process with ultraviolet Protein Detection instrument and registering instrument record eluent stream.

3) wash post, treat that sample all enters resin soon after, contain 10 with the 0.1M phosphate buffered saline buffer ^-3M MgSO ₄(pH6.5) wash post, flow velocity 3mL/ minute is zero to the OD280nm light absorption value, and needing damping fluid approximately is 5～6 times of column volumes.

4) wash-out contains 10 with the 0.1M phosphate buffered saline buffer ^-3M MgSO ₄Carry out the target protein wash-out with 0.5M NaCl (pH6.5), flow velocity 3mL/ minute, monitor the protein stream artificial situation and collect maximum albumen wash-out part (target protein peak generally appear at add behind the elutriant soon) with ultraviolet Protein Detection instrument, collect the elutriant that contains target protein.

5) with in the elutriant dislocation dialysis tubing, distilled water is carried out dialysed overnight, during change distilled water for several times, purpose is in order to remove wherein contained NaCl.

6) dialyzate can obtain target protein dry powder by lyophilize, and purity of protein can reach 99% (SDS-PAGE electrophorogram through the LabWork software analysis, U.S. UVP company product).More than, but prolonged preservation under the room temperature.Look the application target difference, can be processed into various formulations, as foodstuff additive, injection etc.

The bioactive detection of embodiment 9:T4 N,O-Diacetylmuramidase recombinant protein

Utilize colorimetry to carry out the bacteriolyze specific activity, concrete operations are as follows:

1. substrate preparation: the micrococcus (Micrococcus Lysodeikticus) of taking out freezing from-72 ℃ of Ultralow Temperature Freezers is (available from Chinese common micro-organisms DSMZ, bacterium numbering 1.634), behind a little bacterium liquid of inoculating needle picking, at LB (5g yeast extract, 10g Tryptones, 10g NaCl, 20g agar, add water to 1L, pH7.0) rule on the solid plate substratum, be inverted overnight incubation for 37 ℃.

The single bacterium colony of picking micrococcus cell cultures is inoculated in the 5mL LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night, and all transfer next day in 100mL LB liquid nutrient medium, and 37 ℃ of shaking culture are to cell concn OD _600nmValue is 1.602.Nutrient solution is transferred in the 100mL centrifuge tube, and 4 ℃ of 5000rpm centrifugal 10 minutes behind the ice bath, collecting precipitation.Suspend and washed cell with 0.05M Tris-HCl (pH7.4) damping fluid 50mL, the same centrifugal after, collecting precipitation.Cell is suspended in the identical Tris-HCl damping fluid of 10mL once more, and changes and to be displaced in the drying bottle, place-20 ℃ of freezing 1 weeks (refrigerated storage is essential to strengthening the cell susceptibility), and then carry out lyophilize.Cell dry powder can place prolonged preservation under the moisture eliminator room temperature in confined conditions.If cell dry powder is insensitive to the T4 N,O-Diacetylmuramidase, also cell dry powder can be placed 37 ℃ to preserve so that strengthen the susceptibility of cell.

Get micrococcus lyophilized powder 40mg, be suspended in phosphate buffered saline buffer (the 5.54g Na of 100mL 0.2M again ₂HPO ₄12H ₂O, 0.704g NaH ₂PO ₄2H ₂O, adding distil water be to 100mL, pH7.4), and its OD _350nmValue is about 1.0 (if 40mg micrococcus lyophilized powder is suspended in the phosphoric acid buffer of 50mL again its OD ₃₅₀Value is about 1.6), standby.

2. substrate is hatched: get the some milligrams of the pure product of T4 N,O-Diacetylmuramidase recombinant protein of above-mentioned preparation, respectively get 300uL after being diluted to different working concentrations respectively, with the above-mentioned 2.7mL micrococcus substrate for preparing, negative control is a distilled water respectively.After placing 37 ℃ of incubators to hatch 1h each test tube, measure the light absorption value (adding 300 μ L distilled water with the phosphate buffered saline buffer of 2.7ml 0.2M returns to zero) of solution under the 350nm wavelength, measurement result sees Table 3:

The active detection of table 3 T4 N,O-Diacetylmuramidase recombinant protein bacteriolyze

T4 N,O-Diacetylmuramidase working concentration (mg/mL)	??OD 350The nm light absorption value	Contrast CK-
			??0	??1.721	??1.693
??0.0005	??1.645	??1.715
			??0.0010	??1.554	??1.673
??0.0015	??1.515	??1.681
			??0.0020	??1.443	??1.696
??0.0025	??1.402	??1.666
			??0.0030	??1.364	??1.697
??0.0035	??1.269	??1.672
			??0.0040	??1.242	??1.673

Annotate: numeral is the mean value of three test-results in the table

The present invention is first according to the mould codon of having a preference for of Rui Shi wood, synthetic a brand-new T4 lysozyme gene, and merge with the encoding sequence of Rui Shi mould cellobiohydrolase 1 secreting signal peptide of wood (17aa); The promotor of mould cellobiohydrolase 1 gene of Rui Shi wood and the terminator sequence of this gene have been cloned; And utilize above-mentioned various elements, on the basis of plasmid pPIC9K, rebuild brand-new, a mould efficient expression vector of Rui Shi wood induction type and secretor type; After in the T4 lysozyme gene being imported the mould cell of Rui Shi wood, by methods such as resistance screening, active detections, obtained to stablize and to efficiently express the mould engineering strain of reorganization Rui Shi wood of T4 N,O-Diacetylmuramidase, again through sequence of operations such as high density fermentation, recombinant protein purifications, the pure product tool of the reorganization T4 antalzyme protein biological activity of final preparation can be widely used in fields such as medical treatment, food, feed and scientific research.

The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Reference

1、Arisaka?F.，et?al.，The?international?journal?of?biochemistry?&?cellbiology，2003，35(1)：16-21

2、Cellitti?J.，et?al.，Protein?Sci.，2007，16(5)：842-51

3, Jiang Tianyi and Zhu Ping, biotechnology communication, 2007,18 (6): 1050-1052

4、Li?Tao，et?al.，Appl?Microbiol?Biotechnol.，2008，80：573-578

5、Bruno?C.，et?al.，Biotechnol.Lett.，2007，29：1195-1201

6、Hanna?K.，et?al.Biotechnology?and?Bioengineering，2006，94：407-415

7, Xu Jun etc., Acta Biochimica et Biophysica Sinica 2003,35 (5): 454-458

8、Ritva?S.，et?al.，Applied?and?Environmental?Microbiology，1997，63(12)：4938-4940

Sequence table

＜110〉Biological Technology institute, Chinese Academy of Agricultural Sciences

 

＜120〉by the mould method that efficiently expresses and produce the T4 N,O-Diacetylmuramidase in the induction type mode of reorganization Rui Shi wood

 

<130>KHP10112298.4

 

<160>8

 

<170>PatentIn?version?3.5

 

<210>1

<211>164

<212>PRT

＜213〉T4 phage

 

<400>1

Met?Asn?Ile?Phe?Glu?Met?Leu?Arg?Ile?Asp?Glu?Arg?Leu?Arg?Leu?Lys

1???????????????5???????????????????10??????????????????15

Ile?Tyr?Lys?Asp?Thr?Glu?Gly?Tyr?Tyr?Thr?Ile?Gly?Ile?Gly?His?Leu

20??????????????????25??????????????????30

Leu?Thr?Lys?Ser?Pro?Ser?Leu?Asn?Ala?Ala?Lys?Ser?Glu?Leu?Asp?Lys

35??????????????????40??????????????????45

Ala?Ile?Gly?Arg?Asn?Cys?Asn?Gly?Val?Ile?Thr?Lys?Asp?Glu?Ala?Glu

50??????????????????55??????????????????60

Lys?Leu?Phe?Asn?Gln?Asp?Val?Asp?Ala?Ala?Val?Arg?Gly?Ile?Leu?Arg

65??????????????????70??????????????????75??????????????????80

Asn?Ala?Lys?Leu?Lys?Pro?Val?Tyr?Asp?Ser?Leu?Asp?Ala?Val?Arg?Arg

85??????????????????90??????????????????95

Cys?Ala?Leu?Ile?Asn?Met?Val?Phe?Gln?Met?Gly?Glu?Thr?Gly?Val?Ala

100?????????????????105?????????????????110

Gly?Phe?Thr?Asn?Ser?Leu?Arg?Met?Leu?Gln?Gln?Lys?Arg?Trp?Asp?Glu

115?????????????????120?????????????????125

Ala?Ala?Val?Asn?Leu?Ala?Lys?Ser?Arg?Trp?Tyr?Asn?Gln?Thr?Pro?Asn

130?????????????????135?????????????????140

Arg?Ala?Lys?Arg?Val?Ile?Thr?Thr?Phe?Arg?Thr?6ly?Thr?Trp?Asp?Ala

145?????????????????150?????????????????155?????????????????160

Tyr?Lys?Asn?Leu

<210>2

<211>495

<212>DNA

＜213〉artificial sequence

 

<400>2

atgaacatct?tcgagatgtt?gcgcatcgac?gagaggttga?ggttgaagat?ctacaaggac????60

accgagggct?actacaccat?cggcatcggc?cacttgttga?ccaagtcccc?ctccttgaac????120

gccgccaagt?ccgagttgga?caaggccatc?ggcaggaact?gcaacggcgt?catcaccaag????180

gacgaggccg?agaagttgtt?caaccaggac?gtcgacgccg?ccgtcagggg?catcttgagg????240

aacgccaagt?tgaagcccgt?ctacgactcc?ttggacgccg?tcaggaggtg?cgccttgatc????300

aacatggtct?tccagatggg?cgagaccggc?gtcgccggct?tcaccaactc?cttgaggatg????360

ttgcagcaga?agaggtggga?cgaggccgcc?gtcaacttgg?ccaagtccag?gtggtacaac????420

cagaccccca?acagggccaa?gcgcgtcatc?accaccttca?ggaccggcac?ctgggacgcc????480

tacaagaact?tgtaa?????????????????????????????????????????????????????495

 

<210>3

<211>33

<212>DNA

＜213〉artificial sequence

 

<400>3

acagatctaa?ttctggagac?ggcttgttga?atc??????????????????????????????????33

 

<210>4

<211>39

<212>DNA

＜213〉artificial sequence

 

<400>4

ctcgaagatg?ttcatagcac?gagctgtggc?caagaaggc????????????????????????????39

<210>5

<211>42

<212>DNA

＜213〉artificial sequence

 

<400>5

tacaagaact?tgtaaaggtc?accttctcca?acatcaagtt?cg?42

 

<210>6

<211>32

<212>DNA

＜213〉artificial sequence

 

<400>6

acgtcgacca?cgaagagcgg?cgattctacg?gg????????????32

 

<210>7

<211>26

<212>DNA

＜213〉artificial sequence

 

<400>7

aaatgaacat?cttcgagaag?ttgagg???????????????????26

 

<210>8

<211>25

<212>DNA

＜213〉artificial sequence

 

<400>8

aattacaagt?tcttgtaggc?gtccc????????????????????25

Claims (12)

1. the gene of T4 antalzyme protein of encoding is characterized in that, its be according to the codon optimized mistake of the mould preference of Rui Shi wood, coding or to the gene of small part coding T4 antalzyme protein.

2. gene as claimed in claim 1 is characterized in that, described T4 antalzyme protein primary structure is the aminoacid sequence shown in the SEQ ID NO.1.

3. gene as claimed in claim 1 or 2 is characterized in that, it has or has the nucleotide sequence shown in the SEQ ID NO.2 to small part.

4. it is mould that each described gene of claim 1-3 is used to make up the reorganization Rui Shi wood that efficiently expresses and produce the T4 N,O-Diacetylmuramidase.

5. one kind by the mould method that efficiently expresses and produce the T4 N,O-Diacetylmuramidase in the induction type mode of reorganization Rui Shi wood, comprise the synthetic of T4 N,O-Diacetylmuramidase foreign gene, the structure of efficient expression vector, the screening of the mould recon of high expression level wood, the fermentation of the mould engineering bacteria of reorganization Rui Shi wood and the abduction delivering and the purifying of external source recombinant protein, it is characterized in that described T4 N,O-Diacetylmuramidase foreign gene is each described gene of claim 1-3; Adopt mould cellobiohydrolase 1 gene promoter of Rui Shi wood when making up efficient expression vector.

6. method as claimed in claim 5 is characterized in that, when making up efficient expression vector, the T4 lysozyme gene is incorporated in the mould genome of Rui Shi wood as homologous sequence with Rui Shi mould cellobiohydrolase 1 gene promoter of wood or terminator sequence.

7. method as claimed in claim 6 is characterized in that, when making up efficient expression vector, the initial vector of employing is pPIC9K.

8. method as claimed in claim 7 is characterized in that, has added the encoding sequence of mould cellobiohydrolase 1 secreting signal peptide of Rui Shi wood before the 5 ' end of described T4 N,O-Diacetylmuramidase foreign gene.

9. the expression plasmid carrier of the T4 antalzyme protein of recombinating is characterized in that it is plasmid map carrier T4-CBH9K as shown in Figure 2.

10. the engineering strain of an express recombinant T4 antalzyme protein is characterized in that, comprises the described expression plasmid carrier of claim 9.

11. engineering strain according to claim 10 is characterized in that, host cell is that Rui Shi trichoderma strain QM9414 or other can be expressed the Rui Shi trichoderma strain of foreign protein.

12. adopt the method for claim 10 or 11 described engineering strain fermentative production recombinant proteins, comprising:

(1) seed liquor preparation: at first with mycelium inoculation on the PDA substratum, cultivated 6～7 days for 27～28 ℃, again spore is dissolved in the sterilized water, be inoculated in the liquid MM substratum with 1～2% volume ratio, 27～28 ℃ of shaking culture 72～96 hours are as the seed liquor of high density fermentation;

(2) yeast culture: in the wooden mould seed liquor of 8～10% ratio inoculation step (1), 27～28 ℃ of aeration-agitations were cultivated 72～96 hours in the MM substratum, and pH maintains 5.5～6;

(3) protein induced expression: yeast culture is after 72～96 hours, and stream adds the Mierocrystalline cellulose that is dissolved in 50wt% in the MM substratum, makes the substratum cellulose concentration maintain 2～3wt%, dissolved oxygen amount is greater than 20%, temperature maintenance is at 27～28 ℃, and pH maintains 5.5～6, continues the vibration inducing culture 3～4 days;

The MM culture medium prescription of using in said process is: every liter contains: glucose 20g, (NH ₄) ₂SO ₄5g, KH ₂PO ₄15g, MgSO ₄7H ₂O 0.6g, CaCl ₂0.6g, FeSO ₄7H ₂O 0.005g, MnSO ₄H ₂O 0.0016g, ZnSO ₄7H ₂O 0.0014g, CoCl ₂0.002g pH 5.5.

Images