CN101173260A - Representation of high disinfection vitality T4 lysozyme in yeast and producing method thereof - Google Patents

Abstract

The invention relates to a method to express and produce high bactericidal activity T4 lysozyme recombinant protein in yeast, comprising a T4 lysozyme gene which is optimized by codon and has the protein N-terminal sixth amino acid mutated to lysine, and specific culturing condition for yeast fermentation. The invention has the advantages that: as the lysozyme has strong bactericidal activity for various Gram positive and negative bacteria, and has certain resistance to pathogenic fungi and virus, the lysozyme can be widely applied to medicine, food, agriculture, cosmetic and other industries.

Description

Expression and the production method of high disinfection vitality T 4 lysozyme in yeast

The present invention relates in yeast, express and produce the method that possesses the high disinfection vitality T 4 lysozyme recombinant protein, comprising accessing to your password that son is optimized and the albumen n end six amino acid is sported the T4 lysozyme gene of Methionin and specific yeast fermentation incubation growth condition, this N,O-Diacetylmuramidase not only all has very strong fungicidal activity to multiple Gram-positive and negative bacteria, and also may have certain resistance to some pathogenic fungi and virus, therefore can be widely used in industries such as medicine, food, agricultural and makeup.

N,O-Diacetylmuramidase (lysozyme) is found first in nineteen twenty-two that by A.Fleming it is a kind of lytic enzyme that acts on bacteria cell wall specially, so claimed Lysozyme again.N,O-Diacetylmuramidase is a kind of effectively broad spectrum antiseptic-germicide, initial research is thought, N,O-Diacetylmuramidase can act on N-acetylglucosamine in the peptidoglycan and the β-1 between the-acetylmuramic acid, 4 glycosidic links, and owing to the peptidoglycan support in the cell wall is damaged, under the effect that internal penetration is pressed, the cell spalling is opened at last, thereby cause the death of bacterial cell.But recent research is found, the anti-microbial activity of N,O-Diacetylmuramidase be not because its can dissolution of bacteria cell walls, and mainly be owing to there is one or more amphiphatic albumen spirane structures territory in its protein molecular inside, they can pierce through the cytolemma of bacterium, and finally cause the normal physiological metabolism of bacterium to get muddled until death.At the antalzyme protein molecule after high-temperature denatured, though the ability of its hydrolytic bacteria cell walls completely loses, but be exposed on the outside owing to be positioned at intramolecular amphiphilic albumen spirane structure territory, its antibacterial ability is strong all the better, for example, someone causes the protein molecular instability after the six amino acid residue of T4 N,O-Diacetylmuramidase N end is changed into Methionin by methionine(Met), and fungicidal activity has improved 4 times on the contrary ^[1]

N,O-Diacetylmuramidase extensively is present in the middle of natural animal, plant and the microorganism.For example, (human lysozyme is a kind of small molecular basic protein all to contain N,O-Diacetylmuramidase in people's white cell, serum, placenta, saliva, tears, milk, uterus mucus and kidney, it is by monokaryon-scavenger cell secretion, in the human organism, play nonspecific defense reaction), and in the bright juice of papaya, pineapple and Fructus Fici, also all contain abundant N,O-Diacetylmuramidase, by inference, they are also relevant with the disease resistance of plant.

It needs to be noted,, also do not contain the peptidoglycan composition, so the N,O-Diacetylmuramidase in any source is to human body and the equal free of toxic effects of mammalian cell because people and mammalian cell do not have cell wall structure.

The Application Areas of N,O-Diacetylmuramidase comprises the following aspects:

1) N,O-Diacetylmuramidase can be used as sanitas.Itself is a kind of protein, and people and Mammals are not had any toxicity, is the very high food preservatives of a kind of security therefore.Because N,O-Diacetylmuramidase only can act on the cell walls or the cytolemma of target microorganism, and can not act on other material.In use, except noting consumption (generally adding 0.001-0.01%), go back pH value, salt concn, the temperature of GPRS food and in food, add the time of N,O-Diacetylmuramidase and suitable method.

2) preventing dental caries.Carious tooth is modal a kind of in the dental disorder, and major cause is to exist a kind of bacterium that is called Streptococcus mutans in the oral cavity, and it can become sucrose inversion materials such as glucose and mucopolysaccharide.Bacterium in the oral cavity and these materials are bonded together, and the phase mutual coagulation is deposited on dental surface, the formation tartar.Tartar bacterium anaerobically fermenting polysaccharide in the tooth dirt produces multiple organic acid, and that these organic acids can displace from the enamel of dental surface is calcareous, thereby causes carious tooth.If in toothpaste, collutory, clean dose of mouth and chewing gum, manage to add a certain amount of N,O-Diacetylmuramidase, then can kill these pathogenetic bacterias, thereby reach the purpose of prevention and treatment carious tooth.Equally, N,O-Diacetylmuramidase can also be used to making collyrium and the liquid etc. that wets one's whistle.

3) microbiotic substitute.Clinical application shows that N,O-Diacetylmuramidase has preventive and therapeutic effect to grice diarrhoea, and it and immunoglobulin (Ig) have closely on function gets in touch, and can with other life active compounds such as complement acting in conjunction strengthening the activity of antibody, thereby kill bacteria.Shao Chunrong etc. report, the piglet of 7-60 age in days is fed after the lysozyme formulation, and its diarrhea disease percentage significantly reduces, and illustrate that N,O-Diacetylmuramidase has stronger inhibition to the dust Xi Shi intestinal bacteria that cause grice diarrhoea and rotavirus and does ^[2]Bao Chengyu etc. prove that through livestock and poultry such as the broiler chicken of feeding, calf, piglets N,O-Diacetylmuramidase can strengthen animal body immunizing power, are used to prevent and treat digestive tract diseases such as gastro-enteritis, maldigestion ^[3]Research finds that also N,O-Diacetylmuramidase also has stronger restraining effect to streptococcus aureus, ox corynebacterium pyogenes and the intestinal bacteria etc. that cause mastadenitis of cow.

4) in food, beverage and makeup, use as additive.N,O-Diacetylmuramidase integrates pharmacology, health care and anticorrosion three functions, and is without any side effects as a kind of natural protein.For example, if add N,O-Diacetylmuramidase, can replenish the intravital non-specific immunity factor of people in the food, kill the corrupt coccus in the enteron aisle, keep flora normalizing in the enteron aisle, promote bifidus bacillus propagation, strengthen the resistance infection of human body.If join in the beverage, in calcium milk, lactic drink, also can play anti-decayed tooth, tasty and refreshing effect.Particularly humanized milk powder is very important to infant's growth.Lysozyme content in the milk is few, and the lysozyme content in the breast milk is very abundant.For remedying the deficiency of milk powder, can make female emulsifying power milk powder to wherein adding an amount of N,O-Diacetylmuramidase.The adding of N,O-Diacetylmuramidase can be strengthened infant's anti-infection ability.

N,O-Diacetylmuramidase can be divided into three types substantially: c type (hen's egg-white lysozyme), g type (goose albumen N,O-Diacetylmuramidase) and phage lysozyme, they are existing bigger difference aspect substrate kind and the fungicidal activity.The N,O-Diacetylmuramidase of the overwhelming majority of being found all belongs to c type (as people's N,O-Diacetylmuramidase) so far, and, produce at present and go up also all extraction preparations from Ovum Gallus domesticus album of N,O-Diacetylmuramidase commonly used.Phage lysozyme then is coded by various phages self that can the bacterial infection cell, is usually located in the protein coat of phage, and in the process of host cells infected, they are playing an important role aspect the dissolution of bacteria cell walls ^[4]

The T4 N,O-Diacetylmuramidase be by the T4 phage when the ehec infection E.coli produce, it is the highest a kind of N,O-Diacetylmuramidase of fungicidal activity that people found up to now ^[5]But the more important thing is; it not only can act on some Gram-negative pathogenetic bacterias specifically; but also many important Gram-positive pathogenetic bacterias such as streptococcus pneumoniae, streptococcus aureus and ox corynebacterium pyogenes etc. are played germicidal action, its fungicidal activity is respectively 250 times (to negative bacteria) and 6 times (to positive bacteria) (annotate: hen's egg-white lysozyme only has bacteriostatic action to gram-positive bacteria) of c type N,O-Diacetylmuramidase (as people and hen's egg-white lysozyme).

Nineteen eighty-three, people such as Owen clone the T4 lysozyme gene first and have measured its nucleotide sequence ^[6]According to this encoding sequence, known T4 N,O-Diacetylmuramidase is made up of 164 amino-acid residues, and molecular weight is 18700 Da.Under the envrionment conditions of pH 6.0-6.53 and 37 ℃, the T4 N,O-Diacetylmuramidase can stable existence 20 hours, but the optimal pH of this enzyme effect is 7.4-7.7.Its in the animal stomach (sour environment) can keep stable, but mainly brings into play bacteriostatic action in intestines.

1999, people such as D ü ring utilize computer software that the three-dimensional structure of T4 antalzyme protein is analyzed, find that this albumen holding a side to contain 4 αLuo Xuanjiegou territories near C, lay respectively between 115-123 (α 1), 126-134 (α 2), 137-141 (α 3) and 143-155 (α 4) amino-acid residue, wherein α 4 is a typical amphiphilic spirane structure, and its contained clean positive charge with electronegative bacterial cell membrane discern mutually with interaction process in play an important role.Just because of this effect, disturbed the normal physiological metabolism of bacterial cell, thereby reached the purpose of kill bacteria.According to the amino acid residue sequence that forms α 4 spirane structure territories, synthetic this section protein polypeptide.Bacteriostatic experiment shows, though this section polypeptide does not have the function of dissolution of bacteria cell walls, is consistent with complete T4 antalzyme protein on the ability of resisting bacterium and fungi, and it is active even higher than complete T4 antalzyme protein.Recently, people confirm also that by research the T4 N,O-Diacetylmuramidase can make some virally inactivated by a kind of mechanism of non-enzyme effect ^[1]

Although utilizing T4 phage-infect intestinal bacteria to produce the T4 N,O-Diacetylmuramidase is a very sophisticated approach, but this approach only can obtain the natural T4 antalzyme protein of minute quantity at every turn, all right for experimental study, but differ greatly from the desired economic target of suitability for industrialized production, the demand of for this reason, be on a large scale and producing the T4 N,O-Diacetylmuramidase cheaply and satisfying market is sought other new production approach with regard to needs.

The latest developments of genetically engineered research field make and utilize some yeast cell as bio-reactor and efficiently expressing exogenous gene becomes possibility, and what be worth especially proposing is pichia pastoris phaff (Pichia pastoris) expression system.Up to now, people utilize this expression system the foreign protein of successful expression above 300 kinds ^[7], comprise the gene of extensively originating from bacterium, fungi, prokaryotic organism, plant, animal and human's class etc.As bio-reactor, its superiority also is: 1) yeast is a unicellular organism, and genetic background, physiological characteristic are comparatively clear.Its growing nutrient requires simple, the cell density height, and easy handling, and also fermentation technique is comparatively ripe, technology is simple, with low cost; 2) be sole carbon source with methyl alcohol in protein induce expression process, do not secrete toxic substance in no resistance mark, the culturing process, toxicity is little than bacterium, has good security; 3) utilize methanol induction alcohol oxidase promoter (AOX1) to carry out the expression of recombinant protein, no matter in the born of the same parents still secretion all might realize efficiently expressing, and background is expressed seldom when using secretion type expression, product is easy to purifying; 4) yeast is an eukaryote, can carry out a lot of typical higher eucaryote protein translation post-treatment and modify, as the formation of glycosylation, acylization, phosphorylation and protein cleavage, folding, disulfide linkage etc. ^[8,9,10]

Before the present invention, the codon that the gene of coding T4 N,O-Diacetylmuramidase is had a preference for according to yeast of also having no talent carried out optimizing so that it can be more stable and more efficiently at the yeast expression in vivo, greatly increased the biological yield of the T4 N,O-Diacetylmuramidase of recombinating thus; Nobody expressed N and holds six amino acid to be sported the T4 antalzyme protein of the high disinfection vitality of Methionin by methionine(Met) in yeast cell; Reorganization zymic fermentation culture conditions was not carried out optimization yet, thereby for extensive, industrial fermentation are produced this antibiotic recombinant protein and laid the foundation, and this recombinant protein is through partially or completely might being widely used in industries such as medicine, food, agricultural and makeup behind the purifying.

One of purpose of the present invention is that will confirm the to encode gene of T4 N,O-Diacetylmuramidase is only changing under the prerequisite that N holds the 6th aminoacid sequence (changing Methionin into by methionine(Met)), if all encoding sequence is after the codon of having a preference for according to yeast is optimized, the new sequence of gene not only can be expressed in yeast and be produced, and its expression efficiency has great raising than the T4 lysozyme gene of not codon optimization, thereby the biomass that is secreted into external reorganization T4 N,O-Diacetylmuramidase is also increased greatly, and the downstream processing for this recombinant protein brings great convenience also can significantly enough reduce production costs thus.According to a preferential embodiment, T4 lysozyme gene after codon optimized is wanted and can be efficiently expressed in pichia spp and produce, and the T4 N,O-Diacetylmuramidase recombinant protein that is produced not only can be secreted into external, and because the 6th amino-acid residue of this recombinant protein N end changes Methionin into by methionine(Met), cause free more and easier bacterial cell membrane and the interference bacterium eubolism process of being inserted into of some amphiphilic α coilin structural domains that are arranged in T4 lysozyme C end thus, therefore, this T4 N,O-Diacetylmuramidase recombinant protein does not have higher fungicidal activity through the T4 N,O-Diacetylmuramidase recombinant protein of any sudden change than the aminoacid sequence that natural T4 antalzyme protein or other expression systems that are derived from the T4 phage capsid are produced ^[1]

Two of purpose of the present invention provides the suitableeest recombination yeast incubation growth and abduction delivering condition, according to a preferential embodiment, the suitableeest incubation growth and the abduction delivering condition of relevant recombinant yeast pichia pastoris are provided especially, make the increment of this recombination yeast and the secretory volume of recombinant protein T4 N,O-Diacetylmuramidase all reach maximization as much as possible thus, so that lay the foundation for the large-scale industrial production of this recombination yeast, low-cost fermentation and purifying.

The present invention utilizes yeast expression system to produce the T4 N,O-Diacetylmuramidase, and this recombinant protein its active aspect aspect the inhibition pathogenetic bacteria is higher than native protein.According to a preferential embodiment, the method that the invention provides the fermentation process in high density of the method for very big raising T4 N,O-Diacetylmuramidase expression efficiency in pichia spp, the method that improves T4 N,O-Diacetylmuramidase fungicidal activity, recombination yeast exactly and how to use this reorganization T4 antalzyme protein.

The present invention utilizes yeast expression system to produce the T4 N,O-Diacetylmuramidase, if with it through partially or completely being widely used in industries such as medicine, food, agricultural and makeup more behind the purifying.For example, the T4 N,O-Diacetylmuramidase recombinant protein behind the purifying is joined in the toothpaste in certain proportion, behind the life-time service, then can effectively prevent and treat carious tooth.For another example, T4 N,O-Diacetylmuramidase recombinant protein is processed into oral capsule, electuary or other preparation, then has cheapness, safety, effect and determine and characteristics easy to use, more human or animal is protected.According to a preferential embodiment, people or Mammals only need just can obtain the resistivity to people and some bacteriosises of Mammals digestive tube with capsule, electuary or other preparation that the mode of enterally administering utilizes these partial purification products that are derived from the yeast fermentation product or complete purified product (dosage can be determined) to prepare.

The T4 N,O-Diacetylmuramidase recombinant protein of indication of the present invention is expressed in yeast and is produced, the gene that at first is coding T4 N,O-Diacetylmuramidase is come out by complete synthetic, comprising gene order codon optimized mistake or that pass through optimization, then, they are respectively inserted on the Yeast expression carrier that has α-factor signal peptide sequence.On this plasmid vector, also contain an inducible promoter, this promotor is positioned at the upstream of T4 N,O-Diacetylmuramidase encoding sequence, and it handles the expression of T4 lysozyme gene in yeast cell.This plasmid vector is after linearizing, can pass through electro fusion method or LiCl method transformed yeast cell, and then stable integration is to yeast chromosomal, and through after inducing, the T4 N,O-Diacetylmuramidase recombinant protein of expression is secreted in the substratum under the guiding of signal peptide this recombination yeast in the fermentation culture process.

The invention provides the method that greatly improves T4 N,O-Diacetylmuramidase recombinant protein biological yield in yeast cell, according to a preferential embodiment, the invention provides the method that has greatly improved T4 N,O-Diacetylmuramidase recombinant protein biological yield in the pichia spp cell, under native state, can kill multiple Gram-positive and negative pathogenetic bacteria and this recombinant protein is known; In other words, the T4 N,O-Diacetylmuramidase recombinant protein of indication of the present invention has higher fungicidal activity than the natural T4 N,O-Diacetylmuramidase that is derived from the T4 phage; Perhaps more definite theory, T4 N,O-Diacetylmuramidase recombinant protein of the present invention is the same with this T4 N,O-Diacetylmuramidase recombinant protein that is derived from T4 phage or other conventional expression system generation, can be widely used in industries such as medicine, food, agricultural and makeup as additive.For example, as pharmaceutical cpd, the mode by oral, enterally administering or injection can obtain the resistivity to people and some bacteriosises of Mammals digestive tube.

Recombinant protein of the present invention can be the whole of T4 N,O-Diacetylmuramidase native protein.Yet in some specific embodiments, expressed T4 N,O-Diacetylmuramidase also may be the part of this native protein.Sometimes, this T4 N,O-Diacetylmuramidase can have the protein gene of different biological function such as coli heat-sensitive toxin B subunit with another one and expresses respectively simultaneously in same yeast cell or be stitched together and form the fusion rotein co expression.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 the expert of the art knows already.

In yeast cell, the height of T4 antalzyme protein gene expression efficiency, the height of the extraction processing and utilization of this recombinant protein and the related products cost of producing after being directly connected to, according to a preferential scheme of implementing, for improving the expression amount of T4 antalzyme protein in yeast cell, the gene of coding T4 N,O-Diacetylmuramidase can be optimized earlier according to the codon that yeast is had a preference for, so that make this gene more stable in yeast cell, efficient is higher when being transcribed into mRNA and translating into albumen.In addition, in order to improve the fungicidal activity of T4 N,O-Diacetylmuramidase recombinant protein, its partial amino-acid series can be done part and change, according to a preferential embodiment, the 6th amino acid that is positioned at T4 N,O-Diacetylmuramidase N end can be changed into Methionin by methionine(Met), just because of this change, make the C end of this T4 N,O-Diacetylmuramidase recombinant protein freer, and then make this recombinant protein possess higher fungicidal activity, perhaps more definite theory, T4 antalzyme protein of the present invention is the same with this T4 N,O-Diacetylmuramidase recombinant protein that is derived from T4 phage or other conventional expression system generation, can be widely used in medicine as additive, food, industries such as agricultural and makeup.For example, as pharmaceutical cpd, the mode by oral, enterally administering or injection can obtain the resistivity to people and some bacteriosises of Mammals digestive tube.

To the relevant item with other of the method used in the present invention structure of some plasmid vectors very importantly, they play an important role in the processed and applied of yeast cell that obtains indication of the present invention and express recombinant product thereof.The plasmid vector that is used for transformed yeast cell is by dna sequence dna after codon optimized, coding T4 lysozyme gene or derivatives thereof and handle this gene or a inducible promoter that dna sequence dna is expressed at said yeast cell is formed.In some specific embodiments, plasmid vector also comprises a selectivity or marker gene, is mainly used in the screening and the detection of recombinant yeast cell.In some specific embodiments, inducible promoter of the present invention is alcohol oxidase gene promoter (AOX1).As mentioned above, according to some specific embodiment, plasmid vector of the present invention is used to transform pichia spp, and its coded foreign protein is the T4 N,O-Diacetylmuramidase, furtherly, the coded recombinant protein T4 N,O-Diacetylmuramidase of plasmid vector can be killed multiple Gram-positive and negative pathogenetic bacteria.In other words, the T4 N,O-Diacetylmuramidase recombinant protein of indication of the present invention has than the higher sterilizing activity of natural T4 N,O-Diacetylmuramidase that is derived from the T4 phage.Perhaps more definite theory, T4 antalzyme protein of the present invention is the same with this T4 N,O-Diacetylmuramidase recombinant protein that is derived from T4 phage or other conventional expression system generation, can be widely used in industries such as medicine, food, agricultural and makeup as additive.For example, as pharmaceutical cpd, the mode by oral, enterally administering or injection can obtain the resistivity to people and some bacteriosises of Mammals digestive tube.

The present invention also provides the conversion of yeast cell and the screening method of recombinant yeast cell, and it may further comprise the steps: 1) synthetic of T4 lysozyme gene, wherein relate to that the codon of having a preference for according to yeast is optimized this gene codon and the replacing of partial amino-acid; 2) make up a plasmid expression vector, the gene (comprise that gene codon is optimised, change or merge mutually etc.) that wherein contains the gene or derivatives thereof of coding T4 antalzyme protein with other genes, and this gene or derivatives thereof is placed under the manipulation of an inducible promoter; 3) by electro fusion method or LiCl method with above-mentioned plasmid expression vector transformed yeast cell; 4) select the single bacterium colony of transformed yeast cells, be inoculated into respectively one by one on MM and the MD solid plate substratum, those growth on the MD substratum normal but on the MM flat board growth extremely slowly or the yeast list bacterium colony of not growing fully be exactly the recon that contains foreign gene.

According to a specific embodiment, the invention provides through recombinant yeast pichia pastoris incubation growth condition of optimizing and the purification process of recombinating the T4 N,O-Diacetylmuramidase, it has comprised following four-stage: 1) yeast culture, after 22-24 hour cultivation of process, yeast thalline weight in wet base will reach about 95-100g/L; 2) carbon source of feeding, after cultivating 4 hours, the weight in wet base of yeast thalline will arrive about 190-200g/L; 3) abduction delivering adds methyl alcohol, induces yeast cell to efficiently express T4 N,O-Diacetylmuramidase recombinant protein and it is secreted in the substratum; 4) protein purification, fermented liquid is through the centrifuging and taking supernatant liquor, through steps such as ammonium sulfate precipitation and mistake gel molecular sieves, and the purity of T4 N,O-Diacetylmuramidase recombinant protein can reach more than 99%.

The present invention relates to some application problem of T4 N,O-Diacetylmuramidase recombinant protein, for example, related to the moiety problem of a kind of capsule, electuary or other preparation.At least contain in capsule, electuary or other preparation and be derived from zymic T4 N,O-Diacetylmuramidase recombinant protein, and this recombinant protein is as main activeconstituents.For the present invention, the T4 N,O-Diacetylmuramidase recombinant protein that contains in capsule, electuary or other preparation should be able to accurate quantification, so as more effectively to bring into play this T4 N,O-Diacetylmuramidase germicidal action.According to a preferential embodiment of the present invention, the main active ingredient that is derived from pichia spp that is contained in capsule of the present invention, electuary or other preparation is that a N holds six amino acid to be changed by the first propylhomoserin to be T4 N,O-Diacetylmuramidase recombinant protein behind the Methionin, and it can kill multiple Gram-positive and negative pathogenetic bacteria.In other words, the T4 N,O-Diacetylmuramidase recombinant protein of indication of the present invention has than the higher sterilizing activity of natural T4 N,O-Diacetylmuramidase that is derived from the T4 phage.

The present invention has only mentioned pichia yeast expression system when introducing yeast expression system in detail, yet, as the yeast expression system that the expert of the art knew already, many primary yeast expression systems can utilize method provided by the invention to transform, express and produce.Therefore, all these yeast expression systems all should be included within the claim scope of the present invention.

In following example, to describe in detail, used plasmid expression vector is a conformability plasmid expression system in the yeast conversion method that the present invention describes, according to a preferential embodiment, plasmid used in the present invention is expression vector pPIC9K.

The present invention includes following integral part: the 1) synthetic of T4 lysozyme gene wherein comprises gene coded sequence codon optimized mistake or that pass through optimization respectively; With regard to the codon optimized and gene order transformed wherein, it is the full gene DNA nucleotide sequence of the coding T4 N,O-Diacetylmuramidase of the codon had a preference for according to yeast, synthetic, wherein N is held the 6th amino-acid residue to change into Methionin by methionine(Met).Above-mentioned product is inserted directly in the T site of pGEM-T, selects through the Lan Bai system, can obtain to contain external source and insert segmental bacterial clone (recon), the exactness and the integrity of gene that sequential analysis obtains; 2) utilize DNA reorganization skill wood to make up the yeast plasmid expression vector, they contain respectively without the codon optimized and T4 antalzyme protein gene (in contrast) transformed and codon through optimizing and improved T4 antalzyme protein gene or derivatives thereof (comprise with the mutual fusion of other genes etc.); 3) screening of high expression level yeast recon, through electricity fusion or LiCl method above-mentioned yeast plasmid expression vector is imported the yeast recipient cell respectively, on RDB solid plate substratum, select the single bacterium colony of transformed yeast cells, be inoculated into respectively one by one on MM and the MD solid plate substratum, those growth on the MD substratum normal but on the MM flat board growth extremely slowly or the yeast list bacterium colony of not growing fully be exactly the recon that contains foreign gene.In order to screen the high expression level restructuring yeast strains, the yeast recon that obtains is seeded in one by one carries out abduction delivering in the abduction delivering substratum, under identical culture condition, its abduction delivering product is carried out fungicidal activity identify, therefrom can pick out the highest yeast strain of expression efficiency; 4) provide the suitableeest grown cultures of recombination yeast and abduction delivering condition, the high density fermentation of recombination yeast divides feeds and the abduction delivering three phases for yeast culture, carbon source, all gives the suitableeest incubation time and envrionment conditions in each stage.Fermented liquid is through centrifugal, ammonium sulfate precipitation with after crossing the gel molecular sieve and filtering, and can obtain purity and reach T4 N,O-Diacetylmuramidase recombinant protein more than 99%; 5) be that main active ingredient is manufactured capsule, electuary or other preparation with this recombinant protein, under situation, might make the resistivity of human body or other Mammalss acquisition some bacteriosises of digestive tube by enterally administering or drug administration by injection.

In order to describe some preferential embodiment of the present invention in detail, and for referencial use with corresponding drawing:

Codon optimized and the transformation front and back contrast of Fig. 1 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.

Fig. 2 codon is through optimizing and the clone of improved T4 lysozyme gene and the structure of Yeast expression carrier p9KT4M thereof.。

Fig. 3 codon does not pass through the clone of the T4 lysozyme gene of optimizing and transforming and the structure of Yeast expression carrier p9KT4 thereof

Fig. 4 utilizes the height of colony counting method detection resources from the T4 of different recombinant pichia yeast strains N,O-Diacetylmuramidase expression of recombinant proteins efficient.A: handle through the yeast recon abduction delivering product (negative control) that the pPIC9K plasmid transforms; B: handle (negative control) through the BMMY nutrient solution; C-D: handle without the abduction delivering product of the restructuring yeast strains of the T4 lysozyme gene of optimizing through codon; E-F: the abduction delivering product of the restructuring yeast strains of T4 lysozyme gene is handled after codon optimized and change.

Fig. 5 SDS-PAGE detects PP-T4 and PP-T4M high density fermentation expression product and by the T4 N,O-Diacetylmuramidase recombinant protein behind centrifugal, ammonium sulfate precipitation and the gel molecular sieve purifying.1 is standard protein molecular weight marker (Amersham Biosicences product); 2 is the T4 N,O-Diacetylmuramidase recombinant protein behind the purifying, is derived from abduction delivering product behind the PP-T4 high density fermentation; 3 is the T4 N,O-Diacetylmuramidase recombinant protein behind the purifying, is derived from abduction delivering product behind the PP-T4M high density fermentation; 4 is abduction delivering product behind the PP-T4 high density fermentation; 5 be and the PP-T4M high density fermentation after the abduction delivering product; 6 is the yeast recon abduction delivering product (negative control) that transforms through the pPIC9K plasmid.

Be example with Pichia anomala expression T4 N,O-Diacetylmuramidase below, describe some preferential embodiments, but application of the present invention be not limited only to this.

Selection T4 N,O-Diacetylmuramidase is expressed in yeast and production is that therefore, this enzyme can be widely used in the multiple industries such as medicine, food, agricultural and makeup as additive because this N,O-Diacetylmuramidase can be killed multiple Gram-positive and negative pathogenetic bacteria.For example, if with the T4 N,O-Diacetylmuramidase as the main pharmaceutical cpd in capsule, electuary and other preparations, the mode by oral, enterally administering or injection can make people and Mammals obtain resistivity to some predominantly bacteria diseases of digestive tube.Although the T4 N,O-Diacetylmuramidase has very wide application field, its mass-producing, suitability for industrialized production are never solved well.Select pichia yeast expression system to be because it is a present the most frequently used external source expression of recombinant proteins system.

Embodiment 1

1. the synthetic of codon optimized and improved T4 lysozyme gene

The T4 N,O-Diacetylmuramidase is coded by the T4 phage, its codon and prokaryotic organism are more approaching, and yeast belongs to eukaryote, so these two kinds of biologies are existing certain difference aspect the gene codon preference, and this species diversity can have influence on stability and the expression efficiency of T4 lysozyme gene in yeast.For improving the biological yield of T4 N,O-Diacetylmuramidase, dna nucleotide sequence and aminoacid sequence according to the T4 lysozyme gene of having announced already (are seen GenBank, accession number: NY000866), except that the 6th amino-acid residue of albumen n end (changing into present Methionin) by original methionine(Met), under the prerequisite that does not change other aminoacid sequences, (see 1 in the sequence table), the codon of having a preference for according to yeast ^[11], manually design and synthesized the full gene DNA nucleotide sequence of new T4 N,O-Diacetylmuramidase.Transforming Methionin as and will be positioned at albumen n end the 6th locational methionine(Met), is in order further to improve the fungicidal activity of this T4 N,O-Diacetylmuramidase recombinant protein.Meanwhile, for the ease of the structure of Yeast expression carrier after this, in the process of this T4 N,O-Diacetylmuramidase dna nucleotide sequence of synthetic, synthetic and added restriction enzyme site XhoI and yeast Kex2 gene expression product cutting recognition sequence (seeing 2 in the sequence table) before 5 ' of this gene is held first translation initiation codon ATG; Behind 3 ' end terminator codon TAA of this gene, increased restriction enzyme site NotI (said gene splicing and synthetic work by Shanghai Bo Ya biotech company on behalf of finishing).Codon optimized and improved T4 lysozyme gene with transform before compare, changed 139 nucleotide bases wherein, relate to 117 codons altogether, and that G+C content becomes by original 36.6% is present 49.5%, Fig. 1 is seen in contrast before and after the genetic modification.

2. the clone of codon optimized and improved T4 lysozyme gene

The T4 lysozyme gene dna fragmentation of above-mentioned synthetic is directly inserted and is connected in the T site in the pGEM-T plasmid (seeing Promega company product description), according to the method that the said firm provided, by the white screening system of indigo plant, obtain containing the bacterial clone pGEMT4M (see figure 2) of codon optimized and improved T4 lysozyme gene, then, by nucleotide sequence analysis, the gene of determining coding T4 N,O-Diacetylmuramidase is correct and complete (seeing 1 in the sequence table) (dna sequencing by Shanghai Bo Ya biotech company on behalf of finishing).

3. the structure of Yeast expression carrier p9KT4M

During T4 lysozyme gene after synthetic is codon optimized, because in its 5 ' end and 3 ' terminal sequence, introduced XhoI and NotI restriction enzyme site respectively, so the pGEMT4M plasmid DNA is behind XhoI and NotI double digestion, codon optimized and improved T4 lysozyme gene can be scaled off, pass through agarose gel electrophoresis, and then this dna fragmentation of separable acquisition, be inserted into subsequently (XhoI and NotI double digestion) among the plasmid pPIC9K, just be built into yeast inducible expression carrier p9KT4M (see figure 2).

Plasmid pPIC9K is available from American I nvitrogen company, and it is a yeast inducible expression carrier efficiently, under the inducing of methyl alcohol, but efficiently expressing exogenous gene.Because before this expression vector multienzyme is cut the site, contain α-factor signal peptide-coding sequence, with the foreign protein genes amalgamation and expression after, bootable external source recombinant protein is to the yeast exocytosis.In the process outside being secreted into born of the same parents, this signal peptide sequence can fully be cut down by yeast Kex2 or Stel3 gene expression product, thereby can not have influence on the fungicidal activity of T4 N,O-Diacetylmuramidase recombinant protein fully.

Embodiment 2

1. codon is without the synthetic of the T4 lysozyme gene of optimizing

According to the gene order of the T4 N,O-Diacetylmuramidase of having announced already (see GenBank, accession number: NC000866), synthetic the DNA complete nucleotide sequence of coding T4 N,O-Diacetylmuramidase.For ease of making up Yeast expression carrier, in synthetic this gene, before encoding sequence 5 ' end initiator codon ATG, increase XhoI restriction enzyme site and yeast Kex2 gene expression product cutting recognition sequence, behind 3 ' end terminator codon TAA of gene, increased NotI restriction enzyme site (seeing 4 in the sequence table) (said gene splicing and synthetic work by Shanghai Bo Ya biotech company on behalf of finishing).。

2. codon is without the clone of the T4 lysozyme gene of optimizing

The T4 lysozyme gene dna fragmentation of above-mentioned synthetic is directly inserted and is connected in the T site in the pGEM-T plasmid (seeing Promega company product description), according to the method that the said firm provided, by the white screening system of indigo plant, obtain the bacterial clone pGEMT4 (see figure 3) of codon without the T4 lysozyme gene of optimizing, then, by nucleotide sequence analysis, the gene of determining coding T4 N,O-Diacetylmuramidase is correct and complete (seeing 4 in the sequence table) (dna sequencing by Shanghai Bo Ya biotech company on behalf of finishing).

3. the structure of Yeast expression carrier p9KT4

When the full gene DNA nucleotide sequence of synthetic T4 N,O-Diacetylmuramidase, because in its 5 ' end and 3 ' terminal sequence, introduced XhoI and NotI restriction enzyme site respectively, so the pGEMT4 plasmid DNA is behind XhoI and NotI double digestion, this T4 lysozyme gene can be scaled off, and then pass through agarose gel electrophoresis, this dna fragmentation of separable acquisition is inserted into (XhoI and NotI double digestion) among the plasmid pPIC9K subsequently, just is built into yeast inducible expression carrier p9KT4 (see figure 3).

Embodiment 3

1.p9KT4 and the preparation of p9KT4M plasmid DNA

At first use alkaline lysis ^[12]From Bacillus coli cells DH5 α (available from U.S. GIBCO company), extract p9KT4 and p9KT4M plasmid DNA respectively, then with the doubly excessive restriction enzyme BglII of 1-2 respectively enzyme cut above-mentioned plasmid DNA, make it complete linearizing, can 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 after lyophilize, is dissolved in precipitation in the aseptic deionized water again, preserves standby in-20 ℃ of refrigerators.

2. the conversion of yeast cell

The single bacterium colony of picking Pichi strain GS115 (available from American I nvitrogen company), be inoculated in 5mlYPD substratum (1% yeast extract, 2% Tryptones, 2% glucose) in, 30 ℃ of shaking culture are spent the night, therefrom take out the 0.5ml nutrient solution, be inoculated in the 500ml YPD substratum, 30 ℃ of shaking culture are to OD _600nmAbsorbance value is about 1.4-1.5, and 4000rpm collected yeast cell down in centrifugal 2 minutes for 4 ℃.Sedimentary yeast cell is suspended in again in the sterilized water of 500ml ice bath, 4000rpm collected yeast cell down in centrifugal 2 minutes for 4 ℃ once more then.Repeat once above-mentioned washing process.With the 1mol/L sorbyl alcohol of the 25ml ice bath sedimentary yeast cell that suspends again, as above centrifugal collection yeast cell, again with the 1mol/L sorbyl alcohol of the 0.5ml ice bath sedimentary yeast cell that suspends again, therefrom take out 80ul yeast competent cell, respectively with linearization plasmid carrier DNA (4-5ug) thorough mixing of above-mentioned preparation, transfer to then in the aseptic electric shock cup of 0.2cm behind the ice bath.Utilize electric shock instrument Micropulser ^TM(BioRad company product) imports linearizing plasmid DNA in the yeast competent cell, and employed shock parameters is 0.8kv, 11.5uF.After electric shock is finished, the 1mol/L Sorbitol Solution USP that in the electric shock cup, adds the 0.8ml ice bath immediately, fully behind the mixing, this bacterium liquid is applied to RDB[1.34% Yeast Nitrogen Base With Ammonium Sulfatewithout amino acids (YNB) (Sigma company product) with the volume of every plate 200ul, the 1mol/L sorbyl alcohol, 1% glucose, 0.00004% Biotin, 0.005% L-glutamic acid, 0.005% methionine(Met), 0.005% Methionin, 0.005% leucine, 0.005% Isoleucine, 1.5% agar] on the solid plate substratum, dull and stereotyped be inverted also to place 30 ℃ of incubators to be cultured to transform recon occur.

With the yeast list bacterium colony (recon) that newly grows on the aseptic toothpick picking RDB solid plate substratum, difference dibbling MM (1.34% YNB, 0.00004% Biotin, 0.5% methyl alcohol, 1.5% agar) dull and stereotyped and MD (1.34%YNB, 0.00004% Biotin, 2% glucose, 1.5% agar) the solid plate substratum; Cultivated 2 days for 30 ℃, consistent with growth on the MD at MM, its phenotype is Mut ⁺Growth is normal on MD, poor growth or long on MM, and phenotype is Mut ^s

3. the screening of high expression level yeast strain

Picking growth on the MD flat board normal but on the MM flat board poky transformant, be inoculated in the 5mLBMGY[1% yeast extract, 2% Tryptones, 100mmol/L potassium phosphate buffer (pH 7.0), 1.34% YNB, 0.00004% Biotin, 1% glycerine (V/V)] in the liquid nutrient medium (this substratum is carbon source with glycerine), 30 ℃ of shaking tables were cultivated 2 days, and the centrifugal 2min of 4000rpm removes supernatant; With 1mL BMMY[1% yeast extract, 2% Tryptones, 100mmol/L potassium phosphate buffer (pH 7.0), 1.34% YNB, 0.00004% Biotin, 0.5% methyl alcohol] the liquid nutrient medium thalline (this substratum with methyl alcohol as foreign protein genes induced expression thing) that suspends again, 30 ℃ of inducing culture 4 days; 4 ℃ of following centrifugal 5min of 10000rpm, collection contains the supernatant liquor that might contain T4 N,O-Diacetylmuramidase recombinant protein.This supernatant liquor can be directly used in the mensuration of fungicidal activity.

The detection of T4 N,O-Diacetylmuramidase fungicidal activity is carried out according to the colony counting method that D ü ring etc. is reported fully ^[1], the picking strains of streptococcus (the bacterial strain code name: 1.2499, available from DSMZ of Institute of Microorganism, Academia Sinica) single bacterium colony, be inoculated in 5mL LB (5g yeast extract, 10g Tryptones, 10gNaCl, add water to 1L, pH7.0) in the liquid nutrient medium, 37 ℃ of shaking culture are spent the night.Next day, collect inoculum, under the 5000g room temperature centrifugal 5 minutes, collecting precipitation (thalline).Precipitation is suspended in 1XPBS (8gNaCl, 0.2gKH ₂PO ₄, 2.9gNa ₂HPO ₄12H ₂O, 0.2gKCl, adding distil water is to 1L) in the damping fluid, recentrifuge (the same) is collected thalline.Thalline is suspended in the 0.1XPBS damping fluid again, regulates cell concentration to 2 * 10 ⁷Individual/mL.Add 200uL yeast fermentation bacterium liquid in the above-mentioned bacterium liquid of every 200uL, placed 1 hour for 37 ℃, then this bacterium liquid is carried out 10,000 times dilution, the bacterium liquid that therefrom takes out 200uL again is coated on the LB solid plate, 37 ℃ of overnight incubation, next day, the number that forms according to bacterium colony can be judged the height of T4 N,O-Diacetylmuramidase expression amount.

With the suis bacterium liquid handled through the BMMY substratum as negative control, found that, transform the colony counts minimizing (see figure 4) that some restructuring yeast strains methanol induction cultures that obtained can make suis form really through p9KT4 and p9KT4M plasmid DNA.Can reach a conclusion thus, reorganization T4 N,O-Diacetylmuramidase expressed in yeast has fungicidal activity, no matter the codon whether this gene is had a preference for according to yeast carried out optimization.

But it is worthy of note especially, hold the 6th the T4 lysozyme gene behind the amino acid change if contain codon optimized and N in the restructuring yeast strains, the suis colony counts that the subject of knowledge and the object of knowledge forms on the flat board after its methanol induction culture is handled, contain codon than those and reduce (see figure 4) widely without the suis colony counts of optimizing and N holds those restructuring yeast strains of the 6th the unaltered T4 lysozyme gene of amino acid to form, show that thus these two kinds of recombination yeasts on the quantity of secretion reorganization T4 antalzyme protein (or on its fungicidal activity) are what there were significant differences, this means gene codon through optimization and transform after can greatly improve really the T4 lysozyme gene in yeast cell expression efficiency and be secreted into biological yield born of the same parents outside (or owing to this recombinant protein fungicidal activity enhancing).Single its methanol induction culture of yeast strain that is transformed by the pPIC9K plasmid can not make streptococcic colony counts reduce (one of negative control).In addition, some restructuring yeast strains can not make streptococcic bacterium colony form the quantity reduction, and this may fail to express relevant with the T4 lysozyme gene that is imported.

Reduce according to the suis colony counts what, from a plurality of yeast recons of above-mentioned two classes, pick out the yeast strain of T4 N,O-Diacetylmuramidase expression efficiency the highest (the suis colony counts is fewer) respectively, and with its called after PP-T4 (containing codon without the T4 lysozyme gene of optimizing and changing) and PP-T4M (containing the T4 lysozyme gene after codon optimized and N holds the six amino acid change).

Embodiment 4

1. the preparation of seed liquor

Picking PP-T4 and PP-T4M yeast list bacterium colony from the RDB solid plate, be inoculated in respectively in the 10mL YPD liquid nutrient medium, 30 ℃ of shaking culture are spent the night, transfer in 100mL YPD liquid nutrient medium with 10% inoculum size, 30 ℃ of shaking culture 24 hours, transfer in 1L BMGY substratum with 10% inoculum size again, 30 ℃ of shaking culture 24 hours, then with it as seed liquor.

2. the high density fermentation of recombination yeast in the 20L fermentor tank

This fermenting process can be divided into following three phases: 1) the yeast culture stage: held 10L basis fermention medium [10 * BasalSalts (2.67% phosphoric acid in 20L fermentor tank (Shanghai Baoxing biological plant Engineering Co., Ltd product), 0.093% calcium sulfate, 1.82% vitriolate of tartar, 1.49% sal epsom, 0.413% potassium hydroxide)+4% glycerine], the ammoniacal liquor of adding 28% makes the pH value of this substratum maintain (ammoniacal liquor also can be used as the nitrogenous source of yeast bulk-growth simultaneously) about 7.5 earlier before inoculation, again in following ratio, in every liter of basic fermention medium, add 4.37ml trace salt solution PTM1 (0.6% copper sulfate, 0.008% sodium iodide, 0.3% manganous sulfate, 0.02% Sodium orthomolybdate, 0.002% boric acid, 0.05% cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate, 0.025% vitamin H, 0.5% sulfuric acid).The seed liquor that ratio inoculation in 10% prepares before this, 30 ℃ of aeration-agitations were cultivated about 24 hours.Carry out in the process in this stage, along with the growth of thalline, the dissolved oxygen amount in the substratum will reduce gradually by 100%, and after the carbon source in the substratum runs out of, dissolved oxygen amount will be increased to more than 80% once again, and the weight in wet base of thalline will reach 90-95g/L this moment.2) feed the carbon source stage: by peristaltic pump flow feeding liquid, feed supplement liquid is 50% glycerine (wherein containing 12mlPTM1/L), and the stream dosage is 18.15ml/hr/L.30 ℃ of aeration-agitations were cultivated about 4 hours, adjusted air flow in this stage and made dissolved oxygen amount all the time greater than 20%, and the thalline weight in wet base will reach about 180-190g/L this moment.3) the abduction delivering stage: stream adds methyl alcohol (containing 12ml/L PTM1), and the concentration of methyl alcohol is maintained about 0.3% all the time, and dissolved oxygen amount was cultivated 108 hours greater than 20%, 30 ℃ of aeration-agitation all the time, and the reorganization T4 antalzyme protein expression amount of this moment reaches the climax.Peek milliliter fermented liquid descended centrifugal 10 minutes for 4 ℃ through 5000rpm, in PP-T4 and PP-T4M tunning, respectively get the 20ul fermented supernatant fluid respectively and carry out the SDS-PAGE electrophoresis detection, find only in the PP-T4M swimming lane, to have the observable protein band of naked eyes, molecular weight is about about 19KD, it and the molecular weight of T4 N,O-Diacetylmuramidase recombinant protein in the inferring (see figure 5) that matches, the protein band that no naked eyes can observe in the PP-T4 swimming lane according to one's analysis may be relatively lower relevant with this gene expression amount in yeast.

3. the purifying of recombinant protein

After treating that a fermentation period is all over, leave and take the 500mL fermented liquid directly carries out next round as seed liquor (inoculum size is 5%) 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 expression of recombinant proteins amount of thalline is measured.In addition, take turns after fermenting process finishes fully, also get a little bacterium liquid and be coated on the YPD solid plate substratum, and 10 single bacterium colonies of picking arbitrarily therefrom every, and fully according to people's such as Cai Chuanqi method ^[13]Its genomic dna of rapid extraction also carries out PCR to it and detects, found that, the biomass of thalline, the speed of growth and T4 N,O-Diacetylmuramidase Recombinant Protein Expression amount are taken turns kept stable in the fermenting process at each, in addition, the detected result of PCR confirms that also PP-T4 and PP-T4M have good genetic stability (seeing Table 1).

The mensuration of the genetic stability of table 1. recombinant yeast pichia pastoris PP-T4 and PP-T4M bacterial strain and exogenous protein expression stability

After a fermentation period finished, except that staying the 500mL fermented liquid as the seed liquor, remaining fermented liquid descended centrifugal 10 minutes for 4 ℃ through 5000rpm, left and took supernatant.Add solid ammonium sulfate to saturation ratio therein respectively and reach 80%, at room temperature stirred 1 hour, 5000rpm is at room temperature centrifugal 20 minutes then, collecting precipitation, to precipitate and be suspended in again in 10ml 100mM Tris-HCl (pH8.0) damping fluid, and be transferred in the dialysis tubing, 100mM Tris-HC1 (pH8.0) damping fluid at 1L is dialysed under 4 ℃ then, to remove remaining ammonium sulfate, in 24 hours dialysis time, change dialyzate outside three times.After dialysis finished, sucking-off was by dialysate, then according to people's such as Ausubel method in dialysis tubing ^[14], this dialysate to be crossed Sephadex G-75 molecular sieve gel post (Sephadex G-75 is available from AmershamBiosciences company) carry out further purifying, employed column volume is 80X1.5cm.Behind last sample, carry out wash-out, press the volume fraction collection elutriant of 2ml with 100mM Tris-HCl (pH8.0) damping fluid.Detect the lysozyme activity of each part elutriant with colony counting method (see embodiment 3 3), the wash-out that then all can be reduced bacterium colony formation quantity partly pools together, add ammonium sulfate to final concentration once more and reach 80% and precipitate and dialyse, method is the same.Dialysate in the dialysis tubing is transferred to respectively in the drying bottle, and after lyophilize, precipitation is suspended in the deionized water of a certain amount of sterilization again.Detect by SDS-PAGE electrophoresis and gel imaging instrument, find that its purity of protein can reach 99% above (see figure 5).In addition, after T4 N,O-Diacetylmuramidase recombinant protein was handled for a long time through above-mentioned twice ammonium sulfate precipitation, dialysis and column chromatography etc., its fungicidal activity was not subjected to any loss.

(protein determination kit is available from Sigma company to utilize the Lowry method, article No. is P5656) mensuration wherein proteic content (concrete grammar is seen the test kit specification sheets), wherein PP-T4 N,O-Diacetylmuramidase recombinant protein content is about 0.01g/L (mean values of three high density fermentations), and PP-T4M N,O-Diacetylmuramidase recombinant protein content is 0.17g/L (mean values of three high density fermentations), this result shows, the gene of coding T4 N,O-Diacetylmuramidase is after codon is optimized, and its expression efficiency will improve about 17 times before optimizing really at least.

4. the comparison of different recombinant protein fungicidal activities

The T4 N,O-Diacetylmuramidase recombinant protein of above-mentioned purifying is through transferring to identical working concentration with 0.1 * PBS damping fluid, and the same above-mentioned colony counting method (3 among the embodiment 3 seen in concrete operations) that adopts of the detection of fungicidal activity, bacterial strain for examination then is respectively suis (bacterial strain code name: 1.2499), streptococcus aureus (the bacterial strain code name: 1.2465, available from DSMZ of Institute of Microorganism, Academia Sinica) and bacillus coli DH 5 alpha (available from U.S. GIBCO company).Before finding to transform, detected result all can reduce the quantity of three kinds of bacterium colonies that strains tested forms with improved T4 N,O-Diacetylmuramidase recombinant protein, but the most responsive with suis to the T4 N,O-Diacetylmuramidase.And the T4 N,O-Diacetylmuramidase recombinant protein fungicidal activity that N holds six amino acid to change into behind the Methionin obviously is eager to excel 2-4 doubly than unaltered T4 N,O-Diacetylmuramidase, and this matches with the result of study that forefathers report ^[1]

The comparison of the different T4 N,O-Diacetylmuramidase of table 2 recombinant protein fungicidal activity ^{1. 2.}

Bacterial strain	T4 N,O-Diacetylmuramidase (not transforming)	T4 N,O-Diacetylmuramidase (N end 6 is a Methionin)
			Suis	49	18
Streptococcus aureus	75	31
			Intestinal bacteria	19	6

1.: to add the formed colony number of 0.1 * PBS damping fluid is 100, and numeral is handled the remaining relative bacterium colony number that gets off in back for adding different T4 N,O-Diacetylmuramidases in the table, and is the mean value of three tests.

2.: for suis, T4 N,O-Diacetylmuramidase work final concentration is 0.01ug/uL, and for streptococcus aureus and intestinal bacteria, T4 N,O-Diacetylmuramidase work final concentration is 0.1ug/uL.

Embodiment 5

1. contain the capsular preparation of T4 N,O-Diacetylmuramidase recombinant protein

Recombination yeast PP-T4M is after extensive, high density fermentation are produced, can obtain a large amount of, have the highly active T4 N,O-Diacetylmuramidase of the pathogenetic bacteria of a killing recombinant protein, and this albumen does not need to carry out the complicated extraction preparation and the purifying processing of height, fermented supernatant fluid only need pass through simple ammonium sulfate precipitation, to precipitate resuspending then in a small amount of 0.01XPBS damping fluid, to remove residual ammonium sulfate molecule and methyl alcohol, dialysate is T4 N,O-Diacetylmuramidase crude extract after high temperature drying through dialysis.This crude extract can be according to a certain percentage and yam starch or other edible foodstuff starch (starch mainly is as stopping composition) mixing, then their cans become capsule.As the case may be, each capsule can pour into 100-300mg such dry powder, for example, the capsule of a 250mg can contain the reorganization T4 antalzyme protein about 5-10ug.Adult every day 2-3 time, 1-2 grain/time take such capsule, just might obtain resistivity to some digestive tube bacteriosises.

2. contain the preparation of T4 N,O-Diacetylmuramidase recombinant protein injection

Above-mentioned T4 N,O-Diacetylmuramidase recombinant protein is behind the further purifying of process, for example after ion exchange chromatography or the affinity chromatography, can eliminate wherein contained impurity for example carbohydrate, toxin and thermal source fully, the T4 N,O-Diacetylmuramidase recombinant protein of this moment can consider to be processed into injection, as contains recombinant protein 1-5mg/ agent.If this injection is input in human body or the animal body in the mode of injection or intravenous drip; can not only reduce the using dosage and the use cost of T4 N,O-Diacetylmuramidase like this; and more can further improve its diseases prevention and the effect of curing the disease; for example; directly this injection is injected in the mammary gland of milk cow, can reduces the risk that milk cow suffers from mazoitis effectively.

Reference

1.Düring K.，et al.，FEBS Letters，1999，449(2-3)：93-100.

2. Shao Chun honor etc., Jiangsu agricultural sciences, 1996,3:61-62.

3. bag holds jade etc., scientific experimentation and research, 1997,1:2-4.

4.Jollès P and Jollès J，Molecular and Cellular Biochemistry，1984，63：165-189.

5.Tsugita A and Inouye M，The Journal of Biological Chemistry，1968，243(2)：391-397.

6.Owen J E，et al.，J.Mol.Biol.，1983，165：229-248.

7.Cregg J M，et al.，Biotechnology(N.Y.)，2000，11(8)：905-910.

8. fourth Yun Fei etc., life science, 2003,15 (1): 26-30.

9. Liu Zhi deep pool etc., biotechnology, 2004,14 (1): 56-58.

10. Xiao Shengke etc., biotechnology circular, 2004,2:23-30.

11.Sharp P M，et al.，Neucleic Acids Res.，1986，14：5125-5142.

12.Sambrook J and Rusell D W，Molecular Cloing：A Laboratory Manual，2001，ColdSpringer harbor Laboratory Press.

13. Cai's legend and square Rong Xiang, biotechnology journal, 2001,17 (2): 155-160.

14.Ausubel F M，et al.，Short Protocols in Molecular Biology，John Wiley & Sons，Inc.1995，373-374.

Just some the special preferential embodiments that further describe of the present invention are according to the patent application requirement and in order to explain and illustrate the content of this patent. Obviously, do not deviating within the spirit and scope of the present invention, can on this basis, do further improvement and variation.

Sequence table

＜110〉Liu Dehu

＜120〉expression and the production method of high disinfection vitality T 4 lysozyme in yeast

<160>4

<210>1

<211>164

<212>PRT

＜213〉artificial sequence

<400>1

Met Asn Ile Phe Glu Lys 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 Ile Trp Tyr Asn Gln Thr Pro Asn

130 135 140

Arg Ala Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala

145 150 155 160

Tyr Lys Asn Leu

<210>2

<211> 521

<212> DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(510)

<400>2

CTC GAG AAA AGA GAG GCT ATG AAC ATC TTC GAG AAG TTG AGA ATC GAC GAG AGA TTG 57

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

1 5 10 15

AGA TTG AAG ATT TAC AAG GAC ACT GAG GGT TAC TAC ACT ATC GGT ATC GGT CAC TTG 114

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

20 25 30 35

TTG ACT AAG TCC CCA TCC TTG AAC GCT GCT AAG TCC GAG TTG GAC AAG GCT ATC GGT 171

Leu Thr Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile Gly

40 45 50 55

AGA AAC TGT AAC GGT GTT ATC ACT AAG GAC GAG GCT GAG AAG TTG TTC AAC CAA GAC 228

Arg Asn Cys Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu Phe Asn Gln Asp

60 65 70 75

GTT GAC GCT GCT GTT AGA GGT ATC TTG AGA AAC GCT AAG TTG AAG CCA GTT TAC GAC 285

Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala Lys Leu Lys Pro Val Tyr Asp

80 85 90 95

TCC TTG GAC GCT GTT AGA AGA TGT GCT TTG ATC AAC ATG GTT TTC CAA ATG GGT GAG 342

Ser Leu Asp Ala Val Arg Arg Cys Ala Leu Ile Asn Met Val Phe Gln Met Gly Glu

100 105 110

ACT GGT GTT GCT GGT TTC ACT AAC TCC TTG AGA ATG TTG CAA CAA AAG AGA TGG GAC 399

Thr Gly Val Ala Gly Phe Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp

115 120 125 130

GAG GCT GCT GTT AAC TTG GCT AAG TCC ATC TGG TAC AAC CAA ACT CCA AAC AGA GCT 456

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

135 140 145 150

AAG AGA GTT ATC ACT ACT TTC AGA ACT GGT ACT TGG GAC GCT TAC AAG AAC TTG TAA 513

Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Lys Asn Leu *

155 160 165 170

GCG GCC GC 521

<210>3

<211>164

<212>PRT

＜213〉the phage-coded N,O-Diacetylmuramidase of T4

<400>3

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 Ile Trp Tyr Asn Gln Thr Pro Asn

130 135 140

Arg Ala Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala

145 150 155 160

Tyr Lys Asn Leu

<210>4

<211>521

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(510)

<400>4

CTC GAG AAA AGA GAG GCT ATG AAT ATA TTT GAA ATG TTA CGT ATA GAT GAA CGT CTT 57

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

1 5 10 15

AGA CTT AAA ATC TAT AAA GAC ACA GAA GGC TAT TAC ACT ATT GGC ATC GGT CAT TTG 114

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

20 25 30 35

CTT ACA AAA AGT CCA TCA CTT AAT GCT GCT AAA TCT GAA TTA GAT AAA GCT ATT GGG 171

Leu Thr Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile Gly

40 45 50 55

CGT AAT TGC AAT GGT GTA ATT ACA AAA GAT GAG GCT GAA AAA CTC TTT AAT CAG GAT 228

Arg Asn Cys Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu Phe Asn Gln Asp

60 65 70 75

GTT GAT GCT GCT GTT CGC GGA ATT CTG AGA AAT GCT AAA TTA AAA CCG GTT TAT GAT 285

Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala Lys Leu Lys Pro Val Tyr Asp

80 85 90 95

TCT CTT GAT GCG GTT CGT CGC TGT GCA TTG ATT AAT ATG GTT TTC CAA ATG GGA GAA 342

Ser Leu Asp Ala Val Arg Arg Cys Ala Leu Ile Asn Met Val Phe Gln Met Gly Glu

100 105 110

ACC GGT GTG GCA GGA TTT ACT AAC TCT TTA CGT ATG CTT CAA CAA AAA CGC TGG GAT 399

Thr Gly Val Ala Gly Phe Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp

115 120 125 130

GAA GCA GCA GTT AAC TTA GCT AAA AGT ATA TGG TAT AAT CAA ACA CCT AAT CGC GCA 456

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

135 140 145 150

AAA CGA GTC ATT ACA ACG TTT AGA ACT GGC ACT TGG GAC GCG TAT AAA AAT CTA TAA 513

Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Lys Asn Leu *

155 160 165 170

GCG GCC GC 521

Claims (10)

1. one kind by the expression of recombinant yeast and the method for producing the tool high disinfection vitality T 4 lysozyme.

2. as the method in the claim 1, wherein said T4 N,O-Diacetylmuramidase has the aminoacid sequence shown in 1 in the sequence table, and this recombinant protein T4 N,O-Diacetylmuramidase native protein more coded than T4 phage has higher fungicidal activity, if can be widely used in industries such as medicine, food, agricultural and makeup as additive.

3. as the method in the claim 1, the dna sequence dna of coding high disinfection vitality T 4 lysozyme is characterized in that encoding or to the aminoacid sequence of small part coding as shown in sequence table 1.

4. as the described dna sequence dna in the claim 3, it is characterized in that having or have the nucleotide sequence shown in 2 in the sequence table to small part.

5. as the method in the claim 1, need to make up a kind of expression plasmid carrier, it is characterized in that it contains the dna sequence dna of the coding high disinfection vitality T 4 lysozyme described in the claim 3.

6. the expression vector described in right 5, it be meant pPIC9K or other can abduction delivering justacrine recombinant protein to the outer plasmid vector of born of the same parents.

7. as the method in the claim 1, need a kind of yeast host cell, it is characterized in that it comprises the expression vector described in the claim 5.

8. the host cell described in claim 7, it is meant the yeast strain that Pichi strain GS115 or other can expression alien genes.

9. as the method in the claim 1, a kind of method of utilizing pichia spp GS115 to efficiently express and produce the T4 N,O-Diacetylmuramidase described in claim 1, comprising following four-stage:

A: yeast culture

B: the carbon source of feeding

C: abduction delivering

D: protein purification

10. as the method in the claim 1, wherein said high disinfection vitality T 4 lysozyme is behind partial purification or complete purifying, if can be widely used in industries such as medicine, food, agricultural and makeup as additive.

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