CN104313048A - Method for producing lactoferrin by using saccharomyces cerevisiae - Google Patents

Abstract

The invention discloses a method for producing lactoferrin by using saccharomyces cerevisiae. The method comprises the following steps: (1) establishing recombinant bacteria, namely, leading expression cassette to host bacteria, wherein the expression cassette is formed by connecting a promoter Ppgk, alpha-signal peptide gene and encoding gene of the lactoferrin LFcin B; (2) inducing and cultivating the recombinant bacteria to obtain lactoferrin bacteria liquid; and (3) centrifuging the bacteria liquid for collecting supernate, namely precipitating the lactoferrin from the supernate to obtain the lactoferrin. Experiment results show that antimicrobial peptide lactoferrin fermented by using the saccharomyces cerevisiae has obvious antimicrobial effect on escherichia coli DH5a.

Description

A kind of yeast saccharomyces cerevisiae produces the method for lactoferrin

Technical field

The present invention relates to biological technical field, be specifically related to a kind of method that yeast saccharomyces cerevisiae produces lactoferrin.

Background technology

In recent years, China's animal husbandry development is rapid, and its output value accounts for three one-tenth of the general production value of agriculture, has become the mainstay industry in rural economy.But, because feeding environment is poor, production technology falls behind relatively, the situation is tense for animal epidemic, creates serious impact to whole industry.By improving feeding environment, improvement production technology can solve above-mentioned the first two problem; And solve this problem of animal epidemic time, be then typically employed in animal-feed medium-term and long-term, in large quantities add antibiosis usually reach the object of preventing and treating epidemic disease.Antibiotic a large amount of use, serves the effect of " the solution matter of great urgency " in a short time really to control epidemic disease, but long-term abuse of antibiotics, not only can destroy the microecological balance of animal digestive system, and some pathogenic bacterias can be made to produce resistance.Meanwhile, due to microbiotic in animal body residual, threat be also result in the health of the mankind.Based on above negative effect, European Union completely forbade microbiotic in 2006 and uses in animal-feed.China is since accession to WTO, and antibiotic use has had a strong impact on the competitive power of China's livestock industry product in world market; Along with the generally raising of living standards of the people, the quality security problem in the source of livestock product, production and the course of processing also becomes the focus that the common people pay close attention to day by day.Therefore, the Sustainable development of development of new Substitutes For Antibiotic to China's livestock industry plays a very important role.

Antibacterial peptide (antibacterial peptides) is small molecule polypeptide, has the effect resisted the infringement of extraneous pathogenic micro-organism, eliminate vivo mutations cell.Its structure is roughly divided into 5 classes: (1) strand is without the alpha-helix of cysteine residues, or the peptide that the two sections of alpha-helixs connected by random coil form; (2) some amino-acid residue is rich in but not containing the antibacterial peptide of cysteine residues; (3) containing the antimicrobial polypeptide of 1 disulfide linkage; (4) there are more than 2 or 2 disulfide linkage, there is the antibacterial peptide of beta sheet structure; (5) derived by other known function, larger polypeptide and there is the peptide of anti-microbial activity.Antibacterial peptide is encoded by biological specific gene, there is broad spectrum antibiotic activity, especially obvious killing action is had to Resistant strain, but do not destroy zooblast, non-immunogenicity, there is the advantages such as efficient, safety, noresidue, the effect of energy immunity moderation, not disturbing the characteristics such as intestinal beneficial bacterium, be applied to livestock industry, is a kind of Substitutes For Antibiotic of most DEVELOPMENT PROSPECT.

Lactoferrin (Lactoferrin, LF) is found when sepg whey albumen by people such as nineteen thirty-nine Sorensen, and to be separated from cow's milk in nineteen sixty by Groves and to obtain.Bovine lactoferricin (Bovine Lactoferricin is abbreviated as Lfcin B) is 25 amino-acid residues holding (17 ~ 41) to be hydrolyzed from Bovinelactoferrin (bLF) N-through stomach en-, and molecular weight is about 3.1kd.Lfcin is the anti-microbial activity center of bLF, plays an important role (Kanget al, 1996) to bLF anti-microbial activity.Lfcin B is the most obvious part of Lfcin active centre fungistatic effect, and its fungistatic effect is 400 times of Bovinelactoferrin, plays a significant role in maintenance composition of gut flora.Lfcin B in aqueous in two reverse β-pleated sheet structure structures of amphipathic, have heat-resisting, be not easily degraded at digestive tube, the feature of no antigen, can immunity moderation effect, there is broad-spectrum antibacterial action, do not disturb the characteristics such as intestinal beneficial bacterium.It all has anti-microbial effect to many G+ and G-, as intestinal bacteria, Salmonella enteritidis, Pseudomonas aeruginosa, streptococcus aureus, clostridium perfringens etc., but do not act on bifidus bacillus etc. to human body beneficial bacteria, simultaneously can also irritation cell growth, participate in immunomodulatory.Therefore, have broad application prospects in fields such as agricultural, herding, medical treatment.

Natural antibacterial peptide wide material sources, but there is the problems such as cost is high, separation and purification is difficult.And genetic engineering technique is low because having cost, easy and simple to handle, be easy to the features such as separation and purification and the study hotspot become in recent years.The molecular improvement of efficient expression antimicrobial peptides, the foundation of novel fermentation production technology, the exploitation of the special antibacterial peptide product innovation of feed, has become the important goal of this field developers.

Summary of the invention

An object of the present invention is to provide a kind of method of producing lactoferrin.

The method of production lactoferrin provided by the invention comprises the steps:

(1) recombinant bacterium is built: expression cassette is imported Host Strains;

Described expression cassette is formed by connecting by the encoding gene of promotor Ppgk, α-signal peptide gene and lactoferrin Lf cin B successively;

(2) recombinant bacterium described in inducing culture, obtains lactoferrin bacterium liquid;

(3) by centrifugal for described bacterium liquid, supernatant is collected; From supernatant, precipitate lactoferrin, namely obtain described lactoferrin.

In aforesaid method, the described method precipitating lactoferrin from supernatant is for carry out precipitating and concentrating with acetone and Tricholroacetic Acid successively.

In aforesaid method, described Host Strains is yeast saccharomyces cerevisiae INVSc1.

In aforesaid method, the aminoacid sequence of described lactoferrin Lf cin B is as shown in 1-35 amino acids in SEQ ID No.5; The coding gene sequence of described lactoferrin Lf cin B is as shown in the nucleic acid molecule of SEQ ID No.4 1-105 position.

In aforesaid method, the aminoacid sequence of described α-signal peptide is as shown in 1-95 amino acids in SEQ ID No.3; The coding gene sequence of described α-signal peptide is as shown in 1-285 position nucleic acid molecule in SEQ ID No.2.

In aforesaid method, the nucleotide sequence of described promotor Ppgk is as shown in 1-793 position nucleic acid molecule in SEQ ID No.1.

In aforesaid method, by the method that described expression cassette imports in yeast be: the multiple clone site described expression cassette being inserted pYES2 carrier, obtains recombinant vectors; Again recombinant vectors is imported in described host yeast.

Another object of the present invention is to provide a kind of recombinant bacterium.

Expression cassette is imported Host Strains and obtains by recombinant bacterium provided by the invention.

In above-mentioned recombinant bacterium, described expression cassette is formed by connecting by the encoding gene of promotor Ppgk, α-signal peptide gene and lactoferrin Lf cin B successively.

In above-mentioned recombinant bacterium, described Host Strains is yeast saccharomyces cerevisiae INVSc1.

In above-mentioned recombinant bacterium, the aminoacid sequence of described lactoferrin Lf cin B is as shown in 1-35 amino acids in SEQ ID No.5; The coding gene sequence of described lactoferrin Lf cin B is as shown in the nucleic acid molecule of SEQ ID No.4 1-105 position.

In above-mentioned recombinant bacterium, the aminoacid sequence of described α-signal peptide is as shown in 1-95 amino acids in SEQ ID No.3; The coding gene sequence of described α-signal peptide is as shown in 1-285 position nucleic acid molecule in SEQ ID No.2; The nucleotide sequence of described promotor Ppgk is as shown in 1-793 position nucleic acid molecule in SEQ ID No.1.

Last object of the present invention is to provide method and/or the recombinant bacterium described above application in production lactoferrin of production lactoferrin described above.

The Saccharomyces Serevisiae Expression System that the present invention adopts has the following advantages: growth velocity is fast, is easy to high-density culture; The albumen of energy expression-secretion, and product is easy to separation and purification; There is multiple posttranslational modification, as folding in polypeptide, glycosylation, methylate, acetylize etc.; Genetic background is clear, is easy to operate expression vector.

The invention provides a kind of method that yeast saccharomyces cerevisiae produces lactoferrin.Result shows: the present invention utilizes the yeast saccharomyces cerevisiae antibacterial peptide lactoferrin obtained that carries out fermenting to have obvious bacteriostatic action to bacillus coli DH 5 alpha.

Accompanying drawing explanation

Fig. 1 is pYES2-CPS-Blf carrier figure.

Fig. 2 is the pcr amplification of promotor Ppgk gene.

Fig. 3 is that the enzyme that promotor Ppgk connects carrier T cuts qualification.

Fig. 4 is the pcr amplification of α-signal peptide gene.

Fig. 5 is that the enzyme that α-signal peptide gene connects carrier T cuts qualification.

Fig. 6 is the pcr amplification of bovine lactoferrin gene.

Fig. 7 is that lactoferrin-carrier T enzyme cuts qualification.

Fig. 8 is that the enzyme of carrier pYES2-Ppgk transformation of E. coli cuts qualification.

Fig. 9 is that the enzyme of carrier pYES2-CPS transformation of E. coli cuts qualification.

Figure 10 is that the colibacillary enzyme of pYES2-CPS-Blf vector cuts qualification.

Figure 11 is that the enzyme of pYES2-CPS-Blf vector yeast saccharomyces cerevisiae cuts qualification.

Figure 12 is expressing protein Tricine-SDS-PAGE gel electrophoresis figure.

Figure 13 is lactoferrin activity identification.

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

The acquisition of embodiment 1, promotor Ppgk, α-signal peptide gene and bovine lactoferrin gene

1, the acquisition of promotor Ppgk

With Nanyang K bacterial strain (being purchased from Guangdong Microbes Inst) for template, adopt Ppgk-F1 and Ppgk-F2 primer (table 1) amplification promotor Ppgk gene.PCR reaction conditions: 94 DEG C of 5min, 94 DEG C 30s-59 DEG C 30s-72 DEG C of 70s, 30 circulations, 72 DEG C of 7min.After 2% agarose gel electrophoresis, as shown in Figure 2, swimming lane M is marker to result, and swimming lane 1 and 2 is the PCR primer of promotor Ppgk gene.

Reclaim purifying object fragment, be stored in-20 DEG C for subsequent use.The Ppgk gene obtained is connected with carrier T, transformation of E. coli DH5 α bacterial strain, at 37 DEG C, with Age I and Pvu II double digestion screening positive strain (Fig. 3), send positive bacteria liquid to the exactness of company sequencing analysis checking Ppgk sequence.

Sequencing result shows: the carrier T being connected into Ppgk sequence, as shown in 1-793 position Nucleotide in sequence 1, is denoted as Ppgk-T carrier by the sequence of promotor Ppgk.

2, the acquisition of α-signal peptide gene

With plasmid pPIC9K (being purchased from Changsha Yingrun Biological Technology Co., Ltd.) for template, adopt α-F1 and α-F2 primer (table 1) amplification α-signal peptide gene.PCR reaction conditions: 94 DEG C of 5min, 94 DEG C 30s-59 DEG C 30s-72 DEG C of 60s, 30 circulations, 72 DEG C of 7min.After 2% agarose gel electrophoresis, as shown in Figure 4, in the diagram, swimming lane M is marker to result, and swimming lane 1 and 2 is the PCR primer of α-signal peptide gene gene.

Reclaim purifying object fragment, be stored in-20 DEG C for subsequent use.α-the signal peptide gene obtained is connected with carrier T, transformation of E. coli DH5 α bacterial strain, at 37 DEG C, with Kpn I and Not I double digestion screening positive strain (Fig. 5), send the exactness of positive bacteria Ye Song company sequencing analysis α-signal peptide gene sequence.

Sequencing result shows: the carrier T being connected into α-signal peptide sequence, as shown in 1-285 position Nucleotide in sequence 2, is denoted as α-signal peptide-carrier T by the sequence of α-signal peptide gene.

3, the acquisition of bovine lactoferrin gene

According to the gene order of lactoferrin mature peptide in NCBI GenBank, the DNA fragmentation of synthetic lactoferrin mature peptide is also stored in PMD19 Simple plasmid.The sequence of bovine lactoferrin gene, as shown in the Nucleotide of sequence table 3 1-105 position, obtains by synthetic.Synthesize a pair special primer Blf F1 and Blf F2 (table 1) according to its gene order engineer again, with in PMD19 Simple plasmid for template to be increased lactoferrin (Lfcin B) gene fragment by pcr clone.PCR reaction conditions: 94 DEG C of 5min, 94 DEG C 30s-56 DEG C 30s-72 DEG C of 20s, 30 circulations, 72 DEG C of 7min.After 2% agarose gel electrophoresis, as shown in Figure 6, in figure 6, swimming lane M is D2000marker to result, and swimming lane 1-4 is the PCR primer of bovine lactoferrin gene.

Reclaim purifying object fragment, be stored in-20 DEG C for subsequent use.The bovine lactoferrin gene obtained is connected with carrier T, transformation of E. coli DH5 α bacterial strain, at 37 DEG C, with Not I and Xba I double digestion screening positive strain (Fig. 7), send the exactness of positive bacteria Ye Song company sequencing analysis checking bovine lactoferrin gene sequence.

Sequencing result shows: the carrier T being connected into Lfcin 1 B gene sequence, as shown in 1-105 position Nucleotide in sequence 4, is denoted as Blf-T carrier by the sequence of Lfcin 1 B gene.

Table 1, primer sequence

Embodiment 2, structure secreted expression carrier pYES2-CPS-Blf

1, the acquisition of pYES2-Ppgk carrier

With Age I and Pvu II respectively enzyme cut pYES2 carrier (being purchased from Changsha Yingrun Biological Technology Co., Ltd.) and Ppgk-T carrier, product, after 1.5% agarose gel electrophoresis, reclaims Ppgk gene fragment and pYES2 carrier segments; Ppgk gene fragment after being cut by enzyme with T4 ligase enzyme is connected with pYES2 carrier segments, obtains pYES2-Ppgk carrier; By pYES2-Ppgk vector e.colistraindh5α, conversion rear extraction plasmid DNA is carried out enzyme and is cut qualification (Fig. 8).

Carry out sequence verification to pYES2-Ppgk carrier, result to show during the gene order inserted between Age I and Pvu II restriction enzyme site of pYES2-Ppgk carrier is as sequence 1, shown in the Nucleotide of 1-793 position, to show that carrier is correct.

2, the acquisition of pYES2-CPS carrier

With Kpn I and Not I respectively enzyme cut pYES2-Ppgk carrier and α-signal peptide-carrier T, product, after 1.5% agarose gel electrophoresis, reclaims α-signal peptide gene fragment and pYES2-Ppgk carrier segments; α after being cut by enzyme with T4 ligase enzyme-signal peptide gene fragment is connected with pYES2-Ppgk carrier segments, obtains pYES2-CPS carrier; By pYES2-CPS vector e.colistraindh5α, conversion rear extraction plasmid DNA is carried out enzyme and is cut qualification (Fig. 9).

Carry out sequence verification to pYES2-CPS carrier, result to show during the gene order inserted between the Kpn I and Not I restriction enzyme site of pYES2-CPS carrier is as sequence 2, shown in the Nucleotide of 1-285 position, to show that carrier is correct.In sequence 2, the aminoacid sequence of the albumen of 1-285 position nucleic acid molecule coding is as shown in sequence table in sequence table 3.

3, the acquisition of pYES2-CPS-Blf carrier

With Not I, Xba I respectively enzyme cut pYES2-CPS carrier and Blf-T carrier, product, after 1.5% agarose gel electrophoresis, reclaims bovine lactoferrin gene fragment and pYES2-CPS carrier segments; Bovine lactoferrin gene fragment after being cut by enzyme with T4 ligase enzyme is connected with pYES2-CPS carrier segments, obtains pYES2-CPS-Blf carrier, as shown in Figure 1; By pYES2-CPS-Blf vector e.colistraindh5α, conversion rear extraction plasmid DNA is carried out enzyme and is cut qualification (Figure 10).

Carry out sequence verification to pYES2-CPS-Blf carrier, result to show during the gene order inserted between the Not I, Xba I restriction enzyme site of pYES2-CPS-Blf carrier is as sequence 4, shown in the Nucleotide of 1-105 position, to show that carrier is correct.In sequence 4, the aminoacid sequence of the albumen of 1-105 position nucleic acid molecule coding is as shown in sequence table in sequence table 5.

Embodiment 3, recombinant vectors pYES2-CPS-Blf transformed saccharomyces cerevisiae INVSc1

1, yeast saccharomyces cerevisiae INVSc1 competent cell is prepared

1. activated spawn: get 10ul yeast saccharomyces cerevisiae INVSc1 (Bei Nuo bio tech ltd, Shanghai) frozen glycerol stock in 3mlYPD liquid nutrient medium, 30 DEG C, 200rpm incubated overnight.

2. get the above-mentioned activated bacterium liquid of 300ul in 50mlYPD liquid nutrient medium, 30 DEG C, 200rpm cultivates.

3. when above-mentioned bacterial concentration reaches OD600=1.0 ~ 1.3, by the bacterium liquid centrifuge tube of falling 50ml, be placed on ice after 10min, 4 DEG C, 3000rpm, centrifugal 5min collecting cell.

4. the sterilized water re-suspended cell of 50ml precooling is used, 4 DEG C, 3000rpm, collecting cell after centrifugal 5min.

5. repeating step 4..

6. the 1mol/L sorbyl alcohol re-suspended cell of 10ml precooling is used, 4 DEG C, 3000rpm, centrifugal 5min.

7. blow and beat suspension cell gently with the 1mol/L sorbyl alcohol of 160ul precooling, be dispensed in the EP pipe of 1.5ml, often pipe 80ul, be placed in stand-by on ice.

2, in transform plastids pYES2-CPS-Blf to yeast saccharomyces cerevisiae INVSc1 competent cell

1. the pYES2-CPS-Blf plasmid getting 10ul joins in 80ul Saccharomyces cerevisiae competent cell, fully transfers in the electric shock cup of the 0.2cm specification of precooling after mixing, ice bath 5min.

2. dry electric shock cup surface, the parameter corresponding according to electric shock instrument shocks by electricity.

3. in electric shock cup, add the Sorbitol Solution USP of 1ml precooling immediately, be fully transferred in the EP pipe of 1.5ml after mixing.

4. after 30 DEG C of stationary incubation 1h, get 300ul bacterium liquid and coat on SC-U flat board, 30 DEG C, inversion is cultured to and grows mono-clonal.

3, utilize yeast saccharomyces cerevisiae uracil-deficient type minimum synthetic medium (SC-U) plate screening positive strain, get single bacterium colony from the choicest of SC-U flat board rifle and shake bacterium, extract plasmid DNA.

4, by the plasmid transformation escherichia coli DH5 α that previous step is extracted, positive plasmid (Figure 11) is chosen.Secrete peptide upstream design one at α and detect primer, send company to check order, confirm that α secretion peptide and lactoferrin are in same reading frame.Sequencing primer: 5'-GGTACCATGAGATTTCCTTC-3'.The correct recombinant bacterium that checks order, for transforming successful recombination microzyme, is antibacterial peptide lactoferrin saccharomyces cerevisiae engineered yeast.

The expression of embodiment 4, antibacterial peptide lactoferrin saccharomyces cerevisiae engineered yeast and Pichia yeast engineering

One, saccharomyces cerevisiae engineered yeast is induced to express lactoferrin

1, saccharomyces cerevisiae engineered yeast is induced to express lactoferrin

1. transfer 200ul antibacterial peptide lactoferrin saccharomyces cerevisiae engineered yeast in the SC-U liquid nutrient medium of 20ml, 30 DEG C, 200rpm spends the night to shake and cultivates.

2. survey the OD600 value (Y) of bacterium liquid, according to formula X=(Y*20)/0.4, calculate the amount (X) bacterium liquid OD600 being diluted to the SC-U liquid nutrient medium required for 0.4.

3. 4 DEG C, 4000rpm, centrifugal 5min, abandons supernatant, adds SC-U liquid nutrient medium, blow and beat resuspended, mix according to the X value of 2. middle calculating.

4. 30 DEG C, 200rpm, shaking culture 7 ~ 9 days.

2, acetone precipitation albumen:

1. get 10ml bacterium liquid, the centrifugal 5min of 8000rpm, collect supernatant.

2. getting 4ml supernatant adds in the acetone of-20 DEG C of precoolings of 20ml, mixing.

3.-20 DEG C of process 2h are statically placed in.

4. 4 DEG C, 12000rpm, centrifugal 10min, abandons supernatant.

5. the sterilized water dissolution precipitation of 200ul is added.

3, the mensuration of protein concentration

Adopt the Lowry method determination of protein concentration test kit of Solarbio company to carry out concentration determination to the protein sample after precipitation respectively, the sample getting 3 different batches measures.

4, Tricholroacetic Acid precipitation concentration

The test kit of Sheng Gong biotechnology limited-liability company is adopted to carry out precipitation concentration to above-mentioned acquisition protein solution:

1. in above-mentioned protein solution 200ul, 50ul precipitated liquid A is added, vortex oscillation 10s, mixing.

2. 1h is hatched for 4 DEG C.

3. 4 DEG C, the centrifugal 15min of 12000rpm.

4. 600ul washings B vortex oscillation 10s is added.

5. 4 DEG C, after refrigeration 10min, 4 DEG C, the centrifugal 15min of 12000rpm must precipitate.

6. in stink cupboard, outwell supernatant, throw out ventilates and dries 30min.

7. add 20ul lysate C and vortex oscillation 10s, if solution is yellow, adds 1 ~ 5ul distiller liquor D, make it become blueness.

5, Tricine-SDS-PAGE electrophoresis detection expression of results as shown in figure 12, and in fig. 12, M is Premixed Protein Marker, and swimming lane 1 is negative control (empty carrier), and swimming lane 2 is lactoferrin.

Two, Pichia yeast engineering is induced to express lactoferrin

1, the acquisition of Pichia yeast engineering

According to pichia spp access to your password son Preference, optimization design synthetic bovine lactoferrin gene (Lfcin B), bovine lactoferrin gene is connected into pPIC9K carrier, obtain recombinant expression vector pPIC9K-LfcinB (with 6 × histag), by in electroporated for recombinant expression vector pPIC9K-LfcinB Pichia pastoris GS115 (being purchased from Bei Nuo bio tech ltd, Shanghai), obtain the Pichia yeast engineering of expressing lactoferrin peptide gene (Lfcin B).Pichia yeast engineering is coated on YPD flat board.Positive monoclonal is selected in qualification, is inoculated in 5mlYPD liquid nutrient medium, 30 DEG C, 250rpm shaking culture 18-20h; 0.5-1% inoculum size is forwarded to 300mlYPD liquid nutrient medium, 30 DEG C, and 250rpm shaking culture is spent the night to OD600=2 ~ 6 as the kind daughter bacteria of inoculation fermentation tank.

2, Pichia yeast engineering is induced to express lactoferrin

Induction Pichia yeast engineering expresses the detailed step reference of lactoferrin: the expression of antibacterial peptide Cecropin D in pichia spp, purifying and activity identification. Yin Na, Li Hongjun, Peng Mei etc. Products in China magazine volume the 3rd phase March the 21st in 2008: 185 ~ 189.

Carry out expansion fermentation culture by kind of daughter bacteria access 30L fermentor tank, after entering inductive phase, add the expression that 100% methyl alcohol (containing PTM1,12ml/L) induces lactoferrin.

3, the separation and purification of albumen

Utilize the 6 × his tag on carrier, above-mentioned expression product is carried out purifying by the NTA-Ni affinity column specification sheets of QIAGEN company, dialysing with phosphate buffered saline buffer (pH6.0), and using PEG8000 protein concentrate.Sample after concentrated is used for protein concentration and detects.

The lactoferrin concentration that embodiment 5, yeast saccharomyces cerevisiae and pichia spp are induced and expression activitiy

1, lactoferrin concentration compares

The lactoferrin concentration of inducing with yeast saccharomyces cerevisiae and pichia spp is as shown in table 1.

The protein concentration contrast that table 1, two primary yeast fermentations obtain

Note: ug/ml represents the amount of albumen in every milliliter of bacterium liquid

Result shows: the present invention adopt fermentation by saccharomyces cerevisiae gained lactoferrin concentration higher than pichia spp fermentation gained lactoferrin concentration.

2, the expression activitiy of antibacterial peptide lactoferrin

1. E.coli DH5 α is in 37 DEG C, 200rpm incubated overnight.

2. get the bacterium liquid of 200ul E.coli DH5 α, be spread evenly across on the LB flat board of non-added with antibiotic, place Oxford cup gently.

3. the upper cleer and peaceful 200ul yeast saccharomyces cerevisiae supernatant drawn after 200ul sterilized water, 10ug penbritin, 200ul Pichia yeast engineering induction 216h adds Oxford cup, and be placed in 37 DEG C of constant incubator incubated overnight, next day observes fungistatic effect.As shown in figure 13, in fig. 13,1 is penbritin to result; 2 is sterilized water; 3 is pichia spp supernatant; 4 is yeast saccharomyces cerevisiae supernatant.

Result shows: in the cup bacteriostatic experiment of Oxford, and the antibacterial circle diameter of No. 4 samples is greater than No. 3 samples, and the fungistatic effect of the lactoferrin of the fermentation by saccharomyces cerevisiae gained that the present invention adopts is better than the fungistatic effect of the lactoferrin of pichia spp fermentation gained.

Claims (10)

1. produce a method for lactoferrin, comprise the steps:

(1) recombinant bacterium is built: expression cassette is imported Host Strains;

Described expression cassette is formed by connecting by the encoding gene of promotor Ppgk, α-signal peptide gene and lactoferrin Lf cin B successively;

(2) recombinant bacterium described in inducing culture, obtains bacterium liquid;

(3) by centrifugal for described bacterium liquid, supernatant is collected; From supernatant, precipitate lactoferrin, namely obtain described lactoferrin.

2. method according to claim 1, is characterized in that: the described method precipitating lactoferrin from supernatant is for carry out precipitating and concentrating with acetone and Tricholroacetic Acid successively.

3. method according to claim 1 and 2, is characterized in that: described Host Strains is yeast saccharomyces cerevisiae INVSc1.

4., according to the arbitrary described method of claim 1-3, it is characterized in that: the aminoacid sequence of described lactoferrin Lf cin B is as shown in 1-35 amino acids in SEQ ID No.5; The coding gene sequence of described lactoferrin Lf cin B is as shown in the nucleic acid molecule of SEQ ID No.4 1-105 position.

5., according to the arbitrary described method of claim 1-4, it is characterized in that: the aminoacid sequence of described α-signal peptide is as shown in 1-95 amino acids in SEQ ID No.3; The coding gene sequence of described α-signal peptide is as shown in 1-285 position nucleic acid molecule in SEQ ID No.2; The nucleotide sequence of described promotor Ppgk is as shown in 1-793 position nucleic acid molecule in SEQ ID No.1.

6., according to the arbitrary described method of claim 1-5, it is characterized in that: by the method that described expression cassette imports in yeast be: the multiple clone site described expression cassette being inserted pYES2 carrier, obtains recombinant vectors; Again recombinant vectors is imported in described host yeast.

7. a recombinant bacterium, imports Host Strains and obtains by expression cassette;

Described expression cassette is formed by connecting by the encoding gene of promotor Ppgk, α-signal peptide gene and lactoferrin Lf cin B successively;

Described Host Strains is yeast saccharomyces cerevisiae INVSc1.

8. recombinant bacterium according to claim 7, is characterized in that: the aminoacid sequence of described lactoferrin Lf cin B is as shown in 1-35 amino acids in SEQ ID No.5; The coding gene sequence of described lactoferrin Lf cin B is as shown in the nucleic acid molecule of SEQ ID No.4 1-105 position.

9. the recombinant bacterium according to claim 7 or 8, is characterized in that: the aminoacid sequence of described α-signal peptide is as shown in 1-95 amino acids in SEQ ID No.3; The coding gene sequence of described α-signal peptide is as shown in 1-285 position nucleic acid molecule in SEQ ID No.2; The nucleotide sequence of described promotor Ppgk is as shown in 1-793 position nucleic acid molecule in SEQ ID No.1.

10. the arbitrary described method of claim 1-6 and/or the arbitrary described recombinant bacterium of 7-9 are producing the application in lactoferrin.