CN101402954A - Method for expressing recombinant pig lactoferrin N leaf with pichia thermophilic methanol yeast - Google Patents

Abstract

The invention relates to a method for recombining porcine lactoferrin N-lobe by using the expression of pichia methanolica. The method comprises the amplification of PLFN genes, the construction and the identification of a recombinant yeast expression vector, the screening and the detection of recombinant yeast expression vector converted yeast cells PMAD11 and an Ade<+>Mut<s> phenotype transformant, the induced expression of an Ade<+>Mut<s> strain, the purification and the identification of a target protein, the detection of the expression level of the target protein, the flask shaking, the optimization of fermentation conditions of a 101 fermentation tank, the fermentation and other steps. The technology has simple production process and high product purity, and the method can be used for developing feed additives.

Description

A kind of method with pichia thermophilic methanol yeast express recombinant pig lactoferrin N leaf

Technical field

The present invention relates to the method that finishes red methanol yeast expression system express recombinant pig lactoferrin N leaf.

Background technology

(Lactoferrin, LF) nineteen sixty is at first separated acquisition by Groves to lactoferrin from cow's milk, takes on a red color because of combining with iron, so be referred to as " red eggs are white ".At the beginning of finding, LF is considered to a kind of transhipment of and iron and stores relevant protein, so claim Lactotransferrin again.Lactoferrin forms by a peptide chain (the about 80kDa of molecular weight) is folding, it comprises the bladed structure of two structural similitudies, be named as N-leaf (N-lobe respectively, the N-terminal half) and C-leaf (C-lobe, the C-terminal half), two blades are connected by a short peptide chain.Lactoferrin extensively is present in exocrine secretions such as milk, saliva, tear or blood plasma, the neutrophil leucocyte.Further investigation finds that LF is a kind of protein with various biological function, and it not only participates in the transhipment of iron, and have antimicrobial, anti-oxidant, anticancer, regulate function such as immunity system.The investigator finds that many functions of lactoferrin are all closely related with lactoferrin N-Ye, and lactoferricin (LFcin is considered to the anti-microbial activity center of lactoferrin) also is positioned at lactoferrin N-Ye.Forefathers' research thinks that lactoferrin stops its growth by capturing the necessary ion of microorganism growth.But the ionic bond position of lactoferrin is different fully with the sterilization position.Lactoferricin (lactoferricin, Lfcin) be lactoferrin little peptide after the acidol-pepsin hydrolysis in digestive tube, between its 17-42 amino-acid residue in lactoferrin N-Ye, it has wide spectrum antibacterium, antiviral, anti-mycotic activity, and the domestic and international at present research to PLFN yet there are no report.Extract natural LF from the Ruzhong, the method complexity, purity is not high, and the N-leaf that the method by proteasome degradation obtains lactoferrin is very difficult, chemosynthesis PLFN then costs an arm and a leg, and the P.methanolica expression system is a kind of system of high level expression recombinant protein, and it is not only easy and simple to handle, with low cost, and be fit to suitability for industrialized production more.In herding growth, because antibiotic a large amount of excess use, the appearance of Resistant strain, medicine residual, the pollution of environment has become the problem that current people generally are concerned about, and therefore, lactoferrin and polypeptide thereof have wide application prospect.

The patent of application in 2004 " a kind of usefulness finishes the method for red methanol yeast express recombinant lactoferrin and peptide thereof ", application number is 200410024630.9, it to the effect that adopts pichia thermophilic methanol yeast to express pig lactoferrin, do not mention and shake bottle and 10L ferment tank substratum, fermentation parameter such as pH, rotating speed, temperature and methyl alcohol addition manner etc., the contents such as detection method of also not mentioned target protein purifying and expression amount, experimental study through 4 years, we utilize this expression system to express PLFN, determine the relevant fermentation parameter of PLFN recombinant bacterial strain in shaking bottle and 10L fermentor tank, utilized pichia thermophilic methanol yeast expression PLFN and zymotechnique thereof to belong to initiative.

Summary of the invention

The purpose of this invention is to provide a kind of method with pichia thermophilic methanol yeast expression system express recombinant lactoferrin N leaf.

The present invention may further comprise the steps with the method for pichia thermophilic methanol yeast expression system express recombinant lactoferrin N leaf:

1) amplification of pig lactoferrin N leaf (PLFN) gene: get lactation dam mammary gland acinus, the liquid nitrogen freezing grind into powder, extract total RNA and obtain the cDNA of pig lactoferrin N leaf by reverse transcription, a pair of according to PLFN gene expression characteristics design respectively with the primer of a restriction enzyme site, utilize the RT-PCR technology to obtain DNA, directed cloning is gone into the escherichia coli cloning carrier, transformed into escherichia coli clone strain, screening positive clone;

2) structure of recombinant yeast expression vector and evaluation: the positive strain extracting plasmid that filters out more than inciting somebody to action, cut out goal gene and purifying with the DNA restriction endonuclease, the goal gene directed cloning is advanced the transfer vector of crossing with same enzymes double zyme cutting, obtain containing the recombinant transfer vector of lactoferrin N leaf gene, transformed into escherichia coli DH5 α is coated on the LB flat board that contains Amp, the bacterium colony that overnight incubation is grown on the picking flat board immediately, alkaline process extracting plasmid DNA, double digestion and PCR identify;

3) recombinant yeast expression vector transformed yeast cell PMAD11: the linearizing recombinant expression vector, add following reagent in transparent 1.5ml polystyrene sterile tube: 75.0 μ l recombinant expression vectors, final concentration reach 1.0 μ g/ μ L; 3～5 μ L linearizing enzymes, 20.0 μ L damping fluids; Mixing was placed 12～16 hours down for 37 ℃ gently; Carry out the competent preparation of yeast PMAD11 according to Invitrogen Pichia Expression Kit during this period, take out the above-mentioned linearizing recombinant expression plasmid mixed solution of 100 μ L and 1～3 μ g, change 0.2cm electricity revolving cup over to, ice bath 5 minutes, in Bio Rad Gene Pulser electroporation at 750V, 25 μ F, after electricity transforms under the ∞ Ω condition, add 1mlYPAD immediately 28～30 ℃ of static cultivations 1 hour, centrifugal 3 minutes of 1500g room temperature, careful evacuation supernatant, precipitation is resuspended in 1 * YNB of 100 μ L, get 50 μ L bacterium liquid and coat on the MD selection flat board, hatched 3～4 days, observe the growth of transformant in 28～30 ℃ of conditions;

4) Ade ⁺Mut ^sThe screening of phenotype transformant detects: will transform bacterium colony and select on the flat board at MM and MD in corresponding place respectively, screen poor growth on the MM flat board and on the MD flat board well-grown bacterial strain; Inoculate single bacterium colony in 5ml YPD liquid nutrient medium, cultivate after 16～20 hours centrifugal for 28～30 ℃, collect thalline, extract the yeast genes group, and carry out the PCR reaction as template with the pastoris genomic dna that extracts, reaction conditions is as follows: 94 ℃ of pre-sex change 2 minutes, 94 ℃ of 50s, 55 ℃ of 30s, 72 ℃ 2 minutes, totally 35 circulations, last 72 ℃ were extended 10 minutes; Article two, primer is respectively F:5 '-GCGAATTCATGAAGCTCTTCATCCCCGCC-3 ', R:5 '-CCGGATCCCAGGCCCTGGATGGCAGTA AG-3 ';

5) Ade ⁺Mut ^sThe abduction delivering of bacterial strain: the order bacterium colony was cultivated 16～18 hours, and was cultured to OD600=2～10 for 28～30 ℃ in 10～50mlBMDY liquid nutrient medium, centrifugal collection thalline, resuspended with the 5mlBMMY substratum, at 28～30 ℃, 250rpm cultivated 4 days, in inducing process, added 1 time methyl alcohol in per 24 hours, final concentration is 0.5%, 0,24, equi-time point was got 500 μ L fermented liquids in 36,72,96 hours, centrifugal removal thalline, 10%SDS-PAGE and Western blot identification and analysis product;

6) purifying of target protein and content detection: with the 72h fermented liquid at 4 ℃ of centrifugal 15min of following 10000rpm, remove thalline and insolubles, in supernatant, add 51% ammonium sulfate, 4 ℃ of precipitations of spending the night, the centrifugal 10min of 8000rpm, collecting precipitation is dissolved among the appropriate amount of buffer solution A (level pad), with treating sample behind the 0.45 μ m membrane filtration; Install nickel ion metal chelate affinity chromatography post according to process specifications, with going up sample behind the buffer A balance affinity column of 10 times of medium volumes; Treat that sample is not with conjugated protein in 10 times of medium volume buffer A flush away chromatography columns; Adopt the highest yield elution protocol: the damping fluid D wash-out that contains the 20mM imidazoles earlier with 10 times of medium volumes, the damping fluid D wash-out that contains the 250mM imidazoles again with 10 times of medium volumes, 10%SDS-PAGE and Western blot identification and analysis product, one anti-is mouse-anti his monoclonal antibody, and two anti-ly are the sheep anti-mouse igg of alkali phosphatase enzyme mark; Adopt the BCA method purified product to be detected and calculates the target protein expression amount;

7) conditions of flask fermentation optimization.The recombination microzyme that obtains is seeded to shakes bottle, in the shake flask fermentation environment, design by single factor experiment, change the kind or the consumption of a kind of nutritive ingredient in the fermentation basic medium, other nutritive ingredient kind and consumption are constant, the genetic engineering bacterium fermentation condition of optimization expression purpose product.Best carbon source, nitrogenous source, carbon source addition, C/N have been filtered out by single factor experiment.On the basis of single factor experiment, test design L ₁₆(4 ⁵) the orthogonal optimization test, cultivate the fermention medium that has filtered out the suitableeest recombinant bacterial strain protein expression by shake flask fermentation.In addition, test has also been carried out selectivity optimization to the correlative factor (initial pH value, inoculum size) that influences the recombinant bacterial strain protein expression, has determined the shake flask fermentation culture condition.The most suitable substratum and the addition thereof of shake flask fermentation: optimum carbon source glucose (20g/L), optimum nitrogen source peptone (20g/L), vitamin H 4mL/L (4 * 10 ^-5%), MgSO ₄.7H ₂The O addition is 5g/L and KH ₂PO ₄Addition 3g/L.The 50ml substratum of in the 250ml triangular flask, packing into, leavening temperature is that 30 ℃, shaking speed are under the condition of 250rpm, 24h pH value is 4.5 before the fermentation, the pH value is 5.0 behind the 24h, 13h in age is planted in inoculation, the inoculum size of seed culture fluid is under 4% the condition, and it is the most suitable that target protein in the shake flask fermentation environment is expressed.Detect through RPLC under this fermentation condition, recombinant bacterial strain target protein expression amount is 5.8 μ g/mL;

8) 10L ferment tank condition optimizing.From activating picking one single bacterium colony on 24 hours the yeast flat board, insert in four triangular flasks that seed culture medium is housed (sample-loading amount 125ml/500ml triangular flask), temperature is 30 ℃, shaking speed is 250rpm, after back and forth cultivating 13h on the shaking table, be inoculated in the 10L fermentor tank with 4% inoculum size as seed liquor, utilizing the 10L fermentor tank to study influences the fermentation parameter of recombinant bacterial strain growth and target protein expression.

By batch fermentation, optionally studied mechanical stirring rotating speed, initial pH value and temperature, to the influence that recombinant bacterial strain thalli growth and target protein in the fermenting process are expressed, determined the relevant fermentation parameter of recombinant bacterial strain in the 10L fermentor tank.Studied of the influence of different methyl alcohol addition manners, determined best feed supplement mode test strain growth and target protein expression amount.The result shows: the rPLFN engineering bacteria enters logarithmic phase after cultivating 7h, enters logarithmic growth mid-term behind the 13h, and 16h left and right sides logarithmic phase finishes, and begins to enter the stable growth phase, and growth bacterial strain in mid-term flushes, and therefore selecting 13h is inoculation kind of an age.The different vaccination amount has certain influence to thalli growth and target protein expression, and when inoculum size was 4%, the growing state of reorganization bacterium was best, and the expression amount of purpose product is the highest.Inoculation begins to add methyl alcohol behind the 24h and carries out abduction delivering, induces 48h, promptly induces the 48h target protein can reach high expression level amount behind the thalli growth 24h.Fermentation parameter: the mechanical stirring rotating speed is 300～350rpm, adopt the segmentation control strategy, adopt higher mechanical separator speed 350rpm before the 36h, can provide the competent necessary oxygen of thalli growth metabolism that is used for for earlier fermentation, be beneficial to the thalline ramp, adopt 300rpm for assurance target protein higher expression behind the 36h; Fermentation pH value is made as 4.8, and this expresses all favourable to reorganization thalline stable growth and purpose product; 28～30 ℃ of leavening temperatures, 24h selects 30 ℃ of the temperature of suitable thalli growth before the fermentation, selects behind the 24h favourable 28 ℃ of protein expression; Ventilation guarantees the accumulation of normal growth of thalline and purpose product than being 1.0vvm.Behind the fermentation 24h, every 12h replenishes 0.25% methyl alcohol in fermentor tank, is used for the test strain target protein and induces, and the effect of this feed supplement mode is better than the constant current interpolation and adds 0.5% methyl alcohol every 24h.The result shows that test strain is best fermentation parameter in the 10L fermentor tank: the mechanical stirring rotating speed is 300～350rpm, adopts the segmentation control strategy, adopts 350rpm before the 36h, adopts 300rpm behind the 36h; Fermentation initial pH value 4.8; 28～30 ℃ of leavening temperatures, 30 ℃ of the temperature of the suitable thalli growth of the preceding 24h selection of fermentation are selected 28 ℃ behind the 24h; Ventilation is than being 1.0vvm.Behind the fermentation 24h, every 12h replenishes certain density methyl alcohol in fermentor tank, be used for the test strain target protein and induce.In the 10L fermentor tank after fermentation parameter is optimized, recombinant bacterial strain purpose product expression amount is 10.0 μ g/mL after testing;

The invention has the beneficial effects as follows: LF itself has the effect of sterilizing and anti-virus, the effect of product Lfcin after the stomach en-degraded is stronger under one's belt, its mechanism of action uniqueness, be different from traditional microbiotic, LF captures bacterial reproduction by competitiveness and the necessary iron ion of growth reaches the bacteriostatic purpose, Lfcin can punch on bacteria cell wall or the dissolved cell wall, cause the forfeiture of cell transmembrane current potential, entocyte leaks and killing bacteria, it does not disturb duplicating of DNA of bacteria, so bacterium is difficult for developing immunity to drugs to it, experimental results show that PLFN is to intestinal bacteria K88, the bacteriostatic activity of bacterium such as Salmonella choleraesuls and Salmonella typhimurium is higher than PLF.As everyone knows, diarrhoea is to cause the piglet main causes of death, and annual production to raising pigs caused tremendous loss.The piglet ablactation is the high-incidence season of diarrhoea period, and after one of reason was exactly ablactation, piglet had lacked the antimicrobial component LF protection in the milk and caused that the variation of enteron aisle pH value and microecological balance causes suffering from diarrhoea.The present invention is carrier with pMET-B, produces pig lactoferrin N leaf with the pichia thermophilic methanol yeast expression system.Yeast expression system has become the system that efficiently expresses foreign protein, it promptly has the simple of prokaryotic system, have the post-transcriptional control ability of eukaryotic system such as glycosylation etc. again, promptly can be in laboratory operation, also can suitability for industrialized production, be secreted in the substratum oneself protein seldom, the product purity height, active good.The lactoferrin of yeast expression is made fodder additives, play germ-resistant effect simultaneously in that supplement feed is proteic, can prevent and treat the diarrhoea of ablactation piglet effectively, to produce the residual high-quality livestock product of antibiotic-free.

Embodiment

Be that example is introduced concrete operation method below with the pig lactoferrin:

(1) wins DLY lactation pig mammary tissue 50-100 milligram, place the mortar of prior 200 ℃ of bakings and cool to room temperature, add 1/3 mortar hydrops nitrogen, grind into powder also changes it over to 1.5ml Eppendorf pipe, add 1ml Trizol liquid, fully mixing left standstill 5 minutes on ice, add the 0.2ml chloroform again, the tight pipe lid of lid in centrifugal 30 seconds of 4 ℃ of 12000rpm, is got the upper strata water in a new eppendorf pipe behind the thermal agitation mixing, add the 0.5ml Virahol,-20 ℃ left standstill 30 minutes behind the mixing, and centrifugal 10 minutes of 12000rpm abandons supernatant, precipitate with drying at room temperature after 1ml 75% washing with alcohol, after the drying precipitation being dissolved in 20-100 μ L does not have enzyme water, obtains total RNA extracting solution, with total RNA extracting solution of obtaining with suitable primer, carry out reverse transcription (RT) under the effect of M-MLV ThermoScript II, products therefrom is cDNA;

(2) the synthetic upstream and downstream primer of design:

Upstream primer: 5 '-CCGGATCCATGAAGCTCTTCATCCCCGC-3 ',

Downstream primer: 5 '-GCGAATTCTTACAGGCCCTGGATGGCAGTAAG-3 '

The PCR reaction system is as follows:

Sterilization distilled water 33.5 μ L

10 * PCR damping fluid, 5 μ L

25mM?MgCl ₂ 4μL

2.5mM?dNTP 4μL

Each 1 μ L of upstream and downstream primer

cDNA 1μL

Taq enzyme 0.5 μ L

Amplification condition: 94 ℃ of pre-sex change 5 minutes; 94 ℃ 50 seconds, 55 ℃ 30 seconds, 72 ℃ 2 minutes, 35 circulations; 72 ℃ 10 minutes; 4 ℃ of preservations.Agarose gel electrophoresis with 1.0% detects amplified production.The PCR product is all gone up sample 1% agarose gel electrophoresis, and 1Kb left and right sides fragment ,-20 ℃ of preservations are reclaimed in rubber tapping;

(3) product of rubber tapping recovery and plasmid pGEM-T digest with BamH I and EcoR I, add the little centrifuge tube of 200 μ L T4DNA ligase enzyme 1 μ L with 4: 1 ratios, 5 * ligase damping fluid, 1.5 μ L, 4 ℃ of connections are spent the night, and more than connect product 5 μ L and transform 200 μ LCaCl ₂The DH5 α competent cell of method preparation, the rearmounted 37 ℃ of shaking tables of LB liquid nutrient medium 1mL that add 37 ℃ of preheatings, 180rpm vibration 1 hour, prepare screening culture medium simultaneously, Amp (+) LB solid culture primary surface adds the X-gal of 40 μ L20mg/ml and the IPTG of 4 μ L200mg/ml is coated with evenly with aseptic glass rod, putting 37 ℃ of incubators makes it to absorb, centrifugal 5 minutes of above-mentioned bacterium liquid 4000rpm, discard the 1ml supernatant, blowing and beating behind the mixing coating screening culture medium gently with pipettor puts 37 ℃ of incubators and cultivates more than 12 hours, up to obvious and not overlapped single bacterium colony occurring, choose white single bacterium colony adds 5 μ L Amp in 3mL liquid LB substratum, the extracting plasmid, cut the evaluation recombinant plasmid with BamH I and EcoR I enzyme, the screening positive goes out the clone and is inoculated in 37 ℃ of shaking culture of 3ml LBA liquid nutrient medium and spends the night, and the correct back of order-checking alkaline process is the extracting plasmid in a small amount, is connected into after cutting with BamH I and EcoR I enzyme with obtaining recombinant transfer vector pMETB-PLFN with two kinds of restriction endonucleases couple transfer vector pMET-B that cut again;

(4) linearizing pMETB-PLFN recombinant expression vector: in transparent 1.5ml polystyrene sterile tube, add following reagent: reorganization pMETB-PLFN expression vector 75.0 μ l, concentration 1.0 μ g/ μ L; Pst I3～5 μ L; Damping fluid 20.0 μ L, mixing gently spends the night under 37 ℃; Carry out the competent preparation of yeast PMAD11 according to Invitrogen PichiaExpression Kit during this period; taking out 100 μ L mixes with the above-mentioned linearizing recombinant expression plasmid of 1～3 μ g; change 0.2cm electricity revolving cup over to; ice bath 5 minutes; in Bio Rad GenePulser electroporation 750V; 25 μ F; after electricity transforms under the ∞ Ω condition; add 1mlYPAD28～30 ℃ static cultivation 1 hour immediately, centrifugal 3 minutes of 1500g room temperature, careful evacuation supernatant; precipitation is resuspended in 1 * YNB of 100 μ L; get 50 μ L bacterium liquid and coat on the MD selection flat board, hatched 3～4 days, observe the growth of transformant in 28～30 ℃ of conditions.

(5) the order bacterium colony was cultivated 16-18 hour, and was cultured to OD600=2.0～10 for 28～30 ℃ in 10～50mlBMDY liquid nutrient medium, centrifugal collection thalline, resuspended with 5ml BMMY substratum, at 28～30 ℃, 250rpm cultivated 4 days, in inducing process, added 1 methyl alcohol and made its final concentration 0.5% in per 24 hours, 0,24,36,72, equi-time point was got 500 μ L fermented liquids in 96 hours, 10000rpm centrifugal 5min centrifugation thalline and supernatant 10%SDS-PAGE assay products.

(6) extract the thalline genome and carry out the PCR evaluation, supernatant and thalline are carried out SDS-PAGE and Westernblot identification and analysis.

Upstream primer: 5 '-GCGAATTCATGAAGCTCTTCATCCCCGCC-3 '

Downstream primer: 5 '-CCGGATCCCAGGCCCTGGATGGCAGTAAG-3 '

Reaction system:

Sterilization ddH2O 33.5 μ L

10×PCR?Buffer 5.0μL

MgCl2(25mM) 4.0μL

dNTP(2.5mM) 4.0μL

Each (50pM) 1.0 μ L of upstream and downstream primer

Template 1.0 μ L

Taq?plus?DNA?polymerase(5U/μL) 0.5μL

Amplification condition: 94 ℃ of pre-sex change 5 minutes; 94 ℃ 50 seconds, 55 ℃ 30 seconds, 72 ℃ 2 minutes, 35 circulations; 72 ℃ 10 minutes; 4 ℃ of preservations.Agarose gel electrophoresis with 1% detects amplified production.

(7) carry out the 10L ferment tank after identifying correctly.

From activating picking one single bacterium colony on 24 hours the yeast flat board, insert in four triangular flasks that seed culture medium BMDY is housed (sample-loading amount 125ml/500ml triangular flask), temperature is 30 ℃, shaking speed is 250rpm, after back and forth cultivating 13h on the shaking table, be inoculated in the 10L fermentor tank with 4% inoculum size as seed liquor, carry out the Fermentation Conditions of pig lactoferrin N leaf recombinant bacterial strain.Substratum in the 10L fermentor tank: glucose (20g/L), peptone (20g/L), vitamin H 4mL/L (4 * 10 ^-5%), MgSO ₄.7H ₂The O addition is 5g/L and KH ₂PO ₄Addition is 3g/L.

The 10L ferment tank cycle is 72h, and inoculation begins to add methyl alcohol behind the 24h and carries out abduction delivering, induces 48h, promptly induces the 48h target protein can reach high expression level amount behind the thalli growth 24h.Fermentation parameter: the mechanical stirring rotating speed is 300～350rpm, adopts the segmentation control strategy, adopts higher mechanical separator speed 350rpm before the 36h, adopts 300rpm behind the 36h; Fermentation pH value is made as 4.8; 28～30 ℃ of leavening temperatures, 24h selects 30 ℃ of the temperature of suitable thalli growth before the fermentation, selects behind the 24h favourable 28 ℃ of protein expression; Ventilation is than being 1.0vvm.Behind the fermentation 24h, every 12h replenishes 0.25% methyl alcohol in fermentor tank, be used for the test strain target protein and induce.

Recombinant pig lactoferrin N leaf has good antibacterial activity, can express in a large number by yeast expression system, and tunning can be developed as fodder additives.This art production process is simple, and expression product is active high, utilizes pichia thermophilic methanol yeast expression system expression pig lactoferrin N leaf gene and relevant fermentation parameter thereof still not to have relevant report at present, and the present invention still belongs to initiative.

Claims (1)

1. the method with pichia thermophilic methanol yeast expression system express recombinant pig lactoferrin N leaf is characterized in that, is divided into following steps:

1) amplification of pig lactoferrin N leaf gene: get lactation dam mammary gland acinus, the liquid nitrogen freezing grind into powder, extract total RNA and obtain the cDNA of pig lactoferrin N leaf by reverse transcription, a pair of according to PLFN gene expression characteristics design respectively with the primer of a restriction enzyme site, utilize the RT-PCR technology to obtain DNA, directed cloning is gone into the escherichia coli cloning carrier, transformed into escherichia coli clone strain, screening positive clone.

2) structure of recombinant yeast expression vector and evaluation: the positive strain extracting plasmid that filters out more than inciting somebody to action, cut out goal gene and purifying with the DNA restriction endonuclease, the goal gene directed cloning is advanced the transfer vector of crossing with same enzymes double zyme cutting, obtain containing the recombinant transfer vector of lactoferrin N leaf gene, transformed into escherichia coli DH5 α is coated on the LB flat board that contains Amp, the bacterium colony that overnight incubation is grown on the picking flat board immediately, alkaline process extracting plasmid DNA, double digestion and PCR identify.

3) recombinant yeast expression vector transformed yeast cell PMAD11: the linearizing recombinant expression vector, add following reagent in transparent 1.5ml polystyrene sterile tube: 75.0 μ l recombinant expression vectors, final concentration reach 1.0 μ g/ μ L; 3～5 μ L linearizing enzymes, 20.0 μ L damping fluids; Mixing was placed 12～16 hours down for 37 ℃ gently; Carry out yeast PMAD 11 competent preparations according to Invitrogen Pichia Expression Kit during this period; take out the above-mentioned linearizing recombinant expression plasmid mixed solution of 10 μ L and 1～3 μ g; change 0.2cm electricity revolving cup over to; ice bath 5 minutes; in Bio Rad Gene Pulser electroporation at 750V; 25 μ F; after electricity transforms under the ∞ Ω condition; add 1mlYPAD immediately 28～30 ℃ of static cultivations 1 hour, centrifugal 3 minutes of 1500g room temperature, careful evacuation supernatant; precipitation is resuspended in the 1XYNB of 100 μ L; get 50 μ L bacterium liquid and coat on the MD selection flat board, hatched 3～4 days, observe the growth of transformant in 28～30 ℃ of conditions.

4) Ade ⁺Mut ^sThe screening of phenotype transformant detects: will transform bacterium colony and select on the flat board at MM and MD in corresponding place respectively, screen poor growth on the MM flat board and on the MD flat board well-grown bacterial strain; Inoculate single bacterium colony in 5ml YPD liquid nutrient medium, cultivate after 16～20 hours centrifugal for 28～30 ℃, collect thalline, extract the yeast genes group, and carry out the PCR reaction as template with the pastoris genomic dna that extracts, reaction conditions is as follows: 94 ℃ of pre-sex change 2 minutes, 94 ℃ of 50s, 55 ℃ of 30s, 72 ℃ 2 minutes, totally 35 circulations, last 72 ℃ were extended 10 minutes; Article two, primer is respectively F:5 '-GCGAATTCATGAAGCTCTTCATCCCCGCC-3 ', R:5 '-CCGGATCCCAGGCCCTGGATGGCAGTA AG-3 '.

5) Ade ⁺Mut ^sThe abduction delivering of bacterial strain: the order bacterium colony was cultivated 16～18 hours, and was cultured to OD for 28～30 ℃ in 10～50ml BMDY liquid nutrient medium ₆₀₀=2～10, centrifugal collection thalline, resuspended with the 5mlBMMY substratum, at 28～30 ℃, 250rpm cultivated 4 days, in inducing process, added methyl alcohol 1 time in per 24 hours, final concentration is 0.5%, 0,24, equi-time point was got 500 μ L fermented liquids in 36,72,96 hours, centrifugal removal thalline, 10%SDS-PAGE and Western blot identification and analysis product.

6) purifying of target protein and content detection: with the 72h fermented liquid at 4 ℃ of centrifugal 15min of following 10000rpm, remove thalline and insolubles, in supernatant, add 51% ammonium sulfate, 4 ℃ of precipitations of spending the night, the centrifugal 10min of 8000rpm, collecting precipitation is dissolved among the appropriate amount of buffer solution A (level pad), with treating sample behind the 0.45 μ m membrane filtration; Install nickel ion metal chelate affinity chromatography post according to process specifications, with going up sample behind the buffer A balance affinity column of 10 times of medium volumes; Treat that sample is not with conjugated protein in 10 times of medium volume buffer A flush away chromatography columns; Adopt the highest yield elution protocol: the damping fluid D wash-out that contains the 20mM imidazoles earlier with 10 times of medium volumes, the damping fluid D wash-out that contains the 250mM imidazoles again with 10 times of medium volumes, 10%SDS-PAGE and Western blot identification and analysis product, one anti-is mouse-anti his monoclonal antibody, and two anti-ly are the sheep anti-mouse igg of alkali phosphatase enzyme mark; Adopt the BCA method purified product to be detected and calculates the target protein expression amount.

7) conditions of flask fermentation optimization.By the single factor experiment design, change the kind or the consumption of a kind of nutritive ingredient in the fermentation basic medium in the shake flask fermentation environment, other nutritive ingredient kind and consumption are constant, the genetic engineering bacterium fermentation condition of optimization expression purpose product.Best carbon source, nitrogenous source, carbon source addition, C/N have been filtered out by single factor experiment.On the basis of single factor experiment, test design L ₁₆(4 ⁵) the orthogonal optimization test, cultivate the fermention medium that has filtered out the suitableeest recombinant bacterial strain protein expression by shake flask fermentation.In addition, test has also been carried out selectivity optimization to the correlative factor (initial pH value, inoculum size) that influences the recombinant bacterial strain protein expression, has determined the shake flask fermentation culture condition.The most suitable substratum and the addition thereof of shake flask fermentation: optimum carbon source glucose (20g/L), optimum nitrogen source peptone (20g/L), vitamin H 4mL/L (4 * 10 ^-5%), MgSO ₄.7H ₂The O addition is 5g/L and KH ₂PO ₄Addition 3g/L.The 50ml substratum of in the 250ml triangular flask, packing into, leavening temperature is that 30 ℃, shaking speed are under the condition of 250rpm, 24h pH value is 4.5 before the fermentation, the pH value is made as 5.0 behind the 24h, it is 13h that an age is planted in inoculation, the inoculum size of seed culture fluid is under 4% the condition, and it is the most suitable that target protein in the shake flask fermentation environment is expressed.Detect through RPLC under this fermentation condition, recombinant bacterial strain target protein expression amount is 5.8 μ g/mL.

8) 10L ferment tank condition optimizing.Utilizing the 10L fermentor tank to study influences the fermentation parameter of recombinant bacterial strain growth and target protein expression, pass through batch fermentation, mechanical stirring rotating speed, initial pH value and temperature have optionally been studied, to the influence that recombinant bacterial strain thalli growth and target protein in the fermenting process are expressed, determined the relevant fermentation parameter of recombinant bacterial strain in the 10L fermentor tank.Studied of the influence of different methyl alcohol addition manners, determined best feed supplement mode test strain growth and target protein expression amount.The result shows that test strain is best fermentation parameter in the 10L fermentor tank: the mechanical stirring rotating speed is 300～350rpm, adopts the segmentation control strategy, adopts 350rpm before the 36h, adopts 300rpm behind the 36h; Fermentation initial pH value 4.8; 28～30 ℃ of leavening temperatures, 30 ℃ of the temperature of the suitable thalli growth of the preceding 24h selection of fermentation are selected 28 ℃ behind the 24h; Ventilation is than being 1.0vvm.Behind the fermentation 24h, every 12h replenishes 0.25% methyl alcohol in fermentor tank, be used for the test strain target protein and induce.In the 10L fermentor tank after fermentation parameter is optimized, recombinant bacterial strain purpose product expression amount is 10.0 μ g/mL after testing.