CN1584035A - Expressions of recombined swine lactoferrin and its peptide by pichia - Google Patents
Expressions of recombined swine lactoferrin and its peptide by pichia Download PDFInfo
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- CN1584035A CN1584035A CN 200410024630 CN200410024630A CN1584035A CN 1584035 A CN1584035 A CN 1584035A CN 200410024630 CN200410024630 CN 200410024630 CN 200410024630 A CN200410024630 A CN 200410024630A CN 1584035 A CN1584035 A CN 1584035A
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- 102000010445 Lactoferrin Human genes 0.000 title claims abstract description 16
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Abstract
A bichia methanol saccharomyces expressing recombinant swine lactoferrin and its peptide are disclosed. It includes: amplifying LF gene, constructing and identifying recombinant saccharomyces expressing carrier, sieving and detecting PMAD11 or PMAD16 and Ade+Muts phenotypic conversion, inducing expressing Ade+Muts strain, purifying and identifying objective protein, transferring into fermenter to grow after identifying. It achieves simple process and high purity for feed additives.
Description
Technical field
The present invention relates to the method that finishes red methanol yeast expression system express recombinant pig lactoferrin and peptide thereof.
Background technology
Lactoferrin (Lactoferrin, be called for short LF), nineteen sixty is separated acquisition from cow's milk, it is a kind of iron associativity glycoprotein, about molecular weight 80KDa, LF has the effect of transhipment iron ion and sterilizing and anti-virus, and its expression amount in mammary gland is the highest, and the highest with the content in the human milk, the content in the cow's milk is less relatively.Its unique mechanism of action causes people's great attention, and it is added infant formula after separation and purification from milk.Lactoferricin (Lactoferricin is called for short Lfcin) is lactoferrin product behind proteasome degradation in animal digestive tract, and its some biologic activity also is eager to excel than lactoferrin.Natural LF has biological function widely, particularly antibacterial, and antibiotic and sterilizing, antiviral, all there is relevant report aspects such as adjusting organism immune response, but the effect report of LF on animal body is less.Extract natural LF from the Ruzhong, method complexity, purity are not high, cause LF to cost an arm and a leg.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.
Summary of the invention
The purpose of this invention is to provide the method that a kind of usefulness finishes red methanol yeast expression system express recombinant lactoferrin and peptide thereof.
The present invention may further comprise the steps with the method that finishes red methanol yeast expression system express recombinant lactoferrin and peptide thereof:
(1) gets dam mammary gland acinus, the liquid nitrogen freezing grind into powder, extract total RNA and obtain the cDNA of lactoferrin or lactoferricin by reverse transcription, a pair of according to the design of lactoferrin or lactoferricin gene expression characteristics 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 or lactoferricin gene, transformed into escherichia coli DH
5 α, be 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 or PMAD16: 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 PMAD11 or the competent preparation of PMAD16 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 the 5mlYPD liquid nutrient medium, 28～30 ℃ of cultivations are centrifugal after 16～20 hours, collect thalline, extract the yeast genes group with glass bead method, and make template with the pastoris genomic dna that extracts, carry out the PCR reaction with primer general on the yeast vector, reaction conditions is as follows: the pre-sex change of 94C 2 minutes, 94 ℃ of 50s, 55 ℃ of 50s, 72 ℃ 1 minute, totally 35 circulations, last 72 ℃ were extended 10 minutes; Two primers are respectively α-Factor:5-TACTATTGCCAGCATTGCTGC-3 and 3 ' AOXI:GAAGAGAAAAACATTAGTTGGC-3 '.
(5) Ade+Mut
8The abduction delivering of bacterial strain: the order bacterium colony was cultivated 16-18 hour, and was cultured to 0D for 28～30 ℃ in 10～50mlBMDY liquid nutrient medium
600=2.0～10, centrifugal collection thalline, resuspended with the 5mlBMMY substratum, at 28～30 ℃, 255rpm 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 thalline is used the 10%SDS-PAGE assay products;
(6) purifying of target protein and evaluation: express supernatant and remove by filter the thalline residue, in 4 ℃ of ammonium sulfate precipitations with final concentration 70%, centrifugal 15 minutes collecting precipitations of 10000rpm, with concentration is the phosphoric acid buffer dissolving of 10mmol/L, PH7.4, with the PBS of 10mmol/L dialysis 24 hours, during change dialyzate 3 times; Last Sephardex50 sieve chromatography is with above-mentioned phosphoric acid buffer wash-out; Dot ELISA detects LF albumen elution peak, and one anti-ly is mouse-anti his monoclonal antibody, and two anti-ly are the sheep anti-mouse igg of alkali phosphatase enzyme mark;
(7) change the fermentor tank growth over to after identifying correctly.
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, it can punch Lfcin 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.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 a carrier with pMET α-B or pMET-B, produces and the close lactoferrin of natural LF character with the 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.After utilizing fermentor tank to carry out suitability for industrialized production, can produce in a large number, overcome complexity and the expensive problem of extracting LF from the Ruzhong in the past.The lactoferrin of yeast expression is made fodder additives,, can prevent and treat the diarrhoea of ablactation piglet effectively, to produce the residual high-quality livestock product of antibiotic-free in the proteic effect of playing sterilizing and anti-virus simultaneously of supplement feed.
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 1.5mlEppendorf 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 the 1ml75% 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′TCCCCGCGGGCCCCTAAGAAAGGGGTTCGA-3′，
5′-GGACTAGTTGCCTCATCATGAAGGCACAGGC-3′。
The PCR reaction system is as follows:
94 ℃ of 2 clock of sterilization distilled water 36 μ L
72 ℃ of each 1 μ L of upstream and downstream primer 10 minutes
cDNA 2μL
Taq enzyme 1 μ L
The PCR product is all gone up sample 1.0% sugared gel electrophoresis, cut back the 2.1kb fragment under the ultraviolet lamp, place the 1.5mL centrifuge tube, add 600 μ L 6M NaI, 37 ℃ of water-baths are melted fully glue, add 9 μ L binding liquid, mixing, ice bath 15 minutes, centrifugal 5 seconds of 12000rpm, abandon supernatant, 450 μ L, 75% ice washing with alcohol 3 times is abandoned
Clearly, to 37 ℃ of oven for drying residual liquids, precipitation is dissolved in 50 μ L, 0.1 * TE, and centrifugal about 1 minute of 512000rpm changes supernatant and newly manages-20 ℃ of preservations in one;
(3) product of rubber tapping recovery and plasmid pGEM-T add the little centrifuge tube of 200 μ L T4DNA ligase enzyme 1 μ L with 4: 1 ratios, 5Xligase damping fluid 1.5 μ L, and 15 ℃ of connections are spent the night, and more than connect product 5 μ L and transform 200 μ L CaCl
2The DH5 of method preparation
αCompetent cell, 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 L 20mg/ml and the IPTG of 4 μ L 200mg/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 SacII and SpeI 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 SacII and SpeI enzyme with two kinds of restriction endonucleases couple transfer vector pMET that cut again
α-B or pMET-B obtain recombinant transfer vector pMET
α-B-LF. or pMET-B-LF
(4) linearizing pMET
α-B-LF or pMET-B-LF recombinant expression vector: in transparent 1.5ml polystyrene sterile tube, add following reagent: reorganization pMET
α-B-LF or pMET-B-LF expression vector 75.0 μ 1, concentration 1.0 μ g/ μ L; FseI 3～5 μ L; Damping fluid 20.0 μ L, mixing gently spends the night under 37 ℃; Carry out yeast PMAD11 or the competent preparation of PMAD16 according to Invitrogen Pichia Expression 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 Gene Pulser 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 the 5mlBMMY substratum, at 28～30 ℃, 255rpm 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, and centrifugal thalline is used the 10%SDS-PAGE assay products.
(6) express supernatant and remove by filter the thalline residue, in 4 ℃ of ammonium sulfate precipitations with final concentration 70%, centrifugal 15 minutes collecting precipitations of 10000rpm are the phosphoric acid buffer dissolving of 10mmol/L, PH7.4 with concentration, with the PBS of 10mmol/L dialysis 24 hours, during change dialyzate 3 times.Last Sephardex50 sieve chromatography is with phosphoric acid buffer wash-out of the same race.Dot ELISA detects LF albumen elution peak, and one anti-ly is mouse-anti 6 * His monoclonal antibody, and two anti-ly are the sheep anti-mouse igg of alkali phosphatase enzyme mark.The LF elutriant of collecting is pure LF recombination expression product through lyophilize.
(7) change the fermentor tank growth over to after identifying correctly.
Recombinant pig lactoferrin has the biological activity of natural LF, can express in a large number by yeast expression system, and purified product can be developed as fodder additives.This art production process is simple, the product purity height. and utilizing the methanol yeast expression system to express the pig lactoferrin full-length gene does not still have relevant report at present, and the present invention still belongs to initiative.
Claims (1)
1. the method with complete red methanol yeast expression system express recombinant pig lactoferrin and peptide thereof is characterized in that, is divided into following steps:
1) amplification of LF gene: get lactation dam mammary gland acinus, the liquid nitrogen freezing grind into powder, extract total RNA and obtain the cDNA of lactoferrin or lactoferricin by reverse transcription, a pair of according to the design of lactoferrin or lactoferricin gene expression characteristics 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 or lactoferricin gene, transformed into escherichia coli DH
5 α, be 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 or PMAD16: 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 PMAD11 or the competent preparation of PMAD16 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 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) screening of Ade+Muts 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 the 5mlYPD liquid nutrient medium, 28～30 ℃ of cultivations are centrifugal after 16～20 hours, collect thalline, extract the yeast genes group with glass bead method, and make template with the pastoris genomic dna that extracts, carry out PCR reaction with primer general on the yeast vector, reaction conditions is as follows: 94 ℃ of pre-sex change 2 minutes, 94 ℃ of 50s, 55 ℃ of 50s, 72 ℃ 1 minute, totally 35 circulations, last 72 ℃ were extended 10 minutes; Two primers are respectively α-Factor:5-TACTATTGCCAGCATTGCTGC-3 and 3 ' AOXI:GAAGAGAAAAACATTAGTTGGC-3 ';
5) Ade+Mut
sThe abduction delivering of bacterial strain: the order bacterium colony was cultivated 16-18 hour, and was cultured to OD for 28～30 ℃ in 10～50mlBMDY liquid nutrient medium
600=2.0～10, centrifugal collection thalline, resuspended with the 5mlBMMY substratum, at 28～30 ℃, 255rpm 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 thalline is used the 10%SDS-PAGE assay products;
6) purifying of target protein and evaluation: express supernatant and remove by filter the thalline residue, in 4 ℃ of ammonium sulfate precipitations with final concentration 70%, centrifugal 15 minutes collecting precipitations of 10000rpm, with concentration is the phosphoric acid buffer dissolving of 10mmol/L, PH7.4, with the PBS of 10mmol/L dialysis 24 hours, during change dialyzate 3 times; Last Sephardex50 sieve chromatography is with above-mentioned phosphoric acid buffer wash-out; Dot ELISA detects LF albumen elution peak, and one anti-ly is mouse-anti his monoclonal antibody, and two anti-ly are the sheep anti-mouse igg of alkali phosphatase enzyme mark;
7) change the fermentor tank growth over to after identifying correctly.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1995362B (en) * | 2006-12-28 | 2010-06-23 | 浙江工业大学 | Gibberella fujikuroi electroporation genetic transformation method |
| CN102121025A (en) * | 2010-11-04 | 2011-07-13 | 山东大学 | Multi-copy integrated expression vector and preparation method and application thereof in expression of bovine lactoferrin |
| CN102791813A (en) * | 2009-10-08 | 2012-11-21 | 凯旋门生物物理学公司 | Surface-coated structures and methods |
| CN103205435A (en) * | 2013-04-11 | 2013-07-17 | 浙江大学 | Method for expressing lactoferrin improved peptide LF-6 by using intein escherichia coli |
| CN103333225A (en) * | 2013-04-11 | 2013-10-02 | 浙江大学 | Antibacterial peptide, preparation method and applications thereof |
| CN103689247A (en) * | 2013-11-22 | 2014-04-02 | 东北农业大学 | Lactoferrin-expression recombinant lactic acid bacterium composition as weaned piglet feed additive |
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2004
- 2004-05-22 CN CN 200410024630 patent/CN1584035A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1995362B (en) * | 2006-12-28 | 2010-06-23 | 浙江工业大学 | Gibberella fujikuroi electroporation genetic transformation method |
| CN102791813A (en) * | 2009-10-08 | 2012-11-21 | 凯旋门生物物理学公司 | Surface-coated structures and methods |
| CN102121025A (en) * | 2010-11-04 | 2011-07-13 | 山东大学 | Multi-copy integrated expression vector and preparation method and application thereof in expression of bovine lactoferrin |
| CN103205435A (en) * | 2013-04-11 | 2013-07-17 | 浙江大学 | Method for expressing lactoferrin improved peptide LF-6 by using intein escherichia coli |
| CN103333225A (en) * | 2013-04-11 | 2013-10-02 | 浙江大学 | Antibacterial peptide, preparation method and applications thereof |
| CN103689247A (en) * | 2013-11-22 | 2014-04-02 | 东北农业大学 | Lactoferrin-expression recombinant lactic acid bacterium composition as weaned piglet feed additive |
| CN103689247B (en) * | 2013-11-22 | 2015-09-30 | 东北农业大学 | A kind of expression lactoferrin recombination lactic acid bacteria composition as delactational piglets feed addictive |
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