CN103468769A - Selenium-containing recombinant human albumin preparation method - Google Patents

Selenium-containing recombinant human albumin preparation method Download PDF

Info

Publication number
CN103468769A
CN103468769A CN2013101882141A CN201310188214A CN103468769A CN 103468769 A CN103468769 A CN 103468769A CN 2013101882141 A CN2013101882141 A CN 2013101882141A CN 201310188214 A CN201310188214 A CN 201310188214A CN 103468769 A CN103468769 A CN 103468769A
Authority
CN
China
Prior art keywords
selenium
recombinant human
serum albumin
human serum
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN2013101882141A
Other languages
Chinese (zh)
Other versions
CN103468769B (en
Inventor
徐东
杨艳群
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xu dong
Original Assignee
SHENZHEN YATAIXING INDUSTRY Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SHENZHEN YATAIXING INDUSTRY Ltd filed Critical SHENZHEN YATAIXING INDUSTRY Ltd
Priority to CN201310188214.1A priority Critical patent/CN103468769B/en
Publication of CN103468769A publication Critical patent/CN103468769A/en
Application granted granted Critical
Publication of CN103468769B publication Critical patent/CN103468769B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention is suitable for the technical field of the gene engineering, and provides a selenium-containing recombinant human albumin preparation method. The method comprises the steps of constructing a Pichia pastoris engineering strain, carrying out fermentation culture, and purifying. The selenium-containing recombinant human albumin preparation method has the advantages of simple operation, low cost and high yield, a selenium-containing recombinant human albumin prepared through the method has a healthcare effect and overcomes a blood albumin security problem as an albumin supplement, and the selenium-containing recombinant human albumin inhibits cancers, improves the immunity, enhances the tissue restoration capability and has a great market potential as an organic selenium. Compared with present selenium nutriments, the selenium-containing recombinant human albumin has the advantages of clear components, high security, high organic purity and high nutritive value when the selenium-containing recombinant human albumin is sold in the market as a simple nutrition and healthcare product.

Description

Preparation method containing the selenium recombinant human serum albumin
Technical field
The invention belongs to biological technical field, relate in particular to a kind of preparation method containing the selenium recombinant human serum albumin.
Background technology
Human serum albumin content in human plasma is extremely abundant, is the maximum protein of consumption in the recycle system, accounts for 60% of blood plasma total protein, and typical plasma concentration HSA content is 50g/L, and infiltration covers 80% of whole blood plasma.The normal human serum albumin level is every 100mL3.9～4.5g, when albumin level is lower than 3g in every 100mL blood plasma, hydrosarca will occur, when patient is subject to wound and cerebral occurs to raise, during brain oedema pressuring nerve, albumin is most important rescue means.Therefore, human serum albumin is mainly used in surgical blood transfusion and urgent patient's fluid infusion clinically, treatment wound shock, fever, oedema, hypoalbuminemia and hypercythemia etc., and can strengthen the human body resistivity, be important clinical medicine.The Main Function of HSA is maintain the normal osmotic pressure of blood and transport many hydrophilic molecules (as steroid hormone, a large amount of medical molecules such as bile pigment and lipid acid).In addition, human serum albumin also is applied in the preparation of biological product, as clinical and chromatography agent purifying bilirubin, is used as hepatovirus vaccine and stablizer etc.The HSA commodity of selling in the market all extract purifying from people source blood plasma.Yet the human blood source is limited, spreading and the reason of detection technique aspect of the virus such as acquired immune deficiency syndrome (AIDS), hepatitis B, make the situation because using this kind of blood products aids infection and hepatitis happen occasionally, so that the country had forbids the import of external blood products, and this is just stricter to production requirement and the quality control of blood source human serum albumin.
In order to solve the human serum albumin limited contradiction of originating, by genetic engineering technique, human serum albumin gene is cloned on microorganism (bacterium, fungi etc.), animal, plant host to carry out efficient the expression be the promising method of tool, since nearly 20 years, many laboratories and company attempt developing HSA by genetic engineering successively in the world, the domestic research that also has many units to carry out recombination human serum albumin, the HSA gene has been introduced in bacterium, fungi, plant and animal and has been expressed.The expression of rHSA in bacterial expression is mainly the expression in intestinal bacteria E.coli and two kinds of systems of Bacillus subtilus Bacillus subtilis.RHSA expression amount in E coli can reach 7% of total protein of cell, but contains a large amount of disulfide linkage because of in the serum albumin molecule, makes born of the same parents fold outward and is difficult to correctly complete, and product does not have biological activity, and may produce extremely other aminoacid sequence variation; Though thereby the e. coli protein expression amount is high, is applied in production and has little significance.When Bacillus subtilus does to express HSA, product be gathered in cell or outside born of the same parents (outside of cytolemma) cause signal sequence not by enzymolysis, hinder the Secretory Pathway of foreign protein, made that expression amount is not high and the signal peptide clearance is low, thereby also be not suitable for suitability for industrialized production.RHSA also can be rat, mouse, and goat, expressed in the transgenic animal such as milk cow, while testing in vivo, can be expelled to the HSA gene segment in zygote by the microinjection method, and gene carrys out the HSA of the expressed by homologous recombination; Also can transmit HSA DNA is transported in liver cell and expresses by middle acceptor gene; In addition, the rHSA gene is cultivated and also can be carried out secreting, expressing by the cell outer planting, when cultivating, outer planting can induce soon, but the expression regulation of gene is subject to the dual regulation and control of extracellular matrix, complicated operation, FDA does not also ratify at present the recombination human serum albumin listing that any a company produces with transgenic animal, and animal cell culture expensive also makes the expression of zooblast be subject to the restriction of cost; The secreting, expressing that rHSA also can succeed by transgenic technology in potato and tobacco leaf, but with the problem that has equally cost restriction.
Selenium is one of necessary micronutrient element of organism, and it has important biological function.The organism selenium deficiency can cause 40 various disease conditions, as the white muscle disease of Keshan disease, Kaschin-Beck disease and the animal of human body etc., thereby mends selenium organism is very important.Recent research also shows, selenium, as biological anti-oxidant, has the free radical of removing in organism, reduces the effect of body peroxidatic reaction of lipid; Selenium has anticancer and effect that delay senility; Selenium also is improved the effect of immunity of organism vigor.In addition, selenium also has antagonistic effect to heavy metallic salt as toxicity such as Hg, Ni, Pb, Cd.Studies confirm that, Selenoperoxidase (GSH-Px) is to prove conclusively the earliest the selenoprotein of its enzyme function in mammalian body, the selenium intake deficiency, and in blood, the activity of this enzyme can descend.
Up to the present, in human nutrition, the supplementary form of selenium roughly has 2 classes: the 1st class is inorganic selenium, this has received effect preferably at China's Area of Keshan Disease, but the mineral compound of selenium has toxicity more, and using dosage very easily causes adverse side effect accidentally; The 2nd class is the organic selenium compounds of synthetic, for injection, to discharging gradually in vivo selenium, play long-acting, but the mode of injecting drug use has limited its application.
At present or in the protein obtained, contain the less of selenium, be difficult to reach the purpose of mending selenium.
Summary of the invention
In view of this, the invention provides a kind of preparation method containing the selenium recombinant human serum albumin, solve in prior art the human serum albumin preparation cost high, yield poorly, and the poor technical problem of selenium content.
The present invention is achieved in that
A kind of preparation method containing the selenium recombinant human serum albumin, comprise the steps:
Build the Pichia yeast engineering containing the recombinant human serum albumin gene;
This Pichia yeast engineering is carried out to fermentation culture; Add the Sodium Selenite that volume fraction is 0.001%～0.05% in this fermentation culture process in substratum;
By the product purification of fermentation culture, obtain containing the selenium recombinant human serum albumin.
The present invention is containing the preparation method of selenium recombinant human serum albumin, simple to operate, with low cost, productive rate is high, and prepared can be used as the albumin supplement containing the selenium recombinant human serum albumin, play its health-care effect, overcome the problem of blood source albumin security, can be used as again organoselenium and suppress cancer, improve immunizing power, strengthen tissue repairing ability, there is very large market potential.Put on market as simple dietary supplements containing the selenium recombinant human serum albumin, compare with existing selemium nutrition product, its composition is clearer and more definite, and security, organic purity and nutritive value are higher.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
The embodiment of the present invention provides a kind of preparation method containing the selenium recombinant human serum albumin, comprises the steps:
Step S01, build Pichia yeast engineering;
Construction process adopts routine techniques means in the genetically engineered field:
By the clone people rHSA in liver cell source of RT-PCR, product after amplification, after enzyme is cut and reclaimed with electrophoresis, inserts the multiple clone site of plasmid pPIC9K, construction expression plasmid pPIC9K-rHSA, double digestion transforms Pichia pastoris GS115 after identifying positive colony, and PCR identifies positive colony.Specific as follows:
The extraction of total RNA:
A. prepare to extract in advance centrifuge tube, rifle head, mortar and the related reagent etc. of RNA special use:
1. soak the rifle head of centrifuge tube, 10 μ L, 200 μ L and the 1mL of 1.5mL with chloroform, be loaded on respectively with chloroform and wipe away in the vial and rifle head box of putting on the skin, in 121 ℃ of (1.034 * 105Pa) solid pattern sterilizing 20min.
Mortar and grind hammer and clean to dry after, first use alcohol immersion one hour, then soak half an hour with chloroform, first to add liquid nitrogen cooling with front.
3.DEPC the preparation of water: add DEPC in distilled water, make final concentration reach 0.1% (V/V), after firmly shaking up, continue to be placed on shaking table and shake and spend the night with the speed of 100rpm.In 121 ℃ of (1.034 * 105Pa) liquid pattern sterilizing 20min.Cooling rear airtight standby.
B. use Trizol Reagent test kit (INVITROGEN) to carry out the extraction of total RNA, the specification sheets provided by test kit is operated.
1. first human placenta is pulverized in liquid nitrogen.Powder is divided and installs in Eppendorf, every pipe packing 100mg left and right.Every pipe adds 1mL Trizol reagent, mixes, and room temperature is placed 5min.Add chloroform 200 μ L, use forced oscillation 15sec, incubated at room 2～3min.
Under 4 ℃ with the centrifugal 15min of the speed of 12000 * g, mixture be divided into red phenol-chloroform phase (lower phase), white in the middle of mutually and colourless upper strata water.Total RNA is dispersed in the water of upper strata.
3. upper strata water (500 μ L) is transferred in a new Eppendorf pipe, added the 1mL dehydrated alcohol, mix with precipitated rna.
4. more than incubated at room 10min, under 4 ℃, with the centrifugal 10min of the speed of 12000 * g, the RNA of visible white precipitation is affixed on the test tube diapire.
5. abandon supernatant, by 70% washing with alcohol RNA precipitation once, under 4 ℃ with the centrifugal 5min of the speed of 7500 * g.
6. abandon supernatant, seasoning in air, by 60 μ L DEPC water dissolution precipitations ,-70 ℃ save backup.
The sex change agarose gel electrophoresis detects the quality and quantity of total RNA
1. make the sex change agarose gel of one 1.5%: take the 0.9g agarose, add 45mL DEPC water, with after microwave-oven-heating colloidal sol, add successively formaldehyde 10mL, 10 * MOPS6mL, mix, be cooled to fall on the level tray that is plugged comb after 35-40 ℃, place and make it to solidify in 1 hour.
2. centrifugally go out total RNA precipitation, be dissolved in the DEPC water of 20 μ L after the washing drying.
3. sex change 10min. in 65 ℃ of dry baths of constant temperature, ice bath 5min. at once.
4. get 5 μ L total rna solutions, add the sample-loading buffer (loading buffer) of 5 μ L precoolings and the sample buffer (sample buffer) of 10 μ L precoolings, mix.
5. with pipettor, carefully mixed solution is added dropwise in the well of sepharose.
6. with the voltage electrophoresis of 50 volts 90-120 minute (the bromjophenol blue swimming is to the position of glue long 2/3).
7. sepharose is placed in and observes under ultraviolet lamp and take pictures.
RT-PCR amplification HSA cDNA
Human serum albumin cDNA sequence (Genebank V00495) the design upstream and downstream primer of delivering according to Lawn etc., primer sequence is as follows:
SEQ ID NO：1PRIMER13:5’-gatgcacacaagagtgaggtt-3’
SEQ ID NO：2PRIMER14:5’-ttataagcctaaggcagcttg-3’
10 times of gained RNA dilutions are touched to plate as PCR, with ThermoScript II and specific primer P14, the mRNA reverse transcription is become to cDNA, then carry out PCR.The step of reverse transcription and system are: (1) 1.5 or 0.5mL EP pipe in add primer140.5 μ L, RNA2 μ L, dNTP0.5 μ L, distilled water 9 μ L, ice bath after 65C water-bath 5min, slightly centrifugal.(2) add 5 * buffer2 μ L in above-mentioned pipe, 0.1mol/mL DTT1 μ L, Rnase out0.5 μ L, mix rear 42 ℃ of water-bath 2min.(3) add 0.2 μ L SupprscriptTMRT(Invitrogen) mix.(4) 42 ℃ of cultivation 50min in incubator.(5) 70 ℃, 15min deactivation ThermoScript II.
The cDNA of reverse transcription gained of take carries out pcr amplification as template,
Reaction system:
Figure BDA00003216793500061
Reaction conditions is:
Figure BDA00003216793500062
The purifying of the HSA gene fragment of pcr amplification
(1) preparation sepharose, electrophoresis is with the DNA isolation fragment.
(2) after the electrophoresis enough time, under ultraviolet lamp, carefully required DNA fragmentation is scaled off.And remove unnecessary gel as far as possible.
(3) take the weight of sky centrifuge tube, the gel cut with the purpose fragment is contained in the 1.5ml centrifuge tube and claims its weight, obtains the weight of gel piece, determines approx its volume.Generally, the density of gel is 1g/ml, so the relation of the volume of gel and weight can be by following conversion: the weight of gel thin slice be 0.2g its volume be 0.2ml; The Binding Buffer that times gel volume such as adds, be placed in 55 ℃ of-65 ℃ of water-bath temperature to mixture and bathe 7min and melt fully to gel, mixed one time therebetween every 2-3 minute
(4) shift DNA-agarose solution to the HiBind DNA pillar of 700 μ l, and pillar is contained in the 2ml collection tube of a dry Net, under room temperature, the centrifugal 1min of 10,000 * g, discard liquid.
(5) pillar is recovered in collection tube again, added in 300 μ LBinding Buffer to HiBind DNA pillars; Under room temperature, centrifugal 1 minute of 10,000 * g, go to abandon filtrate.
(6) pillar is recovered in collection tube again, added in 700 μ L Wash buffer to HiBind DNA pillars, under room temperature, centrifugal 1 minute of 10,000 * g, go to abandon filtrate.
(7) pillar is recovered in collection tube again, repeated to add in 700 μ L Wash buffer to HiBind DNA pillars, under room temperature, centrifugal 1 minute of 10,000 * g, go to abandon filtrate;
(8) discard liquid, void column is recovered in collection tube again, the centrifugal 1min of 10,000 * g is to dry the liquid of base for post matter remnants.
(9) pillar is contained on the 1.5ml centrifuge tube of a dry Net, adds on 3-50 μ l elutriant or aqua sterilisa on the pillar film, centrifugal 1 minute of 10,000 * g, the solution in centrifuge tube is exactly the DNA product of purifying, is stored in-20 ℃.
The structure of recombinant plasmid pUCMT-HSA
The HSA gene of purifying is connected with the T4DNA ligase enzyme with the pUCMT carrier, connects product and transform the bacillus coli DH 5 alpha recipient bacterium.Coating, containing the SOC agar plate of Amp, IPTG, X-gal, after 37 ℃ of overnight incubation, grows many bluenesss, white colony on flat board.
The evaluation of recombinant plasmid
(1) utilize blue hickie screening, 10 white colonies of picking from each is dull and stereotyped, be inoculated in respectively in the Ep pipe containing 1mL (adding ampicilli) liquid LB substratum.
(2) on 37 ℃ of shaking tables, the velocity fluctuation with 250rpm is cultivated 36h, and the middle pipe lid of opening on clean work station, take a breath 1 time.
(3) room temperature, with the centrifugal 5min of 12,000rpm, is collected bacterial sediment, abandons supernatant, adds the solution I of 100 μ L ice precoolings, and on vortice, thermal agitation suspends precipitation fully.
(4) add 200 μ L solution II, put upside down and mix for several times, room temperature is placed 5min.
(5) add the solution III of 150 μ L ice precoolings, slightly put upside down and mix for several times, ice bath 10min, there will be flocks in clear soln.
(6) room temperature, with the centrifugal 5min of 12,000rpm, is transferred to supernatant in another new Ep pipe.
(7) add the dehydrated alcohol of 2 times of volumes, repeatedly put upside down several times and mix, more than placing 15min under room temperature, with the precipitation plasmid DNA.
(8) room temperature, with the centrifugal 5min of 12,000rpm, is abandoned supernatant.By 75% washing with alcohol DNA precipitation once.
(9) after drying, plasmid DNA is dissolved in to 50 μ L TE(pH8.0) in, and add 1 μ L RNase A (10mg/mL), be placed in 37 ℃ and spend the night to remove RNA.
(10) every kind of recombinant plasmid is carried out to the PCR detection, to determine whether to contain goal gene.
The sequencing of recombinant plasmid
The recombinant plasmid that extracting the positive colony cingula has send the order-checking of Nanjing Jin Sirui Bioisystech Co., Ltd.Adopt the T7 universal primer to be checked order, the reverse complementary sequence that the result obtained is HSA cDNA gene.Obtain the HSA gene order after utilizing software DNAMAN to process, the sequence that in the HSA gene order that sequence alignment result demonstration clone obtains and GENBANK database, registration number is V00495 is in full accord, and result is as follows:
SEQ ID NO：3 1758bp;
Composition 548A;350C;403G;457T;0OTHER
Percentage:31.2%A;19.9%C;22.9%G;26.0%T;0.0%OTHER
Molecular Weight(kDa):ssDNA:544.19dsDNA:1083.68
ORIGIN
1 gatgcacaca agagtgaggt tgctcatcga tttaaagatt tgggagaaga aaatttcaaa
61 gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta
121 aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtcagctgaa
181 aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt
241 cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa
301 tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt
361 gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat
421 gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg
481 tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca
541 aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaagtgt
601 gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc
661 cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa
721 gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt
781 gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa
841 aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct
901 gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct
961 gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat
1021 tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc
1081 tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt
1141 gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag
1201 tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact
1261 ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat
1321 cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta
1381 tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc
1441 ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa
1501 gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag
1561 agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca
1621 aaagaacaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag
1681 gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa
1741 gctgccttag gcttataa
The preparation of linearizing Expression vector pPIC9K-HSA fragment
(1) extract the plasmid in recombinant bacterium DH5 α/pPIC9K-HSA.
(2) Expression vector pPIC9K-HSA extracted with the digestion of restriction enzyme SSal I.Reaction system and reaction conditions are as follows:
In 20 μ L reaction systems, add:
Figure BDA00003216793500101
Aqua sterilisa up to20 μ L, 37 ℃ of reaction 5h.
(3) reclaim test kit with the quick glue of DNA fragmentation and purify linearizing Expression vector pPIC9K-HSA fragment.
(4) use spectrophotometer method, measure the pPIC9K-HSA fragment concentrations reclaimed.
The preparation of pichia spp competent cell
(1) a few days ago, by single colony inoculation of the Pichia pastoris GS115 bacterial strain of conversion use, in the 5mLYPD substratum, 30 ℃ of incubated overnight are to saturated in test.
(2) transform evening before that day, inoculate appropriate incubated overnight liquid at the aseptic triangular flask of 500mL that 100mL YPD substratum is housed, in 30 ℃ of thermal agitation overnight incubation, until cell density reaches 1 * 108 cell/mL(A600nm value at 1.3-1.5).
(3) the culture average mark is installed in two aseptic, the disposable polyphenyl alkene of 50mL pipes to the centrifugal 5min harvested cell of 5000 * g.Pour out nutrient solution, centrifuge tube is inverted to 1min, so that residual nutrient solution flows to end.Every pipe adds 8mL sterilized water re-suspended cell, merges to a polyphenyl alkene pipe.
(4) add 2Ml10 * TE damping fluid, pH7.5. shakes and mixes, then adds 2mL10 * lithium chloride, and rotation mixes, and in 30 ℃, 50r/min shakes 45min.
(5) add 0.5mL 1mol/L DTT, rotation mixes, and in 30 ℃, 50r/min shakes 15min.
(6) the GS115 bacteria suspension is diluted in 50mL water to the centrifugal 5min sedimentation cell of 5000 * g.Pour out supernatant liquor, centrifuge tube is inverted to 1min, so that residual liquid flows to end.
(7) cell precipitation obtained is washed 2 times to the centrifugal 5min sedimentation cell of 5000 * g.Wash successively solution used as follows:
Precipitate for the first time: the 50mL icy water
Precipitate for the second time: the 1mol/L sorbyl alcohol that 10mL is ice-cold
(8) resuspended with appropriate ice-cold 1mol/L sorbyl alcohol, make the final volume of GS115 bacteria suspension be about 0.2mL.
(9) be sub-packed in aseptic, ice-cold 1.5mLEp pipe by every part of 40 μ L, be directly used in and transform or save backup in-80 ℃.
The conversion of Pichia pastoris GS115 competent cell
(1) getting 1 part of GS115 competent cell adds 100ng(to be less than 5 μ L) linearizing expression vector pHBM-HSA fragment, mix.
(2) yeast and transfering DNA mixing are transferred in the electric shock pond in ice-cold 0.2cm gap.
(3) setting (the voltage 1.5kV transformed by general fungi; Electric capacity 25 μ F; Resistance 400 Ω.The electric shock time is 4.2s), the conversion of shocking by electricity.
(4) after the electric shock, add the 1mol/L sorbyl alcohol that 600 μ L are ice-cold immediately in the electric shock pond, mix with the liquid-transfering gun pressure-vaccum, reclaim in the aseptic 1.5mLEp pipe of yeast liquid to.
(5) yeast of recovery is on average coated to 3 containing on the MD flat board of 1mol/L sorbyl alcohol at, bacterium liquid, every 200-600 μ l coating one flat plate.Flat board is placed in to 30 ℃ of cultivations, until single bacterium colony occurs.
The extraction of pichia spp genomic dna
(1) from YPD or MD flat board, picking pichia spp bacterium colony is seeded in the 5mLYPG substratum, and in 30 ℃, the 220r/min shaking culture is spent the night.
(2) yeast cell of getting the 1.5mL incubated overnight is transferred in the 1.5mLEp pipe, the centrifugal 5min of room temperature 12000r/min, the cell that collecting precipitation gets off.
(3) re-suspended cell in 2mLSCED damping fluid (pH7.5), solution is now with the current.
(4) add 0.1-0.3ng zymolyase (mixing before adding), hatch 50min for 37 ℃, obtain and go wall rate<80% to go parietal cell.
(5) add 2ml1%SDS, mix gently, place 5min (0-4 degree) on ice
(6) add 1.5ml5M Potassium ethanoate pH8.9, mix gently
(7), at 4 ℃ of centrifugal 4min of 1000g, retain supernatant DNA precipitation:
(8) shift supernatant from above-mentioned 5, add 2 times of volume ethanol, room temperature is placed 15min, at the centrifugal 20min. of 4 degree 1000g
(9) use 0.7mlTE pH7.4 resuspended precipitation gently, proceed in centrifuge tube.
(10) following operation needs softly, and with the phenol of equivalent: chloroform (1:1v/v) extracting is got, then uses the chloroform of equivalent: primary isoamyl alcohol (24:1) extracting.The upper strata water is proceeded in two centrifuge tubes.
(11) add the 7.5M ammonium acetate of 1/2 volume in every pipe, the ethanol of 2 times of volumes, place 10min on ice, or-20 degree are placed 60min
(12) at 4 ℃ of centrifugal 20min of 10000g, by 1ml70% washing with alcohol precipitation, the dry air precipitation.By the resuspended precipitation of 50ulTE pH7.5.Measure the DNA sample concentration.Two pipes can deposit respectively or be combined deposit in-20 the degree.
The PCR of recombinant yeast pichia pastoris identifies and screening
(1) bacterium colony grown on random picking MD flat board is cultivated, and the picked-up genomic dna extracts unloaded pPIC9K transformant genomic dna as the control group of identifying simultaneously.
(2) take the pastoris genomic dna extracted is template, and Pf1 and Pf4 are primer, carry out the PCR evaluation.PCR reaction system and condition are as follows:
The following reaction system of configuration in the 0.2mL thin-walled tube:
The PCR reaction conditions is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 1min, 65 ℃ of annealing 1min, 72 ℃ are extended 150s, circulate 30 times, and 72 ℃ are extended 10min; 4 ℃ of insulations.
(3) 1% agarose gel electrophoresis are analyzed the PCR product.
(4) choose the recombinant bacterium on the MD flat board, carry out the methanol induction expression, screen the bacterial strain that expression amount is relatively high.
Step S02, fermentation culture;
This Pichia yeast engineering is carried out to fermentation culture; Add the Sodium Selenite that volume fraction is 0.001%～0.05% in this fermentation culture process in substratum, volume fraction is with respect to the volume of substratum, and concentration of sodium selenite is 10mg/L;
Step S03, purifying;
By the product purification of fermentation culture, obtain containing the selenium recombinant human serum albumin.
Preferably, step S02 is as follows:
1, this Pichia yeast engineering is cultivated to 12～18h with YPD substratum (yeast extract paste peptone dextrose culture-medium) under 30 ℃ of conditions, after cultivating, Pichia yeast engineering quantity increases greatly;
2, the Pichia yeast engineering seed liquor after step 1 cultivation being shifted in the BMGY substratum, is 28 ℃ in temperature, under the condition that the pH value is 6, cultivates, and every 8h mass concentration is that 28% strong aqua adjusting pH value is 6.0, and nutrient solution is detected, and reaches bacterium liquid OD 600be 0.6 to get final product, the time is roughly 48 hours;
3, after reaching, in the BMGY substratum, add methanol solution, the pichia spp in substratum is induced, whether detection has drawn containing the selenium recombinant human serum albumin is secreted.
4, confirm to have containing after the secretion of selenium recombinant human serum albumin, add methanol solution and glycerine solution in substratum, add the Sodium Selenite that volume fraction is 0.001%～0.05%, the mass concentration of this glycerine solution is 0.01%～5% simultaneously, and the mass concentration of this methanol solution is 0.01%～5%; The substratum system temperature is adjusted to 28 ℃, pH value is adjusted to 7.0 simultaneously, makes Pichia yeast engineering carry out inducing culture; And, every 12 hours, just add above-mentioned methanol solution, glycerine solution and Sodium Nitrite.The time of this step inducing culture is 96 hours.By the methanol solution and the glycerine solution that use extra fine quality concentration, induced, realize that Pichia yeast engineering can secrete in a large number containing selenium recombinant human serum albumin (target protein), by add Sodium Selenite in substratum, make in the secreted target protein of Pichia yeast engineering and contain a large amount of selenium elements, realized effective combination of target protein and selenium, made greatly to improve containing the value of selenium recombinant human serum albumin.
In step S04, also comprise inactivation step:
Solution after fermentation culture is carried out to the centrifugal treating 10min of 8000rpm, collect supernatant liquor;
By described supernatant liquor, in temperature, be thermal treatment 1～5 hour under 30～80 ℃ of conditions, then all solution after thermal treatment is carried out to the centrifugal treating 20min of 10000rpm, remove the albumen of sex change after thermal treatment, collect supernatant liquor after centrifugal treating, obtain supernatant liquor after thermal treatment.
Then supernatant liquor after above-mentioned thermal treatment is carried out to following purification step:
1, (NH 4) 2sO 4precipitation:
Add solid (NH in supernatant liquor 4) 2sO 4to certain saturation ratio, standing over night, 12000rpm, 4 ℃ of centrifugal 20min, distilled water dissolving for precipitation, dialysis (MWCO=12000);
2, ion exchange chromatography
Comprise CM-Sepharose and DEAE-Sepharose ion exchange chromatography, level pad be respectively the pH5.3(CM-Sepharose ion exchange chromatography with) and pH5.8(DEAE-Sepharose ion exchange chromatography use) the 20mmol/L phosphate buffered saline buffer, sample is adjusted to upper prop after the pH of corresponding level pad.Effluent liquid after CM.Sepharose absorption through the absorption of DEAE-Sepharose post and gradient elution (0～0.5mol/L NaCl, pH5.8), is collected the rHSA Peak Activity again.Above-mentioned two kinds of ion exchange columns all adopt l mol/L NaCl eluant solution to be adsorbed in the residual foreign protein on post.
3, Phenyl-Sepharose hydrophobic chromatography
Phenyl-Sepharose chromatography column 0.85mol/L(NH 4) 2sO 4solution (in 1/4 * PBS damping fluid) balance, 1/4 * PBS damping fluid and water is wash-out respectively, collects the rHSA Peak Activity.With distilled water thoroughly inhale, lyophilize.
4, gel-filtration separation and purification
With Sephacryl S-200 gel-filtration, level pad is 30mmol/L NH4HCO3 (pH7.8).Collect the target protein Peak Activity.
5, the hollow fiber ultrafiltration membrane that concentrating and desalinating selective retention relative molecular mass is 30000, carry out desalination and concentration to target protein.
After above-mentioned purification process, obtain containing the selenium recombinant human serum albumin.
The present invention is containing the preparation method of selenium recombinant human serum albumin, simple to operate, with low cost, productive rate is high, and prepared can be used as the albumin supplement containing the selenium recombinant human serum albumin, play a role in health care, overcome the problem of blood source albumin security, can be used as again organoselenium and suppress cancer, improve immunizing power, strengthen tissue repairing ability, there is very large market potential.Put on market as simple dietary supplements containing the selenium recombinant human serum albumin, compare with existing selemium nutrition product, its composition is clearer and more definite, and security, organic purity and nutritive value are higher.
Below in conjunction with specific embodiment, the above-mentioned preparation method containing the selenium recombinant human serum albumin is described in detail.
Embodiment 1
The embodiment of the present invention, containing selenium recombinant human serum albumin preparation method, comprises the steps:
1, build Pichia yeast engineering
Extract Freshman hepatic tissue RNA, the RT-PCR amplification obtains the rHSA goal gene, amplified production connects cloning vector PMD-18T, build plasmid PMD-18T-rHSA, connection carrier pPIC9K after plasmid double digestion and electrophoresis reclaim, construction expression plasmid pPIC9K-rHSA, double digestion transforms Pichia pastoris GS115 after identifying positive colony, and PCR identifies positive colony;
2, fermentation culture
The positive single colony inoculation of picking is in the 50mlYPD substratum, cultivate 15h for 30 ℃, then will plant daughter bacteria liquid 0.5% is inoculated in the 500ml Erlenmeyer flask containing 100ml BMGY substratum, rotating speed 200rpm, 30 ℃ of culture temperature, every 8h mass concentration is, after 28% strong aqua adjusting pH value is 6.0,48 hours, to detect bacterium liquid OD600=0.724.First add 0.5% analytical pure methyl alcohol, after 12h, add 0.3% glycerine and 0.3% analytical pure methyl alcohol to be induced.Inductive phase, culture temperature was 28 ℃, and pH value is adjusted to 7.0.Every 12h adds glycerine and analytical pure methyl alcohol, and the Sodium Selenite of volume fraction 0.001%, stops fermentation after inducing 96h;
3, deactivation purifying
Collect fermented supernatant fluid: by fermented liquid and the centrifugal 10min of 8000rpm, collect supernatant liquor;
The thermal treatment inactivated proteases: 60 ℃ of thermal treatment 3h, after cool to room temperature, the centrifugal 20min of 10000rpm removes the albumen of sex change after thermal treatment, obtains heat treated supernatant liquor;
(NH 4) 2sO 4fractionation precipitation: add solid (NH in supernatant liquor 4) 2sO 4to certain saturation ratio, standing over night, 12000rpm, 4 ℃ of centrifugal 20min, distilled water dissolving for precipitation, dialysis (MWCO=12000).
CM-Sepharose and DEAE-Sepharose ion exchange chromatography level pad are respectively the 20mmol/L phosphate buffered saline buffer of pH5.3 and pH5.8, and sample is adjusted to upper prop after the pH of corresponding level pad.Effluent liquid after CM.Sepharose absorption through the absorption of DEAE-Sepharose post and gradient elution (0～0.5mol/L NaCl, pH5.8), is collected the rHSA Peak Activity again.Above-mentioned two kinds of ion exchange columns all adopt l mol/L NaCl eluant solution to be adsorbed in the residual foreign protein on post.
Phenyl-Sepharose hydrophobic chromatography Phenyl-Sepharose chromatography column 0.85mol/L(NH4) 2SO4 solution (in 1/4 * PBS damping fluid) balance, 1/4 * PBS damping fluid and water is wash-out respectively, collects the rHSA Peak Activity.With distilled water thoroughly inhale, lyophilize.
Sephacryl S-200 gel-filtration level pad is 30mmol/L NH4HCO3 (pH7.8).Collect the target protein Peak Activity.
The hollow fiber ultrafiltration membrane that concentrating and desalinating selective retention relative molecular mass is 30000, carry out desalination and concentration to target protein.Lyophilize, obtain the seleno human serum albumin.
Embodiment 2
The embodiment of the present invention, containing selenium recombinant human serum albumin preparation method, comprises the steps:
1, build Pichia yeast engineering
The structure of genetic engineering bacterium is with embodiment 1
2, fermentation culture
The positive single colony inoculation of picking is in the 50mlYPD substratum, cultivate 17h for 30 ℃, then will plant daughter bacteria liquid 1.0% is inoculated in the 500ml Erlenmeyer flask containing 100ml BMGY substratum, rotating speed 180rpm, 30 ℃ of culture temperature, every 8h mass concentration is, after 28% strong aqua adjusting pH value is 6.0,48 hours, to detect bacterium liquid OD600=0.688.First add 0.5% analytical pure methyl alcohol, after 12h, add 0.2% glycerine and 0.8% analytical pure methyl alcohol to be induced.Inductive phase, culture temperature was 28 ℃, and pH value is adjusted to 7.0.Every 12h adds glycerine and analytical pure methyl alcohol, and the Sodium Selenite of volume fraction 0.002%, stops fermentation after inducing 96h;
3, deactivation purifying
Collect fermented supernatant fluid: fermented liquid, in the centrifugal 10min of 7000rpm, is collected to supernatant liquor.
The thermal treatment inactivated proteases: 60 ℃ of thermal treatment 2h, after cool to room temperature, the centrifugal 20min of 10000rpm removes the albumen of sex change after thermal treatment, obtains heat treated supernatant liquor.
Purification process, with embodiment 1, obtains containing the selenium recombinant human serum albumin.
Embodiment 3
The embodiment of the present invention, containing selenium recombinant human serum albumin preparation method, comprises the steps:
1, build Pichia yeast engineering
The structure of genetic engineering bacterium is with example 1
2, fermentation culture
The positive single colony inoculation of picking is in the 50mlYPD substratum, cultivate 13h for 30 ℃, then will plant daughter bacteria liquid 1.0% is inoculated in the 500ml Erlenmeyer flask containing 100ml BMGY substratum, rotating speed 180rpm, 30 ℃ of culture temperature, every 12h mass concentration is, after 28% strong aqua adjusting pH value is 6.5,48 hours, to detect bacterium liquid OD600=0.815.First add 0.5% analytical pure methyl alcohol, after 12h, add 0.3% glycerine and 0.6% analytical pure methyl alcohol to be induced.Inductive phase, culture temperature was 28 ℃, and pH value is adjusted to 7.5.Every 12h adds glycerine and analytical pure methyl alcohol, and the Sodium Selenite of volume fraction 0.005%, stops fermentation after inducing 96h;
3, deactivation purifying
Collect fermented supernatant fluid: fermented liquid, in the centrifugal 10min of 7000rpm, is collected to supernatant liquor.
The thermal treatment inactivated proteases: 60 ℃ of thermal treatment 3h, after cool to room temperature, the centrifugal 20min of 10000rpm removes the albumen of sex change after thermal treatment, obtains heat treated supernatant liquor.
Purification process, with embodiment 1, obtains containing the selenium recombinant human serum albumin.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Figure IDA00003216794000011

Claims (5)

1. the preparation method containing the selenium recombinant human serum albumin, comprise the steps:
Build the Pichia yeast engineering containing the recombinant human serum albumin gene;
Described Pichia yeast engineering is carried out to fermentation culture; Add the Sodium Selenite that volume fraction is 0.001%～0.05% in described fermentation culture process in substratum;
By the product purification of fermentation culture, obtain containing the selenium recombinant human serum albumin.
2. the preparation method containing the selenium recombinant human serum albumin as claimed in claim 1, is characterized in that, described fermentation culture step is:
Described Pichia yeast engineering is used under 30 ℃ of conditions to YPD culture medium culturing 12～18h;
Seed liquor is transferred in the BMGY substratum, is 28 ℃ in temperature, under the condition that the pH value is 6, cultivates, and detects bacterium liquid OD 600be to add methanol solution at 0.6 o'clock in substratum;
Temperature is adjusted to 28 ℃, and pH value is adjusted to 5～9 fermentation culture 72～120 hours, every 12 hours, in substratum, adds the Sodium Selenite that methyl alcohol and glycerine mixing solutions and volume fraction are 0.001%～0.05%.
3. the preparation method containing the selenium recombinant human serum albumin as claimed in claim 2, is characterized in that, the mass concentration of described glycerine solution is 0.01%～5%, and the mass concentration of described methanol solution is 0.01%～5%.
4. as claim 1, the 2 or 3 described preparation methods containing the selenium recombinant human serum albumin, it is characterized in that, also comprise following inactivation step before described purification step:
By the solution centrifugal after fermentation culture, collect supernatant liquor;
Be thermal treatment 1～5 hour under 30～80 ℃ of conditions by described supernatant liquor in temperature, collect supernatant liquor after centrifugal treating, obtain supernatant liquor after thermal treatment.
5. the preparation method containing the selenium recombinant human serum albumin as claimed in claim 4, is characterized in that, described purification step is as follows:
By supernatant liquor process (NH after described thermal treatment 4) 2sO 4precipitation, ion exchange chromatography, hydrophobic chromatography, gel-filtration separation and purification and use relative molecular weight are that 5000～100,000 hollow fiber ultrafiltration membrane is carried out desalting treatment, obtain containing the selenium recombinant human serum albumin.
CN201310188214.1A 2013-05-20 2013-05-20 Preparation method containing selenium recombinant human serum albumin Active CN103468769B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310188214.1A CN103468769B (en) 2013-05-20 2013-05-20 Preparation method containing selenium recombinant human serum albumin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310188214.1A CN103468769B (en) 2013-05-20 2013-05-20 Preparation method containing selenium recombinant human serum albumin

Publications (2)

Publication Number Publication Date
CN103468769A true CN103468769A (en) 2013-12-25
CN103468769B CN103468769B (en) 2016-09-07

Family

ID=49793809

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310188214.1A Active CN103468769B (en) 2013-05-20 2013-05-20 Preparation method containing selenium recombinant human serum albumin

Country Status (1)

Country Link
CN (1) CN103468769B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112480240A (en) * 2020-12-24 2021-03-12 河北医科大学第二医院 Separation and purification method of recombinant human serum albumin
CN113322223A (en) * 2021-06-03 2021-08-31 重庆市畜牧科学院 Selenium-enriched yeast genetically engineered bacterium, surface display system thereof and construction method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
陈思: "毕赤酵母表达硒代重组人血清白蛋白", 《北京化工大学学报》, vol. 33, no. 5, 31 May 2006 (2006-05-31), pages 1 - 3 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112480240A (en) * 2020-12-24 2021-03-12 河北医科大学第二医院 Separation and purification method of recombinant human serum albumin
CN113322223A (en) * 2021-06-03 2021-08-31 重庆市畜牧科学院 Selenium-enriched yeast genetically engineered bacterium, surface display system thereof and construction method thereof
CN113322223B (en) * 2021-06-03 2023-09-12 重庆市畜牧科学院 Selenium-enriched yeast genetically engineered bacterium, surface display system thereof and construction method thereof

Also Published As

Publication number Publication date
CN103468769B (en) 2016-09-07

Similar Documents

Publication Publication Date Title
CN101358201A (en) Recombinant human iron-regulatory hormone adenovirus, preparation method and application thereof
CN106540249A (en) A kind of bird flu (H5N1) or the antigen concentrating and purifying process of Porcine reproductive and respiratory syndrome (PRRS) viral vaccine
CN101270158B (en) Target glioma resistant protein, preparation method and application
CN102796758A (en) Recombinant porcine alpha interferon and application thereof in preparing medicines for treating Porcine cytomegalovirus (PCMV)
CN102127164B (en) Method for extracting recombinant human serum albumin from rice seeds
CN104788568A (en) Pichia pastoris genetic engineering hybrid antibacterial peptide CC29 and preparing method thereof
CN103936862A (en) Co-expression of fusion porcine interleukin 4/6 and interleukin 2 genes and application of fusion porcine interleukin 4/6,2 gene in preparation of biological agents
CN103468769A (en) Selenium-containing recombinant human albumin preparation method
CN103232545B (en) A kind of Recombinant Swine Interferon α1-Fc fusion rotein and encoding gene thereof and expression method
CN103275203A (en) Gene engineering preparation and identification method for eriocheir sinensis crustacean hyperglycemic hormone
CN103012577B (en) Recombinant porcine interleukin 4, and encoding gene and expression method thereof
CN104789594A (en) Method for constructing CHO cell strain for stably and efficiently expressing human serum albumin and interleukin II fusion protein
CN107022549A (en) Pelteobagrus fulvidraco beta-defensin gene and its beta-defensin antibacterial peptide and its application
CN106367422A (en) Preparation method of standard product for recombinant ovine interferon Tau biological activity detection
CN103613668B (en) Long-acting fusion interferon for dogs and cats, as well as preparation method and application thereof
CN102199608B (en) Efficient expression of pinellia ternate agglutinin (PTA) in bombyx mori reactor
CN105567714A (en) Recombinant yak lysozyme as well as preparation method and application thereof
CN100536923C (en) Method for expressing human insulin for reducing blood sugar by transgenic lucid ganoderma
CN103012578A (en) Recombinant porcine interleukin 2, and encoding gene and expression method thereof
CN103145845A (en) Expression of Tat and human wild-type P53 fusion protein in Pichia pastoris
CN104531691A (en) Primers for obtaining genes of bovine interferon alpha and preparation method for recombinant bovine interferon alpha
CN103467604B (en) A kind of sleep-inducing peptide fusion protein and application thereof
CN101270157B (en) Genetic engineering target glioma Acp-W1 resistant protein, preparation method and application
CN103705920B (en) Kytoplasm Polyadenylation component binding protein 4 vaccine and Synthesis and applications thereof
CN101260143A (en) Highly effective production method for canine leucocyte interferon

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
ASS Succession or assignment of patent right

Owner name: XU DONG

Free format text: FORMER OWNER: SHENZHEN YATAIXING INDUSTRY LTD.

Effective date: 20150310

C41 Transfer of patent application or patent right or utility model
COR Change of bibliographic data

Free format text: CORRECT: ADDRESS; FROM: 518103 SHENZHEN, GUANGDONG PROVINCE TO: 518000 SHENZHEN, GUANGDONG PROVINCE

TA01 Transfer of patent application right

Effective date of registration: 20150310

Address after: 518000 Baoan District, Shenzhen, Baoan District, the people of the first district of the square building, the first floor of the building on the ground floor, building 13,

Applicant after: Xu Dong

Address before: 518103, Guangdong Province, Shenzhen, Baoan District Baoan district nine District Road Plaza, building 13, building

Applicant before: Shenzhen Yataixing Industry Ltd.

C14 Grant of patent or utility model
GR01 Patent grant