CN1414105A - Method of constructing genetic engineering organism based on in vivo isogenesis recombination - Google Patents
Method of constructing genetic engineering organism based on in vivo isogenesis recombination Download PDFInfo
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Abstract
A method for configuring the genetically engineered biosome by in vivo homologous recombination features that the linear exogenous gene expression unit with the homologous sequence of carrier fragment as both ends and the linear particular expression carrier are used to cotransform particular biosome. The resultant genetically engineered biosome and its usage are also disclosed.
Description
Technical field
The invention belongs to the gene engineering field, be specifically related to the purposes of the genetic engineering organism of a kind of method that makes up the genetic engineering organism and acquisition.
Background technology
Yeast is a class unicellular eukaryote.Human to yeast, especially the application of yeast saccharomyces cerevisiae has a long history, and has accumulated a large amount of experiences.Simultaneously, because people have accumulated rich knowledge to biology of yeast and molecular biology, so, build up in the world behind the bacillus coli gene engineering expression system soon, zymic gene engineering expression system also is built to very soon, at present, it has been one of most important expression system of expression alien gene.Yeast saccharomyces cerevisiae is as unicellular organism, on producing and operating, have the advantages that bacterial expression system is easy to extensive amplification on the one hand, have eukaryotic cell on the other hand again to proteinic translation post-treatment and modification, as: the correct formation of disulfide linkage, the hydrolysis processing of precursor protein, glycosylation etc.At present developed and set up the expression vector of multiple yeast saccharomyces cerevisiae expression system, wherein important with integrated (YIp) carrier and additional build (YEp) carrier.Integrating vector does not contain autonomously replicating sequence (ARS), but is integrated on the karyomit(e) of yeast host bacterium, and the advantage of this class carrier is a good stability, but that its shortcoming is a copy number is low.Additional build carrier owing to contain part or all of sequence from yeast natural plasmid 2 μ, can be in the yeast body self-replicating, it can reach about 30 copies usually at intracellular copy number, but how unstable under non-selection condition.The expression vector of widespread use at present mostly is intestinal bacteria one yeast shuttle plasmid greatly, has the required sequence of the amplification of duplicating in the intestinal bacteria, and this part sequence is proved yeast is had certain toxicity (Kaoru A, Akira N, Hiroyuki A and Yasuji O:Automaticelimination of unnecessary bacterial sequences from yeast vectors.Gene 121 1992 161-165), influence plasmid at intravital stability of host and expression of exogenous gene (Dale L, Carlo V et al:The 2 μ m plasmid as anonselectable, stable, high copy number yeast vector.Plasmid 25 1991 81-95).
Yeast saccharomyces cerevisiae all is considered to study the model animals of homologous recombination always, studies show that, in yeast saccharomyces cerevisiae, linearizing dna fragmentation can effectively improve the occurrence frequency (Martin K:Damagc-induccd recombination inthe yeast Saccharomyces cerevisiae.Mutation Research 451 2000 91-105) of reorganization, and this characteristic makes the technology of utilizing homologous recombination to carry out the genetically engineered operation obtain using widely in yeast saccharomyces cerevisiae.Main application at present comprises: (1) is with specific dna fragmentation recombination and integration (Kaiser to karyomit(e), C.et al.:Methods in Yeast Genetics.ColdSpring Harbor Laboratory Press, 1994 Cold Spring Harbor, NY.); (2) gene interrupt, gene substitution, promptly utilize the DNA reorganization that the fragment in the carrier is inserted in the karyomit(e), cause the gene function disappearance or substitute gene on the karyomit(e) fully by target gene fragment; (3) construction of expression vector.The target gene fragment two ends contain with plasmid sequence on the homologous part, just can be in the yeast body " be connected " with linearizing plasmid and obtain required expression vector (Kevin R.Oldenburg by reorganization, Kham T.Vo, Susan Michaelis and Chris Paddon:Recombination-mediatedPCR-directed plasmid construction in vivo in yeast.Nucl Acids Res 25 (2) 1997 451-452).This method does not rely on whether there is suitable restriction endonuclease sites.Confirm in some zymic gene interrupt experiments: the homology segment of 30-50bp just can carry out reorganization (A in the effective body, Lacroute F, Cullin C.:A simple andefficient method for direct gene deletion in Saccharomyces cerevisiae.Nucleic Acids Res21 (14) 1993 Jul 11 3329-30), that is to say fully and homology segment can be synthesized as the part of PCR primer.Other has some systematic experiments to show that the size of integrating homology segment in the experiment in homology only needs 15bp (the Manivasakam P that just can obtain recombinant clone, Weber SC, McElver J, Schiestl RH.:Micro-homology mediated PCRtargeting in Saccharomyces cerevisiae.Nucleic Acids Res 23 (14) 1995 Jul 25:2799-2800).
But the expression vector that builds up is all bigger, and the stability in cell is waited to improve so far.
Summary of the invention
The object of the present invention is to provide the method for a kind of structure genetic engineering organism based on homologous recombination in the body, this method make the exogenous gene expression unit can be in the genetic engineering organism stable existence.
The present invention also aims to provide the genetic engineering organism that utilizes this method to obtain, especially express the Yeast gene engineering bacterial strain of gene engineering product with commercial value.
What the present invention proposed gives birth to the method for body based on the structure genetic engineering of homologous recombination in the body, be linearizing exogenous gene expression unit and the linearizing particular expression carrier cotransformation specific organism that needed, two ends is had the carrier segments homologous sequence, exogenous gene expression unit and particular expression carrier carry out genetic recombination, the inessential sequence of carrier is replaced by the exogenous gene expression unit, resulting having in the unitary genetic engineering organism of exogenous gene expression, the exogenous gene expression unit can stable existence and high-efficient expression exogenous genes products.
Concrete steps of the present invention are as follows:
1, linearization of carrier: the suitable restriction enzyme of carrier utilization carries out enzyme to be cut, and collects the suitably fragment of size then, and this is the carrier of linearization;
2, obtain two ends and have expression unit with linearizing carrier segments homologous sequence;
3, the suitable organism of cotransformation is distinguished in the carrier and the described expression of step 2 unit of linearization;
4, identify transformant.The transformant that takes a morsel is expressed the analysis of plasmid stability in mensuration that the unit exists situation, expression level and the transformant to them.
Among the present invention, described genetic engineering organism comprises mammalian cell, insect cell, bacterium and fungi.
Among the present invention, described foreign gene comprises coded interference element, hepatitis B virus surface antigen, erythropoietin, human serum albumin, human superoxide dismutase, tumour necrosis factor, Tumor Necrosis Factor Receptors I and II, interleukin 1, the gene of interleukin II and acceptor thereof etc.
Among the present invention, described carrier is to contain based on complete 2 μ plasmids.
Among the present invention, described carrier homologous sequence, its homology segment base number 15-60bp.
Among the present invention, described fungi is a yeast, comprises yeast saccharomyces cerevisiae, kluyveromyces, candiyeast, rhodotorula, schizosaccharomyces pombe etc.
Among the present invention, described bacterium is Bacillus subtilus or intestinal bacteria.
In the above-mentioned yeast, particularly contain yeast gene engineering bacteria DCO4/pHC11R-SA-28 that expresses hepatitis B virus SA-28 fused antigen or the yeast gene engineering bacteria of expressing hepatitis B virus surface antigen and other fused antigens.
In the above-mentioned yeast, particularly contain the yeast gene engineering bacteria DCO4/pHC11R-IFN α 2a of expressing human α 2a or α 2b Interferon, rabbit, or DCO4/pHC11R-IFN α 2b, or express the yeast gene engineering bacteria of other people Interferon, rabbit.
In the above-mentioned yeast, particularly use yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) or Pasteur's pichia yeast (Pichiapastoris).
The employing aforesaid method has obtained the obvious genetic engineering organism of improving of exogenous gene expression cell stability, and the expression of exogenous gene level also significantly improves than additive method.
Description of drawings
Fig. 1: adopt this method to make up the route of yeast gene engineering bacteria.Wherein, (A): the structure of pHC11R-SA-28; (B): the structure of pHC11R-IFN α 2a.
Fig. 2: the PCR of pHC11R-SA-28 transformant identifies.Wherein, the 1-10:PCR template is the total DNA of transformant DCO4/pHC11R-SA-28, the pHC11SA-28:PCR template is the total DNA of transformant DCO4/pHC11SA-28, the pHC11R:PCR template is that carrier segments pHC11R is from connecting DNA, the contrast of pHC11SA-28 plasmid: pcr template is the pHC11SA-28 plasmid DNA, marker: λ-HindII marker.
Fig. 3: the PCR of pHC11R-IFN α 2a transformant identifies.Wherein, the 1-6:PCR template is a transformant DCO4/pHC11R-IFN α 2a transformant, pHC11-IFN α 2a:PCR template is total DNA of transformant DCO4/pHC11-IFN α 2a, the contrast of pHC11-IFN α 2a plasmid: pcr template is a pHC11-IFN α 2a plasmid DNA, marker: λ-HindIII marker.
Fig. 4: the ELISA measurement result of pHC11R-SA-28 transformant expression level.Wherein, Y-axis is the absorbance during ELISA measures, and is directly proportional with the hepatitis B surface antigen activity.Feminine gender is that blank sample is measured, and contrasts the expression product determination of activity of the high expression engineering that transforms into pHC11 SA-28, and 1～10 is the expression product determination of activity of 10 different transformants of pHC11R-SA-28.
Fig. 5: the recombinant conversion sublist reaches interferon alpha 2a protein electrophoresis result.Wherein, 1～6:6 different pHC11R-IFN α 2a transformant expression product, contrast: the contrast of pHC11-IFN α 2a transformant expression product, M: molecular weight Marker.
Fig. 6: plasmid stability in the transformant.
Embodiment
Specifically describe the inventive method below by the example that makes up yeast gene engineering bacteria.
The step that makes up is shown in Fig. 1 (A) and Fig. 1 (B):
1. linearization of carrier pHC11R: carrier pHC11R through SalI and BglI simultaneously enzyme cut, collect the big fragment of 8.7Kbp then, this is the carrier pHC11R of linearization.
2.PCR the expression cells D NA of amplification hepatitis B virus fused antigen SA-28 or people α 2a Interferon, rabbit, the product two ends of acquisition have and linearizing carrier segments homologous 50bp sequence.
3. the carrier pHC11R of linearization and the hepatitis B virus fused antigen SA-28 of pcr amplification are expressed unit or people α A interferon expression unit difference cotransformation yeast saccharomyces cerevisiae DCO4 cir
0Leu2 ade1.
4. the transformant that takes a morsel is expressed the analysis of plasmid stability in mensuration that the unit exists situation, hepatitis B virus fused antigen SA-28 or people α 2a interferon expression level and the transformant to them.Qualification result to transformant is as follows:
Having only in 10 SA-28 transformants not amplify for No. 4 and express unit segment (Fig. 2), can not express SA-28 (Fig. 4), may be connecting certainly of linearization pHC11; All the other nine transformants can both amplify expresses unit segment (Fig. 3), the expression level of SA-28 has difference, wherein No. 1 and No. 7 expression levels are the highest, want that when in former engineering bacteria (Fig. 4) more than 3 times, the stability of plasmid in yeast cell is also apparently higher than former engineering bacteria (Fig. 6).
6 people α 2a Interferon, rabbit transformants all can both amplify expresses unit segment (Fig. 3), and 6 transformants all have the expression of people α 2a Interferon, rabbit as can be seen from the running gel, and expression amount is all apparently higher than former engineering bacteria (Fig. 5).Plasmid stability is also a little more than former engineering bacteria (Fig. 6) in No. 5 transformants.
Further operating process is as follows:
1, the extraction of plasmid DNA.Prepare a small amount of plasmid DNA fast with test kit: (betting office's test kit).
With the 3ml bacterium of incubated overnight, high speed centrifugation 1min thoroughly removes supernatant.
Add 100 μ l Solution I, with the abundant suspension cell of vibrator.Add 200 μ l Solution II, turn upside down immediately or with at the bottom of the finger bomb tube, make the bacterium cracking, room temperature is placed 2mins, makes solution become clarification.Add 400 μ l SolutionIII, turn upside down 5-10 time immediately, make it abundant neutralization, room temperature is placed 2mins.
Take out 2ml sample collection tube and 3S post, on tube wall, put on sample number, the supernatant of back is all transferred in the 3S post.Room temperature is placed 2mins, and room temperature is centrifugal 12,000rpm, 1min.
Take off the 3S post, discard the waste liquid in the collection tube, the 3S post is put into same collection tube, draw 700 μ l Wash Solution to the 3S post, high speed centrifugation 1min, and repeat this step once.Take off the 3S post, discard the waste liquid in the collection tube, the 3S post is put into same collection tube, high speed centrifugation 5mins.
The 3S post is put into clean 1.5ml centrifuge tube, add 50 μ l TE, the centrifugal 5mins of room temperature in 3S post film central authorities.
2, the enzyme of DNA is cut the recovery with carrier segments
With the pHC11R plasmid DNA for preparing, cut with BglI and SalI enzyme.
At the agarose gel electrophoresis isolated fragment, cut off the agar sugar of the large fragment DNA that contains 8.7Kbp, put into the 1.5ml centrifuge tube, add 300～600 μ l S1 liquid by every 100mg agarose, put 55 ℃ of water-bath 10mins, treat that the agar sugar melts fully after, add the Virahol of 1/10 S1 volume, mixing, 55 ℃ of temperature are bathed 1min.
Agarose liquid after melting is moved into adsorption column, and high speed centrifugation 1min outwells liquid in the collection tube, again adsorption column is put into same collection tube, in adsorption column, add 450 μ l W1 liquid, leave standstill 1min after, centrifugal 15secs, outwell liquid in the collection tube, again adsorption column is put into same collection tube, in adsorption column, added 450 μ l W1 liquid, centrifugal 15secs, outwell liquid in the collection tube, again adsorption column is put into same collection tube.
Centrifugal 3mins puts into a clean 1.5ml Eppendorf pipe with adsorption column, and central authorities add 30 μ lT1 liquid at adsorption film, leave standstill 1min, high speed centrifugation 1min ,-20 ℃ of storages.
(this experimental procedure is provided by Shanghai China Shun biotechnology company limited)
3, PCR reaction
The PCR primer design:
Every primer comprises 2 parts: the homology segment about about 50bp, respectively with linearized vector segmental 5 ' section and 3 ' section homology; Another part is the required and template mating section of PCR reaction, approximately about 25bp, in 5 ' primer sequence be the ADH2-GAPDH promoter, fusion (inducible promoter) of yeast saccharomyces cerevisiae or PGK1 promotor (constitutive promoter) 5 ' terminal sequence, in 3 ' primer, be 3 ' sequence of ADH1 terminator.The sequence of synthetic PCR primer is as follows:
5 ' PCR primer:
1. 76nts: underscore partly is a carrier segments BglI end homologous part (50nts),
Italicized item is PADH 2-GAPDH a 5 ' terminal sequence (26nts)
TAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCGGATCCTTCAATATGCGCACATACGC
2. 68nts: underscore partly is a carrier segments BglI end homologous part (44nts),
Italicized item is or PPGK1 5 ' terminal sequence (24nts)
CGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCGGATCCACAGGACGGGTGTGGTCG
3 ' PCR primer:
76nts: underscore partly is carrier segments SalI end homologous part (50nts),
Italicized item is TADH1 a 3 ' terminal sequence (26nts)
AAAGTTCCCTCAAGAATTTTACTCTGTCAGAAACGGCCTTAACGACGTAGThe CCGTGTGGAAGAACGATTACAACAGG reaction system:
Template DNA: 50～100ng
15pM 5 ' primer: 1.0 μ l
15pM 3 ' primer: 1.0 μ l
10mM?dNTP：0.5μl
10×ExTaq?buffer：5μl
5U/μl?ExTaq：1.0μl
Moisturizing to cumulative volume is: 50 μ l response procedures:
95℃ 5mins 1?cycle
94℃ 30secs
58℃ 30secs 30?cycles
72℃ 2mins
72℃ 10mins 1?cycle
4, yeast saccharomyces cerevisiae transforms Lithium Acetate LiAc chemical transformation
Recipient bacterium (the DCO4 cir of 1 single bacterium colony of picking from the flat board
0Leu2 ade1), be inoculated in 3ml YEPD, 30 ℃ of shaking tables are cultivated 16～24hr.
Switching is spent the night bacterium in 50ml YEPD, makes that initial concentration is OD600=0.1, cultivates 4～5hr to OD600=0.4 for 30 ℃.Centrifugal, 5,000rpm, 5mins collects thalline, and uses the sterilized water washed twice.
Resuspended with the 10ml sterilized water, every 1ml packing, 30secs is centrifugal, collects thalline.Every pipe adds 900 μ l sterilized waters, 100 μ l 1M LiAc are resuspended, and 30 ℃, 10mins.
With the quick centrifugal 30secs of thalline, collect thalline, carefully remove LiAc.
Every pipe adds 50%PEG 240 μ l, 1M LiAc 36 μ l, 5mg/ml ssDNA 10 μ l, carrier-pellet segment DNA and PCR fragment (carrier segments and PCR fragment mol ratio are that 1: 6, carrier segments consumption are 1 μ g), and moisturizing to cumulative volume is 360 μ l.Thorough mixing.。
30 ℃ leave standstill 30mins.42 ℃ of water-bath 20mins.8,000rpm, centrifugal 1min abandons supernatant.
Adding 200 μ l sterilized waters beats even.On every selective medium SDA flat board, add 100 μ l bacterium liquid, evenly with aseptic spreading rod coating.Cultivated 3 days for 30 ℃.
(transformation efficiency can reach 0.5-1 * 10
3/ ug carrier segments.)
5, engineering bacterium fermentation method
Engineering bacteria is inoculated into freshly prepd SDA flat board, cultivates 24-36hrs, equivalent is inoculated into 3mlSDA, 30 ℃ of shaking culture 18hrs.
With the 1ml seed liquor be transferred to 25mlYEPE/250ml (inducible promoter, pHC11R-SA-28) in the triangular flask, or in 25mlYEPD/250ml (constitutive promoter, the pHC11R-IFN α A) triangular flask, 30 ℃ of shaking culture 48hrs.
*(SDA：0.67％YNB?2％Glucose?40ug/ml?Adene)
6, simple and easy yeast cell extract preparation
Get 20 OD fermentation thalline and manage in Eppendorf, 5, the centrifugal 5mins of 000rpm abandons supernatant.
Thalline adds 500ul granulated glass sphere, the broken damping fluid * of 500ul.
Place ice bath, the vortex 1min that vibrates, ice bath 1min, 10 times.
12, the centrifugal 15mins of 000rpm draws supernatant, places-20 ℃ of preservations.
* (broken damping fluid (PBS) pH=7.8:NaH
2PO
40.09g Na
2HPO
40.95g be settled to 150ml)
7, the ELISA of SA-28 measures
Prepare: with the micropore serial number.And each sample diluted in proportion: dilute 10 times, 100 times, 1000 times.
Application of sample and enzyme mark: add testing sample or yin, yang and contrast 50 μ l in respective aperture.The enzyme-added mark binding substances 50 μ l in every hole, mixing.Blank hole if (not enzyme-added mark binding substances).
Hatch: put 37 ℃ of isothermal reaction 60mins.
Wash plate: get rid of sample in the plate, pat dry and fill it up with scavenging solution (T-PBS dry powder in the test kit is dissolved in distilled water and orders appearance 500ml).Get rid of scavenging solution behind 15～20secs, repeat 5 times.
Colour developing: every hole adds developer A and each 50 μ l of developer B, 37 ℃ of lucifuge colour developing 15mins.
Stop: every hole adds stop buffer 50 μ l, mixing.
Colorimetric: select wavelength 450nm or dual wavelength 450/630nm, blank well school zero.With microplate reader colorimetric is carried out in every hole, and write down its OD value.Negative control OD value is lower than at 0.05 o'clock by 0.05 calculating, is higher than at 0.05 o'clock and calculates by actual OD.The average OD value of sample well OD value 〉=negative control * 2.1 o'clock, sample HbsAg is positive in this hole; Otherwise, negative.
8, the gel electrophoresis analysis of people α 2a Interferon, rabbit
With the sheet glass washes clean, wipe clean, be fixed on the electrophoresis chamber; Compound concentration is 15% separation gel, and separation gel is injected glass sandwich, and top keeps the glue face smooth with 70% ethanol front cover; After the glue polymerization to be separated, preparation 15% concentrates glue, removes the ethanol on separation gel surface, pours into concentrated glue, inserts the point sample comb.
Get 50 μ l albumen supernatant samples, add 10 μ l, 6 * SDS-PAGE sample preparation liquid, 100 ℃ of water-bath 3-5mins.
After electrophoresis chamber adds electrophoretic buffer, carefully choose and go up the sample comb, draw sample to be analyzed respectively with micro sample adding appliance, inject well.
After application of sample finishes, connect power supply, initial low voltage (30-50V) or the low current used treats that sample after concentrated glue partial concentration becomes a line, strengthens voltage or electric current (60-90V) again, treats can stop electrophoresis when the tetrabromophenol sulfonphthalein indicator arrives bottom margin.
After electrophoresis finished, sledge was opened layer glass gently, took out gel (wear gloves, prevent to pollute the glue face), gel is put in the staining fluid, and vibration dyeing 0.5-2hrs, it is clean to be washed till background with destainer, and band is clear.
Take a picture and preserve or gel drying.
30% (W/V) acr-bis mother liquor, 3mol/L Tris-HCl pH=8.8,0.5mol/L Tris-HCl pH=6.7,10%SDS, TEMED, 10% ammonium persulphate, Tris-Gly electrophoretic buffer (Gly 1.44%Tris 0.6%SDS 0.1%) pH=8.3,0.05% (W/V) Coomassie blue stain liquid, destainer
9, express the pcr amplification of unit segment in the transformant
By kind of each transformant bacterial strain 30 ℃ of shaking culture 16hrs in 3ml SDA nutrient solution, collect the bacterium that spends the night.
With 200 μ l yeast lysate suspension thalline, add 200 μ l granulated glass spherees, vortex thermal agitation 2mins adds the saturated phenol of 100 μ l, 100 μ l chloroform/primary isoamyl alcohol, vortex thermal agitation 2mins, totally 3 times.
10, the centrifugal 10mins of 000rpm collects supernatant.Add isopyknic chloroform/primary isoamyl alcohol extracting.
Add the 3M NaAc of 1/10 volume, the dehydrated alcohol of 2 times of volumes in the supernatant ,-20 ℃ of precipitation 2～3hrs.
70% ethanol is washed once, and the oven dry back adds the dissolving of 20 μ l sterilized waters.-20 ℃ of preservations.
Getting above-mentioned cerevisiae dna extract 10 μ l is pcr template, carries out PCR and identifies.Method is ditto described.
10, the mensuration of plasmid stability in the transformant
Inoculation transformant bacterial strain DCO4/pHC11R-SA-28 and control strain DCO4/pHC11-SA-28, or DCO4/pHC11R-IFN α 2a and control strain DCO4/pHC11R-IFN α 2a be in the 3mlSDA nutrient solution, 30 ℃ of shaking culture 16hrs.
Equivalent is respectively inoculated 100ul to the 25mlYEPD nutrient solution, 30 ℃ of shaking culture 24hrs*.
Measure the OD600 value of two groups of bacterium liquid respectively, with 8,000 times of two groups of bacterium liquid dilutions, the monoploid bacterium is got 100ul coating YEPD flat board.
Left standstill obvious bacterium colony formation on the visible YEPD flat board, about about 300 of every ware 2 days with two groups dull and stereotyped 30 ℃.
Two groups of bacterium make single bacterium colony dibbling SDA flat board with the toothpick of sterilizing respectively, count 100, and 30 ℃ leave standstill cultivation 36hrs, observe dull and stereotyped last two groups of bacteria growing situations, record bacterium colony per-cent.The bacterium liquid of * step is respectively got 100ul be inoculated in other two bottles of YEPD liquid, repeat above-mentioned.
Claims (10)
1. method by homologous recombination construction genetic engineering organism in the body, it is characterized in that needed, two ends are had the linearizing exogenous gene expression unit and the linearizing particular expression carrier cotransformation specific organism of carrier segments homologous sequence, exogenous gene expression unit and particular expression carrier carry out genetic recombination, the inessential sequence of carrier is replaced by the exogenous gene expression unit, resulting having in the unitary genetic engineering organism of exogenous gene expression, the exogenous gene expression unit can stable existence and high-efficient expression exogenous genes products.
2. method according to claim 1 is characterized in that said organism comprises mammalian cell, insect cell, bacterium and fungi.
3. method according to claim 1, it is characterized in that said foreign gene comprises coded interference element, hepatitis B virus surface antigen, erythropoietin, human serum albumin, human superoxide dismutase, tumour necrosis factor, Tumor Necrosis Factor Receptors I and II, interleukin 1, the gene of interleukin II and acceptor thereof etc.
4. method according to claim 1 is characterized in that said carrier, is to contain based on complete 2 μ plasmids.
5. method according to claim 1 is characterized in that said carrier homologous sequence, and its homology segment base number is 15-60bp.
6. method according to claim 2 is characterized in that said fungi is a yeast, comprises yeast saccharomyces cerevisiae, kluyveromyces, candiyeast, rhodotorula, schizosaccharomyces pombe.
7. method according to claim 1, it is characterized in that said genetic engineering organism is a yeast, it contains yeast gene engineering bacteria DCO4/pHC11R-SA-28 that expresses hepatitis B virus SA-28 fused antigen or the yeast gene engineering bacteria of expressing hepatitis B virus surface antigen and other fused antigens.
8. method according to claim 1, it is characterized in that said genetic engineering organism is a yeast, it contains the yeast gene engineering bacteria DCO4/pHC11R-IFN α 2a of expressing human α 2a or α 2b Interferon, rabbit, or DCO4/pHC11R-IFN α 2b, or express the yeast gene engineering bacteria of other people Interferon, rabbit.
9. according to claim 7 or 8 described methods, it is characterized in that said yeast is yeast saccharomyces cerevisiae or Pasteur's pichia yeast.
10. method according to claim 10 is characterized in that concrete steps are as follows:
(1) linearization of carrier: the suitable restriction enzyme of carrier utilization carries out enzyme to be cut, and collects the suitably fragment of size then, and this is the carrier of linearization;
(2) obtain two ends and have expression unit with linearizing carrier segments homologous sequence;
(3) the suitable organism of cotransformation is distinguished in the carrier and the described expression of step 2 unit of linearization;
(4) identify transformant.The transformant that takes a morsel is expressed the analysis of plasmid stability in mensuration that the unit exists situation, expression level and the transformant to them.
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| CN102007212B (en) * | 2008-03-07 | 2013-11-20 | 国立大学法人富山大学 | Homologous recombination method, cloning method, and kit |
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| CN102007212B (en) * | 2008-03-07 | 2013-11-20 | 国立大学法人富山大学 | Homologous recombination method, cloning method, and kit |
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| CN1162546C (en) | 2004-08-18 |
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