CN1285408A - Blue-green alga transgene by using gene integration platform system and method for expressing thymosin 'alpha'1 - Google Patents
Blue-green alga transgene by using gene integration platform system and method for expressing thymosin 'alpha'1 Download PDFInfo
- Publication number
- CN1285408A CN1285408A CN 00124700 CN00124700A CN1285408A CN 1285408 A CN1285408 A CN 1285408A CN 00124700 CN00124700 CN 00124700 CN 00124700 A CN00124700 A CN 00124700A CN 1285408 A CN1285408 A CN 1285408A
- Authority
- CN
- China
- Prior art keywords
- fragment
- gene
- primer
- plasmid
- blue
- 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
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a gene engineering, amplifying out cpc 2 promotor region and integration plateau homologous fragment L and R from cyanophycean Calothrix 7601 chromosome DNA, assembling donor plasmid PUTK containing the elemtns of cpc 2 promotor region, UBT alpha 1 target gene, rbc terminator, Km(1) reporter gene and genetic interation plateau homologous sequence, etc., constructing calothrix 7601 new genetic integration plateau system so as to obtain UBT alpha 1 transgenic algae strain, and the expression product of T alpha 1 gene can be detected from it. Said invention uses cyanophyte as new type gene engineering receptor and bio-reactor to make human T alpha 1 gene obtain high-effective expression in it. It can produce lots of T alpha1, and possesses extensive development vistas.
Description
The present invention relates to a kind of genetically engineered.
Blue-green algae (Cyanophyte) claims cyanobacteria (Cyanobacterium) again, is a kind of special microorganism.The genome of blue-green algae is simple, does not contain chloroplast(id) and Mitochondrial DNA except exposed chromosomal DNA, is convenient to carry out genetic analysis.In recent years, clone a large amount of blue-green algae genes, set up gene transfer system and the heterologous gene expression system of some blue-green algaes.Blue-green algae has been obtained many achievements in research as the recipient bacterium of expression alien gene.Tandeau de Marsac (Mo1.Gen.Genet 1987,209:296～298) to be carrier with pUC303 expressed the prokaryotic organism genus bacillus in unit cell algae Synechococcus sp.PCC7942 (be called for short Synechococcus7942, the rest may be inferred for all the other algae names) kills young mosquito toxin gene; Fukuda etc. (Biotech.Lett 1994,16:1～6) have expressed the ethylene synthetase gene of plant in Synechococcus 7942; Takeshima (Proc.Natl.Acad.Sci USA 1994,91:9685～9689) in Synechococcus 7942, expressed synthetic people superoxide dismutase gene (h-SOD), product accounts for 3% of total protein concentration, and biological activity in the good born of the same parents is arranged; (biotechnology progress, 1994,14 (6): 39～42) in Anabaena 7120, expressed the metallothionein gene (mt-1) of mouse such as Sun Jun.These research work show that the foreign gene that comprises eukaryotic gene can express in blue-green algae.
Thymus gland (Thymus) is maincenter organ important in the immunity system, is responsible for the differentiation and the maturation of T cell, plays an important role in anti-infective, antitumor and anti-autoimmune disorder.Isolated extrasin alpha from the thymosin mixture
1(T α
1), be one 20 octapeptide, it can epidemic prevention suppress the patient infection conditioned pathogen, improves the cell immunocompetent of body, can be used for treating multiple disease such as lupus erythematosus, hepatitis B, acquired immune deficiency syndrome (AIDS) etc., also can be used as the ancillary drug of some cancer of treatment.Present thymosin preparation has been widely used in clinical, but all is polycomponent Zadaxin of extracting from the thymus gland of pig, ox, easily sneaks into some albumen of pig, ox during extraction, may cause the people to produce anaphylaxis after the injection.Though the human thymosin curative effect that adopts the people embryo to extract is pretty good, people's embryonic origin difficulty is difficult to form scale production.But it is with Peptide synthesizer synthesizing thymosins α x, but too expensive.(Zhejiang Medical university journal, 1989,18 (3): such as Wang Zhenxi 106～109) T α
1Gene imports the PWR590 bacterial strain, detects T α with the SPA-ELISA improved method in the bacterial strain that transforms
1Expression; The aminoacid sequence synthetic that Wetzel etc. (Biochemistry1980,19:6096～6104) determine according to Low and Goldstein (1979) T α
1Gene is connected with plasmid pBR322 among the back importing E.coli and expresses, though obtain and natural T α
1Active identical expression product, but expression amount is low.
Purpose of the present invention aims to provide a kind of gene integration platform system that utilizes, with human thymosin alpha
1Positioning integration is in the karyomit(e) of filamentous cyanobacteria Calothrix 7601 after gene and ubiquitin (UB) gene fusion, and starts its expression with ruddiness inductive cpc2 promotor, produces extrasin alpha to utilize transgenic technology
1Method.
Concrete scheme performing step of the present invention is as follows:
The clone of one .cpc2 promoter region (P) and homologous dna fragment (L and R)
Design three couples of PCR primer P1 and P2 respectively according to the promoter region P of the cpc2 operon of filamentous cyanobacteria Calothrix 7601 and the nucleotide sequence at homologous fragment L and these three fragment two ends of R, L1 and L2, R1 and R2, its sequence is as follows:
P1 (5 ' end primer): 5 '-GCGTCGACGAATTGTAAGAA-3 '
SalⅠ????????(20Nts)
P2 (3 ' end primer): 5 '-ATGCATGCTCCAGCATCTCCTA-3 '
SphⅠ?????????(22Nts)
L1 (5 ' end primer): 5 '-CGGAATTCTGCTATTAGTCCGC-3 '
EcoRⅠ?????????(22Nts)
L2 (3 ' end primer): 5 '-CGGTCGACGCCCTCGGTTAAAGGTGT-3 '
SalⅠ??????????(26Nts)
R1 (5 ' end primer): 5 '-CGGTCGACTGTAGCTCCTTAATGTC-3 '
SalⅠ??????????(25Nts)
R2 (3 ' end primer): 5 '-CCTAAGCTTTCTTGATATTCGGCTG-3 '
HindⅢ?????????(25Nts)
Wherein promoter region left end primer P2 has designed a Sph I restriction enzyme site, and its sequence is GCATG ↓ C.
Chromosomal DNA with Calothrix 7601 is a template, and round pcr routinely amplifies fragment P (0.6Kb) respectively, fragment L (1.2Kb) and fragment R (1.2Kb).
By design shown in Figure 1,,,, use T then respectively with Hind III and Sal I while double digestion fragment R and carrier pUC19 with Sph I and Sal I while double digestion fragment P and carrier pUC19 according to the restriction enzyme site in the primer
4Dna ligase connects, and connector transformation receptor bacterium E.coli JM101 obtains white clone with blue white screening, has obtained recombinant plasmid pUCP and pUCR.Press in addition Fig. 1 and design, be connected, obtained recombinant plasmid pULP with behind EcoR I and Sal I double digestion fragment L and the above-mentioned recombinant plasmid pUCP.
Two. the clone of external source donor sheet segment DNA
1. reporter gene (Km
r) and the clone of terminator (rbcS polyA)
Selection contains reporter gene (kalamycin resistance gene-Km
r) and terminator (rbcS polyA) these two segmental plasmid pKYLX71, by design shown in Figure 2, with Xba I and Sal I double digestion pKYLX71, glue reclaims the dna fragmentation of 4.8Kb, be connected with pUCR through same double digestion, connector Transformed E .coli JM101 to contain the two anti-substratum screening of Ap and Km, obtains recombinant plasmid pUKR.
2. goal gene (UBT α
1) acquisition and clone
At first press T α
1The sequences Design of gene following PCR primer T1, T2 and T2 ':
T1 (5 ' end fragment): 5 '-GGGCATGCAAGGATCCGACGCAGCTGTT
SphⅠ????BamⅠ
GACACCAGCTCCGAAATCACCACCAAAGACTTAAAGG-3’(65Nts)
T2 (3 ' end fragment): 5 '-GGGTCGACTAGTTTTCTGCTTCT TCAAC
SpeⅠ
AACTTCTTTTTTTTCCTTTAAGTCTTTGGTGGTGATT-3’??(65Nts)
T2 ' (3 ' end primer): be 5 ' preceding 25 Nucleotide of holding of T2.
Pcr amplification obtains T α routinely
1Fragment.With plasmid pUCUB (with synthetic UB gene clone to constructed plasmid between the Sph I/BamH I site of pUC18 plasmid) is template, primer is U1 (universal primer M13/pUCReverse Sequencing Primer (48)) and U2 (universal primer M13/pUC Sequencing Primer (47)), and the PCR method amplification obtains the UB fragment routinely.Cut T α with BamH I enzyme
1Be connected after the UB fragment, be template with above-mentioned connector again, be that primer amplification obtains UBT α with U1 and T2 '
1Fragment.
By design shown in Figure 3, cut pUKR with Xba I and the incomplete enzyme of Hind III, reclaim the 6.0Kb fragment; With Sph I and Spe I double digestion goal gene UBT α
1With Sph I and Hind III double digestion pULP, above three kinds of endonuclease bamhis are mixed the back connect, transform, with Ap and the two anti-screenings of Km, obtain donor plasmid pUTK.
Three, donor plasmid pUTK electricity swashs conversion Calothrix7601
With EcoR I complete degestion pUTK, obtain wire plasmid pUTK, at voltage 0.4～2.0kv, resistance 100～200 Ω, electricity swashs conversion blue-green algae Calothrix 7601 under the condition of time 2.5～5.0ms, electric capacity 25 μ F, to with the Km screening, obtain to change UBT α through the conversion algae strain after electricity swashs processing through cultivating
1The algae strain of gene.
The strain of conversion algae all surpasses 40ug/ml to the resistance of Km, reaches as high as 80ug/ml, and still remains unchanged substantially after the switching several times.Prove goal gene UBT α through PCR
1Gene has been incorporated on the karyomit(e) of Calothrix 7601, can duplicate with THE REPLICATION OF CHROMOSOME; Confirm that through Western hybridization fusion rotein truly has expression.
The present invention goes out Phycocyanins, C-cpc2 promoter region, integration platform homologous fragment L and R by design pcr amplification from blue-green algae Calothrix 7601 chromosomal DNAs, has assembled to contain cpc2 promotor, UBT α
1The donor plasmid pUTK of elements such as goal gene, rbcS terminator, Kmr reporter gene and gene platform homologous sequence has set up the new gene integration platform system of Calothrix 7601, has obtained commentaries on classics UBT α
1The algae strain of gene, and from transform the algae strain, detect T α
1The expression of gene product.The present invention as novel transgene receptor and bio-reactor, makes people's extrasin alpha with blue-green algae
1Gene is expressed therein, for T α is changeed in the mass production exploitation
1Gene blue algae lays the foundation, and its technology is simple, and cost is low, has very big development prospect.
Fig. 1 is recombinant plasmid pUCP, the structure schema of pULP and pUCR.
Fig. 2 is the structure schema of recombinant plasmid pUKR.
Fig. 3 is the structure schema of donor plasmid pUTK.
Fig. 4 is integrated into Calothrix7601 karyomit(e) synoptic diagram for donor plasmid pUTK.
The invention will be further described by embodiment below.Clone's 1. each segmental pcr amplification of step 1, cpc2 promoter region (P) and homologous dna fragment (L and R)
1.1 the pcr amplification of cpc2 promoter region P (630bp)
At first, Sal I and Sph I on primer, have been designed according to the synthetic cpcB2A2 promoter region (P) of known array design primer
Restriction enzyme site.：
P1 (5 ' end primer): 5 '-GCGTCGACGAATTGTAAGAA-3 '
salⅠ????(20Nts)
P2 (3 ' end primer): 5 '-ATGCATGCTCCAGCATCTCCTA-3 '
SphⅠ??????????(22Nts)
In the aseptic centrifuge tube of 0.5ml, add successively: ddH
2O 37ul, 10 * Taq enzyme buffer liquid 5ul, 4 * dNTP (2.5 mmol/L) 4ul, P1 primer 1ul, P2 primer 1ul, template (blue-green algae genomic dna) 1ul, Taq enzyme (1u/ul) 1ul, cumulative volume 50ul adds paraffin oil 50ul at last in addition and covers.
The PCR reaction parameter is provided with: 94 ℃ of pre-sex change, 5min;
94 ℃ of sex change then, 45S; 50 ℃ of renaturation, 45S; 72 ℃ of extensions, 45S; 30 circulations
Last 72 ℃ of extensions, 5min.
After reaction finishes, get 5ul and carry out 1% agarose gel electrophoresis evaluation, a unique 0.6Kb fragment that meets the expection size on electrophoretogram, occurs, show that this amplified fragments is exactly a desired promoter region fragment.
1.2 the pcr amplification of homologous fragment L (1.2Kb)
Design cpc2 left side homologous region (L) primer equally, also designed Sal I and EcoR I site respectively:
L1 (5 ' end): 5 '-CGGAATTCTGCTATTAGTCCGC-3 '
EeoRⅠ???????????(22Nts)
L2 (3 ' end): 5 '-CGGTCGACGCCCTCGGTTAAAGGTGT-3 '
SalⅠ?????????????(26Nts)
Remove primer and change L1 into, L2, the extension time changes into outside the 75S, and all the other a unique 1.2Kb fragment that meets the expection size occurs all with 1.1 on electrophoretogram, show that this amplified fragments is exactly desired L fragment.
1.3 the pcr amplification of homologous fragment R (1.2Kb)
Design cpc2 right side homologous region (R) primer equally, also designed Sal I and Hind III site respectively:
R1 (5 ' end): 5 '-CGGTCGACTGTAGCTCCTTAATGTC-3 '
SalⅠ??????????(25Nts)
R2 (3 ' end): 5 '-CCTAAGCTTTCTTGATATTCGGCTG-3 '
HindⅢ????????(25Nts)
Remove primer and change R1 into, outside the R2, all the other are all with 1.2.A unique 1.2Kb fragment that meets the expection size on electrophoretogram, occurs, show that this amplified fragments is exactly desired R fragment.2. the clone 2.1 of each fragment PCR products reclaims dna fragmentation from sepharose
The PCR product under ultraviolet lamp, downcuts and contains the segmental gel of required DNA size behind agarose gel electrophoresis, reclaims test kit with glue and reclaims dna fragmentation, and-20 ℃ of preservations are standby.2.2 the connection of PCR product and conversion
(1) according to the restriction enzyme site of PCR fragment two ends designs, carry out the segmental double digestion of PCR, with same double digestion
Carrier (as the pUC19 plasmid) mixes, and adds T
412 ℃ of connections of dna ligase are spent the night.
Wherein press Fig. 1 design, according to the restriction enzyme site in the primer, with Sph I and Sal I while double digestion fragment P
With carrier pUCl9,, use T then respectively with Hind III and Sal I while double digestion fragment R and carrier pUC19
4
Dna ligase connects, connector transformation receptor bacterium E.coli JM101.Press in addition Fig. 1 design, with the EcoR I with
Sal I double digestion fragment L has the segmental plasmid of P to be connected with above-mentioned reorganization, transforms.
(2) (adopt CaCl at the 200ul competent cell
2The method preparation) adds the DNA that 5ul connects, mixing, ice bath 30min in.
(3) 42 ℃ of water-bath 2min, ice bath 2min adds 800ul LB liquid nutrient medium, 37 ℃ of jog 1hr again.
(4) get 200ul and place another pipe, add 4ul IPTG (200mg/ml) and 40ul X-gal (20mg/ml), mix
Even back coating LB flat board (containing Ap 100ug/ml) is cultivated about 10hr for 37 ℃.
2.3 the screening of recon and evaluation
(1) by blue white screening, tentatively chooses some white colonies, be inoculated in the training of 4ml LB (containing Ap 100 ug/ml) liquid
Support in the base, 37 ℃ of shaking culture are spent the night.
(2) extract in a small amount plasmid and carry out enzyme and cut evaluation, the result obtained respectively expection recombinant plasmid pUCP, pUCR and
pULP。PUCP can be become the 3.3Kb fragment by Sal I single endonuclease digestion, is become 2.7Kb and 0.6Kb by the Sph I with Sal I double digestion
Two fragments.PUCR can be become by Sal I or Pst I (the Pst I has only single restriction enzyme site in fragment R) single endonuclease digestion
3.9Kb fragment is become 2.7Kb and two fragments of 1.2Kb by the Hind III with Sal I double digestion.PULP can be by the Sal I
Single endonuclease digestion becomes the 3.9Kb fragment, is become 2.7Kb and two fragments of 1.2Kb with Sal I double digestion by the EcoR I.Distinguish again
With recombinant plasmid pUCP, pUCR, pULP is template amplification fragment P, R and L, and the result has also obtained in advance
The fragment of phase length scale.Show from above experimental result and to contain cpc2 gene promoter district (P) and homologous dna
Clone's of fragment L and R successfully obtains.The clone 1. reporter gene (kantlex-Km of step 2, external source donor sheet segment DNA
r) and the clone of terminator (rbcS polyA)
Plasmid pKYLX71 contains this two fragments.Press Fig. 2 design, with Xba I and Sal I double digestion pKYLX71, glue reclaims the dna fragmentation of 4.8Kb, mixes with the pUCR of the same double digestion of warp, adds T
412 ℃ of connections of dna ligase are spent the night, and connector Transformed E .coli JM101 to contain the two anti-substratum screening of Ap and Km, obtains some clone's.Extract its plasmid respectively and carry out enzyme and cut evaluation, the result has obtained the recombinant plasmid pUKR of the 8.7kb of expection.It can be become the 8.7kb fragment by Sal I single endonuclease digestion, is cut into 6.3Kb and 2.4Kb fragment by the Hind III, is cut into 5.3Kb and 3.4Kb fragment by the Xba I.2. goal gene (UBT α
1) acquisition and clone
2.1 extrasin alpha
1(T α
1) pcr amplification of gene (110bp):
At first press T α
1The sequences Design of gene PCR primer T1, T2 and T2 ':
T1 (5 ' end fragment): 5 '-GGGCATGCAAGGATCCGACGCAGCTGTT
SphⅠ?BamHⅠ
GACACCAGCTCCGAAATCACCACCAAAGACTTAAAGG-3’(65Nts)
T2 (3 ' end fragment): 5 '-GGGTCGACTAGTTTTCTGCTTCTTCAAC
SpeⅠ
AACTTCTTTTTTTTCCTTTAAGTCTTTGGTGGTGATT-3’(65Nts)
T2 ' (3 ' end primer): be 5 ' preceding 25 Nucleotide of holding of T2.
Get T1 and each 2ul of T2 primer, add ddH
2O to 10ul adds paraffin oil 30ul again; Rise to 85 ℃ earlier, 5min
Speed with 1 ℃/lmin is cooled to 25 ℃ then; Add successively again: ddH
2O 30ul, 10 * Taq enzyme buffer liquid 5ul
4 * dNTP (2.5mmol/L) 4ul, Taq enzyme (1u/ul) 1ul
PCR reaction parameter: 94 ℃ of pre-sex change 5min of elder generation; Follow 94 ℃ of sex change 30S; 60 ℃ of renaturation are held concurrently and are extended 20S:
10 circulations; Last 60 ℃ of extension 20S again.
After reaction finishes, get 3ul and carry out the evaluation of 2% agarose gel electrophoresis, obtain the T α of 110bp
1Fragment.2.2 the pcr amplification of UB fragment (345bp):
Removing template changes plasmid pUCUB (by the synthetic UB gene of Dalian Bao Bio-Engineering Company and be cloned into constructed plasmid between the Sph I/BamH I site of pUC18 plasmid) into, primer changes into outside U1 (universal primer M13/pUCReverse Sequencing Primer (48)) and the U2 (universal primer M13/pUC Sequencing Primer (47)), and the remaining reaction component is with 2.1;
PCR reaction parameter: 94 ℃ of pre-sex change 5min of elder generation;
94 ℃ of sex change 30S; 50 ℃ renaturation 30S:72 ℃ is extended 30S; 30 circulations
Last 72 ℃ of extension 3min again.
After reaction finishes, get 5ul and carry out the evaluation of 2% agarose gel electrophoresis, obtain the UB fragment of 345bp.2.3 UBT α
1The PCR and the clone of fragment (388bp):
Cut T α with BamH I enzyme
1Be connected after the UB fragment, be template with above-mentioned connector again, be that primer amplification obtains UBT α with U1 and T2 '
1Fragment.
UBT α
1Segmental PCR reaction, removing template changes UB and T α into
1Connector, primer changes into outside U1 and the T2 ', remaining reaction component and reaction parameter are with 2.2.
According to Fig. 3 design, cut pUKR with Xba I and the incomplete enzyme of Hind III, reclaim the 6.0Kb fragment, wherein comprise terminator, Km screening reporter gene and integrate homologous fragment R: with Sph I and Spe I double digestion goal gene UB-T α
1With Sph I and Hind III double digestion pULP.Above three kinds of endonuclease bamhis are mixed the back connect, conversion, with Ap and the two anti-screenings of Km, the some transformants of picking extract plasmid and carry out the restriction analysis evaluation, have successfully obtained the donor plasmid pUTK of expection.It can be cut into the 10.8Kb fragment by Xba I enzyme, is cut into 8.3Kb and 2.5Kb fragment by Hind III enzyme, is cut into 5.7Kb and 5.1Kb fragment by Sal I enzyme.Step 3, donor plasmid pUTK electricity swash conversion Calothrix7601
Transform Calothrix 7601 with wire pUTK through EcoR I (plasmid pUTK goes up this enzyme site and is positioned at homology segment L and pUC19 segment junction) ' complete degestion gained, if two contrasts, contrast 1 is blank, promptly do not contain the conversion of plasmid, contrast 2 is conversions of the plasmid pKYLX71 of apparatus Kmr gene.
At first cultivate blue-green algae to A
550About=0.6.Get the centrifugal 3ml of being condensed into of 100ml algae liquid, supersound process 30s (power 3).Centrifugal collection frustule, twice of 1.0 mmol/L HEPES (pH7.2) damping fluid centrifuge washing of usefulness precooling.Frustule is suspended in 1.0mmol/L HEPES (pH7.2) damping fluid of precooling.Add DNA to final concentration be 10ug/ml.Get the frustule suspension that 40ul contains DNA, the aseptic disposable electricity that joins precooling swashs in the cup (specification 1mm).Preset each parameter (referring to table 1) of electric exciter, load onto electricity and swash cup, electricity swashs.
Table 1 electricity swashs parameter (positive and negative control only uses two kinds of conditions of C, H)
| Sample number into spectrum | A | ?B | ?C | ?D | ?E | ?F | ?G | ?H | ????I | ????J |
| Voltage (kv) | ????0.4 | ????0.8 | ????1.2 | ????1.6 | ????2.0 | ????0.4 | ????0.8 | ????1.2 | ????1.6 | ????2.0 |
| Resistance (Ω) | ????100 | ????200 | ||||||||
| Time (ms) | ????2.5 | ????5.0 |
| Electric capacity (uF) | ????25 | |
The frustule suspension that electricity is swashed after handling changes cultivation in the 20ml BG.11 liquid nutrient medium (containing Km 20 ug/ml) immediately over to, replenishes 4ug/ml Km every day, to replenish the Km that natural decomposition is fallen in the culturing process.Often shake, cultivated 10 days.Choose well-grown three bottles of algae liquid, each draws 100ul algae liquid, is coated on the BG-11 flat board (to contain 1.5% agar, Km20 ug/ml), cultivates 10 days.Choose 5 best clone's of growth, change in the 20ml BG-11 liquid nutrient medium (containing Km20ug/ml), often shake, cultivated 10 days.From clone algae strain nutrient solution, respectively draw 1ml algae liquid, join respectively in the 20ml BG-11 liquid nutrient medium that contains different concns Km (being respectively 5,10,20,30,40,50,60,70,80 ug/ml), often shake, cultivated lO days.According to upgrowth situation, judge the maximum concentration of respectively cloning the anti-Km of algae strain.Choose the highest clone's of resistance and carry out relatively large cultivation (200ml).Transferring once every 10 days, the guarantor plants.
Choosing is cloned in the sub Km resistance experiment in screening of cloning son and mensuration, and respectively the sub upgrowth situation under different K m concentration of clone differs, and the result shows that five maximum concentrations of cloning the anti-Km of algae strain that supply examination can reach 80ug/ml, and minimum concentration also has 40ug/ml; And the concentration of the anti-Km of control group algae strain is lower than 5ug/ml, and is consistent with the basic resistance of Calothrix 7601.Experimental result shows to have only the blue-green algae that transforms with pUTK clone's of anti-Km to occur, and clone's does not all appear in two contrasts in addition.Though pKYLX71 contains Km
rGene, but do not possess the initial replicon of blue-green algae plasmid, so can not in blue-green algae, duplicate.Although pUTK does not have the initial replicon of blue-green algae plasmid yet, clone's of anti-Km has appearred, and this shows and contains Km
rThe donor dna fragment of gene has been incorporated on the karyomit(e) of Calothrix 7601, duplicates with THE REPLICATION OF CHROMOSOME, and gives expression to the Km resistance.
For the whether success of the transgenosis of verifying blue-green algae, we carry out the PCR evaluation to transforming the algae strain, extract the chromosomal DNA of wild algae strain and conversion algae strain 2 respectively, with it is template, with P1 and T2 ' is primer amplification, and the former takes out of now at nothing as a result, and single band appears in the latter, the about 0.9Kb of its length, this result meet cpc2 promotor (P) and foreign gene to be expressed (UBT α
1) the length sum, tentatively prove P-UBT α
1Gene has been incorporated on the karyomit(e) of Calothrix7601.
For further proving the UBT α that is integrated
1, gene energy effective expression adopts Western hybridization to identify that first antibody adopts T α
1Antibody.Detection of expression at first will be induced blue-green algae Calothrix 7601, when Calothrix 7601 cultivates A
550During=0.8 left and right sides, get 10ml and in ruddiness, handle 30min.Then want sample preparation and electrophoresis detection, the centrifuging and taking frond places a pipe of weighing in advance, and it is heavy to weigh up algae, adds the sample preparation damping fluid of 1 times of amount (V/W) and the sample-loading buffer of 1 times of amount (V/W), and boiling water bath 5min is centrifugal, gets supernatant liquor, and it is standby to put-20 ℃ of preservations.The expression amount of test sample can carry out with SDS-PAGE (SDS-polyacrylamide gel electrophoresis) method: (1) glue: 15% separation gel, 5% concentrates glue (2) electrophoresis: will add to behind the sample boiling water bath 3min in the gel point sample hole, earlier with 50V voltage electrophoresis, treat that bromjophenol blue runs out of
After concentrating glue, voltage is increased to 100V, when bromjophenol blue runs to the gel edge, stop electrophoresis.
Electrotransfer (seal stain): take out glue earlier, being soaked in electricity changes in the damping fluid.The pvdf membrane that clip and gel etc. are big, earlier wetting in anhydrous methanol, change in the damping fluid at electricity again and soak; Other cut six with big filter paper such as film, being dipped in electricity changes in the damping fluid; Sponge in the electroporation also is dipped in electricity to be changeed in the damping fluid.Sponge, filter paper (3), gel, film, filter paper (3), sponge are put well in order, note avoiding bubble, boundary alignment is fixed with sieve tray then, inserts in the electrotransfer groove, and gel is at cathode direction, and film is in anode direction.Adding electricity changes damping fluid, and 4 ℃, 100V shifts 1hr.
Immunology detection: place the TNT damping fluid to wash 3 times film earlier, each 5min.Change in the confining liquid 37 ℃ of jog 30min over to.Put in the TNT damping fluid rinsing 3 times, each 5min.Add the anti-UB antibody of rabbit, 37 ℃ of jog 1hr with the dilution of TNT damping fluid.Put in the TNT damping fluid rinsing 3 times, each 5min.Add goat anti-rabbit igg-HRP, 37 ℃ of jog 1hr with the dilution of TNT damping fluid.Put in the TNT damping fluid rinsing 4 times, each 5min.Add tmb substrate liquid, colour developing 15min, it is positive blue band person to occur, and water washes with termination reaction, and taking-up is dried, and takes pictures.
Shift as stated above, hybridize and develop the color, the result shows that transforming the algae strain has occurred and positive control (T α
1) the much the same band in position, illustrate that transforming the algae strain truly has and give expression to T α
1Albumen.
Claims (3)
1. the utilization gene integration platform system carries out blue-green alga transgene and expresses extrasin alpha
1Method, it is characterized in that going out Phycocyanins, C-cpc2 promoter region, integration platform homologous fragment L and R by design pcr amplification from blue-green algae Calothrix 7601 chromosomal DNAs, assembled and contained cpc2 promotor, UBT α
1Goal gene, rbcS terminator, Km
rThe donor plasmid pUTK of elements such as reporter gene and gene platform homologous sequence has set up the new gene integration platform system of Calothrix 7601, has obtained commentaries on classics UBT α
1The algae strain of gene, its concrete grammar step is as follows:
(1) clone of cpc2 promoter region (P) and homologous dna fragment (L and R)
Design three couples of PCR primer P1 and P2 respectively according to the promoter region P of the cpc2 operon of filamentous cyanobacteria Calothrix 7601 and the nucleotide sequence at homologous fragment L and these three fragment two ends of R, L1 and L2, R1 and R2, its sequence is as follows:
P1 (5 ' end primer): 5 '-GCGTCGACGAATTGTAAGAA-3 '
SalⅠ
P2 (3 ' end primer): 5 '-ATGCATGCTCCAGCATCTCCTA-3 '
SphⅠ
L1 (5 ' end primer): 5 '-CGGAATTCTGCTATTAGTCCGC-3 '
EcoRⅠ
L2 (3 ' end primer): 5 '-CGGTCGACGCCCTCGGTTAAAGGTGT-3 '
SalⅠ
R1 (5 ' end): 5 '-CGGTCGACTGTAGCTCCTTAATGTC-3 '
SalⅠ
R2 (3 ' end): 5 '-CCTAAGCTTTCTTGATATTCGGCTG-3 '
HindⅢ
Wherein promoter region left end primer P2 has designed a Sph I restriction enzyme site, and its sequence is GCATG ↓ C, is template with the chromosomal DNA of Calothrix7601, and round pcr routinely amplifies 0.6kb fragment P respectively, 1.2kb fragment L and 1.2kb fragment R; According to the restriction enzyme site in the primer,, use T then with Sph I and Sal I while double digestion fragment P and carrier pUC19
4Dna ligase connects, and connector transformation receptor bacterium E.coli JM101 obtains white clone with blue screening in vain, gets recombinant plasmid pUCP; With Hind III and Sal I while double digestion fragment R and carrier pUC19, use T then
4Dna ligase connects, and connector transformation receptor bacterium E.coli JM101 obtains white clone with blue screening in vain, gets recombinant plasmid pUCR; Behind EcoR I and Sal I double digestion fragment L and recombinant plasmid pUCP, use T
4Dna ligase connects, and connector transformation receptor bacterium E.coli JM101 obtains white clone with blue screening in vain, gets recombinant plasmid pULP;
(2) clone of external source donor sheet segment DNA:
A. kantlex-Km
rThe clone of reporter gene and rbcS polyA terminator:
Selection contains reporter gene kantlex-Km
rWith two segmental plasmid pKYLX71 of terminator rbcS polyA, with Xba I and Sal I double digestion pKYLX71, glue reclaims the dna fragmentation of 4.8Kb, be connected with recombinant plasmid pUCR through same double digestion, connector Transformed E .coli JM101, to contain the two anti-substratum screening of Ap and Km, obtained the recombinant plasmid pUKR of 8.7kb;
B. goal gene UBT α
1Acquisition and clone:
Press T α 1 gene sequences Design following PCR primer T1, T2 and T2 ':
T1 (5 ' end fragment): 5 '-GGGCATGCAAGGATCCGACGCAGCTGTT
SphⅠ????BarnHⅠ
GACACCAGCTCCGAAATCACCACCAAAGACTTAAAGG-3(65Nts)
T2 (3 ' end fragment): 5 '-GGGTCGACTAGTTTTCTGCTTCTTCAAC
SpeⅠ
AACTTCTTTTTTTTCCTTTAAGTCTTTGGTGGTGATT-3’(65Nts)
T2 ' (3 ' end primer): be 5 ' preceding 25 Nucleotide of holding of T2
With T1 and T2 is primer, and pcr amplification gets T α routinely
1Fragment is a template with plasmid pUCUB, and primer is U1 and U2, and pcr amplification goes out the UB fragment, cuts T α with BamH I enzyme
1Be connected after the UB fragment, be template with this connector again, be that primer amplification obtains UBT α with U1 and T2 '
1Fragment.Cut pUKR with Xba I and the incomplete enzyme of Hind III, reclaim the 6.0Kb fragment, with Sph I and Spe I double digestion goal gene UBT α
1With Sph I and Hind III double digestion pULP, above three kinds of endonuclease bamhis are mixed the back connect, transform, with Ap and the two anti-screenings of Km, obtained donor plasmid pUTK;
(3) donor plasmid pUTK electricity swashs conversion Calothrix 7601
With EcoR I complete degestion pUTK, obtain wire plasmid pUTK, at voltage 0.4～2.0kv, resistance 100～200 Ω, electricity swashs conversion blue-green algae Calothrix 7601 under the condition of time 2.5～5.0ms, electric capacity 25 μ F, to with the Km screening, obtain to change UBT α through the conversion algae strain after electricity swashs processing through cultivating
1The algae strain of gene.
2. carry out blue-green alga transgene and express extrasin alpha as claim 1 described utilization gene integration platform system
1Method, it is characterized in that said plasmid pUCUB for the UB gene clone to constructed plasmid between the Sph I/BamH I site of pUC18 plasmid.
3. carry out blue-green alga transgene and express extrasin alpha as claim 1 described utilization gene integration platform system
1Method, it is characterized in that said primer U1 is universal primer M13/pUC Reverse Sequencing Primer (48), primer U2 is universal primer M13/pUC Sequencing Primer (47).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00124700 CN1116416C (en) | 2000-09-29 | 2000-09-29 | Blue-green alga transgene by using gene integration platform system and method for expressing thymosin 'alpha'1 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00124700 CN1116416C (en) | 2000-09-29 | 2000-09-29 | Blue-green alga transgene by using gene integration platform system and method for expressing thymosin 'alpha'1 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1285408A true CN1285408A (en) | 2001-02-28 |
| CN1116416C CN1116416C (en) | 2003-07-30 |
Family
ID=4590602
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 00124700 Expired - Fee Related CN1116416C (en) | 2000-09-29 | 2000-09-29 | Blue-green alga transgene by using gene integration platform system and method for expressing thymosin 'alpha'1 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1116416C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100430475C (en) * | 2002-02-28 | 2008-11-05 | 宝生物工程株式会社 | Alga-origin promoter, intron and terminator |
| CN108640983A (en) * | 2018-05-17 | 2018-10-12 | 中国科学院微生物研究所 | The application of FvCPC2 albumen and its encoding gene in the growth of regulation and control multiple eating bacterium mycelia and fruit body development |
| CN108977371A (en) * | 2017-05-31 | 2018-12-11 | 中国科学院青岛生物能源与过程研究所 | It can be used for cyanobacteria strains and its application of glycosylglycerol production |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1311115C (en) * | 2002-05-10 | 2007-04-18 | 王新文 | Knitting process of soft shrinkage-preventing knitted pure hemp fabric |
-
2000
- 2000-09-29 CN CN 00124700 patent/CN1116416C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100430475C (en) * | 2002-02-28 | 2008-11-05 | 宝生物工程株式会社 | Alga-origin promoter, intron and terminator |
| CN108977371A (en) * | 2017-05-31 | 2018-12-11 | 中国科学院青岛生物能源与过程研究所 | It can be used for cyanobacteria strains and its application of glycosylglycerol production |
| CN108977371B (en) * | 2017-05-31 | 2021-05-25 | 中国科学院青岛生物能源与过程研究所 | Cyanobacteria strain capable of being used for production of glycerol glucoside and application thereof |
| CN108640983A (en) * | 2018-05-17 | 2018-10-12 | 中国科学院微生物研究所 | The application of FvCPC2 albumen and its encoding gene in the growth of regulation and control multiple eating bacterium mycelia and fruit body development |
| CN108640983B (en) * | 2018-05-17 | 2021-03-30 | 中国科学院微生物研究所 | FvCPC2 protein and application of encoding gene thereof in regulating growth of various edible fungus hyphae and fruiting body development |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1116416C (en) | 2003-07-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101063104A (en) | Engineering bacterium producing 5-glycyl ethylformic acid and construction method thereof | |
| CN1057122C (en) | A recombinant 9 desaturase and a gene encoding the same | |
| CN1116416C (en) | Blue-green alga transgene by using gene integration platform system and method for expressing thymosin 'alpha'1 | |
| CN1080306C (en) | Adjusting and controlling factor for expression of nitrile solution deenzyme and gene thereof | |
| CN1693466A (en) | Engineering bacteria for producing 5-amino acetyl propionic acid and its constructing method | |
| CN1766098A (en) | A kind of mannase and encoding gene thereof and application | |
| CN1188523C (en) | Blue-green alga shuttle plasmid expression vector and method for expressing thymison 'alpha' 1 | |
| CN1216904C (en) | Soybean ethylene response protein transcription factor and its coded gene and application | |
| CN1746304A (en) | The structure of secreting the ammonium engineering bacteria and the application of the sudden change of fixed nitrogen negative regulator gene | |
| CN1908011A (en) | Plant inverse-resistant zinc finger protein, coding gene and application thereof | |
| CN1308452C (en) | Pichia yeast phytase expression system containing reorganized ethyl alcohol oxidase promotor | |
| CN1810971A (en) | Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application | |
| CN1974772A (en) | Sea island cotton receptor analogous protein kinase gene and its application | |
| CN1772908A (en) | Glyphosate acetyl transferase gene and its application | |
| CN101037692A (en) | Method for expressing human insulin by using plant seed oil body | |
| CN1262657C (en) | Method for producing human leukemia inhibitory factor using transgenic plant | |
| CN1485431A (en) | Gene engineering yeast strain of expressing beta-1,3-1,4-glucanase | |
| CN1740320A (en) | Fenneropenaeus chinensis Anti-LPS factor gene and cloning process thereof | |
| CN101067138A (en) | Gene engineering process of preparing human hair papilla cell protein HSPC016 and its expression vector and engineering bacterium | |
| CN1285407A (en) | Heat shock induction exogenous gene high-efficient expressed blue-green alga transgenic expression system and the method for expressing thymosin 'alpha'1 | |
| CN1258595C (en) | Phloem specific expressing promoter | |
| CN1166775C (en) | Application of transgene paddy rice endosperm in genetics demonstration experiment | |
| CN1059236C (en) | High-efficiency conversion system of kluyveromyces fragilis yeast | |
| CN1220777C (en) | Nucleic acids having promoter activity and transgenic plants containing the same | |
| CN1974592A (en) | Specific molecular marker of wild emmer alpha-amylase inhabiting factor and its utilization |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: XIAMEN XIADA KESHENG GENE ENGINEERING CO., LTD. Free format text: FORMER OWNER: XIAMEN UNIVERSITY Effective date: 20040709 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20040709 Address after: 361005 Fujian city of Xiamen province Xiamen University a Yingxue room No. 111 Patentee after: Xiamen Xiamen Kesheng Gene Engineering Co Ltd Address before: Xiamen City, Fujian Province, 361005 South Siming Road No. 422 Patentee before: Xiamen University |
|
| C56 | Change in the name or address of the patentee | ||
| CP03 | Change of name, title or address |
Address after: 361005 Fujian province Xiamen City Siming District No. two Xiamen University Jiageng building room 609 Patentee after: Xiamen Branch Sheng Gene Engineering Co., Ltd. Address before: 361005 Fujian city of Xiamen province Xiamen University a Yingxue room No. 111 Patentee before: Xiamen Xiamen Kesheng Gene Engineering Co Ltd |
|
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20030730 Termination date: 20120929 |