CN1201002C - Broad-spectrum plasmid stabilizing factor PSF-Cxc and separation method and use thereof - Google Patents
Broad-spectrum plasmid stabilizing factor PSF-Cxc and separation method and use thereof Download PDFInfo
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- CN1201002C CN1201002C CNB011383232A CN01138323A CN1201002C CN 1201002 C CN1201002 C CN 1201002C CN B011383232 A CNB011383232 A CN B011383232A CN 01138323 A CN01138323 A CN 01138323A CN 1201002 C CN1201002 C CN 1201002C
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Abstract
The present invention discloses a broad-spectrum plasmid stabilizing factor PSF-Cxc, and a separating method and applications thereof, which relates to gene engineering, more specifically, a plasmid stabilizing technology. The broad-spectrum plasmid stabilizing factor PSF-Cxc (E. coli JM109/pCXC118, CCTCC NO: M201043) provided by the present invention is coded by a section of DNA whose length is 773-bp, the gene contains two open reading frames (ORF), and coded proteins respectively have 145 amino acids and 125 amino acids. The gene of the plasmid stabilizing factor can be cloned to the polycloning sites or other sites of any target plasmid by an enzyme-cutting connection method or an internal recombination method to obtain a new plasmid with high stability. The plasmid stabilizing factor has a strong stabilizing effect on different plasmids in different hosts, and can be extensively applied to the aspects of scientific research, industry, agricultural production, and the like relative to gene engineering. Particularly, the plasmid stabilizing factor is capable of obviously improving the yield of gene engineering products and reducing antibiotic pollution, and has an immeasurable economic value.
Description
Technical field
The present invention relates to genetically engineered; Specifically, the stabilization technique that relates to plasmid.
Background technology
No matter in engineered research field still in industry and agricultural application field, the stability of plasmid all is very important gordian technique.By a large amount of deep researchs, people have had comprehensive understanding to plasmid stable factor and the widespread use thereof of Gram-negative bacteria intestinal bacteria (E.coli) at present.But people but know little about it to the plasmid and the stable factor thereof of gram-positive microorganism.Because the E.coli plasmid as engineering carrier can not be bred effectively, the fundamental research and the applied research of relevant gram-positive microorganism have therefore been limited in gram-positive microorganism.Clavibacter Xyli subsp.cynoaontis (Cxc) is a kind of gram-positive microorganism, can extensively parasitize plant and does not cause tangible plant illness.Portion C xc bacterial strain contains natural plasmid (pCXC100), and we find that pCXC100 can heredity in the Cxc bacterium stably under without any the condition of selecting.We have isolated the broad spectrum plasmid stable factor (PlasmidStabilization Function from Cxc is called for short PSF-Cxc) of the about 0.8Kb of a segment length from pCXC100, and the stabilising effect of this stable factor in other plasmid tested.We find that PSF-Cxc can stablize the plasmid of different sources, and in Cxc and E.coli the same stable effect are arranged.PSF-Cxc can stablize multiple plasmid in Cxc and E.coli, have broad application prospects.Up to the present, we find no any similar bibliographical information or patent report, also find any and its homologous sequence in gene database (GenBank).
Summary of the invention
The objective of the invention is: a kind of broad spectrum plasmid stable factor (PSF-Cxc) is provided; The method of a kind of PSF-Cxc of separation is provided; A kind of method that PSF-Cxc is applied to scientific research, industrial and agricultural production aspect is provided.
Technical scheme of the present invention is:
The method of 1, separating PSF-Cxc:
PCXC100 is cut into the dna fragmentation that can cover whole pCXC100 total length of 10-24.5Kb by restriction enzyme HindIII, EcoR I, Bgl II, Nco I, Nde I, these fragment clonings are obtained plasmid pCXC100 to the corresponding restriction enzyme site on the E.coli plasmid pBR325, pCXC102, pCXC103, pCXC104 and pCXC105.By Detection of Stability, find to have only pCXC101 and pCXC105 energy 100% stably in Cxc and E.coli, to go down to posterity.So determine PSF-Cxc be present in CXC100 that pCXC101 comprises from NcoI in the HindIIIDNA fragment.(Figure 1A)
The contained insertion fragment from Nco I to HindIIIDNA of pCXC101 is carried out enzyme with restriction enzyme HindIII, BglII, Sma I, Xho I and Ava I cuts, and resulting dna fragmentation is cloned into again the corresponding restriction enzyme site of pBR325, obtain plasmid pCXC106, pCXC107, pCXC108, pCXC109 and pCXC110.Detection of Stability finds that have only pCXC106 and pCXC108 can be stabilized in Cxc and the E.coli goes down to posterity, and shows that PSF-Cxc is present in the NcoI-SmaI dna fragmentation that pCXC106 and the common about 5.5Kb of pCXC108 grow.(Figure 1A)
With NcoI digested plasmid pCXC106, the two ends of clearing up this shape material grain with DNase I under the situation that mn ion exists obtain different dna fragmentations by the control digestion time then.The DNA that cleared up carries out the BglII enzyme and cuts, and the endonuclease bamhi of isolated different lengths with its EcoRV and BamHI restriction enzyme site that is cloned into E.coli plasmid pBCSK (+), obtains cloning pCXC111, pCXC112, pCXC113 and pCXC114.Because the dna fragmentation after clearing up does not contain complete Cxc replicon, thereby above clone can not duplicate in Cxc, can only be transformed into and carry out Detection of Stability in the E.coli cell.Detection shows that pCXC113 contains the essential dna fragmentation of PSF-Cxc.(Figure 1B)
Cut pCXC113 with HindIII and KpnI enzyme, handle with the test kit of clearing up of Exonuclease III and S1 nuclease combination then.By controlling the pCXC113 that digestion time has obtained losing the different lengths dna fragmentation, obtain a series of novel plasmids after connecting certainly, pCXC116 and pCXC117 are wherein two.Detection of Stability shows that these two kinds of plasmids all do not contain the PSF-Cxc of function.(Figure 1B)
Use XbaI enzyme cutting pCXC113, fill and lead up sticking terminal with the Klenow enzyme to prevent that Exonuclease III from clearing up plasmid from HindIII one end.Cut the plasmid of this linearize again with the EcoRI enzyme, handle with the test kit of clearing up of Exonuclease III and S1 nuclease combination.By controlling the pCXC113 that digestion time has obtained losing the different lengths dna fragmentation, obtain a series of novel plasmids after connecting certainly, pCXC119 and pCXC120 are wherein two.Detection of Stability shows that pCXC119 can 100% be stabilized in the E.coli cell and goes down to posterity, and pCXC120 can not.So pCXC119 contains the PSF-Cxc of function.(Figure 1B)
Cut pCXC113 with HindIII and EcoRI enzyme, the fragment that obtains is connected to the corresponding restriction enzyme site of pBCSK (+), obtains pCXC118." pCXC118 ", its classification called after E.coli JM109/pCXC118; Its preservation date is December 13 calendar year 2001; Its depositary institution is a Chinese typical culture collection center China. Wuhan. and Wuhan University's postcode 430072; Its deposit number is CCTCC NO:M 201043.
Cut the corresponding DNA fragments on the insertion fragment of pCXC119 with the BamHI enzyme, from connecting, obtain pCXC121 then.Detection of Stability shows that pCXC118 can 100% be stabilized in the E.coli cell and goes down to posterity, and pCXC121 can not.So pCXC118 contains the PSF-Cxc of function.(Figure 1B)
Therefore, we know that pCXC118 and pCXC119 all contain the plasmid stable factor (PSF-Cxc) that derives from pCXC100.Because pCXC118 contains proper restriction site, so be used to make up order-checking subclone and further stability analysis.
2, sequencing analysis PSF-Cxc
The dna fragmentation (773-bp) that plasmid pCXC118 is inserted is cut and is cloned through further enzyme, and respectively to each subclone order-checking, obtains following dna sequence dna:
1
GGATCCCCCG GGCTGCAGGA ATTCGTTGAT TTCCGCGATC AGGCCAATGA
51 GGTCTCTGCT TGGATCTGCC CAGTAGTGAT GAGTCGAGCG AGCGCGATCG
101 TTGATCCCGT CGAAGTGTTG GTTCAGGCGC TTGTGTAGTA CCGAGAAGGC
151 ATGCCCGAAC TCTGCGTCAT CTTCGTAGAG GCGGTCGAAG GTCGGCGATG
201 AGCCTGCTCG GCGTACTGCG CCTTCATGTC CGCGACCGCG AGCTCTAGCG
251 GGCTAGCCAC AGCACTCCCC CGTTCACGAC CCGTACCTCC GGTCAGCTTC
301 CCAGTGGGCG CCCGCGAGTA ACGCCTCGCT CAGCATTCTC GAATCGTCGT
351 CCTTCGTTGT ACCGCTGCTC GAGGCGTTGC ACCTCCGCGT CCAGCGCCGC
401 GGCGATGAAG CCCGAGAACG TCCGATGACC CTCCTGCAGA TGGGTGAGCG
451 TGTACCCGTC CTCAGCCGTG CTCGGCGCTC CTCCCCCACC GTGACAGTCA
501 TCTTGACCTC AGAACGCCGC CCTCGGGGCT CCGAGGATCC TCCCGGCACC
551 GGCACCGGGA CCGTGGGCGC GACACTGTCC GCCACGGACG GCGACTCCGG
601 CACGAAAGCC GGCGCCACCA TCGACGGTGC TGCGGAGGCT TCGCGCTGCG
651 ACCGGATCAG GGATCCCGCA CCGGGACGCG ACGGCGGCGG TGCAACCGTG
701 CGATCAGCCA TTCGTGACCA CGCTCATTTC GGACTCCTTT GCCTGGATAC
751 GTGCGACGAG CTCCGCGGAG ATCGCCTGGA GGTCGTCAGC
GATCAAGCTT。
The line part is an additional restriction enzyme site on the carrier.
This segment DNA sequence has two open reading frame (ORF), and the reading direction of an ORF (#1) is HindIII → SphI, can encoded protein be 145 amino acid; The reading direction of another ORF (#2) is XhoI → HindIII, can encoded protein be 125 amino acid.The long dna fragmentation of this 773-bp not only contains the dna sequence dna of coding plasmid stable factor (PSF-Cxc), also contains to start the required dna sequence dna of plasmid stable factor expression, shows that this stable factor is a kind of dna fragmentation of energy coded protein; So the long dna fragmentation of this 773-bp can independently play stabilising effect in different plasmids.
This sequence contains all DNA sequence and its cloning site on carrier pBCSK (+) of plasmid stable factor (PSF-Cxc).This sequence is to obtain by the subclone order-checking to pCXC118.Sequence on the carrier marks with underscore.
3, detect the stabilising effect of PSF-Cxc to different plasmids
In order to detect the stabilising effect of PSF-Cxc in different plasmids and different hosts, we are cloned into its dna sequence dna respectively on a series of E.coli plasmids and comprise pLARF3, pBR325, pBCSK (+), pUC118 and pUC119, and in E.coli, detect plasmid that does not contain PSF-Cxc and the stability that contains the plasmid of PSF-Cxc.The result show PSF-Cxc existence can all unsettled plasmids become 100% stable, and to the not influence of originally very stable plasmid.Because have only pLARF3 in the above-mentioned plasmid, pLARF3-S and pCXC101 (are formed by the pBR325 structure, contain PSF-Cxc) can in Cxc, duplicate survival, so the stability of these three plasmids in Cxc is detected, the plasmid of finding to have PSF-Cxc be 1 00% stable.These results show that PSF-Cxc all has the stabilising effect of highly significant to different plasmids in different hosts.
Plasmid stable as shown in the table in Cxc and E.coli:
| Plasmid | E.coli | Cxc |
| Generation Plasmid loss Elapsed per generation | Generation Plasmid loss Elapsed per generation | |
| pBR325 pCXC101 pLARF3 pLARF3-S* pBC SK(+) pBC SK(+)-S* pUC118 pUC118-S* pUC119 pUC119-S* | 32 1％ 32 0％ 32 2％ 32 0％ 32 1％ 32 0％ 32 0％ 32 0％ 32 0％ 32 0％ | ND ND 24 0％ 24 1.4％ 24 0％ |
*Mark the plasmid that contains PSF-Cxc (773-bp).Plasmid pCXC101 contains a dna fragmentation (Figure 1A) from pCXC100 that comprises PSF-Cxc 15kb.
4, the application of PSF-Cxc
The gene of this plasmid stable factor (773-bp dna fragmentation) can be cut multiple clone site or other sites that the method for recombinating in the method for connection or the body is cloned into any purpose plasmid by enzyme, to obtain having the novel plasmid of high stability.
(1) aspect molecular biology research, PSF-Cxc can be cloned on the various plasmids of using in the laboratory, comprise prokaryotic organism (E.coli, Cxc and other bacteriums etc.) plasmid and the eukaryote (yeast of usefulness, the cell of laboratory culture etc.) use plasmid, to increase the stability of plasmid, reduce antibiotic application and reduce experimental cost.With the yeast culture is example, and the clone of PSF-Cxc can reduce experimental cost greatly because not needing the auxotroph substratum.Simultaneously yeast in the speed of growth on the perfect medium than about the fast twice on the auxotroph substratum, so use PSF-Cxc can shorten the cycle of some experiments greatly.
(2) aspect industrial production, present many biotechnology medicines are obtained by the goal gene on the E.coli expression plasmid.The plasmid loss that the unstable of plasmid causes makes the purpose product production lower, and the wild effect of output is also more serious.In order to guarantee that the bacterium that contains plasmid becomes dominant microflora, but during fermentation microbiotic to suppress not contain the growth of plasmid bacterium.But antibiotic adding not only increases production cost, simultaneously also because kill the bacterium that does not contain plasmid and slow down bacterial growth and increase energy consumption, and cause certain difficulty for extracting after the fermentation of biotechnology medicine.Therefore, the PSF-Cxc that packs on the fermentation engineering plasmid can save in fermentation all inconvenience that adds microbiotic and bring, and can improve product output, reduce cost and energy consumption.What is more important uses microbiotic that microbiotic is run off in environment in a large number usually in the fermentation, cause serious resistance problem.Therefore replace microbiotic not only simple with PSF-Cxc, can protect environment especially.
(3) aspect agricultural biotechnology engineering: be obtaining of transgenic plant on the one hand, with the PSF-Cxc plasmid loss of genetically engineered plasmid of packing into to avoid causing because can't add microbiotic in the plant.The 2nd, endophyte expression system aspect can guarantee the stability of plasmid in plant with the PSF-Cxc gene engineering expression plasmid of packing into, thereby guarantee the effective expression at whole plants goal gene in vegetative period.
In sum, the present invention has the following advantages and positively effect:
(1) this plasmid stable factor has very strong stabilising effect to different plasmids in different hosts;
(2) this plasmid stable factor is long is 773-bp, can independently bring into play the stabilising effect to plasmid, without any need for other cofactor;
(3) this plasmid stable factor is cloned in the multiple clone site of E.coli plasmid pBCSK (+) at present, can be cloned into easily in any other the purpose plasmid.
(4) this plasmid stable factor can be widely used in aspects such as the scientific effort relevant with genetically engineered, industry and agriculture production.Especially can significantly improve the output of gene engineering product, reduce microbiotic and pollute; Has immeasurable economic worth.
Description of drawings
China typical culture collection center is used for the culture collection of patented procedure and accepts letter of information (receipt):
Classification name E.coli JM109/pCXC118
13 days December calendar year 2001 of preservation date
Depositary institution China. Wuhan. Wuhan University's postcode 430072
Deposit number CCTCC NO:M 201043
The sepn process figure of Fig. 1-stable factor (PSF-Cxc):
A-tentatively determines to contain the dna fragmentation of plasmid stable factor;
B-determines to contain the minimum dna fragmentation of stable factor;
Fig. 2-stable factor (PSF-Cxc) sequential analysis figure.
As Figure 1A, pCXC100 cuts the corresponding restriction enzyme site that the suitable dna fragmentation that obtains is cloned into carrier pBR325 through a series of enzymes, obtains cloning pCXC101,102,103,104 and 105.The contained dna fragmentation of these clones has covered all sections of the pCXC100 of 51Kb length, and the common coverage area of 3-8Kb is arranged in the middle of the adjacent clone.Whether these clones are transformed in Cxc or the E.coli cell, and not containing antibiotic situation down to the youthful and the elderly 24 generations (Cxc) or 32 generations (E.coli), detect plasmid afterwards and also be present in each cell.If each cell after going down to posterity still also has relevant plasmid, we just think that this plasmid contains stable factor, is designated as "+" in last figure; If some cell loss relevant plasmid, we just think that this plasmid does not contain stable factor, is designated as "-".Some clone so can not get transformant in Cxc, can't carry out Detection of Stability because lacked the replicon of natural plasmid pCXC100, is designated as " ND ".Through Detection of Stability, we find to clone right pCXC101 is the clone who contains stable factor and insert the fragment minimum, and we carry out further enzyme to pCXC101 and cut and clone, and obtain the clone pCXC106 shown in the last figure, 107,108,109 and 110.And carry out above-described stability analysis.
Determine to contain the minimum dna fragmentation of plasmid stable factor as Figure 1B.The contained dna fragmentation of plasmid pCXC106 is cut with DNaseIII through enzyme and is cleared up, and obtains pCXC111 to pCXC114.The contained dna fragmentation of plasmid pCXC113 is cut with ExoIII/S1 through enzyme and is cleared up, and obtains pCXC115 to pCXC121.Stability analysis is as described herein.
As Fig. 2, mark contained restriction enzyme site commonly used and its open reading frame that can read respectively from both direction (ORFs) of dna sequence dna (multiple clone site that contains Fig. 2) of PSF-Cxc, and the coded protein sequence of each ORF.
Embodiment
The gene (773-bp dna fragmentation) of coding plasmid stable factor PSF-Cxc is cloned in the middle of the EcoRI and HindIII site of pBCSK (+), can cut multiple clone site or other sites that the method for recombinating in the method for connection or the body is cloned into any purpose plasmid by enzyme, to obtain having the novel plasmid of high stability.
Claims (4)
1, a kind of broad-spectrum plasmid stabilizing factor PSF-Cxc, the dna fragmentation for a kind of energy coded protein is deposited among the intestinal bacteria, it is characterized in that E.coli JM109/pCXC118, CCTCC NO:M 201043.
2, by the described broad-spectrum plasmid stabilizing factor PSF-Cxc of claim 1, it is characterized in that:
Be that a segment length is the 773-bp dna fragmentation, this fragment contains two open reading frame, and an open reading frame encoded protein is 145 amino acid; Another open reading frame encoded protein is 125 amino acid; The long dna fragmentation of this 773-bp also contains the required dna sequence dna of startup plasmid expression.
3, by claim 1 or 2 described broad-spectrum plasmid stabilizing factor PSF-Cxcs, it is characterized in that its dna sequence dna is:
1
GGATCCCCCG GGCTGCAGGA ATTCGTTGAT TTCCGCGATC AGGCCAATGA
51 GGTCTCTGCT TGGATCTGCC CAGTAGTGAT GAGTCGAGCG AGCGCGATCG
101 TTGATCCCGT CGAAGTGTTG GTTCAGGCGC TTGTGTAGTA CCGAGAAGGC
151 ATGCCCGAAC TCTGCGTCAT CTTCGTAGAG GCGGTCGAAG GTCGGCGATG
201 AGCCTGCTCG GCGTACTGCG CCTTCATGTC CGCGACCGCG AGCTCTAGCG
251 GGCTAGCCAC AGCACTCCCC CGTTCACGAC CCGTACCTCC GGTCAGCTTC
301 CCAGTGGGCG CCCGCGAGTA ACGCCTCGCT CAGCATTCTC GAATCGTCGT
351 CCTTCGTTGT ACCGCTGCTC GAGGCGTTGC ACCTCCGCGT CCAGCGCCGC
401 GGCGATGAAG CCCGAGAACG TCCGATGACC CTCCTGCAGA TGGGTGAGCG
451 TGTACCCGTC CTCAGCCGTG CTCGGCGCTC CTCCCCCACC GTGACAGTCA
501 TCTTGACCTC AGAACGCCGC CCTCGGGGCT CCGAGGATCC TCCCGGCACC
551 GGCACCGGGA CCGTGGGCGC GACACTGTCC GCCACGGACG GCGACTCCGG
601 CACGAAAGCC GGCGCCACCA TCGACGGTGC TGCGGAGGCT TCGCGCTGCG
651 ACCGGATCAG GGATCCCGCA CCGGGACGCG ACGGCGGCGG TGCAACCGTG
701 CGATCAGCCA TTCGTGACCA CGCTCATTTC GGACTCCTTT GCCTGGATAC
751 GTGCGACGAG CTCCGCGGAG ATCGCCTGGA GGTCGTCAGC GA
TCAAGCTT。
4, a kind of application rights requires the method for 1 described broad-spectrum plasmid stabilizing factor PSF-Cxc, it is characterized in that:
The gene of this plasmid stable factor two the proteic length of can encoding are the 773-bp dna fragmentation, can cut the method for recombinating in the method for connection or the body by enzyme and be cloned on any purpose plasmid the novel plasmid that among different strain, obtains having high stability.
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