CN106636167A - Thiostrepton-gentamicin resistance gene system as well as resistance expression box and recombinant plasmid containing same - Google Patents
Thiostrepton-gentamicin resistance gene system as well as resistance expression box and recombinant plasmid containing same Download PDFInfo
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Abstract
The invention discloses an independent thiostrepton (tsr)-gentamicin resistance gene (aacC1) system with a nucleotide sequence as shown in SEQ ID NO:1, a resistance expression box and a recombinant plasmid containing the system, as well as a novel recombinant plasmid used for effectively constructing a recombinant mycobacterium without a resistance maker. According to the invention, the condition that thiostrepton THS can be used as a resistance selection marker of mycobacterium is illuminated for the first time, and the working concentration of thiostrepton THS is far lower than those of other selection markers used in the mycobacterium. The resistance expression box is characterized in that a dif sequence is added at two ends of a tsr-aacC1 gene, and can automatically dissociate in the mycobacterium. After the tsr-aacC1 gene is lost, a damaged gene still can express a shortened small peptide without influencing the translation of a downstream gene, thereby effectively avoiding a polar effect. Multiple restriction enzyme cutting sites are further added at two ends of the resistance expression box, so that the resistance expression box can be inserted into a specific sequence, and other sequences can be conveniently and directionally added on two sides of the specific sequence.
Description
Technical field
The invention belongs to genetic engineering field, and in particular to one kind marks recombinant mycobacterium for efficiently building non-resistant
Resistance expression's box and its application.
Background technology
At present during genetic manipulation is carried out to mycobacterium, the resistant maker gene that can be used to screen has very much
Limit, main cause be mycobacterium to Multiple Classes of Antibiotics natural drug resistance, and it requires that the i.e. more stable medicine of low frequency resistance is made
For resistant maker gene.And multiple resistance mark is used in combination, causing the genetic manipulation of mycobacterium needs various heredity to repair
Decorations, the such as stable maintenance of many plasmids and polygenic inactivation, therefore feasibility is extremely difficult.
Aminoglycoside phosphotransferase (APH) gene, it is resistant to kanamycins because of it, and for needing long-term cultivation
Slow growth mycobacterium there is high stability, therefore the resistance screening for being used for mycobacterium as first is marked.So
And, the use of APH is limited due to mycobacterium to the appearance of its spontaneous resistance, although this resistance is low frequency.With it is quick
Growth bacterium is different, and the mycobacterium of slow growth has single rRNA operators, and it is easier to undergo mutation so as to produce
Resistance, such as anti-kanamycins.Additionally, kanamycins in Mycobacterium strain w and Mycobacterium bovis as resistance screening mark
Note is not yet realized.Radford and Hodgson is in reported first hygromycin in 1991 in mycobacterium smegmatis and prapes branch bar
Mark as resistance screening in bacterium BCG, and can be used in other mycobacteriums.Hygromycin is marked as resistance screening, most
Big advantage is for the medicine for clinically using at present is not in crossing drug resistant.Also, hygromycin is used as resistance screening mark
The conversion success rate of note is higher than kanamycins, and its expression efficiency in mycobacterium also significantly larger than derives from Escherichia coli
APH gene pairs kanamycins resistance.
Apramycin in mycobacterium at a slow speed with fast-growth as resistance screening mark using first by Paget and
Davies was reported in 1996, but was clinically not approved for using.Main cause is that apramycin can be caused and its own
There is acetylation, such as kanamycins in close aminoglycoside antibiotics, and its conversion ratio is very low.Additionally, apramycin belongs to
The resistance screening mark of beta-lactam, because it contains beta-lactamase therefore can make mycobacterium produce natural resistance, and blue or green
Mycin is also thus, therefore can not mark as reliable and stable resistance screening in mycobacterium.
The resistance screening that can be used for mycobacterium of other reports is marked with chloramphenicol, but because its stability is low and
High-frequency spontaneous mutation and make it using being restricted, be not particularly suitable for the mycobacterium of slow growth.Streptomysin, sulfamido
Medicine and mercury salt were also once reported and can be used for the resistance screening mark of mycobacterium, but until current, kanamycins, hygromycin
Most common resistance screening mark used in mycobacterium is remained with gentamicin resistance gene.
Because the resistance screening marker number that can be used for mycobacterium is limited, therefore we have developed a kind of acceptable methyl
Chemotaxis protein I and the serine sensitive receptors tsr gene resistant to thiostrepton (THS), as tuberculosis branch bar
Bacterium and the resistance screening marker gene of mycobacterium tuberculosis var bovis BCG-Tice.THS, a kind of antibiotic of thiazoles, in 1954
Separate first and identify to come from remote blue or green streptomycete (Streptomyces azureus), be mainly used in treating the mammary gland of animal
Inflammation, is also used for treating dog sector of breakdown disease, however, its purposes is but because low-solubility and high toxicity are restricted.THS leads to
Cross on the complex formed by 23S rRNA and ribosomal protein L 11 being tightly integrated in bacterium, so as to suppress related egg
The translation of white matter.And tsr genes can encode a kind of RNA transmethylases, the enzyme can be prevented by the 23S rRNA that methylate
THS is attached on ribose composite.Therefore the complete resistances of tsr gene pairs THS, therefore in the cloning vector of various actinomycetes
As resistance screening marker gene.Aminake et al. holds promise for treating malaria in report THS in 2011 and its derivative
Disease, makes parasite quickly be removed because it can target the proteasome of the malarial parasite P.Falciparum of the mankind.
Recently, the research discovery that Westhoff etc. is fastened in breast cancer cell, when THS and taxol/cisplatin chemotherapy medicine connection
When conjunction is used, the expression of Forkhead box M1 (FOXM1) genes will decline, while to from platinum class resistance patient's
The Synergistic killing effect of ascites cells is also remarkably reinforced.In addition, Wada also reported THS equal to 2015 except can be used for
Outside accurate selection markers under the conditions of singly copying in streptomycete, it may also be used for the stability resistance screening mark of thermophilic ground bacillus
Note.However, the data that THS is used for the resistance screening mark of Much's bacillus is but and inadequate, but this is necessary.
The content of the invention
It is an object of the present invention to provide a kind of thiostrepton-gentamicin resistance base for carrying Artificial promoters
Because of tsr-aacC1.
Further object is that providing a kind of for efficiently building the anti-of non-resistant mark recombinant mycobacterium
Property expression cassette.
Further object is that providing a kind of for efficiently building the weight that non-resistant marks recombinant mycobacterium
Group plasmid.
The technical solution used in the present invention is:
A kind of inventor's independent research thiostrepton (tsr)-gentamicin resistance gene (aacC1) resistant gene body
System, its nucleotide sequence such as SEQ ID NO:Shown in 1.
A kind of resistance expression's box for efficiently building non-resistant mark recombinant mycobacterium, containing thiostrepton resistance
Gene and gentamicin resistance gene, and can start thiostrepton resistant gene and gentamicin resistance gene expression
Hsp60 promoters, in addition with dif1 the and dif2 sequences positioned at hsp60 promoters and thiostrepton resistant gene two ends,
Described thiostrepton resistant gene and the nucleotide sequence of gentamicin resistance gene such as SEQ ID NO:Shown in 1, hsp60
Promoter sequence such as SEQ ID NO:Shown in 6, dif1 and dif2 sequences are respectively such as SEQ ID NO:7 and SEQ ID NO:Shown in 8.
Here is named as resistance expression's box " dif2-hsp60-aacC1-tsr-dif1 resistance expression's boxes ".
Preferably, resistance expression's box two ends also added multiple restriction enzyme site.
The nucleotide sequence of resistance expression's box such as SEQ ID NO:4 or SEQ ID NO:Shown in 5.
Recombinant plasmid containing the above dif2-hsp60-aacC1-tsr-dif1 resistance expression's box.
Preferably, the nucleotide sequence of the recombinant plasmid such as SEQ ID NO:Shown in 2.
A kind of new recombinant plasmid for efficiently building non-resistant mark recombinant mycobacterium, it contains:Promoter, bite
Phage incorporation site, integrase gene, ampicillin resistance gene Amp R, M13 primers, replication origin and described
Resistance expression's box.It is in the direction of the clock, Lac Z promoters, Lac Z operators, M13 reverse primers, claim 2~5 times
Resistance expression's box, bacteriophage integration site described in one, integrase gene, M13 forward primers, Escherichia coli replication orgin
(f1), ampicillin resistance gene promoter, ampicillin resistance gene Amp R, mycobacterium replication orgin ori M according to
It is secondary to link together.
The promoter is Hsp60 promoters, Amp R promoters and Lac Z promoters, and the replication origin is
Escherichia coli replication orgin f1 and mycobacterium replication orgin ori M.
Preferably, the nucleotide sequence of the new recombinant plasmid such as SEQ ID NO:Shown in 3.
The invention has the beneficial effects as follows:
(1) present invention illustrates first thiostrepton THS and can mark as the resistance screening of mycobacterium, and its work
Make concentration far below other resistance markers used in mycobacterium, as shown in table 2 and table 3;Additionally, in thiostrepton
(tsr) in-gentamicin resistance gene (aacC1) system, gentamicin resistance gene is used for Escherichia coli and shame dirt branch bar
The resistance screening mark of bacterium, GEN is marked for M.smegmatis mc as resistance screening2The 155 transformation efficiency such as institute of table 1
Show;And thiostrepton resistant gene is then used for the resistance screening mark of Much's bacillus and mycobacterium tuberculosis var bovis, THS makees
Mark for resistance screening as shown in table 1 for the transformation efficiency of M.bovis BCG and M.tuberculosis H37Rv;
(2) thiostrepton (tsr)-gentamicin resistance gene (aacC1) resistant gene system carries short promoter
Hsp60, can express in Escherichia coli and mycobacterium, and the transcription and translation of downstream gene is not affected during orientation insertion;
(3) resistance expression's box of the invention with the addition of dif sequences at tsr-aacC1 genes two ends, can be in mycobacterium
In dissociate automatically.After tsr-aacC1 genes are lost, destroyed gene may continue to express the small peptide for shortening, and not affect downstream
The translation of gene, so as to polar effect can be prevented effectively from;
(4) resistance expression's box two ends of the invention also added multiple restriction enzyme site so that the expression cassette both may be inserted into
To in particular sequence, it is also convenient in its two side positionings addition other sequences.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, may be used also
To obtain other accompanying drawings according to these accompanying drawings.
Fig. 1 is the structural representation of plasmid p60GTE;
Fig. 2 is plasmid p60GTE restriction enzyme site figures;
Fig. 3 is the structure flow chart of plasmid p60GTE;
Fig. 4 is the structural representation of integrative plasmid p60GTI;
Fig. 5 is the structure flow chart of integrative plasmid p60GTI..
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than the embodiment of whole.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
Molecular biology experiment technology employed in following examples include PCR amplifications, plasmid extraction, plasmid convert,
DNA fragmentation connection, digestion, gel electrophoresis etc., if no special instructions, generally conventionally operate, and specifically can be found in《Molecule
Cloning experimentation guide》(third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitangs etc. are translated,
2002, Beijing:Science Press), or according to the condition proposed by manufacturer.
PCR reactions pfu enzymes used, dNTP and related reagent are bought from Shanghai life work biology and are had in following examples
Limit company;Antibiotic thiostrepton and gentamicin are purchased from Shanghai Sigma-Aldrich;E. coli competent
DH5a is bought in Guangzhou Dongsheng bio tech ltd, and article No. is C1042;DNA coupled reactions are precious biological using Takara
The T4DNA connection kits of company, model:D6020A;Extraction of plasmid DNA kit (BSC01M1), DNA QIAquick Gel Extraction Kits
(BSC02M1), PCR purification kits (BSC03M1) are purchased from Bo biotech firms.
Used PCR primer and DNA fragmentation are responsible for conjunction by Shanghai Jierui Biology Engineering Co., Ltd in following examples
Into.
Embodiment 1 builds plasmid p60GTE
The structural representation of plasmid p60GTE is shown in Fig. 1, and restriction enzyme site figure is shown in Fig. 2, and it contains above-mentioned tsr-aacC1 bases
Cause.As seen from Figure 1, plasmid p60GTE contains successively in the direction of the clock:Escherichia coli replication orgin (ori E), tsr-aacC1
Resistant gene, promoter hsp60 (for starting thiostrepton-gentamicin resistance gene, i.e. tsr-aacC1) is main to use
In plasmid screening.
The plasmid p60GTE contains thiostrepton-gentamicin resistance gene (tsr-aacC1), such as SEQ ID NO:
Described in 1, the 2760-3145 sequences on the gene are promoter sequence (hsp60), for starting the table of tsr-aacC1 genes
Reach.The hsp60 sequences such as SEQ ID NO of tsr-aacC1 gene orders two ends connection:Shown in 6.
In addition Not I restriction enzyme sites, XbaI enzyme cutting are sequentially connected respectively at the two ends of hsp60-aacC1-tsr resistance systems
Site, HindIII restriction enzyme sites, XhoI restriction enzyme sites, this 4 restriction enzyme sites can be used to cut the hsp60-aacC1- on plasmid
Tsr resistance systems.
1) the structure flow chart of plasmid p60GTE see in Fig. 3, this flow process will using to plasmid p60Lux N (by this laboratory
Oneself builds, and plasmid map is shown in Fig. 3, and concrete construction method sees reference document 22).Contain original hygromycin in plasmid p60Lux N
Resistant gene (Hyg*), about long 1.7kb.
Concrete grammar is as follows:
1. the structure of plasmid p60Gm
With restriction enzyme Nde I and Pst I digested plasmid p60Lux N, purify QIAquick Gel Extraction Kit and reclaim 7.9kb's
Fragment;
Synthetic DNA fragment aacC1 is (such as SEQ ID NO:Shown in 9, conjunction is responsible for by Shanghai Jierui Biology Engineering Co., Ltd
Into), the aacC1 two ends of synthesis carry Nde I and Pst I restriction enzyme sites, so, by the aacC1 and matter after purified recovery
Grain p60Lux N carry out fragment connection, convert E. coli competent DH5 α, using the LB solid plates containing gentamicin resistance
Positive colony is filtered out, after cultivating in picking monoclonal to LB fluid nutrient mediums, plasmid is extracted, digestion identifies correct clone
As plasmid p60Gm.
2. the structure of plasmid p60GTE
With restriction enzyme PstI and Hind III digested plasmid p60Gm, purifying QIAquick Gel Extraction Kit recovery
The fragment of 6.7kb;
Synthetic DNA fragment tsr is (such as SEQ ID NO:Shown in 10, conjunction is responsible for by Shanghai Jierui Biology Engineering Co., Ltd
Into), the tsr two ends of synthesis carry Pst I and Hin restriction enzyme sites, so, by the tsr and plasmid p60Gm after purified recovery
Fragment connection is carried out, E. coli competent DH5 α are converted, using the LB solid plates containing gentamicin resistance the positive is filtered out
Clone, after cultivating in picking monoclonal to LB fluid nutrient mediums, extracts plasmid, and digestion identifies correct clone and is plasmid
p60GTE。
The structure of the integrative plasmid p60GTI of embodiment 2 and application
The structural representation of integrative plasmid p60GTI is shown in that Fig. 4, plasmid p60GTI contain successively in the direction of the clock:Lac
Z promoters and Lac Z operators (can be used for blue hickie screening, and the entirely expression of plasmid backbone behind starting), M13 is reverse
Primer (for PCR amplification identifications), dif2-hsp60-aacC1-tsr-dif1 resistance expression's boxes, bacteriophage integration site attP,
Integrase gene Int (can give expression to integrase, make plasmid enter Mycobacterium tuberculosis genes group by attP integrations), and M13 is positive
Primer (for PCR amplification identifications), Escherichia coli replication orgin (f1), ampicillin resistance gene promoter, ammonia benzyl mould
Plain resistant gene Amp R (for plasmid screening), mycobacterium replication orgin ori M.dif2-hsp60-aacC1-tsr-dif1
Resistance expression's box two ends are sequentially connected respectively HindIII restriction enzyme sites, XhoI restriction enzyme sites, can be used to cut on plasmid
Dif2-hsp60-aacC1-tsr-dif1 resistance expression's boxes.
The structure flow process of integrative plasmid p60GTI is shown in Fig. 5, and plasmid pUCDHmke used in this structure flow process is by this
Laboratory oneself builds, and plasmid map is shown in Fig. 5, and concrete construction method sees reference document 23;Plasmid pMH94 is by U.S. John Hope
Jin Si universities WilliamBishai laboratories give, and plasmid map is shown in Fig. 5, and concrete construction method sees reference document 24.
Concrete operations are as follows:
1. the structure of plasmid pUCDGT
With restriction enzyme Xba I digested plasmid pUCDHmKE, purify QIAquick Gel Extraction Kit and reclaim the fragment of 2.7kb and (contain
dif2-ΩHYG-dif1);
With restriction enzyme Xba I digested plasmid p60GTE, the fragment that QIAquick Gel Extraction Kit reclaims 1.8kb is purified
(hsp60-aacC1-tsr);Two fragments are attached after reaction, E. coli competent DH5 α are converted, using big containing celebrating
The LB solid plates of chloramphenicol resistance filter out positive colony, after cultivating in picking monoclonal to LB fluid nutrient mediums, extract plasmid,
Digestion identifies correct clone and is plasmid pUCDGT (containing dif2-hsp60-aacC1-tsr-dif1).
2. the structure of plasmid p60GTI
With restriction enzyme HindIII digested plasmid pUCDGT, purify QIAquick Gel Extraction Kit and reclaim the fragment of 1.8kb and (contain
dif2-hsp60-aacC1-tsr-dif1);
With restriction enzyme HindIII digested plasmid pMH94, the fragment that QIAquick Gel Extraction Kit reclaims 6.1kb is purified;By two
Individual fragment is attached after reaction, converts E. coli competent DH5 α, is sieved using the LB solid plates containing gentamicin resistance
Positive colony is selected, after cultivating in picking monoclonal to LB fluid nutrient mediums, plasmid is extracted, digestion identifies correct clone i.e.
For plasmid p60GTI.
3. convert
It is proceeded into respectively mycobacterium smegmatis (method for transformation by the method for electricity conversion for plasmid p60GTI and p60GTE
See reference document 25), and Much's bacillus and mycobacterium tuberculosis var bovis BCG (H37Rv tuberculosis reference cultures, method for transformation
See reference document 26), obtain corresponding transformant.
Gained transformant is incubated 12 hours in 37 DEG C of incubators, bacterium is fully brought back to life and is made its resistance expression.Filtered with band
After the rifle point pressure-vaccum of core is mixed, dispense into tubule, 10000rpm is centrifuged 1 minute precipitation transformed bacteria, abandons supernatant, uses 0.6mL
After 7H9 culture mediums are resuspended, with 500 μ L/ plates 7H11 (mycobacterium smegmatis containing corresponding antibiotic is laid on:GEN 5μg/mL;Tuberculosis point
Branch bacillus and mycobacterium tuberculosis var bovis BCG:The μ g/mL of THS 5 and 10) in culture plate.Simultaneously after 100 times of dilution, 500 μ L/ plates paving
Plate, lucifuge is in 37 degree of incubator cultures.
The dirt of the shame containing p60GTI and p60GTE plasmids can be obtained on the flat board containing corresponding antibiotic (GEN and THS)
Mycobacterium, and Much's bacillus and mycobacterium tuberculosis var bovis BCG, this step demonstrates the dif2-hsp60- of the present invention
AacC1-tsr-dif1 resistance expressions box can be expressed successfully in mycobacterium.
The mycobacterium smegmatis with p60GTI and p60GTE plasmids that will be obtained, and Much's bacillus and prapes
Mycobacterium BCG transformant carries out liquid Secondary Culture, culture 3 days after (for mycobacterium smegmatis) or after 14 days (for
Much's bacillus and mycobacterium tuberculosis var bovis BCG), plate (the μ g/mL of THS 10), institute are paved after the bacterium solution of culture is diluted
The bacterium colony that 80% is there are about on the flat board for obtaining lost THS resistances, i.e., cannot grow on the flat board containing sulphur Streptothrix (THS),
The dif2-hsp60-aacC1-tsr-dif1 resistance expressions box that this step is demonstrated in this patent can be successfully automatic in mycobacterium
Dissociation, and have higher efficiency.THS is marked for M.bovis BCG and M.tuberculosis as resistance screening
The transformation efficiency and GEN of H37Rv marks the transformation efficiency such as institute of table 1 for M.smegmatis mc2155 as resistance screening
Show.
Table 1:THS marks the conversion for M.bovis BCG and M.tuberculosis H37Rv as resistance screening
Efficiency and GEN are marked for M.smegmatis mc as resistance screening2155 transformation efficiency
Transformation efficiency:It is defined as every micrograms of DNA and converts the clone's number for successfully growing;
As a result it is to calculate what is come by the average clone's number grown in three parallel flats.
Table 2:THS is for the MIC (minimal inhibitory concentration) of wild type and recombinant mycobacterium
Table 3:GEN is for wild type and recombinant mycobacterium M.smegmatis mc2155 MIC
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Within god and principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (10)
1. a kind of thiostrepton (tsr)-gentamicin resistance gene (aacC1) system, it is characterised in that:Its nucleotide sequence
Such as SEQ ID NO:Shown in 1.
It is 2. a kind of to build resistance expression's box that non-resistant marks recombinant mycobacterium for efficient, it is characterised in that:Containing sulphur chain
Silk rhzomorph resistant gene and gentamicin resistance gene, thiostrepton resistant gene and gentamicin resistance gene table can be started
The hsp60 promoters that reach, positioned at dif1 the and dif2 sequences at hsp60 promoters and thiostrepton resistant gene two ends, it is described
Thiostrepton resistant gene and gentamicin resistance gene nucleotide sequence such as SEQ ID NO:Shown in 1, hsp60 starts
Subsequence such as SEQ ID NO:Shown in 6, dif1 and dif2 sequences are respectively such as SEQ ID NO:7 and SEQ ID NO:Shown in 8.
3. resistance expression's box according to claim 2, it is characterised in that:Resistance expression's box two ends are additionally provided with multiple restriction enzyme
Site.
4. resistance expression's box according to claim 3, it is characterised in that the nucleotide sequence of resistance expression's box is such as
SEQ ID NO:4 or SEQ ID NO:Shown in 5.
5. a kind of recombinant plasmid, it is characterised in that:Containing arbitrary resistance expression's box in claim 2~4.
6. recombinant plasmid according to claim 5, it is characterised in that:The nucleotide sequence of the recombinant plasmid such as SEQ ID
NO:Shown in 2.
It is 7. a kind of to build the new recombinant plasmid that non-resistant marks recombinant mycobacterium for efficient, it is characterised in that:Containing opening
Mover, bacteriophage integration site, integrase gene, ampicillin resistance gene Amp R, M13 primers, replication origin with
And resistance expression's box described in any one of claim 2~4.
8. new recombinant plasmid according to claim 7, it is characterised in that start containing Lac Z successively in the direction of the clock
It is son, Lac Z operators, M13 reverse primers, resistance expression's box described in any one of claim 2~5, bacteriophage integration site, whole
Synthase gene, M13 forward primers, Escherichia coli replication orgin (f1), ampicillin resistance gene promoter, ampicillin
Resistant gene Amp R, mycobacterium replication orgin ori M.
9. the new recombinant plasmid according to claim 7 or 8, it is characterised in that:The promoter is Hsp60 promoters, Amp
R promoters and Lac Z promoters, the replication origin is Escherichia coli replication orgin f1 and mycobacterium replication orgin
ori M。
10. the recombinant plasmid according to claim 7 or 8, it is characterised in that the nucleotide sequence of the recombinant plasmid is such as
SEQ ID NO:Shown in 3.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040254100A1 (en) * | 2000-01-31 | 2004-12-16 | Vermeulen Mary W. | Use of thiostrepton as an anti-mycobacterial agent |
CN103451181A (en) * | 2013-06-06 | 2013-12-18 | 中国科学院广州生物医药与健康研究院 | Resistance expression cassette for efficiently constructing recombinant mycobacterium without resistance markers |
-
2016
- 2016-10-17 CN CN201610902985.6A patent/CN106636167A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040254100A1 (en) * | 2000-01-31 | 2004-12-16 | Vermeulen Mary W. | Use of thiostrepton as an anti-mycobacterial agent |
CN103451181A (en) * | 2013-06-06 | 2013-12-18 | 中国科学院广州生物医药与健康研究院 | Resistance expression cassette for efficiently constructing recombinant mycobacterium without resistance markers |
Non-Patent Citations (5)
Title |
---|
FENG YANG ET AL.: "Efficient construction of unmarked recombinant mycobacteria using an improved system", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
JULIUS MUGWERU ET AL.: "A Cassette Containing Thiostrepton, Gentamicin Resistance Genes, and dif sequences Is Effective in Construction of Recombinant Mycobacteria", 《FRONTIERS IN MICROBIOLOGY》 * |
KATHRYN E.A.LOUGHEED ET AL.: "New anti-tuberculosis agents amongst known drugs", 《TUBERCULOSIS》 * |
甄永苏: "《抗肿瘤药物研究与开发》", 30 September 2004, 化学工业出版社 * |
邓名荣等: "一个硫链丝菌素抗性基因标记、用于DNA导入链霉菌的pSET152衍生载体(英文)", 《微生物学通报》 * |
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