CN108103172A - A kind of detection method of plasmid genetic stability of Streptococcus suis extracellular factor gene and application - Google Patents
A kind of detection method of plasmid genetic stability of Streptococcus suis extracellular factor gene and application Download PDFInfo
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Abstract
Detection method and application the present invention provides a kind of plasmid genetic stability of Streptococcus suis extracellular factor gene, belong to bioengineering field.The host strain bacterium solution that the present invention passes through the plasmid for culture Streptococcus suis extracellular factor gene of recovering, and carry out the secondary culture of continuous multi-generation, certain algebraically is spaced to sample and detect, by the genetic stability of the host strain of the plasmid of the Streptococcus suis extracellular factor gene that detects artificial preservation, provide reliable support for research pig hemolysin gene and ensure；Above-mentioned detection method is applied in the plasmid research of Streptococcus suis extracellular factor gene, or the research and culture of pig hemolysin gene provide reference and uses for reference, and have important application value.
Description
Technical field
The present invention relates to bioengineering field, in particular to a kind of plasmid of Streptococcus suis extracellular factor gene
The detection method of genetic stability and application.
Background technology
II type bacterium of high-pathogenicity porcine hammer (Streptococcus suisserotype 2, SS2) can cause a series of urgency
The communicable diseases such as property septicemia, meningitis and arthritis, disable, lethality it is higher, be a kind of important infecting both domestic animals and human venereal disease
It is former.
Extracellular factor (Extracellular protein factor, EF) is the outstanding feature virulence factor of SS2
One of with important protective antigens.
Streptococcus suis has generation in various regions, and strong influence and larger economic loss, mesh are caused to pig breeding industry
Preceding no effective antibody vaccine, therefore the research of Streptococcus suis extracellular factor is needed to strengthen, few at present be directed to contains
There is the research of the stability of the artificial preservation of host strain of the plasmid of Streptococcus suis extracellular factor gene.
The content of the invention
The first object of the present invention is the detection for providing the plasmid genetic stability of Streptococcus suis extracellular factor gene
Method by this detection method, can have the genetic stability of pig hemolysin gene more reliable understanding, be Streptococcus suis
The research of extracellular factor gene provides reliable support.
The second object of the present invention is the plasmid genetic stability for providing above-mentioned Streptococcus suis extracellular factor gene
Application of the detection method in the research of pig hemolysin gene.
In order to realize the above-mentioned purpose of the present invention, using following technical scheme：
A kind of detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene, including：
The host strain bacterium solution of the plasmid containing Streptococcus suis extracellular factor gene is cultivated in fluid nutrient medium；
Continuous passage culture host strain bacterium solution；
The host strain bacterium solution of interval sampling secondary culture simultaneously carries out genetic stability detection.
The detection method of the plasmid genetic stability of above-mentioned Streptococcus suis extracellular factor gene is in Streptococcus suis cell
Application in extrinsic factor gene studies.
Beneficial effects of the present invention are：The present invention cultivates the plasmid containing Streptococcus suis extracellular factor gene by recovering
Host strain bacterium solution, and carry out the secondary culture of continuous multi-generation, be spaced certain algebraically and sample and detect, pass through detection and manually protect
The genetic stability of the host strain of the plasmid of the Streptococcus suis extracellular factor gene of Tibetan, to study Streptococcus suis extracellular factor
Gene provides reliable support and ensures；Above-mentioned detection method is applied in Streptococcus suis extracellular factor gene studies,
Reference and reference can be provided for the research and culture of Streptococcus suis extracellular factor gene, there is important application value.
Description of the drawings
It in order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of scope, for those of ordinary skill in the art, without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the colonial morphology figure for the 5th generation Escherichia coli that experimental example 4 of the present invention provides；
Fig. 2 is the colonial morphology figure for the 15th generation Escherichia coli that experimental example 4 of the present invention provides；
Fig. 3 is the colonial morphology figure for the 25th generation Escherichia coli that experimental example 4 of the present invention provides；
Fig. 4 is the colonial morphology figure for the 40th generation Escherichia coli that experimental example 4 of the present invention provides；
Fig. 5 is the 5th generation colibacillus PCR electrophoresis result figure that experimental example 4 of the present invention provides；
Fig. 6 is the 15th generation colibacillus PCR electrophoresis result figure that experimental example 4 of the present invention provides；
Fig. 7 is the 25th generation colibacillus PCR electrophoresis result figure that experimental example 4 of the present invention provides；
Fig. 8 is the 40th generation colibacillus PCR electrophoresis result figure that experimental example 4 of the present invention provides；
Fig. 9 is the 5th generation Escherichia coli biochemical test result figure that experimental example 4 of the present invention provides；
Figure 10 is the 15th generation Escherichia coli biochemical test result figure that experimental example 4 of the present invention provides；
Figure 11 is the 25th generation Escherichia coli biochemical test result figure that experimental example 4 of the present invention provides；
Figure 12 is the 40th generation Escherichia coli biochemical test result figure that experimental example 4 of the present invention provides.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Hemolysin is the Major Virulence Factors of Streptococcus suis, and after pig-pig infection streptococcus, hemolysin plays a role, serious shadow
Ring its growth development；Tremendous influence is brought to pig-breeding, therefore is exactly necessary to the detection research of pig hemolysin；And contain
The genetic stability of the especially artificial storage conditions of genetic stability of the plasmid of Streptococcus suis extracellular factor gene is also must
It wants.
Below to a kind of detection of the plasmid genetic stability of Streptococcus suis extracellular factor gene of the embodiment of the present invention
Method and application are specifically described.
A kind of detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene, including：
The host strain bacterium solution of the plasmid containing Streptococcus suis extracellular factor gene is cultivated in fluid nutrient medium；
Continuous passage culture host strain bacterium solution；
The host strain bacterium solution of interval sampling secondary culture simultaneously carries out genetic stability detection.
Common cloning vector pEASY-Blunt is selected as carrier, by Streptococcus suis extracellular factor gene：
CATGCTAACACAGTGACAGAAGCAGAGACAGCTGTAG
CACCAGCTAACCAAGACCTTGGAAATGAGACTAAAACGGAAG
AAGAACCCAAGGAACCAATCGAAGCAGTTCGCACGGACATGG
AAAACCGTGCAGCTGAAATCTTGCCGGAGGCGCTGAATGCTAG
TGTAACAAACCAAGCACCAGTTATTCCGACTATTGGAGATCTTC
CTAAAGATGCGAGTGGTCAGAATGTTCATGGTAAGGCAACGGA
TAATAAGATTTATCGTGTTGTATACGTTTTTGGTAATGTAGCAGG
GACTACGGAGACAGAAGATGGTAAACAAAATGTTGCTCCAACA
TTTAACAGAAATGATGCAACTAAAACTTTTCCAATCACAGATCC
AGATAGCGACATTCAAACTATTTCATACGAAGTTCCAGCTGATA
TTGCAAGCTATACCTTGGATGATCCAAACTCAATTGTTACTAAT
GGCACCTCACCTGGTCCAGTATCTTACTTAGATGGTCCAAATGG
GTCAGCCACTCTCACACAAGATGGTTATCTAAC clones are connected on cloning vector pEASY-Blunt.
It is detected using primer, detection primer is：
EF sense primer gene orders：5’ CACAGTGACAGAAGCAGAGACAG3’；
EF anti-sense primer gene orders：5’CATCTTGTGTGAGAGTGGCTG3’.
The fragment length of primer amplification is 530bp.
Further, the inoculum concentration of host strain bacterium solution is the 3%-7% of the volume of fluid nutrient medium.
Host strain and fluid nutrient medium keep a rational inoculative proportion, it is ensured that bacterium solution can recover faster and into
Enter logarithmic phase, the time of saving experiment that can be larger, accelerate experiment process.
Further, host strain bacterium solution is glycerine bacterium solution.
Usual glycerol stock is low-temperature preservation, since glycerine has antifreeze effect, by adding glycerine, can be effectively protected
Bacterial strain is not destroyed in cryogenic conditions；And since glycerine has the effect of moisturizing, can preferably ensure the moisture of bacterium solution, it protects
Demonstrate,prove the activity of strain.
Further, the preparation of glycerine bacterium solution includes：
The plasmid of the gene of extracellular factor containing Streptococcus suis is transformed into competent escherichia coli cell, is drawn by identification
Analyte detection is identified, obtains positive recombination bacillus coli；
Recombination bacillus coli is cultivated, and adds glycerine and glycerine culture presevation is made.
Further, competent escherichia coli cell is one in TOP10 cells, DH5 α cells and BL21 (DE3) cell
Kind.
Since the growth of Escherichia coli is rapid, using Escherichia coli as host strain, pass through culture and the passage of simply recovering
Culture, can comparatively fast obtain substantial amounts of bacterium solution, you can to obtain substantial amounts of Streptococcus suis extracellular factor gene；Escherichia coli
It is the host strain of common clone and expression, has the advantages that more.
Further, the plasmid of the gene of extracellular factor containing Streptococcus suis and the volume ratio of competent escherichia coli cell are
2-6:100。
By controlling the ratio of plasmid and competent escherichia coli cell, the transformation efficiency of plasmid can be improved.
Further, the plasmid concentration of the gene of extracellular factor containing Streptococcus suis is 2.5-7.1ng/ μ L.
Further, method for transformation is：Mix the plasmid of the gene of extracellular factor containing Streptococcus suis and Escherichia coli impression
State cell, places 25-37min on ice；Heat shock 40-60s, places 2-3min on ice.
Further, the temperature of heat shock is 42 DEG C.
The recombinant plasmid for being connected with Streptococcus suis extracellular factor gene can be efficiently transformed into Escherichia coli by heat shock
In competent cell, recombinant host bacterium is then cultivated, reaches the mesh of the plasmid of transformed clone Streptococcus suis extracellular factor gene
's.
The detection method of above-mentioned Streptococcus suis extracellular factor gene genetic stability is in the research of pig hemolysin gene
Application.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene, pig chains
The detection method of pneumoniae cells extrinsic factor gene genetic stability, including the plasmid of Streptococcus suis extracellular factor gene is converted
Into host strain cell, glycerol stock then is made in the host strain of the plasmid containing Streptococcus suis extracellular factor gene, is passed through
Culture and passage glycerol stock, the genetic stability of identification passage glycerol stock.
The competent cell of DH5 α is selected to implement certainly in others as competent escherichia coli cell in the present embodiment
In mode other competent escherichia coli cells can also be selected to be tested, such as TOP10 cells or BL21 (DE3) cell.
The conversion of plasmid is as follows：
1.1 take the competent cell of 100 μ L bacillus coli DH 5 alphas to be added in EP pipes, and melt on ice；
1.2 take the plasmid (pEASY-Blunt-EF) for the Streptococcus suis extracellular factor gene that 2 μ L concentration are 7.1ng/ μ L
It is added in the competent cell of the bacillus coli DH 5 alpha melted in EP pipes, places 25min on ice；
1.3 by EP pipes under 42 DEG C of temperature conditionss heat shock 40s, by EP pipes fast transfer to cooled on ice 2min；
1.4 add in the LB liquid medium without antibiotic of 200 μ L, 150rpm cultures 45min in EP pipes；
1.5 take the bacterium solution even spread of 50 μ L to LB solid mediums (Amp+) on, 37 DEG C of culture 16h.
The identification of positive host strain
From the LB solid mediums (Amp of step 1.5+) on single spots of picking 10 respectively, cultivated in LB fluid nutrient mediums,
Then bacterium solution PCR identifications are carried out, the positive sample with band is enlarged culture.
It prepares glycerol stock and carries out culture presevation
2.1 prepare the spare of 30% glycerine；
2.2 take the logarithm, and thalline were collected by centrifugation by bacterium solution 400 the μ L, 2000rpm in growth period；
2.3 add in the fresh LB fluid nutrient mediums of 200 μ L, and thalline is dispelled to form bacteria suspension；
2.4 add in 30% isometric glycerine, mixing.
Embodiment 2
The present embodiment provides a kind of detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene, pig chains
The detection method of the plasmid genetic stability of pneumoniae cells extrinsic factor gene is included the matter of Streptococcus suis extracellular factor gene
Grain is transformed into host strain cell, and the host strain of the plasmid containing Streptococcus suis extracellular factor gene then is made glycerine
Bacterium, by cultivating and passing on glycerol stock, the genetic stability of identification passage glycerol stock.
The competent cell of TOP10 is selected in the present embodiment as competent escherichia coli cell, certainly other real
Applying in mode can also select other competent escherichia coli cells to be tested, such as DH5 α cells or BL21 (DE3) cell.
The conversion of plasmid is as follows：
1.1 take the competent cell of 100 μ L Escherichia coli TOP10 to be added in EP pipes, and melt on ice；
1.2 take the plasmid (pEASY-Blunt-EF) for the Streptococcus suis extracellular factor gene that 6 μ L concentration are 0.8ng/ μ L
It is added in the competent cell of the Escherichia coli TOP10 melted in EP pipes, places 37min on ice；
1.3 by EP pipes under 42 DEG C of temperature conditionss heat shock 60s, by EP pipes fast transfer to cooled on ice 3min；
1.4 add in the LB liquid medium without antibiotic of 200 μ L, 150rpm cultures 45min in EP pipes；
1.5 take the bacterium solution even spread of 50 μ L to LB solid mediums (Amp+) on, 37 DEG C of culture 16h.
The identification of positive host strain
From the LB solid mediums (Amp of step 1.5+) on the single spot of picking 10 respectively, cultivated in LB fluid nutrient mediums,
Then bacterium solution PCR identifications are carried out；Positive sample with band is enlarged culture.
It prepares glycerol stock and carries out culture presevation
2.1 prepare the spare of 30% glycerine；
2.2 take the logarithm, and thalline were collected by centrifugation by bacterium solution 400 the μ L, 2000rpm in growth period；
2.3 add in the fresh LB fluid nutrient mediums of 200 μ L, and thalline is dispelled to form bacteria suspension；
2.4 add in 30% isometric glycerine, mixing.
Embodiment 3
The present embodiment provides a kind of detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene, pig chains
The detection method of the plasmid genetic stability of pneumoniae cells extrinsic factor gene is included the matter of Streptococcus suis extracellular factor gene
Grain is transformed into host strain cell, and the host strain of the plasmid containing Streptococcus suis extracellular factor gene then is made glycerine
Bacterium, by cultivating and passing on glycerol stock, the genetic stability of identification passage glycerol stock.
The competent cell of TOP10 is selected in the present embodiment as competent escherichia coli cell, certainly other real
Applying in mode can also select other competent escherichia coli cells to be tested, such as DH5 α cells or BL21 (DE3) cell.
The conversion of plasmid is as follows：
1.1 take the competent cell of 100 μ L Escherichia coli TOP10 to be added in EP pipes, and melt on ice；
1.2 take the plasmid (pEASY-Blunt-EF) for the Streptococcus suis extracellular factor gene that 4 μ L concentration are 2.1ng/ μ L
It is added in the competent cell of the Escherichia coli TOP10 melted in EP pipes, places 31min on ice；
1.3 by EP pipes under 42 DEG C of temperature conditionss heat shock 45s, by EP pipes fast transfer to cooled on ice 3min；
1.4 add in the LB liquid medium without antibiotic of 200 μ L, 150rpm cultures 45min in EP pipes；
1.5 take the bacterium solution even spread of 50 μ L to LB solid mediums (Amp+) on, 37 culture 16h.
The identification of positive host strain
From the LB solid mediums (Amp of step 1.5+) on the single spot of picking 10 respectively, cultivated in LB fluid nutrient mediums,
Then bacterium solution PCR identifications are carried out；Positive sample with band is enlarged culture.
Pcr amplification reaction body：2 μ L of bacterium solution are as template, 2.5 μ L of PCR reaction buffers, dNTPS0.5 μ L, sense primer
0.25 μ L, 0.25 μ L and Taq archaeal dna polymerase of anti-sense primer, 0.25 μ L, add water to supply to 25 μ L.Response procedures are：94 DEG C pre-
3min is denatured, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 40s, 35 cycle, 72 DEG C of extension 3min.
It prepares glycerol stock and carries out culture presevation
2.1 prepare the spare of 30% glycerine；
2.2 take the logarithm, and thalline were collected by centrifugation by bacterium solution 400 the μ L, 2000rpm in growth period；
2.3 add in the fresh LB fluid nutrient mediums of 200 μ L, and thalline is dispelled to form bacteria suspension；
2.4 add in 30% isometric glycerine, mixing.
Embodiment 4
The present embodiment provides a kind of detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene, this realities
Apply the glycerine for the Escherichia coli that the plasmid containing Streptococcus suis extracellular factor gene being prepared in embodiment 3 is taken in example
Bacterium is tested as strain, is as follows：
1.1 add in the LB fluid nutrient mediums (ampicillin containing 100mg/L) of 3mL in test tube；
1.2 are inoculated into glycerol stock strain in test tube according to 3% inoculum concentration；
1.3 under conditions of 37 DEG C 120rpm shaken cultivations；
1.4 carry out continuous passage culture, and the sample in the 5th generation, the 15th generation, the 25th generation and the 40th generation is taken to be tested respectively.
Colonial morphology compares
Appropriate eosin methylene blue culture medium is prepared, 121 DEG C of autoclave sterilization 15min prepare tablet in an aseptic environment,
5,15,25,40 generation bacterium solutions is taken to carry out line culture respectively, 37 DEG C of cultures for 24 hours, observe colonial morphology.
As a result as shown in Fig. 1 to Fig. 4, from colony morphological observation, in the 5th generation, the 15th generation, the 25th generation and the 40th generation, contain pig
The colonial morphology of the E. coli SampLes of the plasmid of Streptococcal cells extrinsic factor gene is consistent, without apparent difference, and equal energy
Single spot is enough grown, illustrates that the Escherichia coli of the plasmid containing Streptococcus suis extracellular factor gene still possess vigor.
PCR Molecular Identifications
Take E. coli SampLes that the 5th generation, the 15th generation, the 25th generation and the 40th generation contain Hepatitis E virus plasmid respectively into
Row PCR reacts, the PCR reaction systems and response procedures of PCR reaction systems and response procedures reference embodiment 3.
PCR reaction results are as shown in Fig. 5 to Fig. 8, stripe size 530bp, meet the size of expected purpose segment, M in figure
Represent DNAMarker, 1 is detection sample, and 2 be negative control；The PCR product electrophoresis knot of the Escherichia coli in 5 generations is passed in Fig. 5
Fruit, Fig. 6 are the PCR product electrophoresis result of the Escherichia coli in 15 generations of passage；The PCR of Escherichia coli in Fig. 7 for 25 generations of passage is produced
Object electrophoresis result, Fig. 8 are the PCR product electrophoresis results of the Escherichia coli in 40 generations of passage；Pass on 5 generations, 15 generations, 25 generations and 40 generations
Escherichia coli can still detect the plasmid containing Streptococcus suis extracellular factor gene, illustrate extracellular containing Streptococcus suis
The Escherichia coli of the plasmid of factor gene can keep its genetic stability in 5 generations of passage, 15 generations, 25 generations and 40 generations.
The biochemical reaction experiment of Escherichia coli
The Escherichia coli of plasmid containing Streptococcus suis extracellular factor gene are being passed on into 5 generations, 15 generations, 25 generations and 40
It is sampled after generation, carries out indole experiment, MR (methyl red) experiments, VP experiments and citrate experiment and identified.
The 5th generation biochemical test result such as Fig. 9 institutes of the Escherichia coli of plasmid containing Streptococcus suis extracellular factor gene
Show, it is the front and rear comparison diagram of indole experiment that wherein figure is opened on figure upper left side two, and it is comparison diagram before and after VP experiments that figure is opened in upper right side two；
It is the front and rear comparison diagram of MR experiments that wherein figure is opened in figure lower left two, and it is comparison diagram before and after citrate experiment that figure is opened in lower right two；
Its genetic stability can be kept in 5 generations of passage by showing the Escherichia coli of the plasmid containing Streptococcus suis extracellular factor gene.
The 15th generation biochemical test result of the Escherichia coli of plasmid containing Streptococcus suis extracellular factor gene is as schemed
Shown in 10, it is the front and rear comparison diagram of indole experiment that wherein figure is opened on figure upper left side two, and it is right before and after VP is tested that figure is opened in upper right side two
Than figure；It is the front and rear comparison diagram of MR experiments that wherein figure is opened in figure lower left two, and it is right before and after citrate is tested that figure is opened in lower right two
Than figure；Showing the Escherichia coli of the plasmid containing Streptococcus suis extracellular factor gene can keep its heredity steady in 15 generations of passage
It is qualitative.
The 25th generation biochemical test result of the Escherichia coli of plasmid containing Streptococcus suis extracellular factor gene is such as
Shown in Figure 11, it is the front and rear comparison diagram of indole experiment that wherein figure is opened on figure upper left side two, and it is before and after VP is tested that figure is opened in upper right side two
Comparison diagram；It is the front and rear comparison diagram of MR experiments that wherein figure is opened in figure lower left two, and it is before and after citrate is tested that figure is opened in lower right two
Comparison diagram；Its heredity can be kept in 25 generations of passage by showing the Escherichia coli of the plasmid containing Streptococcus suis extracellular factor gene
Stability.
The 40th generation biochemical test result of the Escherichia coli of plasmid containing Streptococcus suis extracellular factor gene is as schemed
Shown in 12, it is the front and rear comparison diagram of indole experiment that wherein figure is opened on figure upper left side two, and it is right before and after VP is tested that figure is opened in upper right side two
Than figure；It is the front and rear comparison diagram of MR experiments that wherein figure is opened in figure lower left two, and it is right before and after citrate is tested that figure is opened in lower right two
Than figure；Showing the Escherichia coli of the plasmid containing Streptococcus suis extracellular factor gene can keep its heredity steady in 40 generations of passage
It is qualitative.
Embodiment 5
The present embodiment provides a kind of detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene, this realities
The glycerol stock for applying the Escherichia coli for the plasmid that the Streptococcus suis extracellular factor gene being prepared in embodiment 3 is taken in example is made
It is tested, is as follows for strain：
1.1 add in the LB fluid nutrient mediums (ampicillin containing 100mg/L) of 3mL in test tube；
1.2 are inoculated into glycerol stock strain in test tube according to 10% inoculum concentration；
1.3 under conditions of 37 DEG C 120rpm shaken cultivations；
1.4 carry out continuous passage culture, and the sample in the 5th generation, the 15th generation, the 25th generation and the 40th generation is taken to be tested respectively.
The genetic stability detection of the Escherichia coli of the plasmid of the Streptococcus suis extracellular factor gene of continuous passage, including
Colonial morphology compares, Gram's staining and PCR identification and biochemistry detection.
In conclusion the embodiment of the present invention by the plasmid of Streptococcus suis extracellular factor gene by being transformed into large intestine bar
Then glycerol stock is made in bacterium, by cultivating glycerol stock and continuous passage, measure the Streptococcus suis extracellular factor gene in different generations
Plasmid Escherichia coli genetic stability, the research for Streptococcus suis extracellular factor gene provides reliable guarantee.
Embodiments described above is part of the embodiment of the present invention, instead of all the embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without creative efforts
Every other embodiment, belongs to the scope of protection of the invention.
SEQUENCE LISTING
<110>Hanbang Medical Science ＆ Technology Co., Ltd., Harbin City
<120>A kind of detection method of plasmid genetic stability of Streptococcus suis extracellular factor gene and application
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 548
<212> DNA
<213> Streptococcus suisserotype
<400> 1
catgctaaca cagtgacaga agcagagaca gctgtagcac cagctaacca agaccttgga 60
aatgagacta aaacggaaga agaacccaag gaaccaatcg aagcagttcg cacggacatg 120
gaaaaccgtg cagctgaaat cttgccggag gcgctgaatg ctagtgtaac aaaccaagca 180
ccagttattc cgactattgg agatcttcct aaagatgcga gtggtcagaa tgttcatggt 240
aaggcaacgg ataataagat ttatcgtgtt gtatacgttt ttggtaatgt agcagggact 300
acggagacag aagatggtaa acaaaatgtt gctccaacat ttaacagaaa tgatgcaact 360
aaaacttttc caatcacaga tccagatagc gacattcaaa ctatttcata cgaagttcca 420
gctgatattg caagctatac cttggatgat ccaaactcaa ttgttactaa tggcacctca 480
cctggtccag tatcttactt agatggtcca aatgggtcag ccactctcac acaagatggt 540
tatctaac 548
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
cacagtgaca gaagcagaga cag 23
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
catcttgtgt gagagtggct g 21
Claims (10)
1. a kind of detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene, which is characterized in that including：
The host strain bacterium solution of the plasmid containing Streptococcus suis extracellular factor gene is cultivated in fluid nutrient medium；
Host strain bacterium solution described in continuous passage culture；
Interval samples the host strain bacterium solution of the secondary culture and carries out genetic stability detection.
2. the detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene according to claim 1,
It is characterized in that, the inoculum concentration of the host strain bacterium solution is the 3%-7% of the volume of the fluid nutrient medium.
3. the detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene according to claim 1,
It is characterized in that, the host strain bacterium solution is glycerine bacterium solution.
4. the detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene according to claim 3,
It is characterized in that, the preparation of the glycerine bacterium solution includes：
The plasmid of the gene of extracellular factor containing Streptococcus suis is transformed into competent escherichia coli cell, by identifying that primer is examined
Identification is surveyed, obtains positive recombination bacillus coli；
The recombination bacillus coli is cultivated, and adds glycerine and glycerine culture presevation is made.
5. the detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene according to claim 4,
It is characterized in that, the competent escherichia coli cell is one kind in TOP10 cells, DH5 α cells and BL21 (DE3) cell.
6. the detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene according to claim 4,
It is characterized in that, the plasmid of the Streptococcus suis extracellular factor gene is 2- with the volume ratio of the competent escherichia coli cell
6:100。
7. the detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene according to claim 6,
It is characterized in that, the concentration of the Streptococcus suis extracellular factor gene is 2.5-7.1ng/ μ L.
8. the detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene according to claim 4,
It is characterized in that, the method for the conversion is：Mix the plasmid of the Streptococcus suis extracellular factor gene and the Escherichia coli
Competent cell places 25-37min on ice；Heat shock 40-60s, places 2-3min on ice.
9. the detection method of the plasmid genetic stability of Streptococcus suis extracellular factor gene according to claim 8,
It is characterized in that, the temperature of the heat shock is 42 DEG C.
10. the detection of the plasmid genetic stability such as claim 1-9 any one of them Streptococcus suis extracellular factor genes
Application of the method in the research of pig hemolysin gene.
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| CN201810007564.6A CN108103172A (en) | 2018-01-04 | 2018-01-04 | A kind of detection method of plasmid genetic stability of Streptococcus suis extracellular factor gene and application |
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| WO2002006493A1 (en) * | 2000-07-19 | 2002-01-24 | Cheil Jedang Corporation | Recombinant plasmid psp1, microorganisms transformed therewith, and method for producing an alkaline protease vapk |
| CN103555710A (en) * | 2013-10-29 | 2014-02-05 | 贵州省福泉市福中福农牧有限公司 | Method for improving stability of recombinant plasmid by chemical-mutagenesis saccharomyces cerevisiae genetically engineered bacterium |
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| WO2002006493A1 (en) * | 2000-07-19 | 2002-01-24 | Cheil Jedang Corporation | Recombinant plasmid psp1, microorganisms transformed therewith, and method for producing an alkaline protease vapk |
| CN103555710A (en) * | 2013-10-29 | 2014-02-05 | 贵州省福泉市福中福农牧有限公司 | Method for improving stability of recombinant plasmid by chemical-mutagenesis saccharomyces cerevisiae genetically engineered bacterium |
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