CN103966248A - Bacterium gene point mutation molecular cloning technology based on homologus recombination - Google Patents
Bacterium gene point mutation molecular cloning technology based on homologus recombination Download PDFInfo
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Abstract
The invention relates to a bacterium gene point mutation molecular cloning technology based on homologus recombination. According to the technology, a gene segment C is inserted between a target original bacterium gene A and a gene B adjacent to the gene A, the insertion site of the inserted gene segment is positioned between promoters of the target gene A and the gene B adjacent to the gene A, and the expression of the two genes is not influenced; a plasmid is taken as a carrier, and is converted after double enzyme digestion and connection, so that the two segments are connected with the gene segment C to obtain a new plasmid; finally, the new plasmid is converted into an original bacterium. The situation that the target gene can be directly subjected to point mutation in the original bacterium is realized, the living environment of genes is not changed, and the expression of the genes is not influenced.
Description
Technical field
The present invention relates to molecular biosciences field, especially a kind of bacterial gene point mutation molecule clone technology based on homologous recombination.
Background technology
Point mutation (Gene Point Mutation), for 1 or 1 pair of base in DNA chain change, comprises base substitution (conversion, transversion) and phase shift mutation (loss or 1 of slotting people or several base).Point mutation is to cause one of important factor of bacterium proterties change, as point mutation causes the variation of microbiotic target position enzyme, thereby causes bacterium to produce antibiotics resistance.As the point mutation of: (1) bacterium Quinolone resistance determining region (QRDRs) coding target enzyme, cause the change of drug effect target position, reduce the avidity of quinolones and resistance.(2) the 45th Gly of gonococcus mtrR gene (GGC → GAC) Asp, 14 His (CAC → CGC) Arg and the 51st Phe (TTC → GTC) Val sudden change are closely related with the multi-drug resistant of gonococcus epidemic strain.(3) 23SrRNA Gene A 2143G point mutation is relevant to the resistance of clarithromycin to helicobacter pylori.(4) point mutation of rpsL gene is closely related to Rifampin, ethambutol, vazadrine and Streptomycin sulphate resistance with mycobacterium tuberculosis respectively.
Mainly to adopt order-checking, native polyacrylamide gel electrophoresis (PAGE) to the research of point mutation at present, PCR-SSCP (strand conformation polymorphism analysis, Single-Strand ConformationPolymorphism) etc. method carry out point mutation mensuration, by carrying and the comparative analysis of not carrying point mutation bacterial strain object proterties, assert the mechanism of action of point mutation.But this method can only, by point mutation bacterial strain and the not proterties comparison between point mutation bacterial strain, infer that point mutation is relevant to proterties, can not study intuitively the result of point mutation.In addition, also there is report, in protein function research, adopt point mutation test kit to build mutation gene, study the function of point mutation in order to study the variation of point mutation front and back protein function.But the proterties of bacterium as drug-resistant intensity be the complex process that polygene, polyprotein participate in, and this method is confined to indivedual genes and albumen, is difficult to whole phenomenons of explaining that point mutation causes in bacterium internal medium.So the Perfected process of research point mutation function is: directly goal gene is carried out to point mutation in original bacterium, and do not change the living environment of gene, but also do not have a kind of desirable technology to carry out point mutation to bacterium at present.
Homologous recombination (Homologus Recombination) refers to and occurs between the DNA molecular that contains homologous sequence between sister chromatid or on same karyomit(e) or reconfiguring within molecule, and it is also one of method of gene knockout.There is at present the mode of employing " upstream fragment-insertion gene-downstream fragment " to make target gene inactivation.On homologous recombination principle, also can be applied to and build in point mutation bacterial strain.But key is the fragment of homologous recombination to original bacterium, except carrying point mutation, can not disconnects any gene and not affect gene (containing goal gene) and express.
Summary of the invention
The object of the present invention is to provide a kind of bacterial gene point mutation molecule clone technology based on homologous recombination, realize and directly goal gene is carried out to point mutation in original bacterium, and do not change the living environment of gene, do not affect the expression of gene.
In order to realize foregoing invention object, the present invention adopts following technical scheme:
A kind of bacterial gene point mutation molecule clone technology based on homologous recombination, be adjacent and between gene B, insert gene fragment C in the original bacterium Gene A of object, the insertion point of gene fragment of inserting, between the promotor of goal gene A gene B adjacent with it, and does not affect the expression of 2 genes; Then adopt 2 fragments of PCR method amplification, one of them fragment is: the inner A of goal gene starts to the upstream fragment between goal gene A promotor and B gene promoter; Another fragment is: starting to the downstream fragment of B gene inside between A gene promoter and B gene promoter; Taking plasmid as carrier, transform two above-mentioned fragments are connected with gene fragment C and obtain a new plasmid through double digestion and after connecting again, this plasmid is containing " fragment-gene fragment C-downstream, upstream fragment "; Finally new plasmid is converted into original bacterium.
The present invention includes following steps:
(1) choosing of cloning vector and bacterial strain: choose suitable plasmid vector, confirm its restriction enzyme site, select suitable bacterial strain as clone bacterium;
(2) selection of insertion gene fragment;
(3) preparation of upstream fragment and downstream fragment: for goal gene point mutation position and insertion gene fragment position and adjacent gene design primer; Every upstream and downstream primer adds corresponding restriction enzyme site and protection base, and the upstream primer of the downstream primer of upstream fragment and downstream fragment is base sequence complementation, and primer location is adjacent at goal gene between the promotor of gene;
(4) preparation of point mutation bacterial strain after homologous recombination: upstream fragment, insertion gene fragment, downstream fragment are converted into clone bacterium through 3 double digestions and after connecting respectively, and obtaining two ends is the plasmid of antibiotic-screening gene containing original bacterium gene fragment, centre.
Described original bacterium is Shigella flexneri, intestinal bacteria, salmonella, cholera bacteria or campylobacter jejuni etc.
Described insertion gene fragment is antibiotic-screening gene.
A kind of bacterial gene point mutation molecule clone technology based on homologous recombination of the present invention, realizes and directly goal gene is carried out to point mutation in original bacterium, and do not change the living environment of gene, does not affect the expression of gene.Bacterial gene point mutation is to cause one of important factor of bacterium proterties change.The invention provides a kind of molecule clone technology of bacterial gene point mutation, described technology verified and succeeded by the gyrA point mutation of Shigella flexneri, and this technology can promote the use of other bacterium as bacteriums such as intestinal bacteria, salmonella, cholera, campylobacter jejunis.This technology can build in laboratory a point mutation bacterial strain with 10 days left and right time, can promote the use of in laboratory.
Brief description of the drawings
Fig. 1: build gyrA point mutation schematic diagram; (A). the site that kantlex drug resistant gene inserts, arrow represents the position (gyrAF1 (F1), gyrAR1 (R1), gyrAF2 (F2), gyrAR2 (R2)) of primer; (B) sequence of gyrA transgenation point.
Embodiment
Below by specific embodiment, the present invention is described further, but the present invention is not by embodiment is limited.
Embodiment 1: bacillary dysentery is a kind of serious enteric infection disease, and Epidemic Scope is wide, velocity of propagation is fast, sickness rate is high, the annual morbidity approximately 1.65 hundred million in the whole world, wherein 1.63 hundred million occurs in developing country, annual dead 1,000,000 people.Shigella is the pathogenic bacteria of bacillary dysentery, and fluoroquinolones (taking Ciprofloxacin as representative) is the choice drug that treatment bacillary dysentery is recommended by the World Health Organization (WHO, 2004).But shigella fluoroquinolone resistance is and increases the weight of trend, now there are some researches show the closely related of Shigella flexneri quinolones resistance and gyrA Ser-83 → Leu (TCG → TTG) sudden change, also have certain relation with the point mutation in 87 sites, but the point mutation in shigella gyrA Ser-83 → Leu and 87 sites directly causes it also not come to a conclusion to the much variations of Ciprofloxacin MIC.So this research department is by building point mutation (83 site TCG → TTG of gyrA gene, 87 site GAC → GGC) bacterial strain, the impact with the point mutation of studying 2 sites on fluorine quinoline promise resistance, specific as follows:
The gyrA point mutation bacterial strain of artificial constructed Shigella flexneri 2a301.Adopt the method for antibiotic-screening homologous recombination to build the gyrA point mutation bacterial strain of Shigella flexneri 2a301, kantlex drug resistant gene box inserts between gyrA gene and its downstream gene (ubiG), as shown in Figure 1, working method is (time that completes 5 stages is about 10 days) as follows for primer and drug resistant gene box insertion point:
1. downstream fragment proceeds to plasmid.Taking Shigella flexneri 2a301 as template, amplify PCR product fragment (792bp, downstream primer) with primer gyrAF2 and gyrAR2 in table 2; Downstream fragment and plasmid pUC19, after KpnI and EcoRI double digestion, connect and are converted into bacillus coli DH 5 alpha, and producing bacterial strain pUC19:gyrAF2R2 confirms after pcr amplification.
2. antibiotic-screening gene proceeds to plasmid, and is connected with downstream fragment.With KanF and KanR primer amplification aphA3 gene (kantlex resistance screening-gene), extract pUC19:gyrAF2R2 plasmid.PCR product and plasmid with KpnI and XbaI double digestion after, connect and be converted into bacillus coli DH 5 alpha, screen with the LB substratum containing kantlex, produce pUC19:gyrAF2R2:Kan bacterial strain, after pcr amplification, confirm.
3. upstream fragment-antibiotic-screening gene-downstream fragment is connected.Bacterial strain 1 and bacterial strain 13 (in table 1) are Hangzhou fluorine quinoline promise resistance shigella strain isolated, wherein 83 sites of the gyrA gene of bacterial strain 13 ((TCG → TTG, Serine → leucine); Bacterial strain 1, except 83 sites, also has sudden change (GAC → GGC, aspartic acid → glycine) on 87 sites.1. taking gyrAF1 and gyrAR1 as primer, respectively taking 3 bacterial strains such as Fu Shi 2a301, bacterial strain 1, bacterial strains 13 as pcr template, amplification obtains 3 kinds of upstream fragments.2. extract pUC19:gyrAF2R2:Kan plasmid, plasmid and 3 kinds of above upstream fragments are converted into bacillus coli DH 5 alpha through XbaI and the connection of PstI double digestion, to screen containing kantlex 50 μ g ml-1 and containing the flat board of Ampicillin Trihydrate 100 μ g ml-1.3. obtain the bacterial strain of 3 types by 3 kinds of above upstream fragment clones, be respectively pUC19:gyrAF2R2:Kan:gyrAF1R1-301 (upstream fragment is taking Fu Shi 2a301 as template), pUC19:gyrAF2R2:Kan:gyrAF1R1-1 (upstream fragment is taking bacterial strain 1 as template), pUC19:gyrAF2R2:Kan:gyrAF1R1-13 (upstream fragment is taking bacterial strain 13 as template).
4. " upstream fragment-antibiotic-screening gene-downstream fragment " homologous recombination, to original bacterium, obtains point mutation strain.1. taking bacterial strain pUC19:gyrAF2R2:Kan:gyrAF1R1-301/1/13 as template, taking gyrAR1 and gyrAR2 as primer, amplify 2560bpPCR fragment.2. after purifying, electricity is converted in Shigella flexneri 2a301, with LB plate screening homologous recombination containing kantlex 50 μ g ml-1, wherein the bacterial strain of the acquisition taking pUC19:gyrAF2R2:Kan:gyrAF1R1-301 as template is Kan301 (not contrasting strain containing the screening antibiotics resistance of point mutation); The bacterial strain obtaining taking bacterial strain 1 and 13 as template is Kan301:83Leu87Gly and Kan301:83Leu (containing the screening antibiotics resistance mutant strain of point mutation).3. after pcr amplification order-checking, confirm said mutation strain.
5. pair point mutation bacterial strain carries out the research of object proterties.Obtain 4 bacterial strains such as 1 original bacterium, 1 contrast strain and 2 point mutation strains by above step, be specially: S.flexneri2a301 (original bacterium), Kan301 (does not contain the screening antibiotics resistance of point mutation, contrast strain), Kan301:83Leu (containing the screening antibiotics resistance mutant strain 1 of 83 locus gene point mutation), Kan301:83Leu87Gly (containing the screening antibiotics resistance mutant strain 2 of 83 and 87 dibit point gene point mutation).
Adopt constant broth dilution method, measure the minimal inhibitory concentration (MIC) of above-mentioned 4 class bacterial strains to fluoroquinolone antibiotics Ciprofloxacin, result shows: S.flexneri2a301 (original bacterium) and Kan301 bacterial strain (contrast strain) are all 0.03125 μ gmL to the MIC of Ciprofloxacin
-1.We think that between gyrA and ubiG gene, inserting kantlex screens antibiotic resistance gene, does not affect object proterties (i.e. the MIC on Ciprofloxacin).And Kan301:83Leu (mutant strain 1) and Kan301:83Leu87Gly bacterial strain (mutant strain 2) are all 0.125 μ gmL-1 to the MIC of Ciprofloxacin, be S.flexneri2a301 (original bacterium) and Kan301 bacterial strain (contrast strain) MIC 4 times.
Based on above result, reach a conclusion: 1. the point mutation in Shigella flexneri gyrA gene 83 sites directly cause bacterial strain to the MIC of Ciprofloxacin from 0.03125 μ gmL
-1rise to 0.125 μ g mL
-1.2. the whether point mutation in gyrA gene 87 sites, the impact of the point mutation that does not directly affect gyrA gene 83 sites on MIC.
Bacterial strain used in this research of table 1
| Shigella flexneri title | General introduction |
| S.flexneri2a301 | Clinical separation strain, NCBI sequence number: NC_004337 |
| Kan301 | Do not contain the kantlex resistance S.flexneri2a301 mutant strain of point mutation |
| Kan301：83Leu | Containing the kantlex resistance S.flexneri2a301 mutant strain of 83 locus gene point mutation |
| Kan301：83Leu87Gly | Containing the kantlex resistance S.flexneri2a301 mutant strain of 83 and 87 dibit point gene point mutation |
| 1 | In Hangzhou Region of Zhe Jiang Province fluoroquinolone resistance Shigella flexneri clinical separation strain (gyrA83:TTG; 87:GGC) |
| 13 | In Hangzhou Region of Zhe Jiang Province fluoroquinolone resistance Shigella flexneri clinical separation strain (gyrA83:TTG; ) |
Primer sequence used in this research of table 2
arepresent restriction enzyme site.
The present embodiment is verified and is succeeded by the gyrA point mutation of Shigella flexneri, realize in original bacterium and directly goal gene has been carried out to point mutation, and do not change the living environment of gene, do not affect the expression of gene, use 10 days left and right time can build in laboratory a point mutation bacterial strain by the technology of the present invention, can promote the use of in laboratory.
Claims (4)
1. the bacterial gene point mutation molecule clone technology based on homologous recombination, it is characterized in that being adjacent and between gene B, inserting gene fragment C in the original bacterium Gene A of object, the insertion point of gene fragment of inserting, between the promotor of goal gene A gene B adjacent with it, and does not affect the expression of 2 genes; Then adopt 2 fragments of PCR method amplification, one of them fragment is: the inner A of goal gene starts to the upstream fragment between goal gene A promotor and B gene promoter; Another fragment is: starting to the downstream fragment of B gene inside between A gene promoter and B gene promoter; Taking plasmid as carrier, transform two above-mentioned fragments are connected with gene fragment C and obtain a new plasmid through double digestion and after connecting again, this plasmid is containing " fragment-gene fragment C-downstream, upstream fragment "; Finally new plasmid is converted into original bacterium.
2. a kind of bacterial gene point mutation molecule clone technology based on homologous recombination according to claim 1, is characterized in that comprising the steps:
(1) choosing of cloning vector and bacterial strain: choose suitable plasmid vector, confirm its restriction enzyme site, select suitable bacterial strain as clone bacterium;
(2) selection of insertion gene fragment;
(3) preparation of upstream fragment and downstream fragment: for goal gene point mutation position and insertion gene fragment position and adjacent gene design primer; Every upstream and downstream primer adds corresponding restriction enzyme site and protection base, and the upstream primer of the downstream primer of upstream fragment and downstream fragment is base sequence complementation, and primer location is adjacent at goal gene between the promotor of gene;
(4) preparation of point mutation bacterial strain after homologous recombination: upstream fragment, insertion gene fragment, downstream fragment are converted into clone bacterium through 3 double digestions and after connecting respectively, and obtaining two ends is the plasmid of antibiotic-screening gene containing original bacterium gene fragment, centre.
3. a kind of bacterial gene point mutation molecule clone technology based on homologous recombination according to claim 1 and 2, is characterized in that described original bacterium is Shigella flexneri, intestinal bacteria, salmonella, cholera bacteria or campylobacter jejuni.
4. a kind of bacterial gene point mutation molecule clone technology based on homologous recombination according to claim 1 and 2, is characterized in that described insertion gene fragment is antibiotic-screening gene.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1321184C (en) * | 1994-03-15 | 2007-06-13 | 中山大学 | Anti-glyphosate gene obtained by gene optimization and its expression carrier |
| CN1995331A (en) * | 2006-12-29 | 2007-07-11 | 东北农业大学 | Baby pig hydropsy colon bacius gene mutant and its construction method |
| CN102703424A (en) * | 2012-06-19 | 2012-10-03 | 南京师范大学 | Method for recombination-mediated escherichia coli genome point mutation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1321184C (en) * | 1994-03-15 | 2007-06-13 | 中山大学 | Anti-glyphosate gene obtained by gene optimization and its expression carrier |
| CN1995331A (en) * | 2006-12-29 | 2007-07-11 | 东北农业大学 | Baby pig hydropsy colon bacius gene mutant and its construction method |
| CN102703424A (en) * | 2012-06-19 | 2012-10-03 | 南京师范大学 | Method for recombination-mediated escherichia coli genome point mutation |
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Application publication date: 20140806 |