CN108841849A - Carrier and method for riemerella anatipestifer genome point mutation - Google Patents
Carrier and method for riemerella anatipestifer genome point mutation Download PDFInfo
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- CN108841849A CN108841849A CN201810367550.5A CN201810367550A CN108841849A CN 108841849 A CN108841849 A CN 108841849A CN 201810367550 A CN201810367550 A CN 201810367550A CN 108841849 A CN108841849 A CN 108841849A
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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Abstract
The present invention relates to a kind of carrier and method for riemerella anatipestifer genome point mutation, the carrier obtains plasmid pBAD24 by cfx gene cloning to plasmid pBAD24 restriction enzyme site KpnI and XbaI::Then EXpheS* is connected into plasmid pBAD24 again by cfx::At XbaI the and SalI restriction enzyme site of cfx, the template vector of gene mutation is obtained, pBAD24 is named as::cfx+EXpheS*；Then mutated gene upstream sequence and mutated gene downstream sequence are connected into pBAD24 respectively::The cfx upstream region of gene of cfx+EXpheS* carrier and the downstream EXpheS* obtain the carrier for being used for the point mutation of riemerella anatipestifer genome；The carrier is capable of the carry out riemerella anatipestifer genome point mutation of simple and effective, and then can be used for the research of the gene function of riemerella anatipestifer.
Description
Technical field
The invention belongs to field of biotechnology, it is related to also relating to for the carrier of riemerella anatipestifer genome point mutation
And using the carrier to the method for riemerella anatipestifer genome point mutation.
Background technique
Riemerella anatipestifer is a kind of important birds pathogenic bacteria.At present due to lacking corresponding gene editing work
Have, so the gene function and pathogenic mechanism research to riemerella anatipestifer are less and do not go deep into.Gene editing technology is to grinding
The function of studying carefully gene is most important.Be presently available for riemerella anatipestifer gene editing technology be mainly gene missing and
Covering, however if necessary to studying the key area or key amino acid of functional gene, then need to functional gene into
Row point mutation.One of method is that point mutation is generated by the method for over-lap PCR, then by this gene containing mutation sites
It is cloned into shuttle plasmid and transgene gene-deleted strain is studied.However after this method can not reflect point mutation completely
Genome itself the case where.In addition, there is the risk lost in a replication process in shuttle plasmid.For the defect more than overcoming,
It is badly in need of a kind of method for riemerella anatipestifer genome point mutation.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of loads for riemerella anatipestifer genome point mutation
Body；The second object of the present invention is to provide the method using the carrier in riemerella anatipestifer genome point mutation.
In order to achieve the above objectives, the present invention provides the following technical solutions：
1. being used for the carrier of riemerella anatipestifer genome point mutation, the carrier is by cfx gene cloning to plasmid
PBAD24 restriction enzyme site KpnI and XbaI obtain plasmid pBAD24::Then EXpheS* is connected into plasmid pBAD24 again by cfx::
At XbaI the and SalI restriction enzyme site of cfx, the template vector of gene mutation is obtained, pBAD24 is named as::cfx+EXpheS*；So
Mutated gene upstream sequence and mutated gene downstream sequence are connected into pBAD24 respectively afterwards::The cfx base of cfx+EXpheS* carrier
Because of upstream and the downstream EXpheS*, that is, obtain the carrier for being used for the point mutation of riemerella anatipestifer genome.
Preferably, the EXpheS* is by with pLMF03::PheS* plasmid is as template, SEQ ID NO.8 and SEQ ID
Sequence shown in NO.9 is that primer carries out PCR amplification, i.e. acquisition EXpheS*；The pLMF03::PheS* plasmid is by such as SEQ ID
Sequence shown in NO.5 is connected into shuttle plasmid pLMF03 and obtains.
Preferably, the cfx gene is made by following methods：Using plasmid pLMF03 as template, SEQ ID NO.6 and
Sequence shown in SEQ ID NO.7 is that primer carries out PCR amplification, obtains cfx gene.
Preferably, mutated gene upstream sequence sequence as shown in SEQ ID NO.10 and SEQ ID NO.11 is to draw
Object, riemerella anatipestifer genomic DNA are that template carries out PCR amplification and obtains；The mutated gene downstream sequence is by SEQ ID
Sequence shown in NO.12 and SEQ ID NO.13 be primer, riemerella anatipestifer genomic DNA be template carry out PCR amplification and
?.
2. mutated gene will be contained in carrier in the method for riemerella anatipestifer genome point mutation using the carrier
Upstream sequence, cfx gene, EXpheS* and mutated gene downstream sequence sequence amplification come out, the method for passing through Natural Transformation turns
Enter in riemerella anatipestifer wild strain RA ATCC, be coated on the GCB plate containing 1 μ g/ml Cefoxitin cfx, is screened
The positive colony of first time homologous recombination out is named as RA ATCC Δ RA0C_1912::Cfx+EXpheS* will contain mutation base
Because the sequence of point mutation is transferred to RA ATCC Δ RA0C_1912 by the method for Natural Transformation::In cfx+EXpheS*, applied
On the GCB plate containing 13mM p-cl-Phe, the positive colony of second of homologous recombination is filtered out.
Preferably, the sequence containing mutated gene upstream sequence, cfx gene, EXpheS* and mutated gene downstream sequence
Column are by using the carrier as template, sequence shown in SEQ ID NO.10 and SEQ ID NO.12 is primer amplification acquisition.
Preferably, the sequence containing mutated gene point mutation is obtained by following methods：Respectively with SEQ ID NO.13 and SEQ
ID NO.14, SEQ ID NO.15 and SEQ ID NO.16 are primer, and riemerella anatipestifer genomic DNA is template progress
After PCR amplification obtains product, then using amplified production as template, SEQ ID NO.13 and SEQ ID NO.16 is that primer is melted
It closes PCR and obtains catastrophe point mutant nucleotide sequence.
The beneficial effects of the present invention are：The present invention disclose for riemerella anatipestifer genome point mutation carrier and
Method provides a kind of method that simple and effective carries out the point mutation of riemerella anatipestifer genome, and then can be used for pest of duck
In Mo Shi bacillus gene function research.And this method has biological generality, for the gene function for studying other bacteriums
Provide Technical Reference.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out
Explanation：
Fig. 1 is that (1 swimming lane indicates pheSup segment to amplification pheSup and pheSdown fragment electrophoretic figure, and 2 swimming lanes indicate
PheSdown segment, M indicate Marker).
Fig. 2 is that (1 swimming lane is fusion segment to fusion DNA vaccine electrophoresis result in figure；M indicates Marker).
Fig. 3 is recombinant plasmid pLMF03::PheS* qualification result such as figure (A：PCR qualification result is carried out, 1 swimming lane is in figure
Recombinant plasmid pLMF03::pheS*；B：Double digestion identification, restriction enzyme site NcoI, SpeI；1 swimming lane is double digestion recombinant plasmid
pLMF03::PheS*, 2 swimming lanes are double digestion empty plasmid pLMF03；3 swimming lanes are the empty plasmid without carrying out digestion
PLMF03, M indicate Marker).
Fig. 4 is bacterial strain R.anatipestife pLMF03 and R.anatipestife pLMF03::PheS* is to p-Cl-
(No. 1 is R.anatipestifer pLMF03 to the sensibility qualification result of Phe in figure, and No. 2 are R.anatipestifer
pLMF03::pheS*)。
Fig. 5 is pBAD24::Cfx+EXpheS* plasmid construction flow chart.
Fig. 6 is pBAD24::Cfx and pBAD24::Qualification result (the A of cfx+EXpheS*:With KpnI and XbaI double digestion
Plasmid pBAD24::Cfx, 1 swimming lane are the electrophoretogram carried out after double digestion；B：With XbaI and SalI double digestion plasmid
pBAD24::Cfx+EXpheS*, 1 swimming lane are the electrophoretogram carried out after double digestion, and M indicates Marker).
Fig. 7 is that (1 swimming lane is amplified fragments RA0C_1912up+cfx+EXpheS*+RA0C_ to recombination plasmids detection result
1912down；M indicates Marker).
Fig. 8 is positive colony RA ATCC Δ RA0C_1912::(1,2 swimming lanes distinguish table to cfx+EXpheS*PCR testing result
It is shown as wild strain RA ATCC and cfx resistant mutant strain RA ATCC Δ RA0C_1912::cfx+EXpheS*；A figure is 16SrDNA,
B figure is RA0C_1912, and C figure is cfx+EXpheS*).
Fig. 9 is that warm segment PCR schemes (fusion DNA vaccine electrophoretogram：1 swimming lane is RA0C_1912up+RA0C_1912*+RA0C_
1912down segment).
Figure 10 is that (odd number swimming lane is RA ATCC Δ to positive colony RA ATCC11845RA0C_1912*PCR testing result
RA0C_1912::Cfx+EXpheS*, even number swimming lane be RA ATCC11845RA0C_1912*, 1,2 swimming lanes be 16S rDNA, 3,4
Swimming lane is RA0C_1912*, and 5,6 swimming lanes are cfx+EXpheS*).
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.
The present invention utilizes the mutant of the pheS gene on riemerella anatipestifer R.anatipestife ATCC genome
As the label that reversely screens of one kind, and then invents a kind of template plasmid containing the reversed selection markers and utilize this template matter
Grain invents a kind of method in genome point mutation.Its principle is：PheS is α-subunit of phenylalanyl tRNA synthase,
After 301 alanine (A) of PheS sport glycine (G), the substrate specificity of Phenylalanyl-tRNA synthetase will be reduced
Property, the halogenated analogs fenclonine (p-cl-Phe) of phenylalanine will be transported to by Phenylalanyl-tRNA synthetase
In cell, and p-cl-Phe effect toxic to cell will lead to cell death.Therefore after adding p-cl-Phe in the medium,
The bacterium for carrying the pheS gene of mutation will be dead.
The template plasmid containing pheS mutated gene is constructed, by the method for Natural Transformation, using homologous recombination and reversely
The principle of screening to carry out point mutation to riemerella anatipestifer genome.
The present invention by riemerella anatipestifer R.anatipestife ATCC be mutated RA0C_1912 gene for, for
Bright concrete operation method of the invention.
The generation of embodiment 1.pheS mutated gene
Primer pheS up p1 is used respectively：5'-catgccatggcaatgttagaatacattgacgcatatc-3'(SEQ
ID NO.1), pheS up p2：5'-cccataccaaatccatagccg-3'(SEQ ID NO.2)；pheS down p1:5'-
Cggctatggatttggtatggg-3 ' (SEQ ID NO.3), pheS down p2:5'-
Ggactagtcccagacttgttgattttggacg-3 ' (SEQ ID NO.4) amplify pheSup part (914bp) and
The part pheSdown (418bp), PCR amplification condition are as follows：(1) 98 DEG C of initial denaturation 30s；(2) 98 DEG C of denaturation 10s, (3) 55 DEG C are moved back
Fiery 30s, (4) 72 DEG C of extension 30s, (2)-(4) recycle 30 times；(5) 72 DEG C of extensions 7min, (6) 22 DEG C of preservation ∞；Amplification is such as
Shown in Fig. 1, then the two segments are merged using the method for fusion DNA vaccine, fusion results are as shown in Fig. 2, obtain pheS
Mutated gene, i.e. pheS*, nucleotide sequence as shown in SEQ ID NO.5, wherein coding amino acid the 301st bit codon by
GCT sports GGA.
Embodiment 2. examines whether the mutated gene can be used as reversed selection markers
Fusion segment is cloned into shuttle plasmid pLMF03 (Genbank：KU997673 in), recombinant plasmid pLM is obtained
F03::PheS*, using sequence shown in SEQ ID NO.1 and SEQ ID NO.4 carry out PCR amplification, and with NcoI and Sp e I into
Row digestion identification, qualification result are as shown in Figure 3.R.anatipestife A is transferred to by way of engagement transfer again
TCC, while to turn shuttle plasmid pLMF03 empty carrier as control, respectively obtain bacterial strain R.anatipestife pLMF03::
PheS* and bacterial strain R.anatipestife pLMF03.Respectively by bacterial strain R.anatipestife pLMF03 with
R.anatipestife pLMF03::PheS* is in the GCB/cfx of various concentration p-cl-Phe (0mM, 10mM, 13mM), and 37 DEG C
Incubator culture 18h, as a result as shown in Figure 4.The results show that carrying pheS mutant when p-cl-Phe concentration is 13mM
Bacterial strain do not grow, and compare well-grown, show R.anatipestife pLMF03::PheS* is shown to p-Cl-Phe
Sensibility, and it is insensitive to p-Cl-Phe to compare R.anatipestife pLMF03.Therefore, mutated gene pheS* can make
For the label reversely screened.
Embodiment 3. is used for the template vector pBAD24 of gene mutation::The building of cfx+EXpheS*
It uses plasmid pLMF03 as template, uses primer：cfx p1：5'-
Cggggtacctgaccccgaagcagggttatgc-3 ' (SEQ ID NO.6), cfx p2：5'-
gctctagagcaaagcaagtgcagtttaagattttactg-3'(SEQ ID NO.7)；The cfx of tape starting is amplified
Carry out and use pLMF03::PheS* is as template primer EXpheS*p1：5'-
Gctctagagcatttcaaaaatttaacttaaaccactg-3 ' (SEQ ID NO.8), EXpheS*p2：5'-
Acgcgtcgaccctttttttgttacttatagcg-3 ' (SEQ ID NO.9), by the High-expression promoter of the plasmid and
PheS* is amplified together to be come, and product is named as EXpheS*.Cfx segment is first cloned into plasmid pBAD24, digestion position later
Point is KpnI and XbaI, which is named as pBAD24::Cfx, digestion verification result is as shown in A in Fig. 6.Again by EXpheS* piece
Section is cloned into pBAD24::Cfx, restriction enzyme site are XbaI and SalI, which is named as pBAD24::Cfx+EXpheS*, digestion
For verification result as shown in B in Fig. 6, specific building process is as shown in Figure 5.
The foundation of embodiment 4, riemerella anatipestifer genome point mutation process
For by RA0C_1912 gene mutation
(1) upstream of the gene about 800bp and downstream about 800bp come out to (primer is respectively by corresponding primer amplification：
RA0C_1912upP1:5'-ctagctagctagggcatttatggtacaatagaactag-3'(SEQ ID NO.10)；RA0C_
1912upP2:5'-cggggtaccccgtaggtactattattttctag-3'(SEQ ID NO.11)；RA0C_
1912downP1:5'-aactgcagaaccaatgcattggtgaggctaattatactcgtac-3'(SEQ ID NO.12)；
RA0C_1912downP2:5'-cccaagcttgggcccttagctgtaccgtagcctg-3'(SEQ ID NO.13)；Then will
Fragment upstream and segments downstream are cloned into pBAD24 respectively::On cfx+EXpheS*, recombinant plasmid pBAD24 is constructed::RA0C_
1912up+cfx+EXpheS*+RA0C_1912down.The recombinant plasmid is used to go out segment RA0C_1912up+ as template amplification
Cfx+EXpheS*+RA0C_1912down, result figure is as shown with 7.
(2) RA0C_1912up+cfx+EXpheS*+RA0C_1912down segment is transferred to by the method for Natural Transformation
In riemerella anatipestifer wild strain RA ATCC.It is coated on the GCB plate containing 1 μ g/ml Cefoxitin cfx, is filtered out
The positive colony RA ATCC Δ RA0C_1912 of first time homologous recombination::Cfx+EXpheS*, and 16S is identified with the method for PCR
RDNA, RA0C_1912 and cfx+EXpheS*, as a result as shown in Figure 8.
(3) utilize the method for fusion DNA vaccine by the RA0C_1912 (RA0C_1912*) of mutation and the upstream of the gene
767bp and downstream 841bp is fused into RA0C_1912up+RA0C_1912*+RA0C_1912down segment, primer RA0C_
1912upP1：5 '-gctaatgtgataaaatttcacgagggcgg-3 ' (SEQ ID NO.14), RA0C_1912*upP2：5'-
gaacatttttaagtagatgacctc-3'(SEQ ID NO.15)；RA0C_1912*downP1：5'-
Gaggtcatctacttaaaaatgttc-3 ' (SEQ ID NO.16), RA0C_1912downP2：5'-
Cccttagctgtaccgtagcctgtattttgg-3 ' (SEQ ID NO.17), amplification is as shown in figure 9, obtained fusion
The AAT of script is sported termination codon TAA in fusion sequence as shown in SEQ ID NO.18 by segment.
(4) RA0C_1912up+RA0C_1912*+RA0C_1912down segment is transferred to cf by the method for Natural Transformation
X resistant mutant strain RA ATCC Δ RA0C_1912::In cfx+EXpheS*.It is coated in the G CB containing 13mM p-cl-Phe
On plate, the positive colony RA ATCC11845RA0C_1912* of second of homologous recombination is filtered out, and reflected with the method for PCR
Fixed, electrophoretogram is as shown in Figure 10.
(5) raw work is sent to be sequenced the RA0C_1912* sequence of amplification, the results showed that the mutational site RA0C_1912 with design
Unanimously, determine the success of RA0C_1912 point mutation.
The above result shows that the segment of EXpheS* gene will be contained as reversed screening mark using the method for Natural Transformation
Note can be used for the point mutation of riemerella anatipestifer genome.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
<110>Sichuan Agricultural University
<120>Carrier and method for riemerella anatipestifer genome point mutation
<160> 18
<170> SIPOSequenceListing 1.0
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<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 1
catgccatgg caatgttaga atacattgac gcatatc 37
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<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 2
cccataccaa atccatagcc g 21
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 3
cggctatgga tttggtatgg g 21
<210> 4
<211> 31
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 4
ggactagtcc cagacttgtt gattttggac g 31
<210> 5
<211> 1311
<212> DNA
<213>Artificial sequence (Artificial sequence)
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atgttagaat acattgacgc atatctacaa gaggtaaagc aatttcagtc ttcaaacaaa 60
gatgaaatag agcaattcag aataaaattt aacggtaaaa aaggaatttt aaatgccatc 120
tttgatgagt ttaaacaagt tccgaacgaa caaaaaaagg cttttggaca aaaaataaat 180
gttctaaaac aagctgttgc cgaaaaacta gaagagctta aaaatgcaac cgcctctagt 240
attgttgtag aaaaagaaga tttaactcgt cctgggtatc ctttggaatt ggggagcaga 300
caccctatca acttagttaa gaatagaatt atcgagatat ttaagtccat agggtttgcc 360
gtgtcagacg gtccagaaat agaggacgac tggcacaact ttacagccct taacctcccc 420
gaatatcacc ctgctagaga tatgcaggat acgtttttca tagagcagaa tcctgacacc 480
ctccttagaa cacacacttc gtctgtgcag ataagacata tggaacagaa ccaacctcca 540
atgcgtattc tatcaccggg tagagtattt agaaatgagg ctatttcttc tcgttcgcat 600
tgtattttcc accagataga aggactttat attgatgaaa aggtaagttt tgcggatttg 660
aagcaaacca tacagttttt cactacggag ctttttggaa agtctaaaat tagaatgaga 720
ccgtcttatt tcccattcac cgagccaagt gcggaggttg atgtttattg gggacttaac 780
tccgaaacag actaccgaat tactaaaggt actggttggc tagaaattat gggctgtggt 840
atggtagacc ctgctgtgct taaaaatgta aatataaacc ctgataaata cagcggctat 900
ggatttggta tgggaataga gcgaatcgtg atgctcctct accaaatgag cgacattcgt 960
atgttctttg aaaacgatgt aagaatgtta gaacagttta aaacgctata agtaacaaaa 1020
aaaggaaact taaaatttta agtttccttt tttattttta atgggtatat gctgcaaagg 1080
cttcttctaa ggaagcatat ttgcctttga actcctctat tggagaatct tgcataatgt 1140
tgcccttgtg tatcagtatc actctactgc acaacgcctc tacctcctgc ataatatgtg 1200
tagatagaat aaccgttttt ttacgcccta tttctttaat cacttctcgt atctctaaaa 1260
tttggttagg gtctagtccg ttggttggct cgtccaaaat caacaagtct g 1311
<210> 6
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<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 6
cggggtacct gaccccgaag cagggttatg c 31
<210> 7
<211> 38
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 7
gctctagagc aaagcaagtg cagtttaaga ttttactg 38
<210> 8
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<212> DNA
<213>Artificial sequence (Artificial sequence)
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gctctagagc atttcaaaaa tttaacttaa accactg 37
<210> 9
<211> 32
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 9
acgcgtcgac cctttttttg ttacttatag cg 32
<210> 10
<211> 37
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 10
ctagctagct agggcattta tggtacaata gaactag 37
<210> 11
<211> 32
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 11
cggggtaccc cgtaggtact attattttct ag 32
<210> 12
<211> 43
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 12
aactgcagaa ccaatgcatt ggtgaggcta attatactcg tac 43
<210> 13
<211> 34
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 13
cccaagcttg ggcccttagc tgtaccgtag cctg 34
<210> 14
<211> 29
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 14
gctaatgtga taaaatttca cgagggcgg 29
<210> 15
<211> 24
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 15
gaacattttt aagtagatga cctc 24
<210> 16
<211> 24
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 16
gaggtcatct acttaaaaat gttc 24
<210> 17
<211> 30
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 17
cccttagctg taccgtagcc tgtattttgg 30
<210> 18
<211> 2076
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 18
gctaatgtga taaaatttca cgagggcggt tggattacgg tagttcttgc tggttttata 60
ggtatttgta tgtatgcttg gtataatggt agaatgataa aaaacagatt tataaaattt 120
gtaaaactag agaattatat ttctacgatt agagatttaa aactagatga tagtgtgcct 180
aaatatgcta ctaacctagc cttctttagc cgtgctaaaa gggaagatga aatagagtct 240
aaaattattt attctataat tagggctcaa cctaagagag ctgatcatta ttttatactg 300
aacatcatca atcaagaaaa cccttatact ttcaaatatg aaatagatga agtattacct 360
ggaacgattt ataaaatcaa tttcttatta gggtttaaaa tagatagaag aattaacgat 420
tatttccaag atgtgctgga agatatgatg aactctggaa ttatctctga taagagtaat 480
tatccatcgc ttaggagtca taatattcct ccagatatga agtatgtgat tatagataat 540
gtctatataa atgacaatct tttcaccata aaggaaaaaa ttacgatgaa tatttataac 600
tttgtaaaaa aattaggtag caatgatttt aaagcctttg ggcttgccac tcataatgtg 660
gtggtagaat ctgctccact tttgtattca gcagctggag acaagcgtat acaaatggaa 720
aattttaaaa caagtaacta aataaatcta gaaaataata gtacctaatg gaacatcaag 780
agaaagatat agaaataata aaagacgctc taaagcagta tttacaagaa aacggattta 840
gaaacacgcc tgaacgctat actatattag aggaaattta taaacttgat gaacattttt 900
aagtagatga cctctacctc attatgctca ataaaaagta tcatgtgagt aaagccacca 960
tctacaatag tatagaaatt tttctagatg cagggcttat tcgtaagcac cagtttggcg 1020
aaaaatctat gacttcagct tcttatgaga aatctttctt cgataaacag cacgaccatt 1080
tggtaatcta taaaaaggaa ggtagcaaag aaattgcaga aatcatagaa ttttgcgacc 1140
ctcgtatcca aggaattaaa gattctatag aagaaatatt tggcgtgaaa atagaaaatc 1200
attcgcttta tttctacggt cataaaaaag cataatgagg ctaattatac tcgtactaat 1260
gtgcttaggg ttttctgcat tggctcagca gaaagagaaa gcctctacaa caccattggt 1320
aaaagatgcg tattttaaac caaaccctaa taccaaaggt actgttgcag gaaatgaaga 1380
aaagttaaag catattcact cagactcttt ggttagacgt cctgatttat atgaaggtaa 1440
tcctgtattt attggtaatg tagagtttca acatcaaggc tcagttttaa aggcagataa 1500
ggttgtattt taccagaatg acaactttgt aaaagcaata ggtaatgtag tgcttaccac 1560
tgccgaaggc aaccgcatta cttcccaaga aatggaatac gatagtaaaa ctcaaagagg 1620
tatcgcaaga aaaaatgtgg tgcttacaga ccctcagcaa accataaaaa cagaaacact 1680
ttattacgac cgtttaccca atacagctta ttttaattcg ggtggaacca tttataatgg 1740
taaaaatact atttggacgc aagtgggcac ctataacatc aacacacaga cgatagatgt 1800
ttcgggtaat gtaagtattg acaacgataa atatcgtgta gaaggttcta aaatcattca 1860
aaatcaaaaa actaatatag cagaattttt aggagcaaca aaagttatca ataaacaaaa 1920
tcctagaaat tatgtttata cagaaaaggg gcgttacctt atgacttcca aagaggtgta 1980
tctcaataaa aactctagaa tacattacaa tgggaaagtc ctcacgggag aaaccatgta 2040
ttataaccaa aatacaggct acggtacagc taaggg 2076
Claims (7)
1. being used for the carrier of riemerella anatipestifer genome point mutation, it is characterised in that：The carrier by cfx gene cloning extremely
Plasmid pBAD24 restriction enzyme site KpnI and XbaI obtain plasmid pBAD24::Then EXpheS* is connected into plasmid again by cfx
pBAD24::At XbaI the and SalI restriction enzyme site of cfx, the template vector of gene mutation is obtained, pBAD24 is named as::cfx+
EXpheS*；Then mutated gene upstream sequence and mutated gene downstream sequence are connected into pBAD24 respectively::Cfx+EXpheS* is carried
The cfx upstream region of gene of body and the downstream EXpheS* obtain the carrier for being used for the point mutation of riemerella anatipestifer genome.
2. being used for the carrier of riemerella anatipestifer genome point mutation according to claim 1, it is characterised in that：It is described
EXpheS* is by with pLMF03::PheS* plasmid as template, sequence shown in SEQ ID NO.8 and SEQ ID NO.9 be primer into
Row PCR amplification, i.e. acquisition EXpheS*；The pLMF03::PheS* plasmid is connected into shuttle as the sequence as shown in SEQ ID NO.5
Plasmid pLMF03 and obtain.
3. being used for the carrier of riemerella anatipestifer genome point mutation according to claim 1, it is characterised in that：It is described
Cfx gene is made by following methods：Using plasmid pLMF03 as template, sequence shown in SEQ ID NO.6 and SEQ ID NO.7 is
Primer carries out PCR amplification, obtains cfx gene.
4. being used for the carrier of riemerella anatipestifer genome point mutation according to claim 1, it is characterised in that：It is described prominent
Change gene upstream sequence sequence as shown in SEQ ID NO.10 and SEQ ID NO.11 is primer, riemerella anatipestifer genome
DNA is that template carries out PCR amplification and obtains；The mutated gene downstream sequence is as shown in SEQ ID NO.12 and SEQ ID NO.13
Sequence is primer, and riemerella anatipestifer genomic DNA is that template carries out PCR amplification and obtains.
5. the method using any one of Claims 1 to 44 carrier in riemerella anatipestifer genome point mutation, feature
It is：By in carrier containing mutated gene upstream sequence, cfx gene, EXpheS* and mutated gene downstream sequence sequence amplification
Out, it is transferred in riemerella anatipestifer wild strain RA ATCC11845, is coated in containing 1 μ by the method for Natural Transformation
On the GCB plate of g/ml Cefoxitin cfx, the positive colony of first time homologous recombination is filtered out, is named as RA ATCC Δ
RA0C_1912::Sequence containing mutated gene point mutation is transferred to RA by the method for Natural Transformation by cfx+EXpheS*
ATCC ΔR A0C_1912::In cfx+EXpheS*, it is coated on the GCB plate containing 13mM p-cl-Phe, is filtered out
The positive colony of second of homologous recombination.
6. method of the carrier in riemerella anatipestifer genome point mutation according to claim 5, it is characterised in that：It is described
Sequence containing mutated gene upstream sequence, cfx gene, EXpheS* and mutated gene downstream sequence is by using the carrier as mould
Plate, sequence shown in SEQ ID NO.10 and SEQ ID NO.12 are primer amplification acquisition.
7. method of the carrier in riemerella anatipestifer genome point mutation according to claim 5, it is characterised in that：Contain
The sequence of mutated gene point mutation is obtained by following methods：Respectively with SEQ ID NO.13 and SEQ ID NO.14, SEQ ID
NO.15 and SEQ ID NO.16 is primer, after riemerella anatipestifer genomic DNA carries out PCR amplification acquisition product for template,
Then using amplified production as template, SEQ ID NO.13 and SEQ ID NO.16 is that primer carries out fusion DNA vaccine acquisition point mutation sequence
Column.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110938577A (en) * | 2019-11-01 | 2020-03-31 | 四川农业大学 | Riemerella anatipestifer CH-1 strain fur gene deletion attenuated vaccine candidate strain, construction method and application |
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