CN103436478A - Salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof - Google Patents
Salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof Download PDFInfo
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Abstract
The invention discloses a salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof. The salmonella enteritidis double knockout attenuated mutant is obtained by knocking out crp gene and spiC gene of salmonella enteritidis C50041. The invention further discloses a preparation method as well as application of the salmonella enteritidis double knockout attenuated mutant. Further attenuation of the salmonella enteritidis attenuated mutant is realized. A foundation is laid for researching salmonella enteritidis attenuated live vaccines and live vector vaccines.
Description
Technical field
The present invention relates to microorganism field, be specifically related to that a kind of Salmonella enteritidis is dual-gene knocks out attenuation mutant and preparation and application thereof.
Background technology
Salmonella is a kind of pathogenic bacterium of important food source property Zoonosis bacteriosis, to study one of the most extensive bacterium, wherein two kinds of serotypes of Salmonella enteritidis and Salmonella typhimurtum account for leading in many developed countries, poultry, Salmonella enteritidis and Salmonella typhimurtum are considered to most important two kinds of serotypes.Salmonella can cause the asymptomatic inapparent infection of bird, but, at the young bird that is less than two week age, the possibility of the outburst that causes high lethality rate and whole body systemic infection is arranged.The pollution of Salmonella enteritidis to bird product is its primary pollution source that enters people's food chain for a long time.And it is a very important misfortune source that the chicken caecum carries salmonella, because it can cause level infection, through the fecal pollution eggshell, pollutes meat product during butchering, and may pollute ovary.
Domestic normal employing medicine is controlled method, has caused the series of problems such as bacterial drug resistance and drug residue.Recent years, many countries turn to vaccine prevention and control salmonellosis gradually, obtained good effect, in recent years, the research that the novel Attenuated Salmonella that utilizes genetic engineering technique that virulent strain virulence associated gene excision is built is made vaccine receives much concern, and novel attenuated live vaccine security is good, be difficult for reversion; Do not lose immunogenicity in attenuation, living vaccine can be bred in the immune animal body, can stimulate body to produce comprehensive systemic immunity reaction and local immune response, can produce immunne response to the plurality of antigens of pathogenic agent; Immunizing power is solid, and duration of immunity is long, is more having superiority aspect the infection of prevention and control salmonella than inactivated vaccine and subunit vaccine, thereby is being comparatively desirable vaccine.
Existing scholar adopts transposon radom insertion method to identify the research (Geng Shizhong of some white dysentery Salmonellas virulence associated gene functions, Jiao Xinan, Chen Xiaojuan, Pan Zhiming, Zhang Hui, Chen Xiang, insertion Mutagenesis in Salmonella pullorum by Mini-Tn 5 Transposon [J], journal of animal science and veterinary medicine, 2008,39 (5): 621-626); The row method of Red homologous recombination method and efficient restructuring suicide is applied to gene knockout and Gene Replacement, is widely applied.
Summary of the invention
The purpose of this invention is to provide a kind of Salmonella enteritidis attenuation mutant and preparation and application thereof.
At first the present invention provides a kind of Salmonella enteritidis attenuation mutant, for obtaining after the crp gene by Salmonella enteritidis (Salmonella enteritidis) C50041 and spiC gene knockout.
The reading frame sequence of the crp gene of wild-type Salmonella enteritidis C50041 is SEQ ID NO:1.
Further, as embodiment specifically enumerates, the polynucleotide passage that in the crp gene of wild-type Salmonella enteritidis C50041, sequence is SEQ ID NO:2 is lacked, the polynucleotide passage that to have replaced to sequence be SEQ ID NO:3.
In SEQ ID NO:2,626-637bp part ttccgtcagaat is non-coding area sequence.
The entire reading frame of the spiC gene order of wild-type Salmonella enteritidis C50041 is as SEQ ID NO:4; In embodiments of the present invention, above-mentioned crp and spiC gene order are by complete disappearance.
Further, as embodiment specifically enumerates, in the spiC gene of wild-type Salmonella enteritidis C50041, the polynucleotide passage that sequence is SEQ ID NO:5 is lacked, the polynucleotide passage that to have replaced to sequence be SEQ ID NO:6.
In SEQ ID NO:5,35-418bp is the spiC entire reading frame.
Further, for ease of screening, Salmonella enteritidis attenuation mutant of the present invention inserts resistant gene at spiC gene complete reading frame disappearance place, as kalamycin resistance gene.More accurate, as the embodiment of the present invention is enumerated, can insert kalamycin resistance gene at the entire reading frame disappearance place of the spiC of wild-type C50041 gene.This Salmonella enteritidis attenuation mutant has kalamycin resistance.
Further, the sequence of described kalamycin resistance gene is SEQ ID NO:6.
The present invention further provides the construction process of described Salmonella enteritidis attenuation mutant, comprised the following steps:
1) take Salmonella enteritidis C50041 as starting strain, through adopting the Red homologous recombination method by Salmonella enteritidis crp gene knockout, obtain mutant strain △ C50041 (crp);
2), with the spiC gene of restructuring suicide plasmid method knockout mutant strain △ C50041 (crp) and insert kalamycin resistance gene, obtain mutant △ C50041 (crp, spiC/Km) and be Salmonella enteritidis attenuation mutant of the present invention.
Further, step 1) in, adopt the Red homologous recombination method, the polynucleotide passage that in the crp gene of wild-type Salmonella enteritidis C50041, sequence is SEQ ID NO:2 has been replaced to the polynucleotide passage that sequence is SEQ ID NO:3.
Further, step 2) in, adopt restructuring suicide plasmid method, the polynucleotide passage restructuring that in the spiC gene of mutant strain △ C50041 (crp), sequence is SEQ ID NO:5 is replaced with to the kalamycin resistance gene that sequence is SEQ ID NO:6.
Step 2) specifically comprise the following steps:
A) amplify upstream and downstream homology arm spiC12 and the spiC34 fragment of spiC gene from Salmonella enteritidis C50041 genome; By the spiC12 of acquisition and spiC34 fragment respectively the T clone be building up to the pMD18-T carrier, obtain pMD-spiC12, and pMD-spiC34;
B) Hind III and XhoI difference double digestion for pMD-spiC12 step a) obtained and pMD-spiC34, reclaim pMD-spiC12 and spiC34, and two recovery products are connected, and builds plasmid pMD-Δ spiC; (the splicing fragment that Δ spiC is spiC12 and spiC34)
Insert the Km resistant gene between the spiC12 of the recombinant plasmid c) step b obtained and spiC34 fragment, build plasmid pMD-Δ spiC/Km, cut and obtain upstream and downstream homology arm spiC12 and the spiC34 fragment that two ends are respectively the spiC gene with BamH I single endonuclease digestion enzyme, centre is the recombinant fragment of Km resistant gene;
D) recombinant fragment step c obtained is cloned into suicide plasmid pGMB151, obtains recombination suicide vector pGMB151 Δ spiC/Km(and is stored in E.coli Spy372);
E) recombination suicide vector steps d obtained is transformed into appropriate host E.coli χ 7213 as the donor bacterium, the mutant strain △ C50041 (crp) that the step 1) of take obtains carries out conjugal transfer and restructuring as recipient bacterium, utilize resistance and the screening of sucrose susceptibility to obtain mutant strain △ C50041 (crp, spiC/Km).
The present invention further discloses the purposes of Salmonella enteritidis attenuation mutant of the present invention on the living vaccine for preparing the birds enteritis salmonellosis.
Described birds enteritis salmonellosis is the chicken intestinal diorder salmonellosis.
The invention also discloses a kind of chicken intestinal diorder Salmonellas disease vaccine, comprise Salmonella enteritidis attenuation mutant of the present invention.
Also can further comprise adjuvant in described vaccine.
The present invention realizes the further attenuation of Salmonella enteritidis attenuated strain.For developing Salmonella enteritidis attenuated live vaccine and live vector vaccine, lay the foundation.
The accompanying drawing explanation
The pcr amplification product electrophoretogram of Fig. 1 .spiC gene.
M:DL2000bp DNA marker；
1,2: be the pcr amplification result of bacterial strain C50041spiC gene.
Fig. 2. plasmid pMD-spiC12, the pMD-spiC34 enzyme is cut the evaluation electrophoretogram.
M1：DNA marker(λ-EcoT14)；
M2:DL2000DNA marker；
The band that for the 1-2:pMD-spiC12 plasmid, EcoR I, Xho I enzyme are cut: the band that the 3-4:pMD-spiC34 plasmid adopts BamH I enzyme to cut.
Fig. 3. recombinant plasmid pMD-△ spiC enzyme is cut the evaluation electrophoretogram.
M:DNA marker(λ-EcoT14)
Lane1-2: the band that plasmid pMD-△ spiC cuts with Xho I enzyme.
Fig. 4. recombinant plasmid pMD-△ spiC/Km enzyme is cut the evaluation electrophoretogram.
M:DNA marker(λ-EcoT14)；
The band that 1-4:Plasmid pMD-△ spiC/Km adopts Xho I enzyme to cut;
The band that 5-8:Plasmid pMD-△ spiC/Km cuts with BamH I enzyme.
Fig. 5. the enzyme of restructuring pGMB151-Δ spiC/Km is cut the evaluation electrophoretogram.
M:DNA marker(λ-EcoT14)；
The band that 1-3:Plasmid pGMB151-△ spiC/Km cuts with BamH I enzyme.
Fig. 6 .pMD-crpU plasmid figure.
Fig. 7 .pMD-crpD plasmid figure.
Fig. 8 .pMD-△ crp plasmid figure.
Fig. 9 .pMD-△ crp/Cm plasmid figure.
Figure 10. target practice fragment PCR amplification electrophorogram,
M:DL2000marker, 1:crp-YZ-F/R primer amplification fragment.
Figure 11 .crp-U-F/R, crp-D-F/R primer proof diagram
M:DL2000marker, 1,5: negative control, 2,3: the checking of transformant crp-U primer, 6,7: the checking of transformant crp-D-F/R primer, 4,8:C50041 (WT) positive control.
Figure 12 .crp-YZ-F/R primer proof diagram
M:DL2000marker, 1,2: transformant crp-yz-F/R proof diagram, 3:C50041 (WT) positive control.
Figure 13. crp-YZ-F/R primer PCR proof diagram after the removal resistant gene
M:DL2000maker,1,2,3,4:△C50041(crp)。
Figure 14. △ C50041(crp, spiC/Km) PCR qualification result electrophoretogram
M:DL2000bp DNA marker；
The 1-2:C50041 wild-type;
3-5: △ C50041(crp, spiC/Km) use primer △ spiC-YZ – F/R.
Figure 15. △ C50041(crp, spiC/Km) PCR qualification result electrophoretogram;
M:DL2000ladder; 1-4: △ C50041(crp, spiC/Km) use primer △ crp-YZ-F/R.
Figure 16. the lasting Carriage of bacterium in liver.
Figure 17. the lasting Carriage of bacterium in spleen.
Figure 18. the immunity test result.
Left figure is mutant strain Δ C50041(crp, spiC/Km) the immunity test result
Right figure is contrast PBS immunity test result.
Embodiment
The concrete construction process of Salmonella enteritidis attenuation mutant of the present invention is: spiC upstream and downstream sequence is connected respectively to kalamycin resistance gene (KmR) two ends, junction fragment is built up to suicide plasmid pGMB151, import Salmonella enteritidis △ C50041 (crp) through conjugal transfer, salmonella typhi spiC Gene Partial disappearance in wherein inserting KmR, obtain the transformant called after △ C50041 (crp, spiC/Km) of anti-kantlex.The 1 age in days Hai Lanbai chick of take is model, the difference of virulence between icp gene deletion mutantion strain and wild strain, the lethality rate test-results shows, △ C50041 (crp, spiC/Km) virulence of mutant strain, significantly lower than wild-type C50041, is potential Salmonella enteritidis attenuation candidate living vaccine.
Said C50041 in the present invention, refer to that deposit number is CMCC50041.Depositary institution Chinese medicine microbial strains preservation administrative center.
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is in order to describe specific specific embodiments, rather than in order to limit the scope of the invention; In specification sheets of the present invention and claims, unless explicitly pointed out in addition in literary composition, singulative " ", " one " and " this " comprise plural form.
When embodiment provides numerical range, unless should be understood that the present invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology of using in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.The concrete grammar used in embodiment, equipment, material, grasp and record of the present invention according to those skilled in the art to prior art, can also be with the method to described in the embodiment of the present invention, equipment, material is similar or any method, equipment and the material of the prior art that is equal to are realized the present invention.
Unless otherwise indicated, in the present invention, disclosed experimental technique, detection method, preparation method all adopt the routine techniques of molecular biology, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and the association area of the art routine.These technology are existing perfect explanation in existing document, specifically can be referring to MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987and periodic updates; The series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
The structure of C50041 transgenation strain
C50041 source: Chinese medicine microbial strains preservation administrative center, deposit number CMCC50041
1.C50041 evaluation:
1.1. Bacteria Identification
1.1.1 after the dyeing of dyeing microscopic examination gram's method, observe thalli morphology under ordinary optical microscope, measure the bacterium size.The gramstaining result shows that microscopy can be observed the short and small straight-bar bacterium of the redness that is dispersed in arrangement, and size is about 1 μ m * 4 μ m.
1.1.2 biochemical test
Single colony inoculation biochemical identification pipe of picking pure culture, observations after 37 ℃ of cultivation 24h.
2.C50041 the structure of transgenation strain
2.1 target-gene sequence is measured
2.1.1 the amplification of target gene
According to the salmonella genome sequence of having delivered on GenBank, design specific primer with primer-design software Primer5.0, as shown in table 2.The Salmonella enteritidis C50041 genomic dna of take is template, PCR method amplification spiC.50 μ l pcr amplification systems are as table 1:
Table 1, spiC gene PCR amplification system
Reaction conditions is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 45s, 64 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 30 circulations, 72 ℃ are extended 10min, 4 ℃ of preservations.
Table 2: amplified target gene primer sequence
2.1.2T-A clone and evaluation
Reaction utilizes 1% agarose gel electrophoresis detection after finishing, as Fig. 1, test kit reclaims purified pcr product, and reclaimer operation is undertaken by Fragment purification Kit specification sheets.Reclaim product and be connected with pMD18-T carrier (purchased from Takara Bio Inc), linked system (10 μ L): reclaim product 4.5 μ L, pMD18-T carrier 0.5 μ L, SolutionI5.0 μ L, 16 ℃ of metal baths connections are spent the night.To connect product and be transformed into intestinal bacteria competence DH5 α, 37 ℃ of cultivations on the solid LB of Amp, IPTG and X-gal substratum, through blue hickie screening, the single hickie bacterium colony of picking is cultivated, extract plasmid PCR (the primer: spiC-F/R) identify, PCR method is the same, and positive is sent order-checking, obtains spiC gene reading frame sequence to be: SEQ ID NO:4.
2.2 the structure of suicide plasmid
2.2.1 primer
According to the salmonella genome sequence of having delivered on GenBank, design specific spiC upstream and downstream aligning primer with primer-design software Primer5.0, as table 3
Table 3: gene knockout primer table
2.2.2spiC the amplification of upstream and downstream homology arm spiC12, spiC34 fragment
The Salmonella enteritidis C50041 genomic dna of take is template, PCR method amplification spiC upstream and downstream homology arm spiC12, spiC34 fragment.The pcr amplification system of spiC12, spiC34 (50 μ L) is the same.
The PCR reaction conditions is as follows:
94℃ 5min
94 ℃ of 45sec, 64 ℃ of 45sec, 72 ℃ of 60sec, 30 circulations
72℃ 10min
Reaction utilizes 1% agarose gel electrophoresis detection after finishing, test kit reclaims purified pcr product, and reclaimer operation is undertaken by Fragment purification Kit specification sheets.Reclaim product and be connected with the pMD18-T carrier, linked system (10 μ L): reclaim product 4.5 μ L, pMD18-T carrier 0.5 μ L, SolutionI5.0 μ L, 16 ℃ of metal baths connections are spent the night.To connect product and be transformed into intestinal bacteria competence DH5 α, 37 ℃ of cultivations on the solid LB of Amp, IPTG and X-gal substratum, through blue hickie screening, the single hickie bacterium colony of picking is cultivated, and extracts plasmid enzyme restriction and identifies, as Fig. 2.Positive colony send the order-checking of Hua Da Polymorphism, and the plasmid that checks order correct is called after pMD-spiC12, pMD-spiC34 respectively.
2.2.3 the splicing of homologous recombination fragment and evaluation
2.2.3.1spiC12 and the splicing of spiC34 fragment
Extract pMD-spiC12, pMD-spiC34 plasmid in a small amount, carry out double digestion with XhoI and Hind III restriction enzyme respectively, enzyme is cut system (20 μ L): plasmid (pMD-spiC12/pMD-spiC34) 8 μ L, Hind III1 μ L, XhoI1 μ L, 10 * M Buffer2 μ L, the ddH of sterilizing
2o8 μ L, 37 ℃ of water-bath enzymes are cut 2h.Plasmid pMD-spiC12 double digestion is a fragment (about 3.5kb), and plasmid pMD-spiC34 double digestion is two fragments (about 520bp and 2.7kb).Enzyme is cut product through 1% agarose gel electrophoresis, and test kit reclaims the 3.5kb fragment of purifying pMD-spiC12 and the 520bp fragment of pMD-spiC34, and both 16 ℃ of metal baths are connected and spend the night.Linked system (10 μ L): T4DNA ligase enzyme 1 μ L, 10 * connection Buffer1 μ L, the 3.5kb fragment 2 μ L of pMD-spiC12, the 520bp fragment 6 μ L of pMD-spiC34.Connect product and transform DH5 α, coating Amp(100 μ g/mL) the LB solid plate, picking list bacterium colony is cultivated, and the extraction plasmid carries out enzyme and cuts evaluation, as Fig. 3.Identify correct plasmid called after pMD-Δ spiC.
2.2.3.2Km the splicing of resistant gene (Km) and pMD-Δ spiC
By recombinant plasmid pMD-Δ spiC and the pMD-Km plasmid that built (with reference to Yangzhou University's academic dissertation document: author Liu Nannan, the method of record structure in " structure and the immunobiology characteristic preliminary study of white dysentery salmonella S06004 △ spiC mutant strain ") carry out respectively XhoI restriction enzyme single endonuclease digestion, agarose gel electrophoresis, reclaim the approximately 4.0kb fragment of purifying pMD-Δ spiC and the approximately 1.4kb fragment of pMD-Km, the T4DNA ligase enzyme connects, connect product and transform DH5 α, containing Amp(100 μ g/mL) and Km(50 μ g/mL) solid LB flat board on cultivate, picking list bacterium colony is cultivated, the extraction plasmid carries out enzyme and cuts evaluation, as Fig. 4.Identify that correct recombinant plasmid called after pMD-Δ spiC/Km(SEQ ID NO:5 is replaced by SEQ ID NO:6).
2.2.3.3 the structure of suicide plasmid and evaluation
By suicide plasmid pGMB151 (laboratory preservation) through BamHI restriction enzyme single endonuclease digestion, enzyme is cut product after 1% agarose gel electrophoresis, reclaim the about 7800bp carrier segments of purifying pGMB151, plasmid pMD-Δ spiC/Km is cut into individual chip through Aat II digestion with restriction enzyme enzyme, after reclaiming purifying, use again the BamHI digestion with restriction enzyme, reclaim about 2.7kb fragment, reclaim fragment with the about 7800bp of pGMB151 and be connected through the T4DNA ligase enzyme.Connect product and be transformed into the E.coli.Spy372 competent cell, at Amp(100 μ g/mL), Sm(100 μ g/mL), Km(50 μ g/mL) solid LB flat board on cultivate.Picking list bacterium colony is cultivated, and extracts plasmid, and enzyme is cut evaluation, as Fig. 5.Identify correct recombinant plasmid called after pGMB151-Δ spiC/Km.
2.3 Salmonella enteritidis Δ C50041(crp, spiC/Km) screening and the evaluation of mutant strain
2.3.1 structure Salmonella enteritidis Δ C50041(crp):
2.3.1.1 primer
According to the salmonella genome sequence of having delivered on GenBank, design specific crp upstream and downstream aligning primer with primer-design software Primer5.0, as table 4
Table 4:
2.3.1.2crp upstream and downstream homology arm crpU, crpD fragment T clone
The Salmonella enteritidis C50041 genomic dna of take is template, PCR method amplification crp upstream and downstream homology arm crpU, crpD fragment.
The PCR reaction conditions is as follows:
Reaction utilizes 1% agarose gel electrophoresis detection after finishing, test kit reclaims purified pcr product, and reclaimer operation is undertaken by Fragment purification Kit specification sheets.Reclaim product and be connected with the pMD18-T carrier, linked system (10 μ L): reclaim product 4.5 μ L, pMD18-T carrier 0.5 μ L, SolutionI5.0 μ L, 16 ℃ of metal baths connections are spent the night.To connect product and be transformed into intestinal bacteria competence DH5 α, 37 ℃ of cultivations on the solid LB of Amp, IPTG and X-gal substratum, through blue hickie screening, the single hickie bacterium colony of picking is cultivated, and extracts plasmid PCR and identifies.Positive colony send the order-checking of Hua Da Polymorphism, and the correct plasmid that checks order is ordered respectively pMD-crpU, pMD-crpD, and the plasmid schematic diagram is respectively: Fig. 6,7.
2.3.1.3 the splicing of homologous recombination fragment and evaluation
2.3.1.3.1crpU and the splicing of crpD fragment
Extract pMD-crpU, pMD-crpD plasmid in a small amount, carry out double digestion with XhoI and Hind III restriction enzyme respectively, enzyme is cut system (20 μ L): plasmid (pMD-crpU/pMD-crpD) 8 μ L, Hind III1 μ L, XhoI1 μ L, 10 * M Buffer2 μ L, the ddH of sterilizing
2o8 μ L, 37 ℃ of water-bath enzymes are cut 2h.Plasmid pMD-crpU double digestion is a fragment (about 3.5kb), and plasmid pMD-crpD double digestion is two fragments (about 925bp and 2.7kb).Enzyme is cut product through 1% agarose gel electrophoresis, and test kit reclaims the 3.5kb fragment of purifying pMD-crpU and the 925bp fragment of pMD-crp34, and both 16 ℃ of metal baths are connected and spend the night.Linked system (10 μ L): T4DNA ligase enzyme 1 μ L, 10 * connection Buffer1 μ L, the 3.5kb fragment 2 μ L of pMD-crpU, the 925bp fragment 6 μ L of pMD-crpD.Connect product and transform DH5 α, coating Amp(100 μ g/mL) the LB solid plate, picking list bacterium colony is cultivated, and the extraction plasmid carries out PCR, enzyme is cut evaluation.Identify correct plasmid called after pMD-Δ crp, schematic diagram is: Fig. 8.
2.3.1.3.2Cm resistant gene (Cm
r) with the splicing of pMD-Δ crp
By recombinant plasmid pMD-Δ crp and the pMD-Cm built
rplasmid is (with reference to Yangzhou University's academic dissertation document: author Hu Jiao, in " structure of Salmonella choleraesuls C500 △ asd gene-deleted strain and as the preliminary study of vaccine live vector ", the method for record builds) carry out respectively XhoI restriction enzyme single endonuclease digestion, agarose gel electrophoresis, approximately 4.0kb fragment and the pMD-Cm of recovery purifying pMD-Δ crp
rapproximately 1.1kb fragment, the T4DNA ligase enzyme connects, and connects product and transforms DH5 α, containing Amp(100 μ g/mL) and Km(50 μ g/mL) solid LB flat board on cultivate, the cultivation of picking list bacterium colony, PCR, enzyme are cut evaluation.Identify that correct recombinant plasmid called after pMD-Δ crp/Cm(SEQ ID NO:2 is replaced by SEQ ID NO:3), schematic diagram is: Fig. 9.
2.3.1.3.3 the amplification of target practice fragment
The pMD-Δ crp/Cm of take is template, uses the crp-YZ-F/R primer amplification, and agarose gel electrophoresis reclaims the purpose band of 1700bp, and as Figure 10, glue reclaims purified product and uses as the target practice fragment.
2.3.1.4 screening and the evaluation of Salmonella enteritidis Δ C50041 (crp) mutant strain
2.3.1.4.1 competent preparation
Plasmid pKD46 (laboratory preservation) electricity that carries the Red recombinase system is transformed in wild strain C50041 competence and builds bacterial strain C50041(pKD46), in the dull and stereotyped upper 30 ℃ of overnight incubation of penbritin, picking list colony inoculation spends the night containing 30 ℃ of shaking culture of LB substratum of Amp.Get 200 μ l cultures and be inoculated into 5ml containing in the LB substratum of Amp, and add the L-arabinose that final concentration is 60mM, 30 ℃ of shaking culture are to OD600 ≈ 0.6, ice bath 10min, 4 ℃, the centrifugal 10min collection of 1500rpm bacterium, precipitation is washed 3 times with 10% glycerine of ice bath precooling, resuspended with 10% glycerine of 25 μ l precoolings, is competent cell.
2.3.1.4.2 electricity transforms
The target practice fragment of 100ng (volume of PCR product can not surpass competent cell 10%) is joined in the eppendorf pole cup that diameter is 1mm with 20 μ l competent cell mixtures, ice bath 2min, 5ms shocks by electricity under the parameter of voltage 1.8KV, add the nonresistant liquid SOC of 1ml substratum immediately in pole cup, pipettor blows gently after even and is transferred in the 1.5mL dactylethrae, cultivate 2h for 220rpm37 ℃, get half and coat the dull and stereotyped upper 37 ℃ of incubated overnight of the LB that contains paraxin, screening CmR transformant, use crpU-F/R, crpD-F/R and the checking of crp-YZ-F/R primer PCR, as Figure 11, 12, verify correct bacterium colony called after △ C50041 (crp/Cm).
2.3.1.4.3Cm knocking out of resistant gene
Get 37 ℃ of joltings and cultivate Δ C50041 (crp/Cm
r) bacterium (OD600 be about 0.4-0.6), take out ice bath 15min-20min, 4000r/min, 10min is centrifugal, abandons supernatant.After the resuspended washing of sw of bacterial sediment use 30mL precooling 2 times, according to thalline, how much use the resuspended packing competence of 10mL left and right 10% glycerine bacterium 100 μ L/ dactylethraes.-70 ℃ save backup.Temperature sensitivity plasmid pCP20(laboratory is preserved) electricity is transformed into Δ C50041 (crp/Cm
r) bacterium: get 1 μ L pCP20 plasmid extraction liquid, with 100 μ L Δ C50041 (crp/Cm
r) mixing of electric shock competence bacterium, transfer in the click cup of precooling electric shock (voltage 1.8kv).The SOC substratum that adds immediately preheating after electric shock, 30 ℃ of jolting 2h cultivate.Coat on the LB flat board containing the two resistances of Amp, Cm, 30 ℃ of cultivations, the screening positive transformant, the positive bacteria of acquisition is transferred in nonresistant common LB liquid, cultivate to remove the pCP20 plasmids for 42 ℃, 2～3 generations of continuous passage, separate single bacterium colony in the line of the LB of nonreactive solid plate, single bacterium colony is carried out to full bacterium PCR checking, select to meet and expect that the bacterium colony of size of band carries out Amp, the Cm resistance detects, choose the equal sensitivity (Amp of two kinds of microbiotic
scm
s), and the crp-YZ-F/R primer PCR verifies correct bacterium colony, as Figure 13, through order-checking, detects and meets expection, called after Δ C50041 (crp).
2.3.2 homologous recombination
Recombinant plasmid pGMB151-Δ spiC/Km is transformed into to E.coli χ 7213, at Amp(100 μ g/mL), Sm(100 μ g/mL), Km(50 μ g/mL), DAP(50mg/mL) solid LB flat board on cultivate, positive bacteria called after χ 7213(pGMB151-Δ spiC/Km).Take χ 7213(pGMB151-Δ spiC/Km) be the donor bacterium, Salmonella enteritidis Δ C50041(crp) be recipient bacterium, carry out conjugal transfer.Homologous recombination for the first time: picking recipient bacterium Δ C50041(crp respectively) LB substratum, donor bacterium χ 7213(pGMB151-Δ spiC/Km) in containing Amp(100 μ g/mL), Sm(100 μ g/mL), Km(50 μ g/mL), DAP(50mg/mL) LB substratum, 37 ℃, after 200r/min jolting incubated overnight, bacterium liquid is washed twice rear suspension with aseptic PBS, in donor bacterium and recipient bacterium ratio, be that 5:1 mixes recipient bacterium with the donor bacteria suspension, the aseptic filter membrane of 0.22 μ m is affixed on the LB solid plate containing DAP, the suspension of mixing is dripped on filter membrane, dull and stereotyped in 37 ℃ of overnight incubation (about 24h).With aseptic PBS, the lawn of growing on filter membrane is washed down, is coated the g/mL containing Amp(100 μ), Km(50 μ g/mL), Sm(100 μ g/mL) three kinds of antibiotic LB flat boards, 37 ℃ of overnight incubation.
Homologous recombination for the second time: aseptic 10mM MgSO for homologous recombination gained positive bacteria for the first time
4solution is washed twice rear suspension, and dilution spread is in the solid LB substratum without NaCl (containing Km50 μ g/mL, 10% sucrose), 37 ℃ of overnight incubation (approximately being no less than 24h), screening Km
rsm
ramp
rpositive bacterium colony, by containing repeatedly going down to posterity (5-7 generation) without NaCl liquid LB substratum of 10% sucrose, aseptic 10mM MgSO for bacterium liquid
4solution is washed twice rear suspension, and coating, containing the flat board without NaCl of Km, 10% sucrose, is screened Km
rsm
samp
sbacterium.Application primer (Δ spiC-YZ-F/R; Δ crp-YZ-F/R) bacterium of above screening carried out to the PCR checking, as Figure 14,15, verify that correct bacterial strain is identical with expection through the order-checking evaluation, and do not undergo mutation.Determine correct bacterium called after △ C50041(crp, spiC/Km).
Safety testing: the lethality rate test of 1 age in days Hai Lanbai chick
△ C50041(crp, spiC/Km by embodiment 1 preparation) mutant strain and wild strain C50041(CMCC:50041) the virulence comparison
The cultivation of bacterium: bacterium is 37 ℃ of standing cultivation 24h on the LB flat board, and 10 full single bacterium colonies of picking are full plate line on the LB flat board, and 37 ℃ of standing cultivation 5h, scrape the bacterium on the LB flat board with sterilizing PBS, and bacterial concentration is adjusted to 10
9cFU/ml.
1 age in days Hai Lanbai chick is divided into 10 groups at random, each bacterial strain 5 group, every group of 14 chickens.By concentration, be 10
9the bacterium liquid of CFU/ml is diluted to every chicken muscle injection 0.1ml bacterial suspension of 5 extent of dilution with sterilizing PBS, observes death condition in fortnight, calculates mortality ratio.Test-results shows, the virulence of △ C50041 (crp, spiC/Km) mutant strain all significantly reduces (table 5).Table 5: wild strain C50041 and the mutant strain lethality rate to 1 age in days SPF chicken:
C50041 and △ C50041(crp, Δ spiC/Km) two bacterium are respectively with 10
7the amount of bacteria intramuscular inoculation of CFU, each group respectively after infection 1d, 4d, 7d, 10d, 13d, 16d, 20d, 24d, 28d cut open and kill experimental chicken, asepticly get its spleen, part liver, bacterial count.In chicken liver, Figure 16 is shown in the bacterial count variation, and bacterium bacterial count Changing Pattern in spleen is shown in Figure 17.Wild-type C50041 infected group is in inoculation all death afterwards on the 6th, but in detectable time point, content of molds in liver and spleen is all apparently higher than mutant bacteria, mutant bacteria all finally is completely removed in liver and spleen, in liver, mutant bacteria can't detect in the 10th day after infection, in spleen, at 13 days, can't detect.
Immunogenicity experiments: IgG antibody test
On the basis of embodiment 3, collect attenuated strain Δ C50041(crp, spiC/Km) serum of immune group and PBS control group, carry out indirect ELISA and detect IgG antibody.Specific practice is referring to document: strolls, and brilliant swallow, Gong Jiansen, Lv Xiaojuan, Dan Yanju, Liu Xuexian. adopt the dry coated method ELISA of full bacterium antigen to detect the research [J] of fowl cholera serum antibody. Chinese poultry resource, 2009,31 (5): 31-33.
Result is as Figure 18.Result shows: within the 1st week, can stimulate and produce IgG antibody after immunity, within first 2 weeks, peak rapidly, slightly descend after the 2nd week.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all can, under spirit of the present invention and category, be modified or be changed above-described embodiment.Therefore, such as in affiliated technical field, have and usually know that the knowledgeable, not breaking away from all equivalence modifications that complete under disclosed spirit and technological thought or changing, must be contained by claim of the present invention.
SEQUENCE LISTING
<110 > Yangzhou University
<120 > Salmonella enteritidis is dual-gene knocks out attenuation mutant and preparation and application thereof
<130>
<160> 20
<170> PatentIn version 3.3
<210> 1
<211> 633
<212> DNA
<213 > Salmonella enteritidis C50041
<400> 1
atggtgcttg gcaaaccgca aacagacccg actcttgaat ggttcttgtc tcattgccac 60
attcataagt acccgtcaaa gagcacgctg attcaccagg gtgaaaaagc agaaacgctg 120
tactacatcg ttaaaggctc cgtggcagtg ctgatcaaag atgaagaagg gaaagaaatg 180
atcctttctt atctgaatca gggtgatttt attggtgaac tgggcctgtt tgaagaaggc 240
caggaacgca gcgcctgggt acgtgcgaaa accgcatgtg aggtcgctga aatttcctac 300
aaaaaatttc gccaattaat ccaggtcaac ccggatattc tgatgcgcct ctcttcccag 360
atggctcgtc gcttacaagt cacctctgaa aaagtaggta acctcgcctt ccttgacgtc 420
accgggcgta tcgctcagac gctgctgaat ctggcgaaac agcccgatgc catgacgcac 480
ccggatggga tgcagatcaa aatcacccgt caggaaatcg gccagatcgt cggctgctcc 540
cgcgaaaccg ttggtcgtat tttgaaaatg ctggaagatc aaaacctgat ctccgcgcat 600
ggcaagacca tcgtcgtcta cggcacccgt taa 633
<210> 2
<211> 637
<212> DNA
<213 > Salmonella enteritidis C50041
<400> 2
tggcaaaccg caaacagacc cgactcttga atggttcttg tctcattgcc acattcataa 60
gtacccgtca aagagcacgc tgattcacca gggtgaaaaa gcagaaacgc tgtactacat 120
cgttaaaggc tccgtggcag tgctgatcaa agatgaagaa gggaaagaaa tgatcctttc 180
ttatctgaat cagggtgatt ttattggtga actgggcctg tttgaagaag gccaggaacg 240
cagcgcctgg gtacgtgcga aaaccgcatg tgaggtcgct gaaatttcct acaaaaaatt 300
tcgccaatta atccaggtca acccggatat tctgatgcgc ctctcttccc agatggctcg 360
tcgcttacaa gtcacctctg aaaaagtagg taacctcgcc ttccttgacg tcaccgggcg 420
tatcgctcag acgctgctga atctggcgaa acagcccgat gccatgacgc acccggatgg 480
gatgcagatc aaaatcaccc gtcaggaaat cggccagatc gtcggctgct cccgcgaaac 540
cgttggtcgt attttgaaaa tgctggaaga tcaaaacctg atctccgcgc atggcaagac 600
catcgtcgtc tacggcaccc gttaattccg tcagaat 637
<210> 3
<211> 115
<212> DNA
<213 > artificial sequence
<400> 3
ctcgagatgg gaattagcca tggtccatat gaatatcctc cttagttcct attccgaagt 60
tcctattctc tagaaagtat aggaacttcg aagctgctcc agcctacacc tcgag 115
<210> 4
<211> 384
<212> DNA
<213 > Salmonella enteritidis C50041
<400> 4
atgctggcag ttttaaaagg cattgcatta attcaggata tcaaggccga aggtaatagc 60
cgatcctgga taatgactat tgatgggcat cctgccagag gagaaatttt ctcagaagca 120
ttttctattt ctttgttctt aaatgacctg gaaagcttac ctaagccttg tcttgcctat 180
gtgacactac tgcttgcagc acacccggac gtccatgatt atgctataca gctcacagcg 240
gatgggggat ggttaaacgg ttattatacc acaagtagta gctctgagct tattgctatt 300
gagatagaaa aacacctggc tttaacttgc attttaaaaa atgtaatacg caatcaccat 360
aaactttatt cgggtggggt ataa 384
<210> 5
<211> 508
<212> DNA
<213 > Salmonella enteritidis C50041
<400> 5
atagaaactc ccatttatgt ctgaggaggg attcatgctg gcagttttaa aaggcattcc 60
attaattcag gatatcaggg ccgaaggtaa tagccgatcc tggataatga ctattgatgg 120
gcatcctgcc agaggagaaa ttttctcaga agcattttct atttctttgt tcttaaatga 180
cctggaaagc ttacctaagc cttgtcttgc ctatgtgaca ctactgcttg cagcacaccc 240
ggacgtccat gactatgcta tacagctcac agcggatggg ggatggttaa acggttatta 300
taccacaagt agtagctctg agcttattgc tattgagata gaaaaacacc tggctttaac 360
ttgcatttta aaaaatgtaa tacgcaatca ccataaactt tattcgggtg gggtataaaa 420
tggtagtaaa taaacgttta atcttaattt tactatttat actcaataca gcaaagagtg 480
atgagttatc atggaaaggt aatgactt 508
<210> 6
<211> 1506
<212> DNA
<213 > kalamycin resistance gene
<400> 6
ctcgagatgg gaattagcca tggtccatat gaatatcctc cttagttcct attccgaagt 60
tcctattctc tagaaagtat aggaacttca gagcgctttt gaagctgggg tgggcgaaga 120
actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc 180
cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg 240
gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag 300
gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg 360
gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg 420
atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc 480
caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg 540
catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc 600
cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg 660
tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc 720
atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc 780
cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc 840
tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc 900
attcaggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc 960
cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc 1020
ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac 1080
gatcctcatc ctgtctcttg atcagatctt gatcccctgc gccatcagat ccttggcggc 1140
aagaaagcta tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct 1200
ggcaattccg gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc 1260
ccactgcaag ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag 1320
tagctgacat tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtgttccgc 1380
ttcctttagc agcccttgcg ccctgagtgc ttgcggcagc gtgggggatc ttgaagttcc 1440
tattccgaag ttcctattct ctagaaagta taggaacttc gaagcagctc cagcctacac 1500
ctcgag 1506
<210> 7
<211> 32
<212> DNA
<213 > artificial sequence
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taggatccat gctggcagtt ttaaaaggca tt 32
<210> 8
<211> 35
<212> DNA
<213 > artificial sequence
<400> 8
tactcgagtt ataccccacc cgaataaagt ttatg 35
<210> 9
<211> 41
<212> DNA
<213 > artificial sequence
<400> 9
aaagaattcg tcgacggatc cgcgacactg ccgttcccca g 41
<210> 10
<211> 34
<212> DNA
<213 > artificial sequence
<400> 10
aaactcgagc accctttatg ccagacaaat gcca 34
<210> 11
<211> 33
<212> DNA
<213 > artificial sequence
<400> 11
aaactcgagc aaattcgctc acaaccacat ccg 33
<210> 12
<211> 46
<212> DNA
<213 > artificial sequence
<400> 12
aaagaattcg tcgacggatc cgctgcagta atcacagata gcagcc 46
<210> 13
<211> 20
<212> DNA
<213 > artificial sequence
<400> 13
accggtggtc caggcggaat 20
<210> 14
<211> 23
<212> DNA
<213 > artificial sequence
<400> 14
gctttcctcc agttgcctgt tgc 23
<210> 15
<211> 28
<212> DNA
<213 > artificial sequence
<400> 15
taggatccgc gatgctccag ggctcgtt 28
<210> 16
<211> 29
<212> DNA
<213 > artificial sequence
<400> 16
tactcgagag caccatgcgc ggttatcct 29
<210> 17
<211> 31
<212> DNA
<213 > artificial sequence
<400> 17
tactcgaggg cgcgttttat catgcgccat t 31
<210> 18
<211> 28
<212> DNA
<213 > artificial sequence
<400> 18
taggatccgc gcgtcggcag acgatgat 28
<210> 19
<211> 20
<212> DNA
<213 > artificial sequence
<400> 19
<210> 20
<211> 20
<212> DNA
<213 > artificial sequence
<400> 20
Claims (8)
1. a Salmonella enteritidis attenuation mutant, for obtaining after the crp gene by Salmonella enteritidis C50041 and spiC gene knockout.
2. Salmonella enteritidis attenuation mutant as claimed in claim 1, it is characterized in that, in the genome of described Salmonella enteritidis attenuation mutant, the polynucleotide passage that in the crp gene of Salmonella enteritidis C50041, sequence is SEQ ID NO:2 lacked, the polynucleotide passage that to have replaced to sequence be SEQ ID NO:3; Simultaneously, the polynucleotide passage that in the spiC gene of described Salmonella enteritidis C50041, sequence is SEQ ID NO:5 is lacked restructuring and is replaced with the kalamycin resistance gene that sequence is SEQ ID NO:6.
3. the construction process of Salmonella enteritidis attenuation mutant as claimed in claim 1, comprise the following steps:
1) take Salmonella enteritidis C50041 as starting strain, through adopting the Red homologous recombination method by Salmonella enteritidis crp gene knockout, obtain mutant strain △ C50041 (crp);
2), with the spiC gene of restructuring suicide plasmid method knockout mutant strain △ C50041 (crp) and insert kalamycin resistance gene, obtain mutant △ C50041 (crp, spiC/Km) and be described Salmonella enteritidis attenuation mutant.
4. construction process as claimed in claim 3, it is characterized in that, step 1) in, adopt the Red homologous recombination method, the polynucleotide passage that in the crp gene of wild-type Salmonella enteritidis C50041, sequence is SEQ ID NO:2 has been replaced to the polynucleotide passage that sequence is SEQ ID NO:3.
5. construction process as claimed in claim 3, it is characterized in that, step 2) in, adopt restructuring suicide plasmid method, the polynucleotide passage that in the spiC gene of mutant strain △ C50041 (crp), sequence is SEQ ID NO:5 is just recombinated and replaced with the kalamycin resistance gene that sequence is SEQ ID NO:6.
6. the purposes of the described Salmonella enteritidis attenuation mutant of claim as arbitrary as claim 1-3 on the living vaccine for preparing the birds enteritis salmonellosis.
7. as claim, 6 described purposes, is characterized in that, described birds enteritis salmonellosis is the chicken intestinal diorder salmonellosis.
8. a chicken intestinal diorder Salmonellas disease vaccine, comprise the described Salmonella enteritidis attenuation mutant of the arbitrary claim of claim 1-3.
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| CN103789337A (en) * | 2014-02-18 | 2014-05-14 | 扬州大学 | Suicide vector pGMB152 for blue-white selection and application thereof |
| CN104560853A (en) * | 2015-01-07 | 2015-04-29 | 四川农业大学 | Riemerella anatipestifer CH1 attenuated strain, as well as construction method and application thereof |
| CN104651288A (en) * | 2015-01-07 | 2015-05-27 | 四川农业大学 | Pasteurella multocida attenuated strain deficient in crp as well as construction method and application of attenuated strain |
| CN104560853B (en) * | 2015-01-07 | 2018-03-02 | 四川农业大学 | One plant of riemerella anatipestifer CH1 attenuated strain and its construction method and application |
| CN105018400A (en) * | 2015-06-03 | 2015-11-04 | 安徽农业大学 | Salmonella enteritidis strain with ssrAB gene deletion and construction method thereof |
| CN105018400B (en) * | 2015-06-03 | 2020-02-11 | 安徽农业大学 | Salmonella enteritidis ssrAB gene-deleted strain |
| CN106190943A (en) * | 2016-08-02 | 2016-12-07 | 重庆理工大学 | A kind of dual-gene disappearance Salmonella enteritidis, its construction method and the vaccine containing this dual-gene disappearance Salmonella enteritidis |
| CN106190943B (en) * | 2016-08-02 | 2019-10-08 | 重庆理工大学 | It is a kind of it is dual-gene missing Bacterium enteritidis, its construction method and contain this it is dual-gene missing Bacterium enteritidis vaccine |
| CN106754535A (en) * | 2016-12-31 | 2017-05-31 | 山东信得科技股份有限公司 | The weak malicious Bacterium enteritidis of one kind restructuring |
| CN106754535B (en) * | 2016-12-31 | 2020-07-24 | 山东信得科技股份有限公司 | Recombinant salmonella enteritidis with weak toxicity |
| CN115851771A (en) * | 2022-08-03 | 2023-03-28 | 扬州大学 | Salmonella gallinarum attenuated isolate not expressing Peg pili and application thereof |
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Application publication date: 20131211 |