CN106754594A - A kind of Salmonella choleraesuls attenuated carrier bacterium and its construction method - Google Patents
A kind of Salmonella choleraesuls attenuated carrier bacterium and its construction method Download PDFInfo
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- CN106754594A CN106754594A CN201611138726.7A CN201611138726A CN106754594A CN 106754594 A CN106754594 A CN 106754594A CN 201611138726 A CN201611138726 A CN 201611138726A CN 106754594 A CN106754594 A CN 106754594A
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Abstract
A kind of Salmonella choleraesuls attenuated carrier bacterium and its construction method, belong to animal bacteria gene engineering technology field.Attenuated carrier bacterium of the present invention is to have lacked ΔmanA、Δcrp::TT araC PBAD、ΔrelA::araC PBAD lacI TT、ΔsopBAnd ΔasdAThe Salmonella choleraesuls of C78 3 of gene, are named as rSC0016.Method of the present invention using the suicide vector of non-resistant mark as Salmonella choleraesuls carrier is built, can avoid using the carrier of antibiotic marker, can use safely in clinical practice；In addition exogenous antigen is carried using balanced lethal system, it is ensured that the vaccine strain for only carrying exogenous antigen could survive producing vaccine and entering in host, improve the Immune efficiency of vaccine.
Description
Technical field
The invention belongs to animal bacteria gene engineering technology field.
Background technology
Gene engineering method causes weak salmonella typhimurium as vector expression foreign gene, is widely used in the mankind and moves
The Therapy study of the various diseases of thing, achieves good result.But the Salmonella carrier in many reports, otherwise make to subtract
Poisonous carrier excessive attenuation, loses immunogenicity, though or remain good immunogenicity, carrier bacterial strain is but attenuated not
It is enough, lack security.
Salmonella choleraesuls are the important pathogens of pig.On with Salmonella choleraesuls as vehicle delivery pig other
The research of pathogen antigen, it is existing at home and abroad to report on a small quantity, such as：The Chen Huanchun academician laboratory of Hua Zhong Agriculture University is with hog cholera
Salmonella China attenuated vaccine type strain C500 is vehicle delivery mycoplasma hyopneumoniae antigen；Military Medical Science Institute military affairs beast
The painting Changchun laboratory for curing research institute is vehicle delivery swine fever using Salmonella choleraesuls China attenuated vaccine type strain C500
The antigen of virus.These results of study have following common ground：One is these candidate vaccine strains malicious by force to hog cholera Salmonella
Strain attack poison（Attack toxic agent amount>10×LD50）100% protection can be produced；Although two is have certain to the poison of attacking of exogenous pathogen
Effect, but protective efficacy is not ideal enough.Its reason may be the use of Salmonella choleraesuls attenuated vaccine type strain C500
It is carrier, although Salmonella choleraesuls attenuated vaccine type strain C500 is to the enough attenuations of host, but the immunogene of its generation
Property seem also to be not enough to induction preferably for the immune response of exogenous antigen.Therefore in the urgent need to developing a kind of vaccine carrier,
Vaccine carrier should be made to be attenuated enough, it is ensured that the complete security of animal, vaccine carrier is induced outstanding immunogene again
Property, the pathogen infection of carrier bacterium and the exogenous antigen of its carrying is resisted in high quality.
This laboratory finds during early-stage Study, with arabinose regulation and control Salmonella choleraesulscrpVirulence base
Attenuated carrier bacterium because produced by（Chinese patent application CN 104498418A）Can induce preferable to adult BALB/c mouse
Immunogenicity, but after attenuated strain is immune, with 20 × LD of high dose50Wild type streptococcus suis 2-type（SS2）Than with 10 ×
LD50SS2 attack poison protective efficacy it is low, while attenuated strain still has the duplication of attenuated strain, table in the liver and spleen of mouse
Also there is certain risk and deficiency in bright security and immunogenicity as attenuated strain.
sopBGene is encoded by salmonella SPI-1, and the knockout of the gene can reduce salmonella typhimurium and cause
The inflammatory reaction of host intestine, reduce salmonella typhimurium to host immune system stress, mitigate to host immune system
Damage, so as to improve the security to host, meanwhile, host can be improved to salmonella typhimurium and its external source for carrying
The immunogenicity of antigen.Different animals have different susceptible salmonellas, conversely, same salmonella is for different animals
Have different pathogenic, the effect of the virulence gene contained by it also can be different.Lacked in Salmonella choleraesulssopBGene,
Whether inflammatory reaction caused by identical reduction Salmonella choleraesuls infection can be reached, Salmonella choleraesuls is improved to pig
Immunogenicity, up to the present yet there are no and reported both at home and abroad.
The content of the invention
For prior art defect, present invention aim at providing, a kind of higher with security and immunogenicity is stronger
The Salmonella choleraesuls attenuated carrier bacterium of feature.
Attenuated carrier bacterium of the present invention is to have lacked ΔmanA、Δcrp::TT araC PBAD、ΔrelA::araC
PBAD lacI TT、ΔsopBAnd ΔasdAThe C78-3 Salmonella choleraesuls of gene, are named as rSC0016.
RSC0016 is deposited in the China General Microbiological strain positioned at city of BeiJing, China Chaoyang District North Star West Road 1 institute 3
Preservation administrative center (CGMCC), preservation day is on June 20th, 2016, and deposit number is CGMCC NO.12645, the biomaterial
Classification And Nomenclature be:Salmonella choleraesuls are attenuated, Latin name is:Salmonella choleraesuis。
The present invention is malicious in arabinose regulation and control Salmonella choleraesuls carrier with C78-3 Salmonella choleraesuls as parent
On the basis of power gene expression, due to having knocked out Salmonella choleraesuls insopB Gene, makes carrier bacterium possess the peace of attenuation
During omnicharacteristic, the ability of the immunogenicity of carrier bacterium induction host is improved, carrier bacterium is turned into the epidemic disease of safety and efficient protectiveness
Miao Zhu；Carrier bacterium is knocked out simultaneouslyasd Gene, makes the prokaryotic expression exogenous antigen that carrier bacterium carries with itasd+ Carrier
Form balanced lethal system, it is to avoid use the prokaryotic expression carrier with antibiotic marker.
Invention achieves following beneficial effect：
1st, immunogenicity is strong：
Arabinose regulation and control Salmonella choleraesuls carrier virulence gene is postponed into missing and postpones the technology of expression exogenous antigen
Cause host inflammation with Salmonella choleraesuls are knocked outsopBGenetic method is combined, and carrier bacterium is possessed the Special safety of attenuation
Property when, improve carrier bacterium induce host immunogenicity.It is demonstrated experimentally that containing ΔsopBRSC0016 plants of ratio of mutation is not contained
ΔsopB RSC0011 plants（ΔmanA、Δcrp::TT araC PBAD、ΔrelA::araC PBAD lacITT and ΔasdA）
The immune response for being induced is more preferable.For example：The rSC0016 (pS-SaoA) of the SaoA albumen of streptococcus suis 2-type is carried, can be with
The attack of the wild type streptococcus suis 2-type of 100% resistance high dose, it was demonstrated that rSC0016 can induce perfect immunogene
Property, this has great market applicability and creates significant economic benefit for the various diseases of pig.
Δ is not contained more thansopB RSC0011 plants（ΔmanA、Δcrp::TT araC PBAD、ΔrelA::araC
PBAD lacITT and ΔasdA）It is published in (Zhenying Jib, Jing Shangb, Yuan Li, Shifeng in 2015
Wang, Huoying Shi, Live attenuated Salmonella enterica serovar Choleraesuis
vaccinevector displaying regulated delayed attenuation and regulateddelayed
antigen synthesis to confer protection againstStreptococcus suis in mice,
Vaccine 33 (2015) 4858–4867)。
2nd, greater security：
Compared to not containing ΔsopB RSC0011 plants of mutation（ΔmanA、Δcrp::TT araC PBAD、ΔrelA::araC
PBAD lacITT and ΔasdA）, the present invention contains ΔsopB The rSC0016 of mutation is more attenuated, thus with safety higher
Property, it is suitable as vaccine carrier and weanling piglet is immunized.
Another object of the present invention is the construction method for providing above-mentioned Salmonella choleraesuls attenuated carrier bacterium.
Construction method of the present invention is comprised the following steps：
1）Build and contain ΔsopBThe suicide vector of mutator：
Genome with Salmonella choleraesuls C78-3 expands homology arm fragment as template by primer, knocks out hog cholera sramana
In the gene order of Salmonella C78-3sopB Genetic fragment, and by the gene order for knocking out Salmonella choleraesuls C78-3
'ssopB Fragment gene after genetic fragment is connected in suicide plasmid pRE112, obtains missing ΔsopB Gene order is committed suiside
Plasmid；Δ will be lacked againsopB Gene order suicide plasmid is converted into engineering bacteria Escherichia coli χ 7213, is obtained and is contained ΔsopB
The suicide vector of mutator, is named as χ 7213（pS006）；
2）Build and introduce ΔsopBRSC0013 plants of mutator：
To have lacked ΔmanA、ΔPcrp::TT araC PBAD crpAnd ΔrelA::araC PBAD lacI The C78-3 of TT genes
Salmonella choleraesuls are recipient bacterium, with containing ΔsopBThe suicide vector of mutator is donor bacterium, by donor bacterium and recipient bacterium
Engaged, through culture, purifying, obtain and introduce ΔsopBRSC0013 plants of (Δ of mutatormanA, Δ Pcrp::TT araC
PBAD crp, ΔrelA::araC PBAD lacI TT, ΔsopB）；
3）Build Salmonella choleraesuls attenuated carrier bacterium rSC0016：
To introduce ΔsopBRSC0013 plants of mutator is recipient bacterium, with containing ΔasdASuicide vector χ 7213 (pS004) is
Recipient bacterium, donor bacterium and recipient bacterium are engaged, and through culture, purifying, obtain Salmonella choleraesuls attenuated carrier bacterium
rSC0016。
Method of the present invention using the suicide vector of non-resistant mark as Salmonella choleraesuls carrier is built, can keep away
Exempt from, using the carrier of antibiotic marker, to use safely in clinical practice；In addition external source is carried using balanced lethal system
Antigen, it is ensured that the vaccine strain for only carrying exogenous antigen could improve epidemic disease in production vaccine and into survival in host
The Immune efficiency of seedling.
Further, step 1 of the present invention）In, the genome with Salmonella choleraesuls C78-3 as template, by drawing
Thing P1 and P2 are expanded through PCRpipCHomology arm gene order；Genome with Salmonella choleraesuls C78-3 as template, by
Primer P3 and P4 are expanded through PCRpipDThe homology arm sequence of gene；WillpipCHomology arm gene order andpipDGene it is same
Source arm sequence is with bases longs 1:1 ratio is mixed to prepare mix products；Using mix products as template, using primer P1 and draw
It is the homology arm fragment of 493bp that thing P4 carries out Overlap PCR amplifications and obtains length, and the homology arm fragment is connected to
PM18T cloning vectors, the JM109 competent cells of conversion extract above-mentioned bacterial plasmid, then through KpnI and SacI digestions, connection
Into the suicide plasmid carrier pRE112 with chloramphenicol and sucrose selection markers, obtain lacking ΔsopBGene order
PRE112 carriers.
The nucleotide sequence of described primer P1, P2, P3 and P4 is respectively：
P1：5’- CGCAGAGCTCATATCACCTATAATTATC - 3’；
P2：5’- CATATAGTTACCTCAAGACAGCGTTTTTAATATTCCTG - 3’；
P3：5’- CAGGAATATTAAAAACGCTGTCTTGAGGTAACTATATG - 3’；
P4：5’- ATTAGGTACCAGCAGTATTGTCTGCGTCAGC - 3’.
The step 2）In, take and lacked ΔmanA、ΔPcrp::TT araC PBAD crpAnd ΔrelA::araC
PBAD lacI The C78-3 Salmonella choleraesuls of TT genes were cultivated in LB fluid nutrient mediums through 10-12 hours, and acceptor is obtained
Bacterium bacterium solution；Δ will be containedsopBThe suicide vector of mutator was cultivated in LB liquid through 10-12 hours, and the culture of donor bacterium is obtained
Bacterium solution；It is described to contain Δ for culturesopBIn the LB liquid of the suicide vector of mutation, 2,6- diaminopimelic acids（DAP）Content is
50ug/mL, chloromycetin content is 25mg/mL.
The step 2）In, after purification, picking single bacterium is cultivated to cloud in falling within LB fluid nutrient mediums, and 10 times of series are dilute
Release in coating LB solid mediums；Rule respectively at LB plates, the LB plates containing chloramphenicol from solid medium picking single bacterium colony, in
Cultivated through 10-12 hours under 37 DEG C of environment, the introducing Δ that then picking only grows in LBsopBRSC0013 plants of mutation.
The step 3）In, rSC0013 plants is taken in LB fluid nutrient mediums, cultivated through 10-12 hours, prepared acceptor
Bacterium bacterium solution；Take containing ΔasdASuicide vector χ 7213 (pS004) was cultivated in LB fluid nutrient mediums through 10-12 hours, is obtained and is supplied
Body bacterium cultivates bacterium solution；It is described to contain Δ for cultureasdAIn the LB fluid nutrient mediums of suicide vector χ 7213 (pS004), 2,6- bis-
Diaminopimelic acid（DAP）Content is 50ug/mL, and chloromycetin content is 25mg/mL.
Experiment proves that the present invention postpones attenuation and postpones the carrier of expression exogenous antigen in arabinose regulation and control virulence gene
Upper structure contains ΔsopBThe Salmonella choleraesuls carrier of mutator, can reduce Salmonella choleraesuls attenuated strain
Virulence, the inflammatory reaction that mitigation attenuated strain causes is significantly improved entrained by Salmonella choleraesuls to the damaging action of host
Exogenous antigen induces the immunogenicity of host, so that containingsopBThe pig of the carrying Streptococcus suis SaoA albumen of mutator is suddenly
Random salmonella vaccine Candidate Strain rSC0016（pS-SaoA）Complete protection can be provided the infection of SS2 and SS7 street strains
And cross-protection.
Brief description of the drawings
Fig. 1 is ΔsopBGene delection builds figure.
Fig. 2 is ΔsopBMutation suicide vector figure.
Fig. 3 is containing ΔsopBThe PCR identification electrophoretograms of the χ 7213 (pS006) of mutation.Wherein, swimming lane 1：DL2000 DNA
marker；Swimming lane 2-7：Positive findings（I.e.：It is accredited as after the positive sample of mutation is further purified and randomly selects the PCR of bacterium colony
Electrophoretogram；" positive findings " for appearing below, unless specifically indicated, is such case）.
Fig. 4 is to containΔsopBThe PCR identification electrophoretograms of the rSC0013 of mutation.Wherein, swimming lane 1：DL2000 DNA
marker；Swimming lane 2-3：RSC0017 positive findingses；Swimming lane 4-6：RSC0013 positive findingses.
Fig. 5 is containing ΔasdAIt is mutated the PCR identification electrophoretograms of rSC0016.Wherein, swimming lane 1：DL2000 DNA marker；
Swimming lane 2-6：RSC0016 positive findingses.
Fig. 6 be different virus strain infection rabbit ileums rheuminess object product and inoculation after time chart.In figure, ##：With
Street strain C78-3 plants is compared（p<0.01）, * *：Compare with LB blank control groups（P<0.01）.
Fig. 7 is the rheuminess thing total number of bacteria of different virus strain infection rabbit ileums.In figure, #：With street strain C78-3 plants of ratio
Compared with（p<0.05）, * *：Compare with LB blank control groups（P<0.01）.
Fig. 8 is the pathological change of rabbit ileum after inoculation street strain C78-3（H.E is dyeed）Picture.
Fig. 9 is the pathological change of rabbit ileum after inoculation rSC0016（H.E is dyeed）Picture.
Figure 10 is the pathological change of rabbit ileum after inoculation LB（H.E is dyeed）Picture.
Figure 11 is amplificationsaoAThe PCR identification electrophoretograms of gene.Wherein, swimming lane 1：DL2000 DNA Marker；Swimming lane 2-
6：AmplificationsaoAGene PCR electrophoretogram.
Figure 12 is the digestion identification electrophoretogram of pS-SaoA recombinant expression plasmids.Wherein, swimming lane 1：DL10000 DNA
Marker；Swimming lane 2-4：Positive findings restriction enzyme digestion and electrophoresis figure.
Figure 13 is the digestion identification electrophoretogram of recombinant plasmid.Wherein, swimming lane 1：DL10000 DNA Marker；Swimming lane 2：
rSC0016(pS-SaoA)；Swimming lane 3：rSC0011(pS-SaoA)；Swimming lane 4：rSC0018(pS-SaoA).
Figure 14 is that Western-blot detects SaoA in rSC0016 (pS-SaoA), rSC0018 (pS-SaoA), rSC0011
Expression electrophoretogram in (pS- SaoA) bacterial strain.Wherein, swimming lane 1：Albumen Marker；Swimming lane 2：rSC0011(pS-SaoA)；Swimming
Road 3：rSC0016(pS- SaoA)；Swimming lane 4：rSC0018(pS-SaoA).
Figure 15 is that pS-SaoA plasmids pass 10-50 for restriction enzyme digestion and electrophoresis figure in rSC0016 (pS-SaoA).Wherein, swimming lane 1：
DL10000 DNA marker；Swimming lane 2-6：RSC0016 (pS-SaoA) 10-50 generations.
Figure 16 is attenuated strain colonization ability comparative experiments figure in BALB/c mouse liver.
Figure 17 is attenuated strain colonization ability comparative experiments figure in BALB/c mouse spleen.
Figure 18 is attenuated strain colonization ability comparative experiments figure in BALB/c mouse enteron aisle sends Yi Ershi to tie.
Figure 19 is the IgG figures of anti-SaoA in immune attenuated strain BALB/c mouse serum.
Figure 20 is the IgG figures of anti-salmonella OMPs in immune attenuated strain BALB/c mouse serum.
Figure 21 is the IgA figures of anti-SaoA in immune attenuated strain BALB/c mouse vaginal washing fluid.
Figure 22 is the figure of cell factor IFN-γ content in the immune attenuated strain BALB/c mouse lung of ELISA detections and spleen.
Figure 23 is the figure of IL-4 contents in the immune attenuated strain BALB/c mouse lung of ELISA detections and spleen.
Figure 24 is the figure of the immune attenuated strain BALB/c mouse Cytokine of Serum IFN-γ content of ELISA detections.
Figure 25 is that IL-4 contains spirogram during ELISA detections are immunized attenuated strain BALB/c mouse serum.
Figure 26 is the antibody for Streptococcus suis SS2 SaoA albumen in the immune attenuated strain experiment Swine serum of ELISA detections
IgG level views.
Figure 27 is resisted for Salmonella choleraesuls outer membrane protein OMPs during the immune attenuated strain of ELISA detections tests Swine serum
Body IgG level views.
Figure 28 is the mucous membrane for Streptococcus suis SS2SaoA in the immune attenuated strain test pig nasal cavity cotton swab of ELISA detections
Antibody I gA level views.
Figure 29 is the figure of cell factor IFN-γ content in the immune attenuated strain test pig lung of ELISA detections and spleen.
Figure 30 is the figure of cell factor IL-4 contents in the immune attenuated strain test pig lung of ELISA detections and spleen.
Figure 31 is the figure of cell factor IL-17 contents in the immune attenuated strain test pig lung of ELISA detections and spleen.
Figure 32 is the figure of the immune attenuated strain test pig Cytokine of Serum IFN-γ content of ELISA detections.
Figure 33 is the figure of cell factor IL-4 contents in the immune attenuated strain test pig lung of ELISA detections and spleen.
Figure 34 is the figure of cell factor IL-17 contents in the immune attenuated strain test pig lung of ELISA detections and spleen.
Figure 35 is the figure that immune attenuated strain test pig compares through the survival rate that SS2 attacks poison.
Figure 36 is that PBS control group test pig attacks malicious histopathology change photo through SS2.
Figure 37 is that PBS control group test pig attacks malicious pathologic change photo through SS2.
Figure 38 is that immune rSC0018 (pYA3493) strain test pigs attack malicious histopathology change photo through SS2.
Figure 39 is that immune rSC0018 (pYA3493) strain test pigs attack malicious pathologic change photo through SS2.
Figure 40 is that immune rSC0018 (pS-SaoA) strain test pigs attack malicious histopathology change photo through SS2.
Figure 41 is that immune rSC0018 (pS-SaoA) strain test pigs attack malicious pathologic change photo through SS2.
Figure 42 is that immune rSC0016 (pYA3493) strain test pigs attack malicious histopathology change photo through SS2.
Figure 43 is that immune rSC0016 (pYA3493) strain test pigs attack malicious pathologic change photo through SS2.
Figure 44 is that immune rSC0016 (pS-SaoA) strain test pigs attack malicious brain tissue photo through SS2.
Figure 45 is that immune rSC0016 (pS-SaoA) strain test pigs attack malicious lung tissue photo through SS2.
Figure 46 is the variation relation figure of time and body temperature after attacking poison through SS2.
Specific embodiment
In order to illustrate technical scheme and technical purpose, below in conjunction with the accompanying drawings and specific embodiment is to the present invention
It is described further.
First, Salmonella choleraesuls attenuated carrier is built：
1st, build and contain ΔsopBThe suicide vector of mutator and identification：
According to the complete genome sequence of Salmonella choleraesuls in Genbank, wherein Δ is foundsopBGene complete genome sequence and its
Upstream and downstream sequence, primer P1, P2, P3 and P4, the 300bp of above downstream gene or so are designed according to the gene upstream and downstream gene
Fragment is homology arm, is knocked outsopBGene（ATG to TAA）, as shown in Figure 1.
Comprise the following steps that：Genome with Salmonella choleraesuls C78-3 is expanded by primer P1 and P2 as template through PCR
IncreasepipCHomology arm gene order, referred to as fragment 1；Genome with Salmonella choleraesuls C78-3 as template, by primer P3
Expanded through PCR with P4pipDThe homology arm sequence of gene, referred to as fragment 2.By fragment 1 and fragment 2 according to bases longs 1:1 mixes
Close, using above-mentioned mix products as template, carry out Overlap PCR amplifications using primer P1 and primer P4 and obtain length be
The homology arm fragment of 493bp.Reclaim the homology arm fragment and be connected to pM18T cloning vectors, the JM109 competent cells of conversion,
Above-mentioned bacterial plasmid is extracted, then through KpnI and SacI digestions, is connected to the suicide plasmid with chloramphenicol and sucrose selection markers
In carrier pRE112（Fig. 2）, through digestion identification and sequencing, will correctly lack ΔsopBThe pRE112 carriers of gene order
（It is named as pS006）Electricity is converted into engineering bacteria Escherichia coli χ 7213, is named as：χ7213（pS006）, it is standby in -20 DEG C.
The nucleotide sequence of primer P1, P2, P3 and P4 is respectively：
P1：5’- CGCAGAGCTCATATCACCTATAATTATC - 3’.
P2：5’- CATATAGTTACCTCAAGACAGCGTTTTTAATATTCCTG - 3’.
P3：5’- CAGGAATATTAAAAACGCTGTCTTGAGGTAACTATATG - 3’.
P4：5’- ATTAGGTACCAGCAGTATTGTCTGCGTCAGC - 3’.
Identified by PCR using primer P1 and P4 and contain ΔsopBThe suicide vector χ 7213 (pS006) of mutation, containing ΔsopB
The PCR positive fragments size of mutation is 493bp, as seen from Figure 3：Containing ΔsopBThe suicide vector χ 7213 (pS006) of mutator
Successfully construct.
2nd, build and introduce ΔsopBRSC0013 plants of mutator：
Respectively with wild type Salmonella choleraesuls C78, lacked ΔmanA、ΔPcrp::TT araC PBAD crpAnd ΔrelA::araC PBAD lacI The C78-3 Salmonella choleraesuls of TT genes be recipient bacterium, donor bacterium be step 1) in it is obtained
χ 7213（pS006）（Containing ΔsopBThe suicide vector of mutator）.Lacking ΔmanA、ΔPcrp::TT araC
PBAD crpAnd ΔrelA::araC PBAD lacI Δ is introduced in the C78-3 Salmonella choleraesuls of TT genessopBMutation, builds
Obtain rSC0013 plants.
Specific method is as follows：
Obtained according to method preparation in the 0040th to 0101 section of the specification of Chinese patent literature CN 104498418A and lacked ΔmanA、ΔPcrp::TT araC PBAD crpAnd ΔrelA::araC PBAD lacI The C78-3 Salmonella choleraesuls of TT genes,
It is named as Salmonella choleraesuls rSC0010.
Prepare recipient bacterium bacterium solution：Picking Salmonella choleraesuls rSC0010 in the LB fluid nutrient mediums of 5mL, through 10-12
Hour culture.
Prepare donor bacterium bacterium solution：Δ will be containedsopBThe suicide vector χ 7213 (pS006) of mutator is placed in the LB liquid of 5mL
In body（Wherein 2,6- containing 50ug/mL diaminopimelic acids（DAP）, the chloramphenicol of 25mg/mL, through culture in 10-12 hours.
Donor bacterium and recipient bacterium are engaged, and was cultivated through 10-12 hours, repurity to single bacterium colony.
Picking single bacterium is cultivated to cloud in falling within LB fluid nutrient mediums, is serially diluted for 10 times and is coated LB（Containing 5% sucrose）
In solid medium；From solid medium picking single bacterium colony respectively at LB plates, the LB containing chloramphenicol（Chloramphenicol containing 25mg/mL）
Plate is rule, in 37 DEG C of incubated overnights, the single bacterium colony that then picking only grows in LB.
Bacterium colony PCR identification Δs are carried out according to the above primer P1 and P4sopBMutation.ΔsopBThe PCR pieces of gene-deleted strain positive bacteria
Duan great little is 493 bp, by C78-3（ΔsopB）With the introducing Δ being built intosopBMutator (Δ Pcrp::TT
araCPBAD crp, ΔmanA , ΔrelA::araC PBAD lacI TT + ΔsopB）Be respectively designated as rSC0017 plants and
RSC0013 plants.
As seen from Figure 4：RSC0017 plants（2-3 electrophoresis road in Fig. 4）With rSC0013 plants（4-6 electrophoresis road in Fig. 4）Middle success
Introduce ΔsopBMutation.The correct bacterial strain of PCR qualification results is purified and is identified again.To identifying that correct bacterial strain PCR expands again
Increase itsopBGene upstream and downstream sequence, and send the Nanjing Jin Sirui companies to carry out sequencing, the correct bacterial strain of sequencing result is protected
Deposit.
3rd, attenuation Salmonella choleraesuls rSC0016 is built：
In rSC0013 (ΔsmanA, Δ Pcrp::TT araC PBAD crp, ΔrelA::araC PBAD lacI TT, ΔsopB）In
Introduce ΔasdAMutator.
It is specific as follows：
Prepare recipient bacterium bacterium solution：Picking rSC0013 single bacteriums are fallen within the LB fluid nutrient mediums of 5mL, are cultivated through 10-12 hours.
Obtained according to the 0066th~0071 phase method in the specification of Chinese patent literature CN 104498418A and contain ΔasdA
Suicide vector χ 7213 (pYAs004).
Prepare donor bacterium bacterium solution：Picking contains ΔasdASuicide vector χ 7213 (pYAs004) is in the LB fluid nutrient mediums of 5 mL
In (containing 50 μ g/mL 2,6- diaminopimelic acids, 25 mg/mL chloramphenicol), cultivated through 10-12 hours.
Take each 100 μ L coatings of the bacterium solution of donor bacterium and recipient bacterium and be engaged in LB solid mediums (containing 50 mg/mL 2,6-
Diaminopimelic acid) in, 37 DEG C of constant incubator overnight incubations；Scraping lawn turns to be inoculated in LB solid mediums (containing 50 μ g/
ML 2,6- diaminopimelic acid, 25 mg/mL chloramphenicol), cultivated through 10-12 hours under 37 DEG C of environment；It is purified to zygomycete
Single bacterium colony, picking are grown on LB solid mediums (containing 50 μ g/mL 2,6- diaminopimelic acids, 25 mg/mL chloramphenicol)
Several single bacteriums are fallen within LB (containing 50 μ g/mL 2,6- diaminopimelic acids) fluid nutrient medium, 37 DEG C, 220 rpm shaken cultivations
It is in cloud to bacterium solution；100 μ L bacterium solutions are taken, continuous 10 times are diluted to suitable concentration and coat LB solid mediums (containing 50
Mg/mL 2,6- diaminopimelic acid, 5% sucrose) in；Picking single bacterium colony is respectively at common LB solid mediums, containing chloramphenicol
LB solid mediums (containing 25 mg/mL chloramphenicol), the LB solid mediums containing chloramphenicol and 2,6- diaminopimelic acids (contain 50
Mg/mL 2,6- diaminopimelic acid, 25 mg/mL chloramphenicol) in differentiate culture, picking is only containing 2,6- diaminourea heptan two
The single bacterium colony grown in the LB solid mediums of acid.
With the primer Δ publishedasdA-FAnd ΔasdA-RCarry out bacterium colony PCR identification ΔsasdAMutation.ΔasdALack
The PCR fragment size for losing strain positive bacteria is 633bp, is illustrated by Fig. 5：rSC0013(ΔmanA ΔPcrp::TT araC PBAD crp
ΔrelA::araC PBAD lacI TT ΔsopB）Introduce ΔasdAMutation has been successfully constructed.To identifying correct bacterial strain PCR again
Expand itasdAGene upstream and downstream sequence, and send the Nanjing Jin Sirui companies to carry out sequencing, by the correct bacterial strain of sequencing result
It is named as rSC0016.
Primer ΔasdA-F：TGCTCTAGATGTGCATGGCAATCGCCCAAC.
Primer ΔasdA-R：TCCCCCGGGTATCTGCGTCGTCCTACCTTC.
RSC0016 is deposited in the China General Microbiological strain positioned at city of BeiJing, China Chaoyang District North Star West Road 1 institute 3
Preservation administrative center (CGMCC), preservation day is on June 20th, 2016, and deposit number is CGMCC NO.12645, the biomaterial
Classification And Nomenclature be:Salmonella choleraesuls are attenuated, Latin name is:Salmonella choleraesuis。
2nd, phenotypic evaluation is carried out to the attenuation Salmonella choleraesuls rSC0016 for building：
1st, Δ in rSC0016sopBThe phenotypic evaluation of mutation：
The new zealand white rabbit fasting overnight of 8 week old of health is taken, tracheae injection isoflurane anesthesia exposes ileum, every 5-
6cm is pricked with knot, is spaced 1cm, and 3 intestinal segments are ligatured altogether.It is 1 × 10 that every section of ileum injects 1mL total bacterias respectively9CFU's
Salmonella choleraesuls C78-3, rSC0017（C78-3（ΔsopB）, from the step 2 of embodiment 1）Prepare gained）With LB liquid
Body culture medium is blank.Suture abdominal musculature and skin, new zealand white rabbit woollen blanket maintain body temperature on 37 DEG C of left sides
It is right.After operation 8 hours, new zealand white rabbit is injected with yellow Jackets tracheae and implements euthanasia.Rabbit belly is reopened, is received
Secretion in every section of ileum of collection, and compare their volume；Then by above-mentioned secretion be diluted to suitable dilution factor after
Maconkey agar culture medium is coated with separation test bacterial strain, calculates bacteria total amount in ileum secretion；Take each section of ileum good fortune simultaneously
Your Malin's solution is fixed and makes tissue pathological slice, inflammatory reaction of the observation different strains to New Zealand rabbits enteron aisle.Result shows
Show containing ΔsopBRheuminess thing and bacteria total amount are considerably less than street strain C78-3 plants in the ileum of the rSC0017 infection of mutation
（Such as Fig. 6 and Fig. 7, ##：p<0.01；#：p<0.05）, but all it is significantly more than LB blank control groups（Such as Fig. 6 and Fig. 7, * *：P<
0.01）；HE coloration results are displayed that containing ΔsopBInflammatory cell is considerably less than C78-3 open countries in rSC0017 plants of intestinal tissue of mutation
Strain, intestinal villus is more neat compared with street strain's group.Prove ΔsopBMutation can substantially reduce Salmonella choleraesuls street strain and cause
Intestinal inflammatory reaction, as shown in Figure 8,9, 10.Experimental result is the average of three experiment repetitions.Fig. 8 is inoculation street strain
After C78-3, intestinal mucosa inflammatory cell infiltration；After Fig. 9 is for inoculation rSC0016, intestinal mucosa fine hair is complete；Figure 10 is inoculation LB, intestines
Mucous membrane fine hair is complete.
2nd, Δ in rSC0016asdAThe phenotypic evaluation of mutation：
In Chinese Industrial Standards (CIS) low virulent strain C500（Purchased from middle inspection institute）In Δ is introduced by the method for above-mentioned homologous recombinationasdA Mutation,
RSC0018 is named as, there is no arabinose to regulate and control to postpone attenuation and do not have as in following experimentsopBGene delection
Negative control strain.
Picking rSC0016, rSC0018 single bacterium colony are inoculated with 5 mL LB fluid nutrient mediums and diaminourea containing 2,6- heptan two respectively
In the LB fluid nutrient mediums (containing 50 μ g/mL 2,6- diaminopimelic acids) of acid, in 37 DEG C of constant-temperature table 220rpm shaken cultivations
Overnight, the growth conditions that the bacterium is cultivated in two kinds of Different Nutrition conditions are observed.The visible Δ of resultasdAThe bacterial strain of mutation is in nothing
External source 2, does not grow in the environment of 6- diaminopimelic acids, and LB fluid nutrient mediums are limpid transparent.The result shows, rSC0016,
RSC0018 could only grow in the LB fluid nutrient mediums for containing 2,6- diaminopimelic acids, in LB fluid nutrient mediums not
Can growth, thus rSC0016, rSC0018 plant of ΔasdAIt is mutated successfully.
3rd, apply：
Streptococcus suis are a kind of important infectious diseases common to human beings and animals, not only affect pig industry sound development, and also serious harm
The health of the mankind.The pig of current China also has many incomplete aspects with commercialization hammer bacteria vaccine.
In the present embodiment, the Sao for selecting most of Streptococcus suis serological type strains to have（surface antigen
one）As the heterologous antigen of attenuated strain, it is cloned into prokaryotic expression carrier pYA3493, referred to as containssaoAThe protokaryon table of gene
Up to plasmid pS-SaoA.PS-SaoA is converted to the delay attenuation regulated and controled containing arabinose and postpones expression exogenous antigen system
WithsopBWithasdAIn the cholera attenuated Salmonell rSC0016 of gene delection, vaccine candidate strain rSC0016 (pS- are formed
SaoA) bacterial strain, and the evaluation of virulence and immunoprotection efficiency has been carried out in BALB/c mouse and weanling pig, it is the anti-of swine disease
Control provides new thinking.
1st, rSC0016 (pS-SaoA), SC0011 (pS-SaoA), rSC0018 (pS-SaoA) bacterial strain are built：
1）Build asd+Expression SaoA albumen pronucleus expression plasmids pS-SaoA：
With reference to streptococcus suis 2-type bacterium design amplification in GenBanksaoAThe primer of genesaoA- F andsaoA- R, with CVCC3928
Streptococcus suis 2-type is template, PCR amplificationssaoAGene 777bp（See Figure 11）.To be sequenced correctsaoAGene, with protokaryon table
Up to carrier Asd+PYA3493 recovery products after double digestion, and connect, in conversion to engineering bacteria χ 7213.Using in table 3
SaoA-F, saoA-R primer carry out bacterium colony PCR and digestion identification, final to obtain the PCR and all positive (pS- of χ 7213 of digestion identification
SaoA) bacterial strain, the wherein bp of pYA3493 clip sizes 3133 bp, saoA clip sizes 777（Such as Figure 12）, sequencing identification again
SaoA genes, as a result correctly.
Amplification saoAPrimer usedsaoA- F andsaoA- R sequences：
saoA-F：ATGGATCCCAACCTGATGGGGGAC；
saoA-R：GCGTCGACCATTGCTTCCTTAGAG.
2）Build rSC0016 (pS-SaoA), rSC0011 (pS-SaoA), rSC0018 (pS-SaoA), rSC0016
(pYA3493), rSC0011 (pYA3493), rSC0018 (pYA3493) bacterial strain：
The plasmid of χ 7213 (pS-SaoA) and χ 7213 (pYA3493) bacterial strain is extracted, that is, expresses the asd of SaoA albumen+ Protokaryon table
Up to plasmid pS-SaoA and empty carrier plasmid pYA3493.PS-SaoA and pYA3493 plasmids are transformed into attenuation pig by heat shock method
In cholera salmonella rSC0016, rSC0011 and rSC0018 competent cell.
Concrete operations are：Taken under aseptic condition appropriate pS-SaoA or pYA3493 plasmids respectively with rSC0016, rSC0011,
RSC0018 competence bacterias；Ice bath 30min after mixing, 42 DEG C of heat shock 90s, add 700 μ L LB fluid nutrient mediums, in 37 DEG C of constant temperature
Shaking table 100rpm shaken cultivations 45min；The μ L of above-mentioned bacterium solution 200 are taken, LB solid mediums are coated, in 37 DEG C of constant incubator trainings
Support 24h；Picking surface is smooth in linen single bacterium colony, and using saoA-F in table 3, saoA-R primers carry out bacterium colony PCR identifications,
Identified by upgrading grain and digestion；Pure culture digestion identification again is carried out to PCR positive bacterias, digestion result is shown in pYA3493 empty carriers
It is 3113bp, saoA genes are 777bp（As shown in figure 13）.It is final to obtain the PCR and all positive rSC0016 (pS- of digestion identification
SaoA), rSC0016 (pYA3493), rSC0018 (pS-SaoA), rSC0018 (pYA3493), rSC0011 (pS-SaoA) and
RSC0011 (pYA3493) strain.
2nd, detection rSC0016 (pS-SaoA), rSC0011 (pS-SaoA), rSC0018 (pS-SaoA) bacterial strain SaoA albumen
Expression and expression quantity compare：
The correct rSC0016 of qualification result (pS-SaoA), rSC0011 (pS-SaoA), rSC0018 (pS- during being walked on picking
SaoA) the single bacterium colony of bacterial strain, respectively at 37 DEG C of concussion and cultivates of 220rpm in 5 milliliters of LB fluid nutrient mediums to OD600Value is 0.8
When be collected by centrifugation thalline, plus 1mL PBS resuspended.The above-mentioned thalline of instrument cracking to bacterium solution is cracked with ultrasonic cell clarify bright,
12000rpm centrifuging and taking supernatants.Taking the supernatant of equivalent carries out Western Blot identifications.By rSC0016 (pS-SaoA),
RSC0018 (pS-SaoA), the ultrasonic degradation liquid of rSC0011 (pS-SaoA) bacterial strain carry out Western Blot identifications.Result is such as
Figure 14, it is seen that three kinds of bacterial strains can express SaoA albumen, and in the case of identical OD values, equivalent rSC0016 (pS-SaoA) bacterium
Liquid（Label 3 in Figure 14）The SaoA protein contents of middle expression are slightly above rSC0011 (pS-SaoA), are significantly higher than rSC0018 (pS-
SaoA)（Label 2 and 4 in Figure 14）The amount of expression.
3rd, Stability Determination of the pS-SaoA plasmids in rSC0016 (pS-SaoA)：
Picking identifies that right-on rSC0016 (pS-SaoA) single bacterium colony is inoculated in 2mL LB fluid nutrient mediums, in 37 DEG C of constant temperature
Shaking table shaken cultivation.With 12h as a generation, by bacterium solution according to 1:The fresh LB fluid nutrient mediums of 100 inoculations, so continuously reach 50
Generation.The generation single bacterium of picking 10,20,30,40,50 carries out double digestion identification respectively.Result shows, occur after double digestion pYA3493 andsaoABand, be consistent with expected results, as shown in figure 15.Result shows that pS-SaoA recombinant plasmids can be steady in rSC0016
It is fixed to replicate passage.
4th, rSC0016 (pYA3493), rSC0018 (pYA3493), the field planting reality of rSC0011 (pYA3493) and C78-3
Test：
Picking rSC0016 (pYA3493), rSC0018 (pYA3493) and rSC0011 (pYA3493) and C78-3 are in the LB of 5 mL
Fluid nutrient medium, in 37 DEG C of concussion and cultivate 12h, by volume 1:The 100 LB fluid nutrient mediums for being inoculated in 50 mL, in 37 DEG C of perseverances
Shaken cultivation to the OD600 values of bacterium solution are 0.9 or so on warm shaking table, and thalline is collected by centrifugation, and add the resuspended precipitations of PBS, continuous 10
Dilute again.Take continuous 10 times of 100 μ L and be diluted to suitable dilution factor and in counting, another part bacterium solution on Mai Kangkai culture mediums
For Mice Inoculated.48 6 week old female BAl BIcs/c mouse are divided into 4 groups, every group 12, every group of difference oral vaccination 1 × 109's
RSC0016 (pYA3493), rSC0018 (pYA3493), rSC0011 (pYA3493), C78-3（Street strain）；After inoculation
Aseptic 3 livers of mouse, spleen, the enteron aisle of taking of 3d, 7d, 14d, 21d sends Yi Ershi lymph nodules, weighs, grinds, 10 times of dilution groups
Homogenate is knitted, the bacterium solution for taking 100 μ L is counted.Liver, Splenic vessel liquid coating LB solid mediums, mesenteric lymph brief summary homogenate are applied
Bu Maikangkai culture mediums, in 37 DEG C of incubated 24h；According to colony growth result on corresponding culture medium, bacterium in internal organs is calculated
Content.Result shows, because C78-3 is Salmonella choleraesuls velogen strain, in inoculation 3d, in liver, spleen and enteron aisle group
Many C78-3 bacterial strains more than attenuated strain can be separated in Yi Ershi lymph nodules（In Figure 16,17,18, $ $, p<0.01）,
The mouse of 5d, all inoculation C78-3 is all dead；And 3 attenuated strains, with the growth of inoculation time, each attenuated strain is each dirty
Bacterium amount on device is gradually decreased.Field planting quantity and rSC0018 of the rSC0016 (pYA3493) in mouse liver and spleen
(pYA3493) the field planting quantity of bacterial strain is similar, and substantially lower than rSC0011 (pYA3493) bacterial strain field planting quantity（Figure 16,
In 17, ##, p<0.01）, show that the virulence of rSC0016 (pYA3493) and rSC0018 (pYA3493) is less than rSC0011.But
Enteron aisle sends Yi Ershi lymph nodules, the field planting energy of oral rear 7d-21d, rSC0016 (pYA3493) and rSC0011 (pYA3493)
Power is similar, is all significantly stronger than the colonization ability of rSC0018 (pYA3493) strain（In Figure 18, * *, p<0.01）, show rSC0016
(pYA3493) it is strong as the colonization ability of rSC0011 (pYA3493) strain in the colonization ability of lymphoid tissue.
4th, the Efficacy evaluation in BALB/c mouse is carried out to vaccine candidate strain：
Picking rSC0016 (pS-SaoA), rSC0016 (pYA3493), rSC0018 (pS-SaoA), rSC0018 (pYA3493),
RSC0011 (pS-SaoA), rSC0011 (pYA3493) in the LB fluid nutrient mediums of 5 mL, in 37 DEG C of concussion and cultivate 12h, by body
Product compares 1:The 100 LB fluid nutrient mediums for being inoculated in 50 mL, in the OD of shaken cultivation on 37 DEG C of constant-temperature tables to bacterium solution600It is worth and is
0.9 or so, thalline is collected by centrifugation, add the resuspended precipitations of PBS, continuous 10 times of dilutions.Continuous 10 times of 100 μ L are taken to be diluted to suitably
Dilution factor and on Mai Kangkai culture mediums count, another part bacterium solution be used for immune mouse.
The BALB/c mouse of 105 6 week old is divided into 7 groups, every group of 15 mouse.Wherein 1 group oral PBS is used as negative control
Group.Other 6 groups of difference oral immunities 109The rSC0016 (pS-SaoA) of CFU/20 μ L, rSC0016 (pYA3493), rSC0018
(pS-SaoA), rSC0018 (pYA3493), rSC0011 (pS-SaoA), rSC0011 (pYA3493) bacterium solution.Add within 3 weeks after head exempts from
It is strong to be immunized once.3 weeks, 5 weeks venous blood collections under the jaw of mouse after head exempts from, while using PBS washing vaginas, collect vaginal mucosa
Flushing liquor.The blood that will be collected into overnight is obtained serum in 4 DEG C.Serum and vagina mucosa flushing liquor pin are detected with indirect ELISA
To the IgG and IgA antibody potency of the SaoA albumen of streptococcus suis 2-type.0.5d, 3d and 5d after exempting from two, each group is under mouse jaw
Venous blood collection collects serum, and each group 5 mouse of euthanasia during 7d after exempting from two, takes spleen, and lungs are made in homogenate centrifuging and taking
Clearly, detected by the cell and humoral immune response of each strain inducing mouse by the detection kit of cell factor IFN-γ and IL-4
Level；5 weeks after head exempts from, 10 mouse remaining to each group, with 30 × LD50SS2 strains carried out by intraperitoneal injection approach
Poison is attacked, observation 21 days simultaneously records mouse survival situation.
1st, the survey for SaoA protein Is gG and mucosa-immune antibody I gA induced after the immune BALB/c mouse of each attenuated strain
It is fixed：
Enzyme mark version is coated with the SaoA albumen of purifying, the method detection first immunisation of indirect ELISA is directed to for 3 weeks, 5 weeks in serum
IgA potency in the IgG potency and vaginal washing fluid of SaoA, enzyme mark version, indirect ELISA are coated with C78-3 outer membrane proteins OMPs
Method detection serum in induce the IgG potency for Salmonella choleraesuls outer membrane protein OMPs, each sample repeat examine
Survey 3 times.Shown in result as Figure 19,20,21, the blood for SaoA albumen of all immune attenuated strain inductions containing SaoA albumen
The potency of clear IgG and vaginal mucosa antibody I gA is all significantly higher than the antibody of the empty carrier attenuated strain induction for not containing SaoA albumen
Potency（See Figure 19 and 21, * *, P<0.01）；The 3rd week and the 5th week after head exempts from, rSC0016 is immunized（pS-SaoA）Strain and
rSC0011（pS-SaoA）The serum antibody IgG levels and mucoantibody IgA levels for SaoA that strain mouse produces are substantially high
In immune rSC0018（pS-SaoA）Strain（See Figure 19 and 21, #, P<0.05；##, p<0.01）；Head exempts from the 5th week afterwards, is immunized
rSC0016（pS-SaoA）Strain or rSC0016（pYA3493）The OMPs's for Salmonella choleraesuls that the mouse of strain produces
Serum antibody IgG levels are apparently higher than immune rSC0018（pYA3493）The mouse of strain（See Figure 20, #, P ＜ 0.05）.
2nd, the IFN-γ and the level of IL-4 for being induced after the immune BALB/c mouse of each attenuated strain are determined：
Two BALB/c mouses for exempting from rear 7d are taken, whole spleen and whole lung is taken, homogenate is made.12000rpm centrifugations 1min after multigelation
Take supernatant.0.5d, 3d and 5d after secondary immunity are taken, venous blood collection collects serum under jaw.
Above-mentioned tissue is homogenized supernatant and serum as sample, the kit of mouse IL-4, IFN-γ is determined with ELISA
Mouse cytokine level is determined, two exempt from shown in result as Figure 22,23,24,25 of rear 7d, rSC0016 is immunized（pS-SaoA）、
rSC0011（pS-SaoA）And rSC0018（pS-SaoA）Mouse produce IFN-γ and IL-4 levels be all significantly higher than it is immune
The level of the attenuated strain induction containing empty carrier（Figure 22,23,24,25, * *, P ＜ 0.01）；Immune rSC0016（pS-SaoA）It is small
Cell factor IFN-γ level is significantly higher than immune rSC0011 in mouse lungs and spleen（pS-SaoA）Or immune rSC0018（pS-
SaoA）Cell factor IFN-γ level in group mouse lung and spleen（Figure 22, #, p<0.05；##, P ＜ 0.01）；And it is immune
rSC0016（pS-SaoA）And rSC0011（pS-SaoA）The water of cell factor IFN-γ and IL-4 in strain mouse lung and spleen
Averagely it is significantly higher than immune rSC0018（pS-SaoA）The group of strain（Figure 22,23, #, p<0.05；##, P ＜ 0.01）.
Shown in the testing result of the Cytokine of Serum of immune mouse as Figure 24,25, rSC0016 is immunized（pS-SaoA）、
rSC0011（pS-SaoA）And rSC0018（pS-SaoA）Mouse produce IFN-γ and IL-4 levels be all significantly higher than it is immune
The level of the attenuated strain induction containing empty carrier（Figure 24,25, *, p<0.05；*, P ＜ 0.01）；Immune rSC0016（pS-
SaoA）The concentration of IFN-γ and IL-4 reaches peak-peak in 12 hours after exempting from two in the mice serum of group, after booster immunization
Apparently higher than immune rSC0011 in 72h（pS-SaoA）、rSC0018（pS-SaoA）Group（Figure 24,25, #, p<0.05；##, P ＜
0.01）；Immune rSC0011（pS-SaoA）The mice serum IFN-γ and IL-4 concentration of group exempt from two after in 72h concentration it is obvious
Higher than immune rSC0018（pS-SaoA）Group（Figure 24,25, ##, P ＜ 0.01）.
3rd, immune mouse attacks poison：
5 weeks after head exempts from, 10 mouse remaining to each group, with 30 × LD50SS2 strains carried out by intraperitoneal injection approach
Poison is attacked, observation 21 days simultaneously records mouse survival situation.After attacking poison, the mouse of oral immunity rSC0016 (pS-SaoA) is 21d's
Survived in observation period, the survival rate of the mouse of oral immunity rSC0011 (pS-SaoA) is 40%, and oral immunity rSC0018
(pS-SaoA), the mouse of rSC0016 (pYA3493), rSC0011 (pYA3493), rSC0018 (pYA3493) and control group exists
It is all dead in 24h-48h, it is as shown in the table.
SS2 attacks malicious Vaccine effectiveness table：
5th, the Efficacy evaluation in weanling pig is carried out to vaccine candidate strain：
1st, the preparation of vaccine candidate strain and piglet is immune：
Picking rSC0016 (pS-SaoA), rSC0016 (pYA3493), rSC0018 ((pS-SaoA), rSC0018 (pYA3493)
In the LB fluid nutrient mediums of 5 mL, in 37 DEG C of concussion and cultivate 8h, by volume 1:The 100 LB Liquid Cultures for being inoculated in 100 mL
Base, in the OD of shaken cultivation on 37 DEG C of constant-temperature tables to bacterium solution600It is 0.9 or so to be worth, and thalline is collected by centrifugation, and adds PBS resuspended
Precipitation, continuous 10 times of dilutions.Take continuous 10 times of 100 μ L and be diluted to 10-7、10-8、10-9, and in being counted on LB solid mediums, remain
Remaining bacterium solution is used to that piglet to be immunized.
The weanling pig of 65 21 ages in days is divided into 5 groups, every group 13；One group used as blank control group；Remaining 4 groups by oral administration
10 are gavaged respectively9The rSC0016 (pS-SaoA) of CFU, rSC0016 (pYA3493), rSC0018 (pS-SaoA), rSC0018
(pYA3493) strain；Each group head exempt from 3 weeks after booster immunization once, 3 weeks after head exempts from, 5 weeks from the blood sampling of the vena cava anterior of piglet, together
When collect immune swine bronchia mucosal swab.The blood that will be collected into overnight is obtained serum in 4 DEG C；After exempting from two 0.5d, 3d and
5d, each group collects serum from the vena cava anterior blood sampling of piglet；3 piglets of each group euthanasia during 7d, take spleen, lungs after exempting from two
Be made homogenate centrifuging and taking supernatant, serum and spleen to immune swine, lung tissue homogenate supernatant, with porcine cytokine IFN-γ,
The detection of IL-4 and IL-17 detection kits is induced the cell and humoral immune response level of immune swine by each strain；5 after head exempts from
In week, 5 immune swines are taken in each group respectively with 5.0 × 108The SS2 of dosage, takes 5 immune swines with 1.0 × 1010SS7 strains, through ear
Poison is attacked in intravenous injection, and observation 14 days simultaneously records piglet clinical symptoms and survival condition, dead pig is analysed, and gathers sick dead pig
Brain and lung tissue carry out the observation of pathological change.
2nd, the antibody level detection for SaoA albumen that piglet produces after being immunized：
Serum is made in vena cava anterior blood sampling within 3 weeks, 5 weeks after head exempts from, while taking nasal cavity cotton swab.It is coated with the SaoA albumen of purifying
ELISA Plate, for IgA antibody potency, pin in the IgG and nasal membrane of Streptococcus suis SaoA albumen in indirect ELISA detection serum
To the IgG potency of Salmonella choleraesuls carrier OMPs.Result is as shown in Figure 26,27,28.After head exempts from 3 weeks and 5 weeks, it is immunized
Serum antibody IgG potency for SaoA that the piglet of rSC0016 (pS-SaoA) strains and rSC0018 (pS-SaoA) strain produces and
Mucoantibody IgA potency is all remarkably higher than immune empty carrier group (Figure 26,28, * *, P ＜ 0.01)；Immune rSC0016 (pS-
SaoA) the serum antibody IgG potency and mucoantibody IgA potency for SaoA that strain group is produced are all remarkably higher than immune
RSC0018 (pS-SaoA) strain group（Figure 26,28, #, p<0.05；##, P ＜ 0.01）；What the piglet of immune rSC0016 plants of group produced
For carrier bacterium OMPs serum antibody IgG levels also apparently higher than immune rSC0018 plants of group（Figure 27, #, P ＜ 0.05；##, P
＜ 0.01）.
3rd, the level of piglet IFN-γ and IL-4, IL-17 is determined after being immunized：
0.5d, 3d and 5d after booster immunization, serum is collected through vena cava anterior blood sampling respectively；And when 7d every group cut open and kill 3
Piglet, takes spleen, and lungs are made homogenate.Through cell factor in porcine cytokine ELISA kit detection serum and tissue homogenate
The level of IFN-γ, IL-4, IL-17.Result is as shown in Figure 29,30,31,32,33,34：After two exempt from 7 days, in piglet spleen and
In lungs, rSC0016 is immunized（pS-SaoA）And rSC0018（pS-SaoA）IFN-γ, IL-4 the and IL-17 levels of induction all show
Write the bacterial strain higher than immune empty carrier（Figure 29,30,31, * *, p<0.01）；In lungs, rSC0016 is immunized（pS-SaoA）Group
The IFN-γ of generation, IL-4 and IL-17 levels are all significantly higher than immune tradition attenuated strain rSC0018（pS-SaoA）Group (Figure 29,
30th, 31, #, p<0.05；##, P ＜ 0.01)；In spleen, rSC0016 is immunized（pS-SaoA）Cell in attenuated strain group serum
Factor IFN-γ, the concentration of IL-4 are obviously higher than immune traditional attenuated vaccine strain rSC0018（pS-SaoA）Vaccine strain group figure
29th, 30,31, #, p<0.05；##, P ＜ 0.01).
The testing result of immune piglet Cytokine of Serum（See Figure 20）It has been shown that, in 72h after booster immunization, is immunized
rSC0016（pS-SaoA）And rSC0018（pS-SaoA）Piglet produce IFN-γ and IL-4 IL-17 levels it is all significantly high
In the level of the immune attenuated strain induction containing empty carrier（Figure 32,33,34, *, p<0.05；*, P ＜ 0.01）；In booster immunization
Afterwards in 48h, rSC0016 is immunized（pS-SaoA）And rSC0018（pS-SaoA）Piglet produce IL-17 levels be all significantly higher than
The level of the immune attenuated strain induction containing empty carrier（Figure 34, *, p<0.05；*, P ＜ 0.01）；In 72h after booster immunization,
Immune rSC0016（pS-SaoA）The concentration of IFN-γ, IL-4 and IL-17 is apparently higher than immune rSC0018 in the piglet serum of group
（pS-SaoA）Group（Figure 32,33,34, #, p<0.05；##, P ＜ 0.01）.
4th, piglet is immunized attacks poison：
To evaluate ΔsopBThe immunogenicity of Salmonella choleraesuls is being improved, its guarantor attacked streptococcus suis 2 and 7 types is being detected
Shield power, to oral immunity rSC0016 (pS-SaoA), rSC0016 (pYA3493), rSC0018 ((pS-SaoA), rSC0018
(pYA3493) and orally all piglets of the blank control group of equivalent PBS carry out the challenge viral dosage of streptococcus suis 2 and 7 types.Head exempts from
5 weeks each immune groups take 5 piglets afterwards, respectively with 5.0 × 108The SS2 of dosage and 1.0 × 1010SS7 strains carry out ear vein injection
Poison is attacked, observation 14 days simultaneously records piglet clinical symptoms and survival condition.
1 ）SS2 attacks piglet survival rate and pathological characters after poison：
The piglet of immune rSC0016 (pS-SaoA) strain attacks poison 5.0 × 10 through ear vein8After the SS2 of dosage, indivedual piglets are being attacked
There is slight spiritual depressed phenomenon after poison in 48h, be clearly better after 72h, then without obvious pathological symptom, immune swine survival rate is
100%；The son of immune rSC0018 (pS-SaoA) strain, rSC0016 (pYA3493) strains and rSC0018 (pYA3493) strains and PBS groups
Pig is all dead in 108h（As shown in figure 35）.
The pig of immune rSC0016 (pS-SaoA) strain does not have any pathological symptom, pig freedom of movement, and rSC0018 is immunized
(pS-SaoA) most of piglets of strain shown in 24h after poison is attacked it is drowsiness, do not eat, 72h begins with constipation after attacking poison, turn-take,
Forelimb is creeped, four limbs swimming shape, expiratory dyspnea, and arthroncus has limping；Oral PBS blank control groups and rSC0016
(pYA3493) after, the piglet of rSC0016 (pYA3493) groups attacks poison through SS2, the clinical symptoms of appearance are more serious；To blank pair
Dead pig according to group, rSC0018 (pS-SaoA), rSC0018 (pYA3493), rSC0016 (pYA3493) strain analyses discovery, disease
The lung of dead pig has serious cellulosic sample to become, Pulmonary hemorrhage, meningorrhagia etc., with immune rSC0016 (pS-SaoA) strain
Pig has no that the lesion and slight pneumonia of brain form notable contrast after attacking poison.Sick dead pig lung is taken, hepatic tissue separates SS2, can separate
To the SS2 attacked used by poison；Attack 14 days after poison, slaughter the survival pig of all immune rSC0016 (pS-SaoA) strain, cut open inspection take liver,
Spleen, lung, brain tissue, are not separated to and attack strain SS2.
The lungs and brain tissue of the dead pig of collection and survival pig make pathological section, and the visible control group of basis of microscopic observation is young
Pig is filled with the pig meningovascular of immune rSC0018 (pS-SaoA) strain, rSC0018 (pYA3493), rSC0016 (pYA3493) strain
Blood, oedema, inflammatory cell infiltration.Pulmonary venous pleonaemia, alveolar wall thickening, a large amount of inflammatories ooze out in alveolar, and a large amount of inflammatories are thin around blood vessel
Born of the same parents are infiltrated as shown in Figure 36-Figure 45.It is tight that Figure 36 and 37 visible control group piglets attack malicious tissues following MCAO in rats through wild type streptococcus suis 2-type
Reppear blood, lung tissue extravasated blood, bleeding and inflammatory ooze out；Figure 38 and 39 is the empty carrier that immunochemistry weakening strain rSC0018 is carried
Piglet attacks the also severe haemorrhage of malicious tissues following MCAO in rats through wild type streptococcus suis 2-type, and lung tissue extravasated blood, bleeding and inflammatory ooze out；Figure 40
It is that immunochemistry weakening strain rSC0018 carries the SaoA protein carriers piglet of expression Streptococcus suis through wild type pig streptococcus with 41
2 types attack malicious tissues following MCAO in rats bleeding, and lung tissue extravasated blood, bleeding and inflammatory ooze out；Figure 42 and 43 is the sky that attenuated strain rSC0016 is carried
Carrier piglet attacks malicious tissues following MCAO in rats bleeding through wild type streptococcus suis 2-type, lung tissue extravasated blood, bleeding and inflammatory oozes out in alveolar；
And there is not obvious pathological change such as Figure 44-Figure 45 in the brain tissue and lung of the immune most of piglet of rSC0016 (pS-SaoA) groups
It is shown.
2）SS7 attacks piglet survival rate and pathological characters after poison：
5th week after each attenuated strain is immune, 5 immune swines are taken, with 1.0 × 1010The SS7 of dosage attacks poison through ear vein.Result shows
Show, oral immunity rSC0016 (pS-SaoA) groups are within the observation period of 7 days without obvious clinical symptoms, and body temperature holding normal water
It is flat.And immune rSC0018 (pS-SaoA), rSC0018 (pYA3493), the pig of rSC0016 (pYA3493) strain occur in that body
Temperature rise（As shown in figure 46）, and apocleisis is occurred in that, spirit is depressed, the symptom such as asthma, and these symptoms disappear after continuing 5 days,
Temperature recovery is to normal level.
In sum, the present invention postpones attenuation and postpones the carrier of expression exogenous antigen in arabinose regulation and control virulence gene
Upper structure contains ΔsopBThe Salmonella choleraesuls carrier of mutator, can reduce Salmonella choleraesuls attenuated strain
Virulence, the inflammatory reaction that mitigation attenuated strain causes is significantly improved entrained by Salmonella choleraesuls to the damaging action of host
Exogenous antigen induces the immunogenicity of host, so that containingsopBThe pig of the carrying Streptococcus suis SaoA albumen of mutator is suddenly
Random salmonella vaccine Candidate Strain rSC0016（pS-SaoA）Complete protection can be provided the infection of SS2 and SS7 street strains
And cross-protection.
<110>Yangzhou University
<120>A kind of Salmonella choleraesuls attenuated carrier bacterium and its construction method
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<110>Yangzhou University
<120>A kind of Salmonella choleraesuls attenuated carrier bacterium and its construction method
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Claims (6)
1. a kind of Salmonella choleraesuls attenuated carrier bacterium, it is characterised in that：The attenuated carrier bacterium is to have lacked ΔmanA、Δcrp::TT araC PBAD、ΔrelA::araC PBAD lacI TT、ΔsopBAnd ΔasdAThe C78-3 hog cholera sramana of gene
Salmonella.
2. the construction method of Salmonella choleraesuls attenuated carrier bacterium as claimed in claim 1, it is characterised in that including following step
Suddenly：
1）Build and contain ΔsopBThe suicide vector of mutator：
Genome with Salmonella choleraesuls C78-3 expands homology arm fragment as template by primer, knocks out hog cholera sramana
In the gene order of Salmonella C78-3sopB Genetic fragment, and by the gene order for knocking out Salmonella choleraesuls C78-3
'ssopB Fragment gene after genetic fragment is connected in suicide plasmid pRE112, obtains missing ΔsopB Gene order is committed suiside
Plasmid；Δ will be lacked againsopB Gene order suicide plasmid is converted into engineering bacteria Escherichia coli χ 7213, is obtained and is contained ΔsopB
The suicide vector of mutator；
2）Build and introduce ΔsopBRSC0013 plants of mutator：
To have lacked ΔmanA、ΔPcrp::TT araC PBAD crpAnd ΔrelA::araC PBAD lacI The C78-3 of TT genes
Salmonella choleraesuls are recipient bacterium, with containing ΔsopBThe suicide vector of mutator is donor bacterium, by donor bacterium and recipient bacterium
Engaged, through culture, purifying, obtain and introduce ΔsopBRSC0013 plants of mutator；
3）Build Salmonella choleraesuls attenuated carrier bacterium：
To introduce ΔsopBRSC0013 plants of mutator is recipient bacterium, with containing ΔasdASuicide vector χ 7213 (pS004) is
Recipient bacterium, donor bacterium and recipient bacterium are engaged, and through culture, purifying, obtain Salmonella choleraesuls attenuated carrier bacterium.
3. the construction method of Salmonella choleraesuls attenuated carrier bacterium according to claim 2, it is characterised in that the step
1）In, the genome with Salmonella choleraesuls C78-3 is expanded by primer P1 and P2 as template through PCRpipCHomology arm base
Because of sequence；
Genome with Salmonella choleraesuls C78-3 is expanded by primer P3 and P4 as template through PCRpipDGene it is homologous
Arm sequence；
WillpipCHomology arm gene order andpipDThe homology arm sequence of gene is with bases longs 1:1 ratio is mixed to prepare mixing
Product；
Using mix products as template, carry out Overlap PCR amplifications to obtain length using primer P1 and primer P4 is 493bp's
Homology arm fragment, and the homology arm fragment is connected to pM18T cloning vectors, the JM109 competent cells of conversion are extracted above-mentioned
Bacterial plasmid, then through KpnI and SacI digestions, it is connected to the suicide plasmid carrier with chloramphenicol and sucrose selection markers
In pRE112, obtain lacking ΔsopBThe pRE112 carriers of gene order；
The nucleotide sequence of described primer P1, P2, P3 and P4 is respectively：
P1：5’- CGCAGAGCTCATATCACCTATAATTATC - 3’；
P2：5’- CATATAGTTACCTCAAGACAGCGTTTTTAATATTCCTG - 3’；
P3：5’- CAGGAATATTAAAAACGCTGTCTTGAGGTAACTATATG - 3’；
P4：5’- ATTAGGTACCAGCAGTATTGTCTGCGTCAGC - 3’.
4. the construction method of Salmonella choleraesuls attenuated carrier bacterium according to claim 2, it is characterised in that the step
2）In, take and lacked ΔmanA、ΔPcrp::TT araC PBAD crpAnd ΔrelA::araC PBAD lacI The C78-3 of TT genes
Salmonella choleraesuls were cultivated in LB fluid nutrient mediums through 10-12 hours, and recipient bacterium bacterium solution is obtained；
Δ will be containedsopBThe suicide vector of mutator was cultivated in LB liquid through 10-12 hours, and donor bacterium culture bacterium is obtained
Liquid；It is described to contain Δ for culturesopBIn the LB liquid of the suicide vector of mutation, 2,6- diaminopimelic acid contents are 50ug/
ML, chloromycetin content is 25mg/mL.
5. according to claim 2 or 4 Salmonella choleraesuls attenuated carrier bacterium construction method, it is characterised in that the step
Rapid 2）In, after purification, picking single bacterium is cultivated to cloud in falling within LB fluid nutrient mediums, is serially diluted for 10 times and is coated LB solids
In culture medium；From solid medium picking single bacterium colony respectively at LB plates, the LB plates line containing chloramphenicol, passed through under 37 DEG C of environment
Cultivate within 10-12 hours, the introducing Δ that then picking only grows in LBsopBRSC0013 plants of mutation.
6. the construction method of Salmonella choleraesuls attenuated carrier bacterium according to claim 2, it is characterised in that the step
3）In, rSC0013 plants is taken in LB fluid nutrient mediums, cultivated through 10-12 hours, prepared recipient bacterium bacterium solution；
Take containing ΔasdASuicide vector χ 7213 (pS004) was cultivated in LB fluid nutrient mediums through 10-12 hours, and donor is obtained
Bacterium cultivates bacterium solution；It is described to contain Δ for cultureasdAIn the LB fluid nutrient mediums of suicide vector χ 7213 (pS004), 2,6- diaminos
Base pimelic acid content is 50ug/mL, and chloromycetin content is 25mg/mL.
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| CN110373422A (en) * | 2019-07-12 | 2019-10-25 | 扬州大学 | Recombination cracking Salmonella choleraesuis and its construction method and application |
| CN113046384A (en) * | 2021-03-23 | 2021-06-29 | 扬州大学 | Construction method of broad-spectrum antiviral recombinant salmonella |
| CN115340997A (en) * | 2022-08-16 | 2022-11-15 | 扬州大学 | Lpp leader sequence-based recombinant salmonella construction method for targeted presentation and efficient expression of exogenous antigen |
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| CN110373422B (en) * | 2019-07-12 | 2022-05-10 | 扬州大学 | Recombinant cracked salmonella choleraesuis, and construction method and application thereof |
| CN113046384A (en) * | 2021-03-23 | 2021-06-29 | 扬州大学 | Construction method of broad-spectrum antiviral recombinant salmonella |
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