CN107805619A - Streptococcus suis 2-type divIVA gene knockout mutant strains and its application - Google Patents

Abstract

The invention belongs to genetic engineering field, is related to streptococcus suis 2-type divIVA gene knockout mutant strains and its application.The mutant strain Δ divIV preparation methods are：The divIVA genes of encoding D ivIVA albumen on the wild strain 05ZYH33 chromosomes of S.suis 2 are subjected to gene knockout using homologous recombination gene Knockout, combined PCR primer electrophoresis, RT PCR and DNA sequencing confirm, determine that obtained bacterial strain is gene knockout mutant strain, be named as 05ZYH33 Δs divIVA.Carrying out animal experiment study with mutant strain of the present invention, its is pathogenic, the result is that significantly reducing the virulence of experimental animal, can be applied to develop streptococcus suis 2-type attenuated vaccine.

Description

Streptococcus suis 2-type divIVA gene knockout mutant strains and its application

Technical field

The invention belongs to genetic engineering field, is related to streptococcus suis 2-type divIVA gene knockout mutant strains and its application.

Background technology

Streptococcus suis (Streptococcus suis, S.suis) is a kind of zoonosis disease of world-wide prevalence Opportunistic pathogen, pig meningitis, septicemia, arthritis, pneumonia and endocarditis etc. can be not only caused, but also people can be infected and caused a variety of Serious disease, the life security to related practitioner and broad masses cause serious threat.According to the antigenicity of capsular polysaccharide, S.suis can be divided into 35 serotypes, wherein virulence is most for 2 types (Streptococcus suis serotype 2, S.suis 2) By force, clinical recall rate highest.In recent years the prevalences of S.suis 2 are on the rise in the swinery in south China province, when have it is fairly large Break out, it is annual caused by economic loss reach billions of members.Broken out respectively in Jiangsu Province of China and Sichuan Province within 1998 and 2005 Extensive S.suis 2 infects the event of people, causes great life and property loss, and occur a high proportion of, state in patient Inside and outside rare TSS, the state of an illness is dangerous, case fatality rate height (60%~80%), triggers serious public health thing Part.Meanwhile the report for infecting and causing a disease on S.suis in the world in recent years also significantly increases, and have been reported that and be shown in China's perfume Port has been detected by the presence of the bacterium multiple antibiotic resistant strain, and the prevention and control to S.suis 2 bring new challenge.Therefore, Carry out S.suis 2 relevant rudimentary and application study, infect the prevalence in people and animals for prevention and control S.suis 2, further carry High China S.suis prevention and treatment are horizontal significant.

In past nearly half a century, the research for S.suis 2 focuses primarily upon bacterial virulence factors, surface egg Bai Chengfen and signals-modulating element etc., it has been found that including capsular polysaccharide (CPS), muramidase-released protein (muraminidase released protein, MRP), extracellular factor (extracellular factor, EF), hemolysin (suilysin, Sly), glutamte dehydrogenase (glutamate dehydrogenase, GDH), sorting enzyme A (sortase A), Dipeptidyl peptidase Ⅳ (Dipeptidyl Peptidase IV), enolase (Enolase) and Streptococcus suis histidine tripolymer Multiple factors related to Pathogenicity of Bacteria such as albumen (Histidine triad protein of S.suis, Htps).These The discovery of virulence factor is to understanding that S.suis 2 pathogenic course has certain help.But bacterium its life and metabolism are lived The research of dynamic molecular mechanism is less, and the research especially in terms of S.suis cell division mechanism is rarely reported.Cell division, propagation It is the basis of pathogenic bacterial infection host, it is many to participate in fissional protein classes, is not quite similar again in different bacterium.Therefore, Research to S.suis cell division mechanism is for deep its physiological metabolism process of announcement, so as to promote specific aim precautionary measures Research and develop significant.

DivIVA albumen is that the cell division by divIVA gene codes found in a variety of gram-positive bacterias is related Albumen.In recent years in bacillus subtilis, mycobacterium smegmatis, enterococcus faecalis, Listeria monocytogenes and lung Find that the albumen participates in the normal cell division of regulation and control bacterium in scorching streptococcus.Seminar to the people sources of S.suis 2 by separating Velogen strain 05ZYH33 genome analysis, it was found that 1 DivIVA protein coding gene.It is reported that monocytosis Property the listeria spp gene missing the mobility variation of bacterium, biofilm can be caused to be formed and be obstructed and invade cell Reduced capability is contaminated, influences bacterial virulence and pathogenic.But people is there is no so far to the gene function and its ginseng in S.suis 2 With bacterial cell division and pathogenic being studied.

Development has been permitted the pathogenic bacteria pathogenesis of S.suis 2 and the research of prevention and control as domestic and international association area The common choice of more scholars.Vaccine turns into current reply S.suis 2 as a kind of safely and effectively pathogen preventions to be infected Study hotspot.Attenuated live vaccine is traditional vaccine, and based on whole cell, dosage of inoculation is small, and immune effect is preferable, is immunized Power is more lasting.Therefore attenuated live vaccine research turns into an important directions of the new generation vaccines of S.suis 2 research and development.The present invention's is prominent Become bacterial strain and provide important value into the exploitation of attenuated vaccine.

The content of the invention

The purpose of the present invention is the above-mentioned deficiency for prior art, there is provided one plant of streptococcus suis 2 type (Streptococcus suis serotype 2, S.suis 2) divIVA gene knockout mutant strains.

It is a further object of the present invention to provide the application of the mutant strain.

The purpose of the present invention can be achieved through the following technical solutions：

S.suis 2 divIVA gene knockout mutant strains, by the divIVA bases in 2 type Streptococcus suis 05ZYH33 bacterial strains Because from the spectinomycin resistance gene box (Spectinomycin of the complete encoding sequence between the 1st to 690 Resistance cassette, hereinafter referred to as Spc^R) replace.

DivIVA full length genes coded sequence in wherein described 05ZYH33 bacterial strains is as shown in SEQ ID NO.1.

Described spectinomycin resistance gene box sequence is as shown in SEQ ID NO.2.

The method of the described S.suis 2divIVA gene knockout mutant strains of structure, is comprised the steps of：

(1) according to the wild strain 05ZYH33 genome divIVA encoding genes of S.suis 2 as shown in SEQ ID NO.3 DNA sequence upstream, design PCR special primers L1 and L2；According to the wild strains of S.suis 2 as shown in SEQ ID NO.4 The DNA sequence downstream of 05ZYH33 genome divIVA encoding genes, design PCR special primers R1 and R2；Using pSET2 plasmids as Template, design a pair of special primers spc1 and spc2；Wherein primer L1 sequences are as shown in SEQ ID NO.5, and primer L2 sequences are such as Shown in SEQ ID NO.6, primer R1 sequences are as shown in SEQ ID NO.7, and primer R2 sequences are as shown in SEQ ID NO.8, primer Spc1 sequences are as shown in SEQ ID NO.9, and primer spc2 sequences are as shown in SEQ ID NO.10；

(2) using 05ZYH33 genomic DNAs template, respectively using L1/L2 and R1/R2 as primer, amplification obtains both ends difference The target gene divIVA DNA sequence upstream LA fragments of the I restriction enzyme sites of I/BamH containing EcoR and both ends I/ containing Sal respectively The target gene divIVA DNA sequence downstream RA fragments of Sph I restriction enzyme sites；Using pSET2 plasmids as template, with Spc1/Spc2 Expand to obtain the spectinomycin resistance gene box of the both ends I of I/Sal containing BamH restriction enzyme sites respectively for special primer；

L1/L2, R1/R2 and Spc1/Spc2 tri- expands gained PCR primer and detected through 1% agarose electrophoresis to primer to be distinguished The purpose fragment size of acquisition is 1000bp (LA fragments), 961bp (RA fragments) and 1130bp (Spc^R).PCR primer recovery, Purifying, carries out double digestion with EcoR I/BamH I, Sal I/Sph I and BamH I/Sal I respectively, and double digestion product is returned Receive, purifying, freeze standby.

(3) gene knockout carrier pUC::DivIVA structure：By described LA fragments-spectinomycin resistance gene box-RA Gene knockout carrier pUC is obtained between the EcoR I/BamH I restriction enzyme sites of fragment insertion pUC18 carriers::divIVA；Specifically include Following steps：

(a) LA clone：By the LA fragments after EcoR I/BamH I double digestions with being treated with same enzymes double zyme cutting Recombinant plasmid pUC18 be attached, 16 DEG C overnight after by connection product convert DH5a competent escherichia coli cells, through ammonia benzyl After penicillin screening, the clone on picking LB flat boards is incubated at 37 DEG C of shaken overnights in LB fluid nutrient mediums.Next day extracts plasmid DNA, double digestion identification is carried out with EcoR I/BamH I, filter out the positive matter of 1000bp or so sizes DNA fragmentation appearance Grain, is named as pUC18-L.

(b) spectinomycin resistance gene Spc^RClone：By the product after BamH I/Sal I double digestions with using in same The treated recombinant plasmid pUC18-LR of enzyme cutting double digestion is attached, 16 DEG C overnight after connection product converted into DH5a large intestines Bacillus competent cell, after spectinomycin and ampicillin Double Selection, the clone on picking LB flat boards is incubated at LB liquid 37 DEG C of shaken overnights in body culture medium.Next day extracts DNA, carries out double digestion identification with BamH I/Sal I, has filtered out The positive plasmid that 1100bp or so sizes DNA fragmentation occurs, is named as pUC18-LS.

(c) RA clone：Treated by the RA fragments after Sal I/Sph I double digestions and with same enzymes double zyme cutting PUC18-LS carriers are attached, 16 DEG C overnight after by connection product convert DH5a competent escherichia coli cells, through grand mould After element and ampicillin Double Selection, the clone on picking LB flat boards is incubated at 37 DEG C of shaken overnights in LB fluid nutrient mediums. Next day extracts DNA, carries out double digestion identification with Sal I/Sph I, screening has what 960bp or so sizes DNA fragmentation occurred Recombinant plasmid；And matter is done respectively to primer with L1/L2, R1/R2, Spc1/Spc2, L1/Spc2, Spc1/R2, L1/R2 six respectively Grain PCR identifications.Obtained positive recombinant plasmid is named as pUC::divIVA.

(4) gene knockout carrier pUC::DivIVA identification：The positive restructuring gene knockout carrier pUC that will be obtained:: (Hua Da Gene science limited company) is sequenced in divIVA, and sequencing result shows in Spc^RGene both sides have homologous sequence The divIVA target gene of row, gene knockout carrier pUC::DivIVA structure is completely correct.

(5) gene knockout carrier pUC::DivIVA electricity is transformed into 05ZYH33 competence

(a) preparation of the wild strain 05ZYH33 competent cells of S.suis 2：Picking 05ZYH33 single bacterium colonies are inoculated in 3ml THB culture mediums, 37 DEG C of shaking table concussion and cultivates are stayed overnight, next day 1：50 are transferred to 37 DEG C of shaking table shakes in the THY containing DL- threonines Culture is swung to OD₆₀₀For 0.3 or so, bacterium solution ice bath 30min is taken out, 4 DEG C of centrifugation low speed receive bacterium, and are washed with 10% glycerine of precooling Bacterium 3 times, every time no less than 25ml, the 0.3M sucrose further bacterial precipitations of 15% glycerine are finally contained with 0.5ml, and dispense 50 μ L/ manage, be stored in -80 DEG C it is standby.

(b) electricity conversion：10 μ l pUC is added in the competence that 50 μ l are prepared as stated above::After divIVA plasmids Add in electric revolving cup (on ice operate), after 22.5kV/cm, 200 Ω and 25 μ F electricity turn, adding the THB of 0.3M sucrose, (37 DEG C pre- Heat) culture medium 940 μ l, 37 DEG C, it is coated on the THB flat boards containing Spectinomycin resistance after 160rpm concussion and cultivates 2h, 37 DEG C 24-48h is cultivated, picking single bacterium colony is identified；

(6) preliminary screening of 05ZYH33 △ divIVA mutant strains：Streptococcus suis bacterium is selected from spectinomycin THB flat boards Fall, be incubated at 2ml liquid THB (100mg/ml spc respectively^r) culture medium.Bacterium solution is taken as template, with primer Check In1 (sequence is as shown in SEQ ID NO.11) and checkin2 (are located at divIVA gene internals, sequence such as SEQ ID NO.12 institutes Show) enter performing PCR preliminary screening.

If divIVA genes are knocked, PCR amplifications will obtain negative findings, if remaining to amplify expected size The product of (452bp), illustrate that divIVA genes are not knocked.By this method, preliminary screening obtains divIVA gene knockouts Mutant strain, it is named as 05ZYH33 Δs divIVA.

(7) identification of 05ZYH33 △ divIVA mutant strains：

1. assembly PCR is identified：Two outsides that the LA and RA of target gene upstream and downstream homologous sequence are knocked out in divIVA are set again Pair of primers Check Out1/Check Out2 are counted, sequence is as shown in SEQ ID NO.13/SEQ ID NO.14；

Gene knockout carrier pUC::If divIVA and the chromosome of bacterium recombinate, it will 3 kinds of situations occur and occur： A. double crossing over homologous recombination events (double cross-over), i.e. allelic replacement, now spc^RGene substitutes divIVA Gene；B.3 ' end single-swap recombination event (3 ' single cross-over), now whole vector DNA sequence with 3 ' ends it is same Source sequence and be incorporated on the chromosome of bacterium；C.5 ' end single-swap recombination event (5 ' single cross-over), is now carried Body sequence is incorporated on bacterial chromosome with 5 ' end homologous sequences.If generation allelic replacement, with primer Check Out1/Spc2, which enters performing PCR, can amplify 2373p fragment, and entering performing PCR with primer Spc1/Check Out2 can amplify 2344bp fragment, spc can be amplified by entering performing PCR with primer Spc1/Spc2^RGene, and in 05ZYH33, with above primer Negative findings should all be obtained by entering performing PCR.And using 05ZYH33 genomes as template, it can be amplified with primer checkin1/2 452bp purpose fragment, as a result the size of each PCR primer is consistent with theoretical value, and verifies (Hua Da Gene science through DNA sequencing Limited company), confirm the success of △ divIVA mutative symptoms in gene level.

2. RT-PCR is identified：In order to further be verified to △ divIVA mutant strains, Check In1/2 primers point are utilized The other cDNA obtained to mutant strain and street strain's reverse transcription enters performing PCR amplification.Result is positive in wild strain 05ZYH33, explanation DivIVA normal transcriptions；And it is negative in mutant strain Δ divIVA.By the identification of transcriptional level, divIVA bases are successfully obtained Because of knockout mutant strain, Δ divIVA is named as.

Application of the described S.suis 2divIVA gene knockout mutant strains in the attenuated vaccines of S.suis 2 are prepared.

S.suis 2divIVA gene knockout carriers pUC of the present invention::DivIVA is in the structure divIVA genes of S.suis 2 Application in knockout mutant strain.

S.suis 2divIVA gene knockout carriers pUC of the present invention::DivIVA is in the attenuated vaccines of S.suis 2 are prepared Using.

Beneficial effect：

It is spectinomycin resistance gene among structure, both sides are divIVA bases 1. the present invention uses the principle of homologous recombination Because of the gene knockout carrier of upstream and downstream homologous sequence, by the pUC of structure::

DivIVA gene knockout plasmid electricity is transformed into S.suis the last 2 pathogenic strain 05ZYH33 competent cells, by internal Homologous recombination, screen and identify through gene level, transcriptional level and DNA sequencing, successfully obtain mutant strain, be named as Δ divIVA Mutant strain.

2. the present invention to the related biological characteristics of divIVA gene knockout mutant strains and it is pathogenic analyzed, clearly DivIVA genes and pathogenic S.suis 2 relation.It is bent with Δ divIVA strain growths by drawing wild strain 05ZYH33 Line finds that Δ divIVA strain growths slowly substantially lag behind wild strain；Hydrogen peroxide tolerance test shows, Δ divIVA bacterial strains pair Hydrogen peroxide is more sensitive；Cell binding observation indicate that, Δ divIVA bacterial strain abnormal divisions, divide barrier film entanglement, easily Cell mass aggregation is formed, without obvious streptocyte structure, cell pod membrane is thinning, phenomena such as being thinned out；In vitro cell experiment table Bright, Δ divIVA bacterial strains weaken to the anti-phagocytic activity of mouse macrophage；Pathogenic experiment shows in Mice Body, Δ divIVA Bacterial strain is not pathogenic to mouse.The mutant strain provides important clue for the exploitation screening of the attenuated vaccines of S.suis 2, can apply In the exploitation of the attenuated vaccines of S.suis 2.

3. the Δ divIVA that the present invention is built, established for further research S.suis 2 cell division and mechanism of causing a disease Basis, technical support is provided for more effective prevention and control Streptococcus suis.

Brief description of the drawings：

Fig. 1 is gene knockout carrier pUC::DivIVA intersects PCR qualification result figures.

Swimming lane 1,2,3,4,5,6 be respectively using L1/L2, Spc1/Spc2, R1/R2, L1/Spc2, Spc1/R2, L1/R2 as The PCR primer of primer pair, swimming lane 7 are DNA Marker.

Fig. 2 is homologous recombination schematic diagram.

The assembly PCR qualification result figure of Fig. 3 Δ divIVA mutant strains.

Swimming lane 1-8 is with primer Check In1/2, Spc1/Spc2, Check Out1/Spc2, Spc1/Check respectively Out2 is the PCR primer of primer pair.1,3,5,7 is the PCR using 05ZYH33 as template, and swimming lane 2,4,6,8 is with Δ divIVA Mutant strain is the PCR of template, and swimming lane M is DNA Marker.

Fig. 4 is the RNA extractions figure and reverse transcription PCR qualification figure of Δ divIVA mutant strains

A figures are the RNA extraction figures of Δ divIVA mutant strains, and wherein swimming lane 1 is wild strain 05ZYH33 RNA, and swimming lane 2 is Δ DivIVA mutant strains RNA；Swimming lane M is DNA Marker.

B figures are the reverse transcription PCR qualification figure of Δ divIVA mutant strains, and wherein swimming lane M is DNA Marker, swimming lane 1 be with Δ divIVA cDNA mutant strains are template, the PCR primer using Check In1/2 as primer, and 2 be using 05ZYH33cDNA as mould Plate, the PCR primer using Check In1/2 as primer.

Specific implementation method：

Embodiment 1：The structure of gene knockout carrier

(1) according to the upstream and downstream DNA sequence dna of the wild strain 05ZYH33 genome divIVA encoding genes of S.suis 2, design PCR special primers, base sequence are as follows：

L1：5′-CGAATTCGCT TTG CTA AGT TGG TTT-3 ' (SEQ ID NO.5, to introduce at underscore EcoR I restriction enzyme sites)

L2：5′-CGGATCCCTT TCCTCCTAAGTTTTAAC-3 ' (SEQ ID NO.6, to introduce at underscore BamH I restriction enzyme sites)

R1：5′-CGTCGACATT TTA AGC GAG TAG GAG-3 ' (SEQ ID NO.7, to introduce at underscore Sal I restriction enzyme sites)

R2：5′-GCATGCAGA CTT GCT CAA TAG GA-3 ' (SEQ ID NO.8, to introduce at underscore Sph1 restriction enzyme sites)

According to pSET2 plasmid sequences, a pair of special primer Spc1/Spc2 are designed, it is whole as template amplification using pSET2 plasmids Individual spectinomycin resistance gene box, primer sequence are:

Spc1：5’-GGATCCGTTCGTGAATACATGTTATA-3 ' (SEQ ID NO.9, to introduce at underscore BamH I restriction enzyme sites)

Spc2：5’-GTCGACGTTTTCTAAAATCTGAT-3 ' (SEQ ID NO.10, the Sal I at underscore to introduce Restriction enzyme site)

PCR reaction systems are：

PCR reaction conditions are：95 DEG C of pre-degenerations 5min, 94 DEG C of 50s, 54 DEG C of 60s, 72 DEG C of 1min, 30 circulations, finally 72 DEG C of extension 10min, using distilled water as negative control.

L1/L2, R1/R2 and Spc1/Spc2 tri- expands gained PCR primer and detected through 1% agarose electrophoresis to primer to be distinguished The purpose fragment size of acquisition is 1000bp (LA), 961bp (RA) and 1130bp (Spc^R).PCR primer recovery, purifying, respectively Double digestion is carried out with EcoR I/BamH I, Sal I/Sph I and BamH I/Sal I, the recovery of double digestion product, purifying are frozen Deposit standby.

(2) LA clone：By the LA fragments after EcoR I/BamH I double digestions with being treated with same enzymes double zyme cutting Recombinant plasmid pUC18 be attached, 16 DEG C overnight after by connection product convert DH5a competent escherichia coli cells, through ammonia benzyl After penicillin screening, the clone on picking LB flat boards is incubated at 37 DEG C of shaken overnights in LB fluid nutrient mediums.Next day extracts plasmid DNA, double digestion identification is carried out with EcoR I/BamH I, filter out the positive matter of 1000bp or so sizes DNA fragmentation appearance Grain, is named as pUC18-L.

(3) spectinomycin resistance gene Spc^RClone：By the product after BamH I/Sal I double digestions with using in same The treated recombinant plasmid pUC18-L of enzyme cutting double digestion is attached, 16 DEG C overnight after connection product converted into DH5a large intestine bars Bacterium competence cell, after spectinomycin and ampicillin Double Selection, the clone on picking LB flat boards is incubated at LB liquid 37 DEG C of shaken overnights in culture medium.Next day extracts DNA, carries out double digestion identification with BamH I/Sal I, has filtered out The positive plasmid that 1100bp or so sizes DNA fragmentation occurs, is named as pUC18-LS.

(4) RA clone：Treated by the RA fragments after Sal I/Sph I double digestions and with same enzymes double zyme cutting PUC18-LS carriers are attached, 16 DEG C overnight after by connection product convert DH5a competent escherichia coli cells, through grand mould After element and ampicillin Double Selection, the clone on picking LB flat boards is incubated at 37 DEG C of shaken overnights in LB fluid nutrient mediums. Next day extracts DNA, carries out double digestion identification with Sal I/Sph I, screening has what 960bp or so sizes DNA fragmentation occurred Recombinant plasmid；And matter is done respectively to primer with L1/L2, R1/R2, Spc1/Spc2, L1/Spc2, Spc1/R2, L1/R2 six respectively Grain PCR identifications.Obtained positive recombinant plasmid is named as pUC::divIVA.

(5) gene knockout carrier pUC::DivIVA identification：The positive restructuring gene knockout carrier pUC that will be obtained:: (Hua Da Gene science limited company) is sequenced in divIVA, and sequencing result shows in Spc^RGene both sides have homologous sequence The divIVA target gene of row, gene knockout carrier pUC::DivIVA structure is completely correct.

Embodiment 2：The screening and identification of mutant

(1) gene knockout carrier pUC::DivIVA electricity is transformed into 05ZYH33 competence

1. the preparation of the wild strain 05ZYH33 competent cells of S.suis 2：Picking 05ZYH33 single bacterium colonies be inoculated with 3mlTHB culture mediums, 37 DEG C of shaking table concussion and cultivates are stayed overnight, next day 1：50 are transferred to 37 DEG C of shaking tables in the THY containing DL- threonines Concussion and cultivate is to OD₆₀₀For 0.3 or so, 4 DEG C of centrifugation low speed receive bacterium, and wash bacterium 4 times with 10% glycerine of precooling, are no less than every time 25ml, the 0.3M sucrose further bacterial precipitations of 15% glycerine are finally contained with 0.5ml, and dispense 50 μ l/ pipes, be stored in -80 DEG C It is standby.

2. electricity conversion：10 μ l pUC is added in the competence that 50 μ l are prepared as stated above::Add after divIVA plasmids Enter in electric revolving cup (on ice operate), after 22.5kV/cm, 200 Ω and 25 μ F electricity turn, add the THB (37 DEG C of preheatings) of 0.3M sucrose Culture medium 940 μ l, 37 DEG C, it is coated on the THB flat boards containing Spectinomycin resistance after 160rpm concussion and cultivates 2h, 37 DEG C of cultures 24-48h, picking single bacterium colony are identified；

(2) preliminary screening of 05ZYH33 △ divIVA mutant strains：Streptococcus suis bacterium is selected from spectinomycin THB flat boards Fall, be incubated at 2ml liquid THB (100mg/ml spc respectively^r) culture medium.Bacterium solution is taken as template, with primer Check In1 Enter performing PCR preliminary screening with Check In 2 (being located at divIVA gene internals).Its primer sequence is:

Check In1：5′-GACGCAGATGAAGTTGATGACTT-3′(SEQ ID NO.11)

Check In 2：5′-TGAATGTAACTTGCTGTTGGGC-3′(SEQ ID NO.12)

If divIVA genes are knocked, PCR amplifications will obtain negative findings, if remaining to amplify expected size The product of (452bp), illustrate that divIVA genes are not knocked.By this method, preliminary screening obtains divIVA gene knockouts Mutant strain, it is named as 05ZYH33 Δs divIVA.

(3) identification of 05ZYH33 △ divIVA mutant strains：

1. assembly PCR is identified：Two outsides that the LA and RA of target gene upstream and downstream homologous sequence are knocked out in divIVA are set again Pair of primers Check OUt1/Check OUt2 are counted, its primer sequence is:

OUt1:5′-ATATGTCGGAGCAACAAGCAAGAC-3′(SEQ ID NO.13)

OUt2:5′-TTCGATTTCAGCTTCTGCAAGG-3′(SEQ ID NO.14)

Gene knockout carrier pUC::If restructuring (Fig. 2) occurs for divIVA and the chromosome of bacterium, it will 3 kinds of situations occurs Occur：A. double crossing over homologous recombination events (double cross-over), i.e. allelic replacement, now spc^RGene substitutes DivIVA genes；B. 3 ' end single-swap recombination event (3 ' single cross-over), now whole vector DNA sequence with 3 ' hold homologous sequences and are incorporated on the chromosome of bacterium；C.5 ' end single-swap recombination event (5 ' single cross-over), Now carrier sequence is incorporated on bacterial chromosome with 5 ' end homologous sequences.If generation allelic replacement, uses primer Check OUt1/Spc2, which enter performing PCR, can amplify 2373bp fragment, and entering performing PCR with primer Spc1/Check Out2 can expand Increase the fragment for 2344bp, spc can be amplified by entering performing PCR with primer Spc1/Spc2^RGene, and in 05ZYH33, more than Primer, which enters performing PCR, should all obtain negative findings.And using 05ZYH33 genomes as template, it can be expanded with primer Check In 1/2 The 452bp gone out purpose fragment, as a result the size of each PCR primer is consistent (Fig. 3) with theoretical value, and verifies (Hua Da through DNA sequencing Gene science limited company), confirm the success of △ divIVA mutative symptoms in gene level.

2. RT-PCR is identified：In order to further be verified to △ divIVA mutant strains, checkin1/2 primers point are utilized The other cDNA obtained to mutant strain and street strain's reverse transcription enters performing PCR amplification.Result is positive in wild strain 05ZYH33, explanation HtpsA normal transcriptions；And it is negative (Fig. 4) in mutant strain Δ divIVA.By the identification of transcriptional level, successfully obtain DivIVA gene knockout mutant strains, it is named as Δ divIVA.

Embodiment 3：Experiment in vitro

(1) Gram's staining

The specification of the gram staining liquid produced according to Beijing Suo Laibao Science and Technology Ltd operates, and gives respectively 05ZYH33 and Δ divIVA carries out Grain stain, it is found that catenations of the mutant strain Δ divIVA compared to wild strain more dissipates The aggregation of most of thalline is agglomerating, without obvious chain structure, illustrates mutant strain Δ divIVA chaining reduced capability.

(2) growth characteristics

Under same culture conditions, picking Δ divIVA and wild strain 05ZYH33 single bacterium colonies are inoculated in 3mL respectively respectively In THB culture mediums, 37 DEG C of shaken cultivations are stayed overnight.Next day takes out the bacterium being incubated overnight, and determines absorbance at 600nm, uses THB Both are diluted to about 1 × 10 by culture medium⁸CFU/mL concentration.Then 60 μ L mutant strains and wild strain is taken to be inoculated in respectively respectively In THB culture mediums 3mL, in 37 DEG C, 200r/min shaken cultivations, every the separately sampled measure OD of 1 h₆₀₀, using incubation time as Abscissa, OD₆₀₀It is worth for ordinate, draws mutant strain and wild strain growth curve, as a result finds the 4h of 05ZYH33 after inoculation Cell propagation is slowly growth lag phase, and Δ divIVA growth retardation phases longer 6h after inoculation initially enters logarithm life For a long time.05ZYH33 starts fast breeding after exponential phase is entered, and viable count is presented into the increase of geometry multiple, growth curve " J " type, and Δ divIVA viable counts also begin to increase but no 05ZYH33 gathers way soon.The 05ZYH33 after 11h is inoculated with Enter plateau OD with Δ divIVA₆₀₀It is respectively 1.05 ± 0.05,0.43 ± 0.03 that value, which reaches maximum, and number of viable also reaches Peak is respectively 39.5 ± 1.15 × 10⁷CFU/mL、20.12±1.25×10⁷CFU/mL.From 05ZYH33 and Δ after 13h DivIVA progresses into decline phase, OD₆₀₀All begun to decline with viable count.

(3) anti-macrophage phagocytosis experiment

The 05ZYH33 and Δ divIVA (10 with CFSE dye markers⁷CFU) it is added to macrophage Raw264.7 (10: 1) in, 800g, centrifuge 10min after lucifuge gentle agitation incubate altogether effect 2h, washed 3 times with gentle PBS, after add 100 μ g/ml Gentamicin and the dual anti-fresh culture effect 1h of 5 μ g/ml penicillin, it is solid to be eventually adding isometric paraformaldehyde (4%) It is fixed, Flow cytometry intracellular CFSE fluorescence intensities.Flow cytometry shows, Δ divIVA compared with 05ZYH33, its Anti- macrophage phagocytosis killing ability reduces.

(4)H₂O₂Tolerance test

By wild strain and mutant strain Δ divIVA and gradient concentration H₂O₂It is incubated altogether, with being not added with after dilution painting plate count H₂O₂The experimental comparison group of incubation is compared, and calculates the survival rate of bacterium.As a result such as Fig. 5 .3, find in 20~100mM concentration Δ divIVA mutant strain survival rates are below wild strain under gradient, and its significant difference has statistical significance；Mutant strain exists 60mM H₂O₂Survival rate is 0 when more than concentration, shows that Δ divIVA mutant strains oxidation resistance weakens, to H₂O₂Performance is sensitive.

Embodiment 4：Pathogenicity is tested

In order to detect the pathogenic of mutant strain 05ZYH33 Δs divIVA, picking 05ZYH33 and mutation are distinguished on flat board Strain Δ divIVA single bacterium colony, 37 DEG C of shaken cultivations are to mid log phase (OD in THB culture mediums₆₀₀≈ 0.4, about 10⁸CFU dosage) thalline is collected by centrifugation, bacterium is resuspended with sterile PBS buffer.4 week old BALB/c mouse of SPF levels 30, at random It is divided into 3 groups, wild type and mutant strain bacterium solution 1mL (about 10 is injected intraperitoneally respectively⁸CFU/ is only), and set THB negative control groups (1mL/ is only), observes and records mouse invasion and the time-to-live whether there is significant change in time.As a result find, it is wild with lethal dose Dead 7 after strain 05ZYH33 attack mouse 12h, 10 mouse are all dead after 24h, and knock out strain Δ with same dose At the end of the mouse of divIVA attacks to experiment in 7 days, all survivals, and find no any disease symptom.Negative control group 10 State is good.Illustrate that the 05ZYH33 Pathogenicity of Bacteria of divIVA gene knockouts is remarkably decreased, S.suis can be applied to The development of 2 attenuated vaccines.

Sequence table

<110>Chinese People's Liberation Army Medical Research Institute Of Nanjing Military Region

<120>Streptococcus suis 2-type divIVA gene knockout mutant strains and its application

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 690

<212> DNA

<213>Streptococcus suis 05ZYH33 (Streptococcus suis 05ZYH33)

<400> 1

atggcactta cagcattaga attaaaagat aaaacctttg ctacaaaatt tagaggatac 60

gacgcagatg aagttgatga ctttttggac attgtaacac gtgattatga ggatttaatt 120

cgtaaaaatc atgaccaaga gttagaattg aaaaatttgc gagaacgttt ggcttatttt 180

gatgagatga aagaatcatt gagtaaatca gttcttttag ctcaagatac agctgagaaa 240

gtaaaggttg cggctgaaga tcaagcggca aatattatta aacaagctga ctatgatgcg 300

gcaacattgt tacatgaagc taaagataag gcaaatgaaa ttcttcgtaa tgcgactgac 360

aacgcgaaaa aagttgtcat tgaaactgaa gaattaaaaa accagacacg tattttccat 420

cagcgtctaa aatcaacagt agaaagccaa ttatcactgg ttaattcatc tgaatgggag 480

gaaatcctcc gcccaacagc aagttacatt caaacaagtg acgaagcctt tcgtgatgtt 540

cttcataagg ctttggatga agaattacct gttgaagaag aaagtttgga ttatacacgt 600

caattgacac cagaagagat tgcagaatta actcgtcagg cagcagcttt tgagagtgga 660

gaatctgtag tactttcaat agaagaataa 690

<210> 2

<211> 1130

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

gttcgtgaat acatgttata ataactataa ctaataacgt aacgtgactg gcaagagata 60

tttttaaaac aatgaatagg tttacactta ctttagtttt atggaaatga aagatcatat 120

catatataat ctagaataaa attaactaaa ataattatta tctagataaa aaatttagaa 180

gccaatgaaa tctataaata aactaaatta agtttattta attaacaact atggatataa 240

aataggtact aatcaaaata gtgaggagga tatatttgaa tacatacgaa caaattaata 300

aagtgaaaaa aatacttcgg aaacatttaa aaaataacct tattggtact tacatgtttg 360

gatcaggagt tgagagtgga ctaaaaccaa atagtgatct tgacttttta gtcgtcgtat 420

ctgaaccatt gacagatcaa agtaaagaaa tacttataca aaaaattaga cctatttcaa 480

aaaaaatagg agataaaagc aacttacgat atattgaatt aacaattatt attcagcaag 540

aaatggtacc gtggaatcat cctcccaaac aagaatttat ttatggagaa tggttacaag 600

agctttatga acaaggatac attcctcaga aggaattaaa ttcagattta accataatgc 660

tttaccaagc aaaacgaaaa aataaaagaa tatacggaaa ttatgactta gaggaattac 720

tacctgatat tccattttct gatgtgagaa gagccattat ggattcgtca gaggaattaa 780

tagataatta tcaggatgat gaaaccaact ctatattaac tttatgccgt atgattttaa 840

ctatggacac gggtaaaatc ataccaaaag atattgcggg aaatgcagtg gctgaatctt 900

ctccattaga acatagggag agaattttgt tagcagttcg tagttatctt ggagagaata 960

ttgaatggac taatgaaaat gtaaatttaa ctataaacta tttaaataac agattaaaaa 1020

aattataaaa aaattgaaaa aatggtggaa acactttttt caattttttt gttttattat 1080

ttaatatttg ggaaatattc attctaattg gtaatcagat tttagaaaac 1130

<210> 3

<211> 1000

<212> DNA

<213>Artificial sequence (Streptococcus suis 05ZYH33)

<400> 3

gctttgctaa gttggtttcc tagcctatac acaagctcga ttggtcgttt gattcaatgg 60

ctggtagcac cgattttaaa accctttagg cggttaaatc tgcagtttat ggggttggat 120

tggaccgtaa tggcagccat gattgcgctt aatatgggaa cacgcttctt ggttcaatta 180

ttgctcctgt tagcatagtt atgaaaaatg acaaaaactt attgcaacat ttttctaggg 240

aagaaagaga gtttgtagag aaaataatgg atatgtgtca gcaagttgaa gatacgtatt 300

catacagatt aactcacttt ttacatccta gacaagatga aattgtatgc aaaattgcta 360

actactacca gttacaaacg ttttctagtc gtgaccttgt ttcgacagaa cattcgcggg 420

ttattattgc cccgacttat tatgagttgg acatcaagga ctttgagtta accgctcttc 480

atttgtccta tgctagaaaa tttcacacct tatcccattc acaagttctt ggaacttttc 540

tcaatcaact agggattaaa agggagtatt tgggtgatat tttgattgat gatgaacgat 600

tgattgtctt tatcgatcag aaatttgggc agattgcctt acaatctatc actaaagttg 660

ctcgtgtccc tgttaaggga aaggaagaag aatggacgac tgttcgacta ccgattggac 720

aggaatggcg ttcaaaggat gtgttggttt ctagtatgag attggataaa ttgatttcgg 780

tcgcatttaa tctttcaagg gcaactgcaa acaagttgat tgaggctgga catgtgaagt 840

tggattatgt tctgacagaa caaaccagta aattagttga aatagggcaa ttaattagtg 900

ttaggagata tggtagagtt cgcctaaatg aatttttagg tttttctaag caagggaaga 960

taaaattaaa gttagacatt gttaaaactt aggaggaaag 1000

<210> 4

<211> 961

<212> DNA

<213>Artificial sequence (Streptococcus suis 05ZYH33)

<400> 4

attttaagcg agtaggagat ggtggaagcc ctacaatctc tttctatgga tcatcacttt 60

gtttgatctt tcctgaaaat agtaaggaga gacgtccgtt cacgttacga acatagaggg 120

atagaggaaa ctctatctaa actaaggtgg taccacgaac tttcgtcctt atttggcggg 180

agttttttat ttttatgata cgattagaat tagtaaacaa ggataacttt gaagtagtgt 240

tacaagttca acttgcatct gaggaccaac gccgagtggc atcggttgaa tattctttag 300

cccaagcttg gttatataaa gattcgggga tgattctacc atatgctgtg gtatctggca 360

gaaaagttgt tggttttgca atgttatcta ttgaacccaa agataatagt tattatttat 420

ggcgcttgtt gattgataag gattttcaaa atcgtggatg cggaaaagaa gccatccagc 480

aaataattgg aaaggcgaag gcggatccgc tttgtcataa aatttcgata aattatgtca 540

ttggcaatca taaaatgcgg tacattttag aaaaaattgg ttttcaatcc gttggtttag 600

aaggccaaga aatgaaaatg gaattaatta taaaataaag gagttatgat gaaactaaaa 660

gaaacactca atttaggtaa gacagcattt cctatgcgtg caggcttacc aacacgtgag 720

ccagaatggc aaaaggcctg ggatgaagca aacttgtatg ctcgtcgcca agaactcaat 780

gaaggcaaac cagccttcca cctccacgat ggccctccat atgctaacgg aaatatccac 840

gttggacatg ctttaaacaa gatttctaaa gatattatcg ttcgctctaa gtcaatgtct 900

ggtttccgtg ctccatttgt accgggttgg gatacacacg gccttcctat tgagcaagtc 960

t 961

<210> 5

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

cgaattcgct ttgctaagtt ggttt 25

<210> 6

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

cggatccctt tcctcctaag ttttaac 27

<210> 7

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

cgtcgacatt ttaagcgagt aggag 25

<210> 8

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

gcatgcagac ttgctcaata gga 23

<210> 9

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

ggatccgttc gtgaatacat gttata 26

<210> 10

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

gtcgacgttt tctaaaatct gat 23

<210> 11

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

gacgcagatg aagttgatga ctt 23

<210> 12

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

tgaatgtaac ttgctgttgg gc 22

<210> 13

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 13

atatgtcgga gcaacaagca agac 24

<210> 14

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 14

ttcgatttca gcttctgcaa gg 22

Claims (8)

1. Streptococcus suis 2-type 1. (Streptococcus suis serotype 2, S.suis 2) divIVA knock out mutants Strain, it is characterised in that by the divIVA genes in streptococcus suis 2-type 05ZYH33 bacterial strains from the total length between the 1st to 690 Coded sequence spectinomycin resistance gene box is replaced.
2. 2. S.suis 2divIVA gene knockout mutant strains according to claim 1, it is characterised in that described 05ZYH33 Whole coded sequences of divIVA genes in bacterial strain are as shown in SEQ ID NO.1.
3. 3. S.suis 2divIVA gene knockout mutant strains according to claim 1, it is characterised in that described is grand mould Plain resistant gene box sequence is as shown in SEQ ID NO.2.
4. 4. build claim 1 described in S.suis 2divIVA gene knockout mutant strains method, it is characterised in that comprising with Lower step：

   (1) according to the upstream of the wild strain 05ZYH33 genome divIVA encoding genes of S.suis 2 as shown in SEQ ID NO.3 DNA sequence dna, design Specific PCR primers L1 and L2；According to the wild strain 05ZYH33 of S.suis 2 as shown in SEQ ID NO.4 The DNA sequence downstream of genome divIVA encoding genes, design Specific PCR primers R1 and R2；Using pSET2 plasmids as template, Design a pair of special primers Spc1 and Spc2；Wherein primer L1 sequences are as shown in SEQ ID NO.5, primer L2 sequences such as SEQ ID Shown in NO.6, primer R1 sequences are as shown in SEQ ID NO.7, and primer R2 sequences are as shown in SEQ ID NO.8, primer Spc1 sequences As shown in SEQ ID NO.9, primer Spc2 sequences are as shown in SEQ ID NO.10；

   (2), using 05ZYH33 genomic DNAs template, respectively using L1/L2 and R1/R2 as primer, amplification obtains both ends and contained respectively The target gene divIVA DNA sequence upstream LA fragments of EcoR I/BamH I restriction enzyme sites and both ends I of I/Sph containing Sal respectively The target gene divIVA DNA sequence downstream RA fragments of restriction enzyme site；Using pSET2 plasmids as template, using Spc1/Spc2 as primer Amplification obtains the spectinomycin resistance gene box of the both ends I of I/Sal containing BamH restriction enzyme sites respectively；

   (3) gene knockout carrier pUC::DivIVA structure：By described LA fragments-spectinomycin resistance gene box-RA fragments Gene knockout carrier pUC is obtained between the Sal I/Sph1 restriction enzyme sites of insertion pUC18 carriers::divIVA；

   (4) gene knockout carrier pUC::The wild strain 05ZYH33 competent cells of S.suis 2 that divIVA electricity conversions prepare；

   (5) S.suis 2 bacterium colony of the screening with Spectinomycin resistance, combined PCR primer electrophoresis, RT-PCR and DNA sequencing card Real divIVA target gene is replaced by spectinomycin resistance gene box.
5. 5. according to the method for claim 4, it is characterised in that described gene knockout carrier pUC::DivIVA structure bag Containing following steps：

   (a) LA clone：By the LA fragments after EcoR I/BamH I double digestions and the weight treated with same enzymes double zyme cutting Group plasmid pUC18 is attached, and obtains recombinant plasmid pUC18-L；

   (b) clone of spectinomycin resistance gene box：By the product after BamH I/Sal I double digestions with same restriction endonuclease pair The treated recombinant plasmid pUC18-L of digestion is attached, and obtains recombinant plasmid pUC18-LS；

   (c) RA clone：Treated by the RA fragments after Sal I/Sph1 double digestions and with same enzymes double zyme cutting PUC18-LS carriers are attached, 16 DEG C overnight after by connection product convert DH5a competent escherichia coli cells, through grand mould After element and ampicillin Double Selection, double digestion identification is carried out with EcoR I/BamH I, has filtered out 1000bp sizes DNA The positive plasmid that fragment occurs；And respectively with L1/L2, R1/R2, Spc1/Spc2, L1/Spc2, Spc1/R2, L1/R2 six to drawing Thing does plasmid PCR identification respectively, and obtained positive recombinant plasmid is pUC::divIVA.
6. 6. the S.suis 2divIVA gene knockout mutant strains 05ZYH33 △ divIVA described in claim 1 are preparing S.suis Application in 2 attenuated vaccines.
7. 7.S.suis 2divIVA gene knockout carriers pUC::DivIVA is in structure S.suis 2divIVA gene knockout mutant strains In application, wherein described S.suis 2divIVA gene knockout carriers pUC::DivIVA is built by the following method：

   (a) LA clone：By the LA fragments after EcoR I/BamH I double digestions and the weight treated with same enzymes double zyme cutting Group plasmid pUC18 is attached, and obtains recombinant plasmid pUC 18-L；

   (b) clone of spectinomycin resistance gene box：By the product after BamH I/Sal I double digestions with same restriction endonuclease pair The treated recombinant plasmid pUC18-L of digestion is attached, and obtains recombinant plasmid pUC18-LS；

   (c) RA clone：Treated by the RA fragments after Sal I/Sph1 double digestions and with same enzymes double zyme cutting PUC18-LS carriers are attached, 16 DEG C overnight after by connection product convert DH5a competent escherichia coli cells, through grand mould After element and ampicillin Double Selection, double digestion identification is carried out with EcoR I/BamH I, has filtered out 1000bp sizes DNA The positive plasmid that fragment occurs；And respectively with L1/L2, R1/R2, Spc1/Spc2, L1/Spc2, Spc1/R2, L1/R2 six to drawing Thing does plasmid PCR identification respectively, and obtained positive recombinant plasmid is pUC::divIVA.
8. 8.S.suis 2divIVA gene knockout carriers pUC::Applications of the divIVA in the attenuated vaccines of S.suis 2 are prepared, its Described in S.suis 2divIVA gene knockout carriers pUC::DivIVA is built by the following method：

   (a) LA clone：By the LA fragments after EcoR I/BamH I double digestions and the weight treated with same enzymes double zyme cutting Group plasmid pUC18 is attached, and obtains recombinant plasmid pUC18-L.

   (b) clone of spectinomycin resistance gene box：By the product after BamH I/Sal I double digestions with same restriction endonuclease pair The treated recombinant plasmid pUC18-L of digestion is attached, and obtains recombinant plasmid pUC18-LS；

   (c) RA clone：Treated by the RA fragments after Sal I/Sph1 double digestions and with same enzymes double zyme cutting PUC18-LS carriers are attached, 16 DEG C overnight after by connection product convert DH5a competent escherichia coli cells, through grand mould After element and ampicillin Double Selection, double digestion identification is carried out with EcoR I/BamH I, has filtered out 1000bp or so sizes The positive plasmid that DNA fragmentation occurs；It is and right with L1/L2, R1/R2, Spc1/Spc2, L1/Spc2, Spc1/R2, L1/R2 six respectively Primer does plasmid PCR identification respectively, and obtained positive recombinant plasmid is pUC::divIVA.