CN103343102B - Streptococcus suis serotype II htpsC gene knockout mutant strain and application thereof - Google Patents
Streptococcus suis serotype II htpsC gene knockout mutant strain and application thereof Download PDFInfo
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Abstract
The invention belongs the field of gene engineering, and relates to a streptococcus suis serotype II htpsC gene knockout mutant strain and an application thereof. The mutant strain 05ZYH33deltahtpsC has the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.7376. A preparation method of the mutant strain comprises the following step of: carrying out gene knockout on an htpsC gene of coded histidine tripolymer protein Htp of a chromosome of a S.suis II field strain 05ZYH33 by adopting a homologous recombinant gene knockout technology so as to obtain the strain, namely the gene knockout mutant strain through verification of combined PCR (polymerase chain reaction) product electrophoresis, RT (reverse transcription)-PCR and DNA (Deoxyribonucleic Acid) sequencing. By using the mutant strain to perform an animal experiment to research pathogenicity of the strain, results show that the toxicity of the mutant strain to the experiment animal is remarkably reduced, and the mutant strain can be applicable to developing a streptococcus suis serotype II attenuated vaccine.
Description
Technical field
The invention belongs to genetically engineered field, relate to streptococcus suis 2-type htpsC gene knockout mutant strain and application thereof.
Background technology
Streptococcus suis 2-type (S.suis2) is a kind of zoonosis pathogenic bacteria endangering the whole world.This pathogenic bacteria not only can cause pig meningitis, septicemia, sacroiliitis, pneumonia and endocarditis etc., but also can infect people, causes serious threat to the life security of relevant practitioner and broad masses.According to the antigenicity of its capsular polysaccharide, can be divided into 35 serotypes, wherein 2 types (SS2) virulence is the strongest, and clinical recall rate is the highest.In the swinery in south China province in recent years, S.suis2 is popular is on the rise, time have fairly large breaking out, the financial loss caused every year reaches billions of unit.Within 1998 and 2005, break out extensive SS2 respectively in China Jiangsu and Ziyang, Sichuan and infect people's event, cause great life and property loss, and there is a high proportion of, rare toxic shock syndrome in patient both at home and abroad, the state of an illness is dangerous, case fatality rate high (60% ~ 80%), causes serious public health event.It is extremely strong that above-mentioned situation strong indication causes this twice popular SS2 virulence, probably there occurs toxicity variation or obtain the relevant genetic material of virulence.The report infected about S.suis in the world in recent years and cause a disease significantly increases, and has report to be presented at the existence that China Hong Kong has detected this bacterium multiple antibiotic resistant strain, brings new challenge to the prevention and corntrol of S.suis2.Therefore, carry out relevant rudimentary and the applied research of S.suis2 mechanism of causing a disease, popular significant in people and animals is infected for prevention and control S.suis2.But the molecular mechanism of also causing a disease about S.suis infection host is at present still not clear.Therefore, the function of the pathogenic related gene that further exploratory development is new and the effect played in S.suis and host interact thereof, to screening potential vaccine candidate molecule, disclosing its accurate molecular mechanism that development occurs, raising China S.suis prevention and therapy level is significant further.
Research for virulence factors of streptococcus suis is a focus always, is only had capsular polysaccharide (CPS) by the virulence factor definitely confirmed at present.What other was known comprises muramidase-released protein (muraminidase released protein to the pathogenic relevant virulence factor of S.suis, MRP), extracellular factor (extracellular factor, EF), hemolysin (suilysin, Sly), glutamate dehydrogenase (glutamate dehydrogenase, GDH), sorting enzyme A (sortase A), dipeptidyl peptidase Ⅳ (Dipeptidyl Peptidase IV), Hydratase, phosphoenolpyruvate (Enolase) and swine streptococcus Histidine trimer protein (Histidine triad protein of S.suis, Htps) etc.The discovery of these virulence factors has certain help to the pathogenic course understanding S.suis.But well-known, the pathogenic course of bacterium is the interactional complex process of itself and host cell.After bacterium contact host, the defense mechanism of host body must be overcome, particularly will disturb or escape the phagolysis of local and the immunization of humoral immunization mediation, infection could be set up.In long-term evolutionary process, bacterium defines panimmunity escape mechanism, relies on regulation and control oneself expression Multiple components to be escaped the immunity system of host by different mechanism.
Htp family protein is a newfound proteinoid family in suis.In recent years Htp family protein member has all been found in report display A, B, C, G group streptococcus, and also report is seen in the correlative study of this family member's function in streptococcus pyogenes and streptococcus agalactiae, but there is no people to this gene function in S.suis and participate in bacterium cause a disease molecular mechanism study.This seminar is by the genome analysis of the virulent strain 05ZYH33 to the separation of S.suis2 people source, find 3 Histidine trimer proteins (histidine triad protein, Htp) encoding gene, its open reading frame is respectively 05SSU0332,05SSU1267,05SSU1577(called after HtpsA, HtpsB, HtpsC respectively), confirm the existence of Htp family protein in S.suis205ZYH33, and the correlation function carried out this family protein in S.suis2 that takes the lead in is studied at home and abroad.It is reported in streptococcus pneumoniae, this Htp family is by the similar member composition of four sequence height, encode in this bacterium any one single-gene deletion mutantion strain of Pht albumen or dual-gene deletion mutantion strain all can not cause Strain Virulence significantly to change, only have the gene of four members when this albumen of coding all to knock out, Strain Virulence just has obvious decline.
Carry out the common choice research of this pathogenic bacterium pathogenesis and prevention and corntrol having been become to the many scholars of domestic and international association area.Vaccine becomes the study hotspot of current reply S.suis2 infection as a kind of preventions of pathogenic bacteria safely and effectively.Attenuated live vaccine is traditional vaccine, and based on whole cell, dosage of inoculation is little, and immune effect is better, and immunizing power religion is lasting.And be recently that the subunit vaccine of target is because its security is high and the low investigator of enjoying of production cost favors with bacterial surface protein.Subunit vaccine has that component is clear, security good, technology maturation, be convenient to the advantages such as industrialization.This kind of vaccine is often made up of multiple different component, and each component is encoded by the different genes of pathogenic bacteria, thus can avoid the vaccine failure that variation or loss due to individual gene cause.Therefore the research of polyvalent vaccine becomes the trend of S.suis new generation vaccine research and development.The screening of more protective antigen is very important for the exploitation of multivalent subunit vaccine.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a strain streptococcus suis 2-type (Streptococcus suis serotype2) htpsC gene knockout mutant strain is provided.
Another object of the present invention is to provide the application of this mutant strain.
Object of the present invention realizes by following technical scheme:
Streptococcus suis 2-type (Streptococcus suis serotype2) htpsC gene knockout mutant strain 05ZYH33 Δ htpsC, the htpsC gene in this mutant strain in 05ZYH33 bacterial strain from the encoding gene between the 1st to 1000 by spectinomycin resistance gene box Spc
rreplacing, this mutant strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is: CGMCC No.7376, and preservation date is on March 28th, 2013.
HtpsC gene in wherein said 05ZYH33 bacterial strain from the coding gene sequence between the 1st to 1000 as shown in SEQ ID NO.1.
Described spectinomycin resistance gene box Spc
rsequence is as shown in SEQ ID NO.3.
The method of streptococcus suis 2-type (Streptococcus suis serotype2) the htpsC gene knockout mutant strain described in structure, comprises following steps:
(1) according to the DNA sequence upstream of the S.suis2 wild strain 05ZYH33 genome htpsC encoding gene as shown in SEQ ID NO.2, design PCR special primer LA1 and LA2; The DNA sequence downstream of the S.suis2 wild strain 05ZYH33 genome htpsC encoding gene according to such as SEQ ID NO.4, design PCR special primer RA1 and RA2; With pSET2 plasmid for template, design a pair special primer spc-F and spc-R; Wherein primer LA1 sequence is as shown in SEQ ID NO.5, primer LA2 sequence is as shown in SEQ ID NO.6, primer RA1 sequence is as shown in SEQ ID NO.7, primer RA2 sequence is as shown in SEQ ID NO.8, primer spc-F sequence is as shown in SEQ ID NO.9, and primer spc-R sequence is as shown in SEQ ID NO.10;
With 05ZYH33 genomic dna for template, respectively with LA1/LA2 and RA1/RA2 for primer, amplification obtain two ends respectively containing the goal gene htpsC DNA sequence upstream LA fragment of EcoR I/BamH I restriction enzyme site and two ends respectively containing the goal gene htpsC DNA sequence downstream RA fragment of Sal I/Sph I restriction enzyme site; With pSET2 plasmid for template, be that specific primers amplify obtains two ends respectively containing the spectinomycin resistance gene box of BamH I/Sal I restriction enzyme site with spc-F/spc-R;
It is 1005bp (LA), 960bp (RA) and 1130bp that LA1/LA2, RA1/RA2 and spc-F/spc-R tri-pairs of primer amplification gained PCR primer detect through 1% agarose electrophoresis the object clip size obtained respectively.PCR primer reclaims, purifying, carries out double digestion respectively with EcoR I/BamH I, Sal I/Sph I and BamH I/Sal I, by the recovery of double digestion product, purifying, frozen for subsequent use.
(3) structure of gene knockout carrier pUC::htpsC: obtain gene knockout carrier pUC::htpsC by between the Sph I/Kpn I restriction enzyme site of described LA fragment-spectinomycin resistance gene box-RA fragment insertion pUC19 carrier; Specifically comprise following steps:
(a) spectinomycin resistance gene Spc
rclone: the product after BamH I/Sal I double digestion is connected with the pUC18 carrier of same enzymes double zyme cutting process, 16 DEG C spend the night after connection product conversion is entered DH5a competent escherichia coli cell, after amoxicillin screening, the clone on picking LB flat board is incubated at 37 DEG C of shaken overnight in LB liquid nutrient medium.Next day extracts plasmid DNA, carries out double digestion qualification with BamH I/Sal I, by the recombinant plasmid called after pUC18-Spc having about 1100bp size DNA fragmentation to occur.
The clone of (b) RA: the RA fragment after Sal I/Sph I double digestion is connected with the recombinant plasmid pUC18-Spc carrier of same enzymes double zyme cutting process, 16 DEG C spend the night after will connect product conversion DH5a competent escherichia coli cell, after Spectinomycin HCL (Veterinary) and penbritin Double Selection, the clone on picking LB flat board is incubated at 37 DEG C of shaken overnight in LB liquid nutrient medium.Next day extracts plasmid DNA, carries out double digestion qualification with Sal I/Sph I, by the recombinant plasmid called after pUC18-SR having about 960bp size DNA fragmentation to occur.
The clone of (c) LA: the LA fragment after EcoR I/BamH I double digestion is connected with the recombinant plasmid pUC18-SR of same enzymes double zyme cutting process, 16 DEG C spend the night after will connect product conversion DH5a competent escherichia coli cell, after spectinomycin and penbritin Double Selection, the clone on picking LB flat board is incubated at 37 DEG C of shaken overnight in LB liquid nutrient medium.Next day extracts plasmid DNA, carries out double digestion qualification with EcoR I/BamHI, filters out the positive plasmid that about 1000bp size DNA fragmentation occurs; And do plasmid PCR qualification respectively with LA1/LA2, RA1/RA2, spc-F/spc-R, LA1/spc-R, spc-F/RA2, LA1/RA2 six pairs of primers respectively.The positive recombinant plasmid called after pUC::htpsC obtained.
(4) qualification of gene knockout carrier pUC::htpsC: carried out check order (Hua Da Gene science limited-liability company) by the positive recombination knockout carrier pUC::htpsC obtained, sequencing result shows at Spc
rgene both sides have the htpsC goal gene of homologous sequence, and the structure of gene knockout carrier pUC::htpsC is entirely true.
(5) gene knockout carrier pUC::htpsC electricity is transformed into 05ZYH33 competence
1. the preparation of S.suis2 wild strain 05ZYH33 competent cell: the mono-colony inoculation of picking 05ZYH33 and 3mlTHB substratum, 37 DEG C of shaking tables concussion overnight incubation, in the THY that next day, 1:50 was transferred to containing DL-Threonine, 37 DEG C of shaking tables concussions are cultured to OD
600for about 0.3-0.4,4 DEG C of centrifugal low speed receive bacterium, and wash bacterium 4 times with 10% glycerine of precooling, be no less than 25ml at every turn, finally contain the 0.3M sucrose further bacterial precipitation of 15% glycerine with 0.5ml, and packing 50 μ l/ manages, be stored in-80 DEG C for subsequent use.
2. electricity transforms: add add the pUC::htpsC plasmid of 10ul in the competence that 50 μ l are prepared as stated above after in electric revolving cup and (operate on ice), after 22.5kV/cm, 200 Ω and 25 μ F electricity turn, add THB (37 DEG C of preheatings) the substratum 940ul of 0.3M sucrose, 37 DEG C, coat on the THB flat board containing Spectinomycin resistance after 2h is cultivated in 160rpm concussion, cultivate 24-48h for 37 DEG C, picking list bacterium colony is identified;
(6) preliminary screening of 05ZYH33 △ htpsC mutant strain: select swine streptococcus bacterium colony from spectinomycin THB flat board, is incubated at 2ml liquid THB (100mg/ml spc respectively
r) substratum.Get bacterium liquid as template, by primer checkin1(sequence as shown in SEQ ID NO.11) and checkin2(be positioned at htpsC gene internal, sequence is as shown in SEQ ID NO.12) carry out PCR preliminary screening.
If htpsC gene is knocked, pcr amplification will obtain negative findings, if still can amplify the product of expection size (521bp), illustrates that htpsC gene is not knocked.By this method, preliminary screening obtains htpsC gene knockout mutant strain, by its called after 05ZYH33 Δ htpsC.
(7) qualification of 05ZYH33 △ htpsC mutant strain:
1. assembly PCR qualification: knock out at htpsC LA and RA of goal gene upstream and downstream homologous sequence two outside bamboo product pair of primers OUt1/OUt2, sequence is as shown in SEQ ID NO.13/SEQ ID NO.14; If the karyomit(e) of gene knockout carrier pUC::htpsC and bacterium is recombinated, 3 kinds of situations will be there will be and occur: a. double exchange homologous recombination events (double cross-over), i.e. allelic replacement, now spc
rgene replaces htpsC gene; B.3 recombination event (3 ' single cross-over) is changed in ' end single cross, and now whole vector DNA sequence is incorporated on the karyomit(e) of bacterium along with 3 ' end homologous sequence; C.5 recombination event (5 ' single cross-over) is changed in ' end single cross, and now carrier sequence is incorporated on bacterial chromosome along with 5 ' end homologous sequence.If generation allelic replacement, carry out with primer OUt1/Spc2 the fragment that PCR can amplify 2233p, carry out with primer Spc1/Out2 the fragment that PCR can amplify 2187bp, carrying out PCR with primer Spc1/Spc2 can amplify spc
rgene, and in 05ZYH33, carry out PCR with above primer and all should obtain negative findings.And with 05ZYH33 genome for template, the object fragment of the 521bp that can amplify with primer checkin1/2, the size of each PCR primer of result conforms to theoretical value, and through DNA sequencing checking (handsome order-checking), confirms the success of 05ZYH33 Δ htpsC mutative symptom at gene level.
2. RT-PCR qualification: in order to verify 05ZYH33 Δ htpsC mutant strain further, utilize checkin1/2 primer to carry out pcr amplification to the cDNA that mutant strain and street strain's reverse transcription obtain respectively.In wild strain 05ZYH33, result is positive, and htpsC normal transcription is described; And be negative in mutant strain 05ZYH33 Δ htpsC.Through the qualification of transcriptional level, successfully obtain htpsC gene knockout mutant strain, called after 05ZYH33 Δ htpsC.
Beneficial effect:
1. the present invention uses the principle of homologous recombination, be spectinomycin resistance gene in the middle of building, both sides are the gene knockout carrier pUC::htpsC of htpsC gene upstream and downstream homologous sequence, the pUC::htpsC gene knockout plasmid electricity of structure is transformed into streptococcus suis 2-type strong pathogenic strain 05ZYH33 competent cell, pass through In vivo homologous recombination, through gene level, transcriptional level and DNA sequencing screening and identification, successfully obtain mutant strain, called after 05ZYH33 Δ htpsC mutant strain.
2. the present invention is to the related biological characteristic of htpsC gene knockout mutant strain with pathogenicly to analyze, and specify that htpsC gene and the pathogenic relation of S.suis2.05ZYH33 Δ htpsC comparatively wild strain compares, and the arrival logarithmic phase time also slows down relatively, and growth velocity obviously slows down; By the configuration of surface of bacterium under transmission electron microscope observing, find that the pod membrane on mutant strain surface is compared with wild strain, mucous membrane meromixis; Externally obviously decline with the adhesive capacity of Hep-2 cell, its anti-macrophage phagocytic kill capability strengthens slightly, and prompting htpsC gene is relevant to bacterial adhesion function.Animal virulence test-results shows that the virulence of mutant strain 05ZYH33 Δ htpsC declines.Therefore prompting htpsC take part in the pathogenic course of S.suis2, is a kind of new virulence correlation factor.This mutant strain is that the screening of the protective antigen of multivalent subunit vaccine provides important clue, can be applicable to the exploitation of S.suis attenuated vaccine and multivalent subunit vaccine.
3. the 05ZYH33 Δ htpsC of the present invention's structure, for the mechanism of causing a disease studying streptococcus suis 2-type is further laid a good foundation, for more effective prevention and control Streptococcus suis provides technical support.
4. contrary with existing achievement in research, the htpsC gene knockout mutant strain 05ZYH33 Δ htpsC of the S.suis205ZYH33 that the present invention successfully builds, in abdominal injection mouse model virulence experiment, mutagenesis strain 05ZYH33 Δ htpsC virulence declines.Externally obviously decline with the adhesive capacity of people's larynx epithelium Hep-2 cell, prompting htpsC gene is relevant to bacterial adhesion function.The individual gene deletion mutantion strain 05ZYH33 Δ htpsC of the Htp protein family member that the present invention builds causes Strain Virulence to change, and illustrates that HtpsC is a potential virulence factor of swine streptococcus.It is cloned, expresses, purifying; to prepare the candidate molecules of recombinant protein antigen as antigen-immunized animal; carry out cloning, expression and purification; to prepare the candidate molecules of recombinant protein antigen as antigen-immunized animal; find that survival time and the survival rate of mouse all significantly improve; show that HtpsC has immanoprotection action, move towards application for the screening of this bacteria vaccine protective antigen and lay the foundation.
Biomaterial preservation information
05ZYH33 Δ htpsC, Classification And Nomenclature is swine streptococcus Streptococcus suis, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preservation date is on March 28th, 2013, and preserving number is CGMCC No.7376.
Accompanying drawing explanation
Fig. 1 is gene knockout carrier pUC::htpsC intersection PCR qualification result figure.
Swimming lane 1 is 250bp Marker, swimming lane 1,2, and 3,4,5,6 is the PCR primer that is primer pair with LA1/LA2, spc-F/spc-R, RA1/RA2, LA1/spc-R, spc-F/RA2, LA1/RA2 respectively.
Fig. 2 is the assembly PCR qualification result figure of 05ZYH33 Δ htpsC mutant strain
Swimming lane 1 is 250bp Marker, swimming lane 1,3, and 5,7 is take 05ZYH33 as the PCR of template, swimming lane 2,4, and 6,8 is the PCR that are template with 05ZYH33 Δ htpsC mutant strain.
Fig. 3 is RNA extraction figure and the reverse transcription PCR qualification figure of 05ZYH33 Δ htpsC mutant strain
A figure is that the RNA of 05ZYH33 Δ htpsC mutant strain extracts figure, and wherein swimming lane 1 is 05ZYH33 Δ htpsC mutant strain RNA, and swimming lane 2 is wild strain 05ZYH33RNA; B figure is the reverse transcription PCR qualification figure of 05ZYH33 Δ htpsC mutant strain, wherein swimming lane 1 take 05ZYH33cDNA as template, take checkin1/checkin2 as the PCR primer of primer, swimming lane 2 is with 05ZYH33 Δ htpsC cDNA mutant strain for template, take checkin1/checkin2 as the PCR primer of primer.
Embodiment
Embodiment 1: the structure of gene knockout carrier
(1) according to the upstream and downstream DNA sequence dna of S.suis2 wild strain 05ZYH33 genome htpsC encoding gene, design PCR special primer, base sequence is as follows:
LA1:5 '-CG
gAATTCtGAAGCGCAGATAAATC-3 ' (SEQ ID NO.5, underscore place is the EcoR I restriction enzyme site introduced)
LA2:5 '-CG
gGATCCaGTCCAAGGTCAAA-3 ' (SEQ ID NO.6, underscore place is the BamH I restriction enzyme site introduced)
RA1:5 '-C
gTCGACtTTTGTCCTCCTAAGCTC-3 ' (SEQ ID NO.7, underscore place is the Sal I restriction enzyme site introduced)
RA2:5 '-CG
gCATGCaGGTAGCAGAGATAGTAC-3 ' (SEQ ID NO.8, underscore place is the Sph I restriction enzyme site introduced)
According to pSET2 plasmid sequence, design a pair special primer spc-F/spc-R, with pSET2 plasmid for the whole spc expression cassette of template amplification, primer sequence is:
Spc-F:5 '-CC
gGATCCgTTCGTGAATACAT-3 ' (SEQ ID NO.9, underscore place is the BamH I restriction enzyme site introduced)
Spc-R:5 '-CG
gTCGACgTTTTCTAAAATCTGA-3 ' (SEQ ID NO.10, underscore place is the Sal I restriction enzyme site introduced)
PCR reaction system is:
PCR reaction conditions is: 95 DEG C of denaturation 5min, 94 DEG C of 50s, 55 DEG C of 60s, 72 DEG C of 2min, 30 circulations, and last 72 DEG C extend 10min, take distilled water as negative control.
It is 1005bp (LA), 960bp (RA) and 1130bp(Spc that LA1/LA2, RA1/RA2 and spc-F/spc-R tri-pairs of primer amplification gained PCR primer detect through 1% agarose electrophoresis the object clip size obtained respectively
r).PCR primer reclaims, purifying, carries out double digestion respectively with EcoR I/ BamH I, Sal I/Sph I and BamHI/Sal I, by the recovery of double digestion product, purifying, frozen for subsequent use.
(2) spectinomycin resistance gene Spc
rclone: the product after BamH I/Sal I double digestion is connected with the pUC19 carrier of same enzymes double zyme cutting process, 16 DEG C spend the night after connection product conversion is entered DH5a competent escherichia coli cell, after amoxicillin screening, the clone on picking LB flat board is incubated at 37 DEG C of shaken overnight in LB liquid nutrient medium.Next day extracts plasmid DNA, carries out double digestion qualification with BamH I/Sal I, by the recombinant plasmid called after pUC18-Spc having about 1100bp size DNA fragmentation to occur.
(3) clone of RA: the RA fragment after Sal I/Sph I double digestion is connected with the recombinant plasmid pUC18-Spc carrier of same enzymes double zyme cutting process, 16 DEG C spend the night after will connect product conversion DH5a competent escherichia coli cell, after Spectinomycin HCL (Veterinary) and penbritin Double Selection, the clone on picking LB flat board is incubated at 37 DEG C of shaken overnight in LB liquid nutrient medium.Next day extracts plasmid DNA, carries out double digestion qualification with Sal I/Sph I, by the recombinant plasmid called after pUC18-SR having about 960bp size DNA fragmentation to occur.
(4) clone of LA: the LA fragment after EcoR I/BamH I double digestion is connected with the recombinant plasmid pUC18-SR of same enzymes double zyme cutting process, 16 DEG C spend the night after will connect product conversion DH5a competent escherichia coli cell, after spectinomycin and penbritin Double Selection, the clone on picking LB flat board is incubated at 37 DEG C of shaken overnight in LB liquid nutrient medium.Next day extracts plasmid DNA, carries out double digestion qualification with EcoR I/BamH I, filters out the positive plasmid that about 1000bp size DNA fragmentation occurs; And do plasmid PCR qualification respectively with LA1/LA2, RA1/RA2, spc-F/spc-R, LA1/spc-R, spc-F/RA2, LA1/RA2 six pairs of primers respectively, the results are shown in Figure 1, the positive recombinant plasmid called after pUC::htpsC obtained.
(5) qualification of gene knockout carrier pUC::htpsC: carried out check order (Hua Da Gene science limited-liability company) by the positive recombination knockout carrier pUC::htpsC obtained, sequencing result shows at Spc
rgene both sides have the htpsC goal gene of homologous sequence, and the structure of gene knockout carrier pUC::htpsC is entirely true.
Embodiment 2: the selection systems of mutant
(1) gene knockout carrier pUC::htpsC electricity is transformed into 05ZYH33 competence
1. the preparation of S.suis2 wild strain 05ZYH33 competent cell: the mono-colony inoculation of picking 05ZYH33 and 3mlTHB substratum, 37 DEG C of shaking tables concussion overnight incubation, in the THY that next day, 1:50 was transferred to containing DL-Threonine, 37 DEG C of shaking tables concussions are cultured to OD
600for about 0.3-0.4,4 DEG C of centrifugal low speed receive bacterium, and wash bacterium 4 times with 10% glycerine of precooling, be no less than 25ml at every turn, finally contain the 0.3M sucrose further bacterial precipitation of 15% glycerine with 0.5ml, and packing 50 μ l/ manages, be stored in-80 DEG C for subsequent use.
2. electricity transforms: add add the pUC::htpsC plasmid of 10ul in the competence that 50 μ l are prepared as stated above after in electric revolving cup and (operate on ice), after 22.5kV/cm, 200 Ω and 25 μ F electricity turn, add THB (37 DEG C of preheatings) the substratum 940ul of 0.3M sucrose, 37 DEG C, coat on the THB flat board containing Spectinomycin resistance after 2h is cultivated in 160rpm concussion, cultivate 24-48h for 37 DEG C, picking list bacterium colony is identified;
(2) preliminary screening of 05ZYH33 △ htpsC mutant strain: select swine streptococcus bacterium colony from spectinomycin THB flat board, is incubated at 2ml liquid THB (100mg/ml spc respectively
r) substratum.Get bacterium liquid as template, be positioned at htpsC gene internal with primer checkin1 and checkin2() carry out PCR preliminary screening.Its primer sequence is:
Checkin1：5′-GCCATTCGCATCTTCAGCCA-3′（SEQ?ID?NO.11）
Checkin2：5′-TTGAAGGTCCGATCCCGTATGC-3′（SEQ?ID?NO.12）
If htpsC gene is knocked, pcr amplification will obtain negative findings, if still can amplify the product of expection size (521bp), illustrates that htpsC gene is not knocked.By this method, preliminary screening obtains htpsC gene knockout mutant strain, by its called after 05ZYH33 Δ htpsC.
(3) qualification of 05ZYH33 △ htpsC mutant strain:
1. assembly PCR qualification: knock out at htpsC LA and RA of goal gene upstream and downstream homologous sequence two outside bamboo product pair of primers OUt1/OUt2, its primer sequence is:
OUt1:5′-CATTAGCTGTCAGTTCTTCTAAATTCTCTGT-3′（SEQ?ID?NO.13）
OUt2:5′-GACGGAAATATCGTATGTAGTCAGCTG-3′（SEQ?ID?NO.14）
If the karyomit(e) of gene knockout carrier pUC::htpsC and bacterium is recombinated, 3 kinds of situations will be there will be and occur: a. double exchange homologous recombination events (double cross-over), i.e. allelic replacement, now spc
rgene replaces htpsC gene; B.3 recombination event (3 ' single cross-over) is changed in ' end single cross, and now whole vector DNA sequence is incorporated on the karyomit(e) of bacterium along with 3 ' end homologous sequence; C.5 recombination event (5 ' single cross-over) is changed in ' end single cross, and now carrier sequence is incorporated on bacterial chromosome along with 5 ' end homologous sequence.If generation allelic replacement, carry out with primer OUt1/Spc2 the fragment that PCR can amplify 2233p, carry out with primer Spc1/Out2 the fragment that PCR can amplify 2187bp, carrying out PCR with primer Spc1/Spc2 can amplify spc
rgene, and in 05ZYH33, carry out PCR with above primer and all should obtain negative findings.And with 05ZYH33 genome for template, the object fragment of the 521bp that can amplify with primer checkin1/2, the size of each PCR primer of result conforms to theoretical value (Fig. 2), and through DNA sequencing checking (handsome order-checking), confirms the success of 05ZYH33 Δ htpsC mutative symptom at gene level.
2. RT-PCR qualification: in order to verify 05ZYH33 Δ htpsC mutant strain further, utilize checkin1/2 primer to carry out pcr amplification to the cDNA that mutant strain and street strain's reverse transcription obtain respectively.In wild strain 05ZYH33, result is positive, and htpsC normal transcription is described; And be negative in mutant strain 05ZYH33 Δ htpsC.Through the qualification of transcriptional level, successfully obtain htpsC gene knockout mutant strain, called after 05ZYH33 Δ htpsC, and deliver CGMCC preservation, preserving number is CGMCC No.7376, and preservation date is on March 28th, 2013.
Embodiment 3: experiment in vitro
(1) gramstaining
According to the specification sheets operation of the gram staining liquid that Beijing Suo Laibao Science and Technology Ltd. produces, give wild strain 05ZYH33 and mutant strain 05ZYH33 Δ htpsC(CGMCC No.7376 respectively) carry out gram's staining, the catenation that discovery mutant strain 05ZYH33 Δ htpsC compares wild strain is dispersed in more, and the length of chain is shorter than wild strain, mutant strain 05ZYH33 Δ htpsC(CGMCC No.7376 is described) chaining reduced capability.
(2) growth characteristics
Under same culture conditions, respectively picking 05ZYH33 Δ htpsC(CGMCC No.7376) and the mono-bacterium colony of wild strain 05ZYH33 be inoculated in 3mL respectively and contain spectinomycin (100mg/mL) and do not contain in the THB substratum of spectinomycin, 37 DEG C of shaking culture are spent the night.Take out the bacterium of incubated overnight next day, measure 600nm place absorbance, with THB substratum, both are diluted to about 1 × 10
8cFU/mL concentration.Then get 60 μ L mutant strains respectively and wild strain is inoculated in THB substratum 3mL respectively, in 37 DEG C, 200r/min shaking culture, every 1h sampling and measuring OD respectively
600, take incubation time as X-coordinate, OD
600value for ordinate zou, is drawn mutant strain and wild strain growth curve, be found that mutant strain 05ZYH33 Δ htpsC(CGMCC No.7376) growth velocity will lower than the growth velocity of wild strain.
(3) stick and engulf experiment with anti-
Respectively 37 DEG C are cultured to SS2 wild strain 05ZYH33 and the mutant strain 05ZYH33 Δ htpsC(CGMCC No.7376 of logarithmic phase), the centrifugal 10min of 3000g collects bacterium, and wash 3 times with PBS, being resuspended in PBS and making concentration is 10
8the bacterial suspension of CFU/ml.Vital dye Fluoresceincarboxylic acid etheric acid (carboxyfluorescein diacetate, succinimidyl ester, CFDA-SE or be commonly referred to as CFSE) CFSE marks bacterial suspension, hatches 20min for 37 DEG C.4 DEG C of centrifugal 6min of 3000g collect thalline, wash twice with the PBS of 10ml precooling, resuspended to 10
7cFU/ml, places on ice for experiment.Get 37 DEG C and be cultured to logarithmic phase people laryngocarcinoma epithelial cell Hep-2 and scavenger cell RAW264.7, bed board, covers with individual layer after 12h, and PBS washes 3 times, and piping and druming is suspended in ice-cold PBS.The bacterium that CFSE marks is joined in cell suspension, make bacterium and cell quantity than being 100:1,37 DEG C of lucifuge gentle agitation 2h, 3 times are washed with warm PBS, 100ul/ml gentamicin and 5ul/ml penicillin effect 1h, wash 3 times, add isopyknic PBS solution containing 4% (wt/vol) paraformaldehyde and fix.Flow cytomery fluorescence intensity.Flow cytometry shows, 05ZYH33 Δ htpsC(CGMCC No.7376) obviously decline with the adhesive capacity of Hep-2 cell compared with 05ZYH33, its anti-macrophage phagocytic kill capability strengthens slightly.Prompting htpsC gene is relevant to bacterial adhesion function.
Embodiment 4: pathogenicity is tested
In order to detect mutant strain 05ZYH33 Δ htpsC(CGMCC No.7376) pathogenic, respectively picking 05ZYH33 and mutant strain 05ZYH33 Δ htpsC(CGMCC No.7376 on flat board) single bacterium colony, in THB substratum, 37 DEG C of shaking culture are to mid log phase (OD
600≈ 0.4, about 10
8cFU dosage) collected by centrifugation thalline, with the resuspended bacterium of sterile PBS buffer.SPF level female BAl BIc in 4 week age/c mouse 30, is divided into 3 groups at random, respectively abdominal injection wild-type and mutant strain bacterium liquid 1mL (about 10
8cFU/ is only), and establish THB negative control group (1mL/ is only), timely observed and recorded mouse invasion and survival time are with or without considerable change.Found that, after attacking mouse 24h with the wild strain 05ZYH33 of lethal dose, 10 mouse are all dead, and still have 8 survivals after knocking out strain △ htpsC attack mouse 12h with same dose, after being extended down to 72h, dead 5 altogether, survive 5; Survival mice all finds no any disease symptom at the end of observing and testing to 7 days.Negative control group 10 states are all good.Illustrate that the virulence of htpsC gene knockout on 05ZYH33 creates impact.Results of animal shows, htpsC gene is relevant to the virulence of bacterium 05ZYH33, can be applied to the development of streptococcus suis 2-type attenuated vaccine and multivalent subunit vaccine.
Claims (4)
1. streptococcus suis 2-type (
streptococcus suis serotype 2)
htpsCgene knockout mutant strain 05ZYH33 Δ
htpsC, it is characterized in that, in 05ZYH33 bacterial strain
htpsCgene from the encoding gene between the 1st to 1000 by spectinomycin resistance gene box
spc r replacing, the culture presevation of this mutant strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is: CGMCC No.7376, and preservation date is on March 28th, 2013; In described 05ZYH33 bacterial strain
htpsCgene from the coding gene sequence between the 1st to 1000 as shown in SEQ ID NO.1; Described spectinomycin resistance gene box
spc r sequence is as shown in SEQ ID NO.3.
2. build streptococcus suis 2-type according to claim 1 (
streptococcus suis serotype 2)
htpsCthe method of gene knockout mutant strain, is characterized in that comprising following steps:
(1) according to as shown in SEQ ID NO.2
s. suis2 wild strain 05ZYH33 genomes
htpsCthe DNA sequence upstream of encoding gene, design PCR special primer LA1 and LA2; According to as shown in SEQ ID NO.4
s. suis2 wild strain 05ZYH33 genomes
htpsCthe DNA sequence downstream of encoding gene, design PCR special primer RA1 and RA2; With pSET2 plasmid for template, design a pair special primer spc-F and spc-R; Wherein primer LA1 sequence is as shown in SEQ ID NO.5, primer LA2 sequence is as shown in SEQ ID NO.6, primer RA1 sequence is as shown in SEQ ID NO.7, primer RA2 sequence is as shown in SEQ ID NO.8, primer spc-F sequence is as shown in SEQ ID NO.9, and primer spc-R sequence is as shown in SEQ ID NO.10;
With 05ZYH33 genomic dna for template, respectively with LA1/LA2 and RA1/RA2 for primer, amplification obtains two ends and contains respectively
ecoRi/
bamHthe goal gene of I restriction enzyme site
htpsCdNA sequence upstream LA fragment and two ends contain respectively
sali/
sphthe goal gene of I restriction enzyme site
htpsCdNA sequence downstream RA fragment; With pSET2 plasmid for template, be that specific primers amplify obtains two ends and contains respectively with spc-F/spc-R
bamHi/
salthe spectinomycin resistance gene box of I restriction enzyme site
spc r ;
(3) gene knockout carrier pUC::
htpsCstructure: described LA fragment, spectinomycin resistance gene box, RA fragment inserted pUC19 carrier
ecoRi
/ Sphgene knockout carrier pUC: is obtained between I restriction enzyme site:
htpsC;
(4) gene knockout carrier pUC::
htpsCelectricity conversion prepares
s. suis2 wild strain 05ZYH33 competent cells;
(5) screening has the swine streptococcus bacterium colony of Spectinomycin resistance, confirms through assembly PCR product electrophoresis, RT-PCR and DNA sequencing
htpsCgoal gene is replaced by spectinomycin resistance gene box, namely obtain streptococcus suis 2-type that preserving number is CGMCC No.7376 (
streptococcus suis serotype 2)
htpsCgene knockout mutant strain 05ZYH33 Δ
htpsC;
Gene knockout carrier pUC: wherein described in step (3):
htpsCbuild especially by following methods:
(a) spectinomycin resistance gene box
spc r clone
:will
bamHi
/ Salproduct after I double digestion connects with the pUC18 carrier of same enzymes double zyme cutting process, obtains recombinant plasmid pUC18-Spc;
The clone of (b) RA: will
sali
/ SphrA fragment after I double digestion connects with the recombinant plasmid pUC18-Spc carrier of same enzymes double zyme cutting process, obtains recombinant plasmid pUC18-SR;
The clone of (c) LA: will
ecoRi
/ BamHlA fragment after I double digestion connects with the recombinant plasmid pUC18-SR of same enzymes double zyme cutting process, 16 DEG C spend the night after will connect product conversion DH5a competent escherichia coli cell, after spectinomycin and penbritin Double Selection, extract plasmid DNA, use
ecoRi
/ BamHi carries out double digestion qualification, filters out the positive plasmid that 1000 bp size DNA fragmentations occur; And do plasmid PCR qualification respectively, the positive recombinant plasmid called after pUC: obtained with LA1/LA2, RA1/RA2, spc-F/spc-R, LA1/ spc-R, spc-F/RA2, LA1/ RA2 six pairs of primers respectively:
htpsC.
3. streptococcus suis 2-type according to claim 1 (
streptococcus suis serotype 2)
htpsCgene knockout mutant strain 05ZYH33 Δ
htpsCpreparing the application in streptococcus suis 2-type attenuated vaccine.
4. streptococcus suis 2-type
htpsCgene knockout carrier pUC::
htpsCstructure streptococcus suis 2-type (
streptococcus suis serotype 2)
htpsCapplication in gene knockout mutant strain, wherein said streptococcus suis 2-type
htpsCgene knockout carrier pUC::
htpsCbuild by the following method:
(a) spectinomycin resistance gene box
spc r clone
:will
bamHi
/ Salproduct after I double digestion connects with the pUC18 carrier of same enzymes double zyme cutting process, obtains recombinant plasmid pUC18-Spc;
The clone of (b) RA: will
sali
/ SphrA fragment after I double digestion connects with the recombinant plasmid pUC18-Spc carrier of same enzymes double zyme cutting process, obtains recombinant plasmid pUC18-SR;
The clone of (c) LA: will
ecoRi
/ BamHlA fragment after I double digestion connects with the recombinant plasmid pUC18-SR of same enzymes double zyme cutting process, 16 DEG C spend the night after will connect product conversion DH5a competent escherichia coli cell, after spectinomycin and penbritin Double Selection, extract plasmid DNA, use
ecoRi
/ BamHi carries out double digestion qualification, filters out the positive plasmid that 1000 bp size DNA fragmentations occur; And do plasmid PCR qualification respectively, the positive recombinant plasmid called after pUC: obtained with LA1/LA2, RA1/RA2, spc-F/spc-R, LA1/ spc-R, spc-F/RA2, LA1/ RA2 six pairs of primers respectively:
htpsC; Wherein, wherein primer LA1 sequence is as shown in SEQ ID NO.5, primer LA2 sequence is as shown in SEQ ID NO.6, primer RA1 sequence is as shown in SEQ ID NO.7, primer RA2 sequence is as shown in SEQ ID NO.8, primer spc-F sequence is as shown in SEQ ID NO.9, and primer spc-R sequence is as shown in SEQ ID NO.10.
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