CN103352015A - HtpsA-gene-knock-out mutant strain of Streptococcus suis serotype 2 and application thereof - Google Patents

Abstract

The invention relates to an htpsA-gene-knock-out mutant strain of Streptococcus suis serotype 2 and application thereof, belonging to the field of genetic engineering. The mutant strain is 05ZYH33 delta htpsA with an accession number of CGMCC No. 7375. A preparation method for the mutant strain comprises the following steps: carrying out gene knockout on an htpsA gene by using a homologous recombination gene knockout technology, wherein the htpsA gene is located on chromosome 05ZYH33 of a wild S. suis 2 strain and codes histidine triad protein Htp; and determining that an obtained strain is a gene-knocked-out mutant strain through combined PCR product electrophoresis, RT-PCR and DNA sequencing identification and naming the obtained gene as 05ZYH33 delta htpsA. Pathogenicity of the mutant strain provided by the invention is researched through animal experiments, and results show that virulence of the mutant strain to a tested animal is substantially reduced; so the mutant strain can be used for developing an attenuated vaccine of Streptococcus suis serotype 2.

Description

Streptococcus suis 2-type htpsA gene knockout mutant strain and application thereof

Technical field

The invention belongs to the genetically engineered field, relate to streptococcus suis 2-type htpsA gene knockout mutant strain and application thereof.

Background technology

Streptococcus suis 2-type (S.suis2) is a kind of zoonosis pathogenic bacteria that endangers the whole world.This pathogenic bacteria not only can cause pig meningitis, septicemia, sacroiliitis, pneumonia and endocarditis etc., but also can infect the people, causes serious threat for relevant practitioner and broad masses' life security.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 recent years S.suis2 is popular in the swinery in south China province is on the rise, the time fairly large breaking out arranged, the financial loss that causes every year reaches billions of units.Broke out extensive SS2 infection people event in China Jiangsu and Ziyang, Sichuan respectively in 1998 and 2005, caused great life and property loss, and a high proportion of, domestic and international rare toxic shock syndrome has appearred among the patient, the state of an illness is dangerous, case fatality rate high (60%～80%) causes serious public health event.Above-mentioned situation points out this twice popular SS2 virulence of initiation extremely strong strongly, the virulence variation has probably occured or obtained the relevant genetic material of virulence.The report that infects 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, has brought new challenge for prevention and the control of S.suis2.Therefore, carry out relevant rudimentary and the applied research of S.suis2 mechanism of causing a disease, infect popular significant in people and animals for prevention and control S.suis2.But, be still not clear about S.suis infection host and pathogenic molecular mechanism at present.Therefore, the effect of the function of the pathogenic related gene that further exploratory development is new and performance in S.suis and host's interaction thereof, to screening potential vaccine candidate molecule, disclosing the accurate molecular mechanism of its genesis, further improve China S.suis prevention and treatment level significant.

Research for virulence factors of streptococcus suis is a focus always, and the virulence factor that has definitely been confirmed at present only has one of capsular polysaccharide (CPS).What other was known comprises muramidase-released protein (muraminidase released protein with 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 of understanding S.suis.But well-known, the pathogenic course of bacterium is the interactional complex process of itself and host cell.After the bacterium contact host, must overcome the defense mechanism of host's body, particularly will disturb or escape the immunization of local phagolysis and humoral immunization mediation, could set up infection.In long-term evolutionary process, bacterium has formed the panimmunity escape mechanism, relies on regulation and control oneself expression Multiple components to escape host's immunity system by different mechanism.

The Htp family protein is a newfound proteinoid family in suis.In recent years report shows in A, B, C, the G group streptococcus has all found Htp family protein member, and the correlative study to this family member's function in streptococcus pyogenes and the streptococcus agalactiae also has been seen in report, studies but there is no the people to this gene function among the S.suis and participate in the pathogenic molecular mechanism of bacterium.This seminar passes through the genome analysis to the virulent strain 05ZYH33 of S.suis2 people source separation, found 3 Histidine trimer proteins (histidine triad protein, Htp) encoding gene, its open reading frame is respectively 05SSU0332,05SSU1267,05SSU1577(respectively called after HtpsA, HtpsB, HtpsC), confirm the existence of Htp family protein among the S.suis205ZYH33, and taken the lead in having carried out the correlation function research to this family protein among the S.suis2 at home and abroad.It is reported in streptococcus pneumoniae, this Htp family is by four member compositions that the sequence height is similar, any one single-gene deletion mutantion strain or the dual-gene deletion mutantion strain of coding Pht albumen can not cause that all Strain Virulence significantly changes in this bacterium, only have four members' that work as this albumen of coding gene all to knock out, Strain Virulence just has obvious decline.Bibliographical information is arranged in 2 type swine streptococcus P1/7, the disappearance of the homologue gene of HtpsA does not produce any impact to parent's virulence.

Carry out the common choice that the research of this pathogenic bacterium pathogenesis and prevention and control has been become the many scholars of domestic and international association area.Vaccine becomes the study hotspot that current reply S.suis2 infects as a kind of safely and effectively pathogenic bacteria preventions.Attenuated live vaccine is traditional vaccine, and take whole cell as the basis, dosage of inoculation is little, and immune effect is better, and the immunizing power religion is lasting.And the subunit vaccine take bacterium surface albumen as target enjoys the investigator to favor because it is safe and production cost is low recently.Subunit vaccine has that component is clear, security good, technology maturation, be convenient to the advantages such as industrialization.This class vaccine often is comprised of a plurality of different components, and each component is by the different genes of pathogenic bacteria coding, thereby can avoid because the variation of individual gene or lose the vaccine failure that causes.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.Mutant strain of the present invention provides important value for the exploitation of attenuated vaccine and multivalent subunit vaccine.

Summary of the invention

The objective of the invention is the above-mentioned deficiency for prior art, a strain streptococcus suis 2-type (Streptococcus suis serotype2) htpsA gene knockout mutant strain is provided.

Another object of the present invention provides the application of this mutant strain.

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

Streptococcus suis 2-type (Streptococcus suis serotype2) htpsA gene knockout mutant strain 05ZYH33 Δ htpsA, it is characterized in that, (Spectinomycin resistance cassette is hereinafter to be referred as Spc by the spectinomycin resistance gene box for the encoding gene of htpsA gene in the 05ZYH33 bacterial strain between the 31st to 1000 ^R) replace, the culture presevation of this mutant strain is in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is: CGMCC No.7375, preservation date are on March 28th, 2013.

The coding gene sequence of htpsA gene in the wherein said 05ZYH33 bacterial strain between the 31st to 1000 is shown in SEQ ID NO.1.

Described spectinomycin resistance gene box sequence is shown in SEQ ID NO.3.

Make up the method for described streptococcus suis 2-type (Streptococcus suis serotype2) htpsA gene knockout mutant strain, comprise following steps:

⑴ design PCR special primer LA1 and LA2 according to the upstream dna sequence dna of the S.suis2 wild strain 05ZYH33 genome htpsA encoding gene shown in SEQ ID NO.2; According to the downstream DNA sequence of the S.suis2 wild strain 05ZYH33 genome htpsA encoding gene shown in SEQ ID NO.4, design PCR special primer RA1 and RA2; Take the pSET2 plasmid as template, design a pair of special primer spc-F and spc-R; Wherein primer LA1 sequence is shown in SEQ ID NO.5, primer LA2 sequence is shown in SEQ ID NO.6, primer RA1 sequence is shown in SEQ ID NO.7, primer RA2 sequence is shown in SEQ ID NO.8, primer spc-F sequence is shown in SEQ ID NO.9, and primer spc-R sequence is shown in SEQ ID NO.10;

⑵ take the 05ZYH33 genomic dna as template, take LA1/LA2 and RA1/RA2 as primer, amplification obtains two ends and contains respectively the goal gene htpsA upstream dna sequence dna LA fragment of Sph I/Sal I restriction enzyme site and the goal gene htpsA downstream DNA sequence RA fragment that two ends contain respectively BamH I/Kpn I restriction enzyme site respectively; Take the pSET2 plasmid as template, amplification obtains the spectinomycin resistance gene box that two ends contain respectively Sal I/BamH I restriction enzyme site take spc-F/spc-R as special primer;

It is 976bp (LA fragment), 847bp (RA fragment) and 1130bp(Spc that LA1/LA2, RA1/RA2 and three pairs of primer amplification gained of spc-F/spc-R PCR product detect the purpose clip size that obtains respectively through 1% agarose electrophoresis ^R).The PCR product reclaims, purifying, carries out double digestion with Sph I/Sal I, BamH I/Kpn I and Sal I/BamH I respectively, and is with the recovery of double digestion product, purifying, frozen for subsequent use.

(3) structure of gene knockout carrier pUC::htpsA: get gene knockout carrier pUC::htpsA between the Sph I/Kpn I restriction enzyme site with described LA fragment-spectinomycin resistance gene box-RA fragment insertion pUC19 carrier; Specifically comprise following steps:

(a) clone of RA: the RA fragment behind the BamH I/Kpn I double digestion is connected with the pUC19 carrier of processing with same enzymes double zyme cutting, 16 ℃ will connect product after spending the night and transform the DH5a competent escherichia coli cell, after the amoxicillin screening, the clone on the picking LB flat board is incubated at 37 ℃ of shaken overnight in the LB liquid nutrient medium.Extract plasmid DNA next day, carry out double digestion with BamH I and Kpn I and identify that the recombinant plasmid called after pUC19-RA that big or small dna fragmentation occurs about 800bp will be arranged.

(b) spectinomycin resistance gene Spc ^RThe clone: the product that Sal I is connected with BamH I double digestion is connected with the recombinant plasmid pUC19-RA that processed with same enzymes double zyme cutting, 16 ℃ will connect product after spending the night and transform the DH5a competent escherichia coli cell, behind spectinomycin and penbritin Double Selection, the clone on the picking LB flat board is incubated at 37 ℃ of shaken overnight in the LB liquid nutrient medium.Extract plasmid DNA next day, carry out double digestion with Sal I and BamH I and identify that the recombinant plasmid called after pUC19-SR that big or small dna fragmentation occurs about 1100bp will be arranged.

(c) clone of LA: the LA fragment behind the Sph I/Sal I double digestion is connected with the recombinant plasmid pUC19-SR that processed with same enzymes double zyme cutting, 16 ℃ will connect product after spending the night and transform the DH5a competent escherichia coli cell, behind spectinomycin and penbritin Double Selection, the clone on the picking LB flat board is incubated at 37 ℃ of shaken overnight in the LB liquid nutrient medium.Extract plasmid DNA next day, carry out double digestion with Sph I and Sal I and identify, filter out the positive plasmid that big or small dna fragmentation occurs about 970bp; And do respectively plasmid PCR with LA1/LA2, RA1/RA2, spc-F/spc-R, LA1/spc-R, spc-F/RA2, six pairs of primers of LA1/RA2 respectively and identify.The positive recombinant plasmid called after pUC::htpsA that obtains.

(4) evaluation of gene knockout carrier pUC::htpsA: the positive recombination knockout carrier pUC::htpsA that will obtain checks order (the large Gene science limited-liability company of China), and sequencing result shows at Spc ^RThe gene both sides have the htpsA goal gene of homologous sequence, and the structure of gene knockout carrier pUC::htpsA is entirely true.

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

1. the preparation of S.suis2 wild strain 05ZYH33 competent cell: the single colony inoculation of picking 05ZYH33 is in the 3mlTHB substratum, 37 ℃ of shaking tables concussion overnight incubation, and next day, 1:50 was transferred to that 37 ℃ of shaking tables concussions are cultured to OD among the THY that contains the DL-Threonine ₆₀₀For about 0.3-0.4,4 ℃ of centrifugal low speed are received bacterium, and wash bacterium 4 times with 10% glycerine of precooling, are no less than 25ml at every turn, contain at last the resuspended bacterial precipitation of 0.3M sucrose of 15% glycerine with 0.5ml, and packing 50 μ l/ manage, be stored in-80 ℃ for subsequent use.

2. electricity transforms: add behind the pUC::htpsA plasmid of adding 10ul in the competence that 50 μ l prepare as stated above (operation on ice) in the electric revolving cup, 22.5kV/cm, after 200 Ω and 25 μ F electricity turn, THB (37 ℃ of preheatings) the substratum 940ul that adds 0.3M sucrose, 37 ℃, after cultivating 2h, the 160rpm concussion coats on the THB flat board that contains the spectinomycin resistance, cultivate 24-48h for 37 ℃, picking list bacterium colony is identified;

(6) preliminary screening of 05ZYH33 △ htpsA mutant strain: select the swine streptococcus bacterium colony from spectinomycin THB flat board, be incubated at respectively 2ml liquid THB (100mg/ml spc ^r) substratum.Get bacterium liquid as template, with primer checkin1(sequence shown in SEQ ID NO.11) and checkin2(to be positioned at the htpsA gene inner, sequence is shown in SEQ ID NO.12) carry out the PCR preliminary screening.

If the htpsA gene is knocked, pcr amplification will obtain negative findings, if still can amplify the product of expection size (354bp), illustrates that the htpsA gene is not knocked.By this method, preliminary screening obtains the htpsA gene knockout mutant strain, with its called after 05ZYH33 Δ htpsA.

(7) evaluation of 05ZYH33 △ htpsA mutant strain:

1. assembly PCR is identified: knock out the LA of goal gene upstream and downstream homologous sequence and pair of primers OUt1/OUt2 is designed in two outsides of RA again at htpsA, sequence is shown in SEQ ID NO.13/SEQ ID NO.14;

If the karyomit(e) of gene knockout carrier pUC::htpsA and bacterium is recombinated, 3 kinds of situations will occur and occur: a. double exchange homologous recombination event (double cross-over), i.e. allelic replacement, at this moment spc ^RGene replaces the htpsA gene; B.3 ' and hold single cross to change recombination event (3 ' single cross-over), this moment, whole carrier DNA sequence was incorporated on the karyomit(e) of bacterium along with 3 ' end homologous sequence; C.5 ' and hold single cross to change recombination event (5 ' single cross-over), this moment, the carrier sequence was incorporated on the bacterial chromosome along with 5 ' end homologous sequence.If the generation allelic replacement carries out the fragment that PCR can amplify 2101p with primer OUt1/Spc2, carry out the fragment that PCR can amplify 1878bp with primer Spc1/Out2, carry out PCR with primer Spc1/Spc2 and can amplify spc ^RGene, and in 05ZYH33, carry out PCR with above primer and all should obtain negative findings.And take the 05ZYH33 genome as template, the purpose fragment of the 354bp that can amplify with primer checkin1/2, the size of each PCR product conforms to theoretical value as a result, and through dna sequencing checking (handsome order-checking), confirms that at gene level 05ZYH33 Δ htpsA mutant strain successfully constructs.

2. RT-PCR identifies: in order further 05ZYH33 Δ htpsA mutant strain to be verified, utilize the checkin1/2 primer respectively the cDNA that mutant strain and street strain's reverse transcription obtain to be carried out pcr amplification.The result is positive among the wild strain 05ZYH33, and the htpsA normal transcription is described; And it is negative in mutant strain 05ZYH33 Δ htpsA.Evaluation through transcriptional level successfully obtains the htpsA gene knockout mutant strain, called after 05ZYH33 Δ htpsA.

At present this 2 type swine streptococcus bacterial classification has been deposited in Chinese microorganism strains preservation management committee common micro-organisms center, preservation center preserving number is: CGMCC No.7375, the Latin formal name used at school of bacterial classification is: Streptococcus suis, preservation date are on March 28th, 2013.

The application of described streptococcus suis 2-type (Streptococcus suis serotype2) htpsA gene knockout mutant strain in preparation streptococcus suis 2-type attenuated vaccine and subunit vaccine.

The application of streptococcus suis 2-type htpsA gene knockout carrier pUC::htpsA of the present invention in making up streptococcus suis 2-type (Streptococcus suis serotype2) htpsA gene knockout mutant strain.

The application of streptococcus suis 2-type htpsA gene knockout carrier pUC::htpsA of the present invention in preparation streptococcus suis 2-type attenuated vaccine or subunit vaccine.

Beneficial effect:

1. the present invention uses the principle of homologous recombination, be spectinomycin resistance gene in the middle of making up, both sides are the gene knockout carrier of htpsA gene upstream and downstream homologous sequence, the pUC::htpsA gene knockout plasmid electricity that makes up is transformed into the strong pathogenic strain 05ZYH33 of streptococcus suis 2-type competent cell, by homologous recombination in the body, through gene level, transcriptional level and dna sequencing screening and identification, successfully obtain mutant strain, called after 05ZYH33 Δ htpsA mutant strain.

2. the present invention learns characteristic and pathogenic the analysis to the associated biomolecule of htpsA gene knockout mutant strain, the relation that clear and definite htpsA gene and S.suis2 are pathogenic.05ZYH33 Δ htpsA(05ZYH33 Δ htpsA) and the wild strain gram's staining found that, the catenation that mutant strain Δ htpsA compares wild strain is dispersed in more, and the length of chain is obviously shorter than the wild strain, and mutant strain 05ZYH33 Δ htpsA(05ZYH33 Δ htpsA is described) the chaining ability weaken; And experiment in vitro demonstration mutant strain 05ZYH33 Δ htpsA(05ZYH33 Δ htpsA) ability of opposing macrophage phagocytic reduces, and animal virulence test-results shows that the virulence of mutant strain obviously descends.This mutant strain can be applicable to the exploitation of S.suis attenuated vaccine and multivalent subunit vaccine for the screening of the protective antigen of multivalent subunit vaccine provides important clue.

3. the 05ZYH33 Δ htpsA(05ZYH33 Δ htpsA that makes up of the present invention), for the further mechanism of causing a disease of research streptococcus suis 2-type is laid a good foundation, for more effective prevention and control Streptococcus suis provides technical support.

4. opposite with existing achievement in research, the htpsA gene knockout mutant strain of S.suis 2 05ZYH33 that this research success makes up, the virulence of mutant strain Δ htpsA obviously descends in abdominal injection mouse model virulence experiment.Illustrate that HtpsA is the important virulence factor of swine streptococcus.Clone, expression and purification; with the candidate molecules of preparing recombinant protein antigen as antigen-immunized animal; survival time and the survival rate of finding mouse all obviously improve; show that HtpsA can be used as protective antigen performance immanoprotection action in the host infection process, and at 2 type streptococcus suis infection Expression In The Process.Anti-HtpS antibody can increase human complement c 3 in the deposition on 2 type swine streptococcus surfaces and strengthen the survival rate of 2 type swine streptococcus in people's whole blood; show that it plays a role in the bacterial immune escape mechanism, move towards application for this bacteria vaccine protective antigen screening and lay the foundation.

Description of drawings:

Fig. 1 is gene knockout carrier pUC::htpsA intersection PCR qualification result figure.

Swimming lane 1,2,3,4,5,6 is respectively the PCR product take LA1/LA2, RA1/RA2, spc-F/spc-R, LA1/spc-R, spc-F/RA2, LA1/RA2 as primer pair, swimming lane 7 is 250bp Marker..

Fig. 2 is the homologous recombination schematic diagram.

The assembly PCR qualification result figure of Figure 30 5ZYH33 Δ htpsA mutant strain.

Swimming lane 1-8 is respectively the PCR product take primer checkin1/2, spc-F/spc-R, OUt1/Spc2, Spc1/Out2 as primer pair.1,3,5,7th, the PCR take 05ZYH33 Δ htpsA mutant strain as template, swimming lane 2,4,6,8th, the PCR take 05ZYH33 as template, swimming lane 9 is 250bp Marker.

Fig. 4 is that RNA extraction figure and the reverse transcription PCR of 05ZYH33 Δ htpsA mutant strain identifies that figure A figure is the RNA extraction figure of 05ZYH33 Δ htpsA mutant strain, and wherein swimming lane 1 is wild strain 05ZYH33RNA, and swimming lane 2 is 05ZYH33 Δ htpsA mutant strain RNA; Swimming lane 3 is 250bp Marker.

B figure is that the reverse transcription PCR of 05ZYH33 Δ htpsA mutant strain is identified figure, wherein swimming lane 1 is 250bp Marker, swimming lane 1 is as template take 05ZYH33 cDNA, PCR product take checkin1/2 as primer, the 2nd, take 05ZYH33 Δ htpsA cDNA mutant strain as template, the PCR product take checkin1/2 as primer.

The biomaterial preservation proves

05ZYH33 Δ htpsA, Classification And Nomenclature is swine streptococcus Streptococcus suis, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the preservation address is No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on March 28th, 2013, and preserving number is CGMCC No.7375.

Specific implementation method

Embodiment 1: the structure of gene knockout carrier

⑴ design the PCR special primer according to the upstream and downstream dna sequence dna of S.suis2 wild strain 05ZYH33 genome htpsA encoding gene, and base sequence is as follows:

LA1:5 '-CG GCATGCGAAAGTGATAAGAGG-3 ' (SEQ ID NO.5, the Sph I restriction enzyme site of underscore place for introducing)

LA2:5 '-C GTCGACTACGGAGCCAACAACT-3 ' (SEQ ID NO.6, the Sal I restriction enzyme site of underscore place for introducing)

RA1:5 '- GGATCCGCGCACAAGCAAGT-3 ' (SEQ ID NO.7, the BamH I restriction enzyme site of underscore place for introducing)

RA2:5 '-AC GGTACCTCAGGATGTTGCATGA-3 ' (SEQ ID NO.8, the Kpn I restriction enzyme site of underscore place for introducing)

According to, the pSET2 plasmid sequence designs a pair of special primer spc-F/spc-R, the whole spectinomycin resistance gene box take the pSET2 plasmid as template amplification, and primer sequence is:

Spc-F:5 '-C GTCGACGTTCGTGAATACATGT-3 ' (SEQ ID NO.9, the Sal I restriction enzyme site of underscore place for introducing)

Spc-R:5 '-CC GGATCCGTTTTCTAAAATCTG-3 ' (SEQ ID NO.10, the BamH I restriction enzyme site of underscore place for introducing)

The PCR reaction system is:

Template (05ZYH33 genomic dna) 2 μ l

Primer 1 (LA1 or RA1) 1 μ l

Primer 2 (LA2 or RA2) 1 μ l

dNTP(10mM each) 4μl

10×PCR buffer 5μl

Ex Taq archaeal dna polymerase 0.4 μ l

Add ddH ₂O

Total 50μl

The PCR reaction conditions is: 95 ℃ of denaturation 5min, and 94 ℃ of 50s, 55 ℃ of 60s, 72 ℃ of 2min, 30 circulations, last 72 ℃ are extended 10min, with the negative contrast of distilled water.

It is 976bp (LA), 847bp (RA) and 1130bp(Spc that LA1/LA2, RA1/RA2 and three pairs of primer amplification gained of spc-F/spc-R PCR product detect the purpose clip size that obtains respectively through 1% agarose electrophoresis ^R).The PCR product reclaims, purifying, carries out double digestion with Sph I/Sal I, BamH I/Kpn I and Sal I/BamH I respectively, and is with the recovery of double digestion product, purifying, frozen for subsequent use.

(2) clone of RA: the RA fragment behind the BamH I/Kpn I double digestion is connected with the pUC19 carrier of processing with same enzymes double zyme cutting, 16 ℃ will connect product after spending the night and transform the DH5a competent escherichia coli cell, after the amoxicillin screening, the clone on the picking LB flat board is incubated at 37 ℃ of shaken overnight in the LB liquid nutrient medium.Extract plasmid DNA next day, carry out double digestion with BamH I and Kpn I and identify that the recombinant plasmid called after pUC19-RA that big or small dna fragmentation occurs about 800bp will be arranged.

(3) spectinomycin resistance gene box Spc ^RThe clone: the product that Sal I is connected with BamH I double digestion is connected with the recombinant plasmid pUC19-RA that processed with same enzymes double zyme cutting, 16 ℃ will connect product after spending the night and transform the DH5a competent escherichia coli cell, behind spectinomycin and penbritin Double Selection, the clone on the picking LB flat board is incubated at 37 ℃ of shaken overnight in the LB liquid nutrient medium.Extract plasmid DNA next day, carry out double digestion with Sal I and BamH I and identify that the recombinant plasmid called after pUC19-SR that big or small dna fragmentation occurs about 1100bp will be arranged.

(4) clone of LA: the LA fragment behind the Sph I/Sal I double digestion is connected with the recombinant plasmid pUC19-SR that processed with same enzymes double zyme cutting, 16 ℃ will connect product after spending the night and transform the DH5a competent escherichia coli cell, behind spectinomycin and penbritin Double Selection, the clone on the picking LB flat board is incubated at 37 ℃ of shaken overnight in the LB liquid nutrient medium.Extract plasmid DNA next day, carry out double digestion with Sph I and Sal I and identify, filter out the positive plasmid that big or small dna fragmentation occurs about 970bp; And do respectively plasmid PCR with LA1/LA2, RA1/RA2, spc-F/spc-R, LA1/spc-R, spc-F/RA2, six pairs of primers of LA1/RA2 respectively and identify, the results are shown in Figure 1.The positive recombinant plasmid called after pUC::htpsA that obtains.

(5) evaluation of gene knockout carrier pUC::htpsA: the positive recombination knockout carrier pUC::htpsA that will obtain checks order (the large Gene science limited-liability company of China), and sequencing result shows at Spc ^RThe gene both sides have the htpsA goal gene of homologous sequence, and the structure of gene knockout carrier pUC::htpsA is entirely true.

Embodiment 2: the screening of mutant and evaluation

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

1. the preparation of S.suis2 wild strain 05ZYH33 competent cell: the single colony inoculation of picking 05ZYH33 and 3mlTHB substratum, 37 ℃ of shaking tables concussion overnight incubation, 1:50 was transferred among the THY that contains the DL-Threonine 37 ℃ of shaking tables and shook and be cultured to OD next day ₆₀₀For about 0.3-0.4,4 ℃ of centrifugal low speed are received bacterium, and wash bacterium 4 times with 10% glycerine of precooling, are no less than 25ml at every turn, contain at last the resuspended bacterial precipitation of 0.3M sucrose of 15% glycerine with 0.5ml, and packing 50 μ l/ manage, be stored in-80 ℃ for subsequent use.

2. electricity transforms: add behind the pUC::htpsA plasmid of adding 10ul in the competence that 50 μ l prepare as stated above (operation on ice) in the electric revolving cup, 22.5kV/cm, after 200 Ω and 25 μ F electricity turn, THB (37 ℃ of preheatings) the substratum 940ul that adds 0.3M sucrose, 37 ℃, after cultivating 2h, the 160rpm concussion coats on the THB flat board that contains the spectinomycin resistance, cultivate 24-48h for 37 ℃, picking list bacterium colony is identified;

(2) preliminary screening of 05ZYH33 △ htpsA mutant strain: select the swine streptococcus bacterium colony from spectinomycin THB flat board, be incubated at respectively 2ml liquid THB (100mg/ml spc ^r) substratum.Get bacterium liquid as template, be positioned at the htpsA gene with primer checkin1 and checkin2(inner) carry out the PCR preliminary screening.Its primer sequence is:

Checkin1：5′-GGCTATGTGACATCACACGGTGA-3′（SEQ ID NO.11）

Checkin2：5′-TCCCGTGTCTTCAATAACATCTGTC-3′（SEQ ID NO.12）

If the htpsA gene is knocked, pcr amplification will obtain negative findings, if still can amplify the product of expection size (354bp), illustrates that the htpsA gene is not knocked.By this method, preliminary screening obtains the htpsA gene knockout mutant strain, with its called after 05ZYH33 Δ htpsA.

(3) evaluation of 05ZYH33 △ htpsA mutant strain:

1. assembly PCR is identified: knock out the LA of goal gene upstream and downstream homologous sequence and pair of primers OUt1/OUt2 is designed in two outsides of RA again at htpsA, its primer sequence is: OUt1:5 '-GAATGGGGAGAAATCAAACGAGTG-3 ' (SEQ ID NO.13) OUt2:5 '-AACAAGATCCGTTGCTTCTCCATC-3 ' (SEQ ID NO.14)

If the karyomit(e) of gene knockout carrier pUC::htpsA and bacterium is recombinated (Fig. 2), 3 kinds of situations will occur and occur: a. double exchange homologous recombination event (double cross-over), i.e. allelic replacement, at this moment spc ^RGene replaces the htpsA gene; B.3 ' and hold single cross to change recombination event (3 ' single cross-over), this moment, whole carrier DNA sequence was incorporated on the karyomit(e) of bacterium along with 3 ' end homologous sequence; C.5 ' and hold single cross to change recombination event (5 ' single cross-over), this moment, the carrier sequence was incorporated on the bacterial chromosome along with 5 ' end homologous sequence.If the generation allelic replacement carries out the fragment that PCR can amplify 2101p with primer OUt1/Spc2, carry out the fragment that PCR can amplify 1878bp with primer Spc1/Out2, carry out PCR with primer Spc1/Spc2 and can amplify spc ^RGene, and in 05ZYH33, carry out PCR with above primer and all should obtain negative findings.And take the 05ZYH33 genome as template, the purpose fragment of the 354bp that can amplify with primer checkin1/2, the size of each PCR product conforms to theoretical value (Fig. 3) as a result, and through dna sequencing checking (handsome order-checking), confirms that at gene level 05ZYH33 △ htpsA mutant strain successfully constructs.

2. RT-PCR identifies: in order further 05ZYH33 △ htpsA mutant strain to be verified, utilize the checkin1/2 primer respectively the cDNA that mutant strain and street strain's reverse transcription obtain to be carried out pcr amplification.The result is positive among the wild strain 05ZYH33, and the htpsA normal transcription is described; And in mutant strain 05ZYH33 Δ htpsA negative (Fig. 4).Evaluation through transcriptional level successfully obtains the htpsA gene knockout mutant strain, called after 05ZYH33 Δ htpsA, and deliver the CGMCC preservation, preserving number is CGMCC No.7375.

Embodiment 3: experiment in vitro

(1) gramstaining

The specification sheets operation of the gram staining liquid of producing according to Beijing Suo Laibao Science and Technology Ltd., give respectively 05ZYH33 and 05ZYH33 Δ htpsA(CGMCC No.7375, carry out gram's staining down together), the catenation that discovery mutant strain 05ZYH33 Δ htpsA compares wild strain is dispersed in more, and the length of chain is obviously shorter than the wild strain, illustrates that the chaining ability of mutant strain 05ZYH33 Δ htpsA weakens.

(2) growth characteristics

Under same culture conditions, the single bacterium colony of picking 05ZYH33 Δ htpsA and wild strain 05ZYH33 is inoculated in respectively 3mL and contains spectinomycin (100mg/mL) and do not contain in the THB substratum of spectinomycin respectively, and 37 ℃ of shaking culture are spent the night.Take out the bacterium of incubated overnight next day, measure 600nm place absorbance, with the THB substratum both are diluted to approximately 1 * 10 ⁸CFU/mL concentration.Then get respectively 60 μ L mutant strains and wild strain and be inoculated in respectively among the THB substratum 3mL, in 37 ° of C, the 200r/min shaking culture is every 1h difference sampling and measuring OD ₆₀₀, take incubation time as X-coordinate, OD ₆₀₀Value is ordinate zou, draws mutant strain and wild strain growth curve, found that both and there was no significant difference.

(3) anti-macrophage phagocytic experiment

05ZYH33 and the 05ZYH33 Δ htpsA(10 with the CFSE dye marker ⁷CFU) join among the scavenger cell Raw264.7 (10:1), 800g, effect 2h is incubated in the soft vibration of lucifuge altogether behind the centrifugal 10min, with gentle PBS washing 3 times, after add again the two anti-fresh culture effect 1h of 100ug/ml gentamicin and 5ug/ml penicillin, add at last isopyknic Paraformaldehyde 96 (4%) fixing, CFSE fluorescence intensity in the Flow cytometry born of the same parents.The Flow cytometry demonstration, 05ZYH33 Δ htpsA compares with 05ZYH33, and its anti-macrophage phagocytic kill capability reduces.

(4) stick experiment

05ZYH33 and the 05ZYH33 Δ htpsA(10 with the CFSE dye marker ⁷CFU) incubate altogether 2h with people's larynx epithelial cell strain HEP-2 (10:1) interaction, the gentle PBS washing of rear usefulness 3 times adds at last isopyknic Paraformaldehyde 96 (4%) and fixes, CFSE fluorescence intensity in the Flow cytometry born of the same parents.Flow cytometry result's demonstration, 05ZYH33 Δ htpsA more easily attaches to the HEP-2 cell than 05ZYH33.

Embodiment 4: the pathogenicity experiment

In order to detect the pathogenic of mutant strain 05ZYH33 Δ htpsA, at the single bacterium colony of flat board difference picking 05ZYH33 and mutant strain 05ZYH33 Δ htpsA, 37 ℃ of shaking culture are to logarithmic growth (OD in mid-term in the THB substratum ₆₀₀≈ 0.4, and approximately 10 ⁸CFU dosage) centrifugal collection thalline is with the resuspended bacterium of aseptic PBS damping fluid.Age in SPF level 4 week, BALB/c mouse was 30, was divided at random 3 groups, and abdominal injection wild-type and mutant strain bacterium liquid 1mL (approximately 10 respectively ⁸CFU/ is only), and establish THB negative control group (1mL/ is only), in time observed and recorded mouse invasion and survival time have or not considerable change.Found that, behind the wild strain 05ZYH33 attack mouse 24h with lethal dose, 10 mouse are all dead, attack all survivals behind the mouse 12h and knock out strain 05ZYH33 Δ htpsA with same dose, still have 8 survivals behind the 24h, be extended down to 48h after, dead 3 altogether, survive 7; When survival mice is observed to experiment end in 7 days, all find no any disease symptom.10 states of negative control group are all good.Illustrate that the htpsA gene knockout has produced impact to the virulence of 05ZYH33, can be applied to the development of streptococcus suis 2-type attenuated vaccine and multivalent subunit vaccine.

Claims (8)

1. streptococcus suis 2-type (Streptococcus suis serotype 2) htpsA gene knockout mutant strain 05ZYH33 △ htpsA, it is characterized in that, htpsA gene in the 05ZYH33 bacterial strain is replaced by the spectinomycin resistance gene box from the encoding gene between the 31st to 1000, the culture presevation of this mutant strain is in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC No.7375, and preservation date is on March 28th, 2013.

2. streptococcus suis 2-type according to claim 1 (Streptococcus suis serotype 2) htpsA gene knockout mutant strain is characterized in that the coding gene sequence of htpsA gene between the 31st to 1000 in the described 05ZYH33 bacterial strain is shown in SEQ ID NO.1.

3. streptococcus suis 2-type according to claim 1 (Streptococcus suis serotype 2) htpsA gene knockout mutant strain is characterized in that described spectinomycin resistance gene box sequence is shown in SEQ ID NO.3.

4. make up the method for streptococcus suis 2-type claimed in claim 1 (Streptococcus suis serotype 2) htpsA gene knockout mutant strain, it is characterized in that comprising following steps:

⑴ design PCR special primer LA1 and LA2 according to the upstream dna sequence dna of the S.suis2 wild strain 05ZYH33 genome htpsA encoding gene shown in SEQ ID NO.2; According to the downstream DNA sequence of the S.suis2 wild strain 05ZYH33 genome htpsA encoding gene shown in SEQ ID NO.4, design PCR special primer RA1 and RA2; Take the pSET2 plasmid as template, design a pair of special primer spc-F and spc-R; Wherein primer LA1 sequence is shown in SEQ ID NO.5, primer LA2 sequence is shown in SEQ ID NO.6, primer RA1 sequence is shown in SEQ ID NO.7, primer RA2 sequence is shown in SEQ ID NO.8, primer spc-F sequence is shown in SEQ ID NO.9, and primer spc-R sequence is shown in SEQ ID NO.10;

⑵ take the 05ZYH33 genomic dna as template, take LA1/LA2 and RA1/RA2 as primer, amplification obtains two ends and contains respectively the goal gene htpsA upstream dna sequence dna LA fragment of Sph I/Sal I restriction enzyme site and the goal gene htpsA downstream DNA sequence RA fragment that two ends contain respectively BamH I/Kpn I restriction enzyme site respectively; Take the pSET2 plasmid as template, amplification obtains the spectinomycin resistance gene box that two ends contain respectively Sal I/BamH I restriction enzyme site take spc-F/spc-R as special primer;

(3) structure of gene knockout carrier pUC::htpsA: get gene knockout carrier pUC::htpsA between the Sph I/Kpn I restriction enzyme site with described LA fragment-spectinomycin resistance gene box-RA fragment insertion pUC19 carrier;

(4) gene knockout carrier pUC::htpsA electricity transforms the S.suis2 wild strain 05ZYH33 competent cell for preparing;

(5) screen the swine streptococcus bacterium colony with spectinomycin resistance, replaced by the spectinomycin resistance gene box through assembly PCR product electrophoresis, RT-PCR and dna sequencing confirmation htpsA goal gene, namely obtain the streptococcus suis 2-type that preserving number is CGMCC No.7375 (Streptococcus suis serotype2) htpsA gene knockout mutant strain 05ZYH33 △ htpsA.

5. method according to claim 4 is characterized in that the structure of described gene knockout carrier pUC::htpsA comprises following steps:

(a) clone of RA: the RA fragment behind the BamH I/Kpn I double digestion is connected with the pUC19 carrier of processing with same enzymes double zyme cutting, gets recombinant plasmid pUC19-RA;

(b) clone of spectinomycin resistance gene box: the product that Sal I is connected with BamH I double digestion is connected with the recombinant plasmid pUC19-RA that processed with same enzymes double zyme cutting, gets recombinant plasmid pUC19-SR;

(c) clone of LA: the LA fragment behind the Sph I/Sal I double digestion is connected with the recombinant plasmid pUC19-SR that processed with same enzymes double zyme cutting, 16 ℃ will connect product after spending the night and transform the DH5a competent escherichia coli cell, behind spectinomycin and penbritin Double Selection, carry out double digestion with Sph I and Sal I and identify, filter out the positive plasmid that big or small dna fragmentation occurs about 970bp; And do respectively plasmid PCR with LA1/LA2, RA1/RA2, spc-F/spc-R, LA1/spc-R, spc-F/RA2, six pairs of primers of LA1/RA2 respectively and identify, the positive recombinant plasmid that obtains is pUC::htpsA.

6. the application of streptococcus suis 2-type claimed in claim 1 (Streptococcus suis serotype2) htpsA gene knockout mutant strain 05ZYH33 △ htpsA in preparation streptococcus suis 2-type attenuated vaccine or subunit vaccine.

7. the application of streptococcus suis 2-type htpsA gene knockout carrier pUC::htpsA in making up streptococcus suis 2-type (Streptococcus suis serotype2) htpsA gene knockout mutant strain, wherein said streptococcus suis 2-type htpsA gene knockout carrier pUC::htpsA makes up by the following method:

(a) clone of RA: the RA fragment behind the BamH I/Kpn I double digestion is connected with the pUC19 carrier of processing with same enzymes double zyme cutting, gets recombinant plasmid pUC19-RA;

(b) clone of spectinomycin resistance gene box: the product that Sal I is connected with BamH I double digestion is connected with the recombinant plasmid pUC19-RA that processed with same enzymes double zyme cutting, gets recombinant plasmid pUC19-SR;

(c) clone of LA: the LA fragment behind the Sph I/Sal I double digestion is connected with the recombinant plasmid pUC19-SR that processed with same enzymes double zyme cutting, 16 ℃ will connect product after spending the night and transform the DH5a competent escherichia coli cell, behind spectinomycin and penbritin Double Selection, carry out double digestion with Sph I and Sal I and identify, filter out the positive plasmid that big or small dna fragmentation occurs about 970bp; And do respectively plasmid PCR with LA1/LA2, RA1/RA2, spc-F/spc-R, LA1/spc-R, spc-F/RA2, six pairs of primers of LA1/RA2 respectively and identify, the positive recombinant plasmid that obtains is pUC::htpsA.

8. the application of streptococcus suis 2-type htpsA gene knockout carrier pUC::htpsA in preparation streptococcus suis 2-type attenuated vaccine, wherein said streptococcus suis 2-type htpsA gene knockout carrier pUC::htpsA makes up by the following method:

(a) clone of RA: the RA fragment behind the BamH I/Kpn I double digestion is connected with the pUC19 carrier of processing with same enzymes double zyme cutting, gets recombinant plasmid pUC19-RA;

(b) clone of spectinomycin resistance gene box: the product that Sal I is connected with BamH I double digestion is connected with the recombinant plasmid pUC19-RA that processed with same enzymes double zyme cutting, gets recombinant plasmid pUC19-SR;

(c) clone of LA: the LA fragment behind the Sph I/Sal I double digestion is connected with the recombinant plasmid pUC19-SR that processed with same enzymes double zyme cutting, 16 ℃ will connect product after spending the night and transform the DH5a competent escherichia coli cell, behind spectinomycin and penbritin Double Selection, carry out double digestion with Sph I and Sal I and identify, filter out the positive plasmid that big or small dna fragmentation occurs about 970bp; And do respectively plasmid PCR with LA1/LA2, RA1/RA2, spc-F/spc-R, LA1/spc-R, spc-F/RA2, six pairs of primers of LA1/RA2 respectively and identify, the positive recombinant plasmid that obtains is pUC::htpsA.

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