CN102776134B - Streptococcus suis Serotype 2 (SS2 for short) double-gene deleted live vaccine and its application - Google Patents
Streptococcus suis Serotype 2 (SS2 for short) double-gene deleted live vaccine and its application Download PDFInfo
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Abstract
The invention relates to the technical field of the zoonotic vaccine preparation, and concretely relates to an SS2 double-gene deleted live vaccine and its application. An SS2 deltaEce1/deltaSalk/R double-gene deleted stain is obtained by continuously deleting an Salk/R gene from a base SS2 field strain SC19 on the basis of the deletion of the a single gene Ece1 through a homologous recombinationmethod, and the SS2 double-gene deleted live vaccine is prepared through adopting a bacterial liquid of the double-gene deleted strain and gelatin as primary materials. The SS2 SS2 deltaEce1/deltaSalk/R strain is preserved in China Center for Type Culture Collection, and has a preservation number being CCTCC NO:M2010360. After the deletion of the Salk/R gene, the SS2 double-gene deleted live vaccine prepared in the invention has the advantages of greatly decreased toxicity, high safety, no risk of strong toxicity return, and high immunogenicity, and can protect immune pigs from the attack of 5*LD50 SS2 strongly-toxic bacterial strains.
Description
Technical field
The invention belongs to animal gene engineering technology field.Relate to the gene vaccine technical field, the present invention is specifically related to the structure of a dual-gene deletion of vaccine strain of streptococcus suis 2-type, vaccine preparation and application.
Background technology
Streptococcus suis 2-type (Streptococcus suis Serotype 2, abbreviation SS2) Streptococcus suis that causes is a kind of important infectious diseases common to human beings and animals, this disease is distributed widely in the world country of respectively raising pigs, not only caused huge financial loss to pig industry, also caused great threat (Lun ZR for simultaneously human health, et al.Streptococcus suis:an emerging zoonoticpathogen.Lancet Infect Dis, 2007,7:201-209).In China, the Streptococcus suis that is caused by streptococcus suis 2-type also extensively exists, and the trend that increases was gradually arranged in recent years, has become a kind of Streptococcus suis that distribution is the widest, harm is the most serious in China at present.1998, the streptococcicosis that the streptococcus suis 2-type that area, China Nantong occurs causes caused 25 people to infect, and 14 people death (Yao Huochun etc. Identification of isolates of swine Streptococcus in Jiangsu Province during 1998. Agricultural University Of Nanjing's journal, 1999,02:67-70).Between 2005 7, August, the streptococcicosis that the streptococcus suis 2-type that area, Ziyang, China Sichuan occurs causes causes 215 people to infect, dead (the Tang J of 38 people, et al.Streptococcal toxic shock syndrome caused by Streptococcus suisserotype 2.PLoS Med, 2006,3:668-676), cause enormous impact not only for the pig industry of China, and bring serious threat to public health, also caused huge pressure for simultaneously people's psychology.
The control of Streptococcus suis is mainly by vaccine inoculation and antibiotic use, but antibiotic life-time service can cause the appearance of drug-fast strain, thereby affects the effect of control, can cause in addition the problem of the aspects such as drug residue and food safety.For transmissible disease, prevention is more even more important, more effective than treatment, also more economically simultaneously.At present clinically mainly by inactivated vaccine carry out immunoprophylaxis (He Kongwang etc. the swine streptococcus Advances on Vaccine. animal doctor's guide, 2009,08:21-22), but inactivated vaccine is difficult to provide completely protection to pig, and production cost is high, and side effect is large, has had a strong impact on its development and application (HOLT ME, et al.Immunization of pigs with killed cultures of Streptococcus suis type 2.Res VetSci 1990,48:23-27; Quessy S, et al.Immunization of mice against Streptococcus suis type 2infection using a live avirulent strain.Can J Vet Res, 1994,58:299-301).Along with popular day by day serious of Streptococcus suis, for the streptococcus suis 2-type that swine streptococcus especially affects and harm is maximum, carry out novel, safe and effective vaccine research and become the task of top priority.
The pathogenic course of bacterium is the interactional complex process of bacterium and host cell, needs the coordinative role of multiple virulence factor.In this process, pathogenic bacterium rely on complicated and accurate signalling system to experience, conduct and respond the variation of external environment, and then regulate corresponding gene expression pattern to make adaptation reaction, comprise the expression of reducing dispensable gene, up-regulated expression in host, survive and pathogenic course in play a crucial role gene (Xu Jianguo. molecular medicine bacteriology, Science Press .2000,179-196.).In the time of in pathogenic bacteria is invaded host, change has all occured in the content of various nutritive substances, temperature, osmotic pressure and pH value etc. in the environment; In addition, some toxicants also appear at (Smith H E in the environment of its existence, et al.Environmentally regulated genes of Streptococcus suis:identification by the use of iron-restrictedconditions in vitro and by experimental infection of piglets.Microbiology, 2001,147:271-280; Huang YT, et al.Streptococcus suis infection.J Microbiology, Immunology and Infection, 2005,58:306-313).The change of environmental factors will inevitably produce huge pressure to the existence of pathogenic bacteria, and pathogenic bacteria is in order in time correctly to catch the variation of various chemical factors in the environment, and regulate rapidly accordingly self structure and physiological behavior, reach the purpose that shakes down with survival and reproduction, the distinctive signalling system that outside atmosphere is continued monitoring of a cover just must be arranged.Think that at present dual signal transduction system (TCS) and density induction (quorum sensing) system plays a part main.Dual signal transduction system is ubiquitous a kind of transmembrane signal transduction system in bacterium, it is experienced external environment on bacterium and changes and make in the process of adaptation reaction and play very important effect (Alexander Y, et al.Signal integration in bacterial two-componentregulatory systems.Genes, 2008,5:2601-2611).The simplest TCS is comprised of two kinds of protein components, and one is histidine kinase (histidine kinase, HK), is usually located on the cytolemma, and its function is identification and the extraneous ambient signal of translation; Another is for being positioned at the intracytoplasmic response regulatory factor (response regulator, RR), major function is that certain local motion form of regulating the expression of some gene or changing bacterium is with response outer signals (Ba-Thein W, et al.The virR/virS locusregulates the transcription of genes encoding extracellular toxin production in Clostridiumperfringens.J Bacteriol, 1996,178:2514-2520).Streptococcus suis 2-type dual signal transduction system Salk/SalR is a pair of dual signal transduction system on the potential 89K pathogenicity island of streptococcus suis 2-type virulent strain 05ZYH33, itself and relevant (the Tang J of salivaricin (a kind of bacteriocin) that mediates the bacterium secretion, et al.Streptococcal toxic shock syndrome caused byStreptococcus suis serotype 2.PLoS Med, 2006,3:668-676).
State Key Laboratory of Agricultural Microbiology Tan Chen doctor (Tan Chen, the immunogenicity research of the pathogenic and 6PGD albumen of streptococcus suis 2-type Ece1, middle National IP Network, 2010, http://epub.cnki.net/grid2008/detail.aspx? QueryID=3﹠amp; CurRec=1) successful structure streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna, various biological experiment data show that its virulence descends obviously, but still have certain remaining virulence, in order to obtain the dual-gene deletion mutantion strain of the higher streptococcus suis 2-type of security, the applicant is take streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna as parent strain, adopt the method disappearance Salk/R gene of homologous recombination, obtain the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R, biological experiment confirms that the dual-gene deletion mycopremna heritability of streptococcus suis 2-type Δ Ece1/ Δ Salk/R that obtains is stable, safe, immunogenicity is good.Further be developed into the dual-gene disappearance living vaccine of streptococcus suis 2-type Δ Ece1/ Δ Salk/R, and its security, immunizing dose, route of inoculation and immunogenicity estimated, every evaluation result shows that the dual-gene disappearance living vaccine of this streptococcus suis 2-type Δ Ece1/ Δ Salk/R can be used as a kind of good vaccine of preventing and treating Streptococcus suis.
Summary of the invention
First purpose of the present invention is further to lack the Salk/R gene on the basis of streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna, obtains the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R safe, that immunogenicity is good.
Second purpose of the present invention is to utilize the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R that makes up to be prepared into the dual-gene disappearance living vaccine of streptococcus suis 2-type.
The 3rd purpose of the present invention is the application of the dual-gene disappearance living vaccine of this streptococcus suis 2-type.
The present invention implements by the following technical programs:
The streptococcus suis 2-type Δ Ece1/ Δ Salk/R bacterial strain (Streptococcussuis II Δ Ece1/ Δ Salk/R) that relates to core biomaterial of the present invention is that the applicant makes up voluntarily, the applicant should dual-gene deletion mycopremna called after swine streptococcus II type (Streptococcus suis II) Δ Ece1/ Δ Salk/R, this bacterial strain is delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on December 21st, 2010, and deposit number is CCTCC NO:M 2010360.
The construction process of dual-gene deletion mutant of the present invention is: the upstream and downstream homology arm of design primer amplification Salk/R gene, be connected to suicide plasmid pSET4s (Daisuke T et al.Construction and characterization ofStreptococcus suis-Escherichia coli shuttle cloning vectors.Plasmid, 2001,45:101-113) the upper recombinant shuttle plasmid that makes up, again the recombinant plasmid electricity is transformed in the streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna, utilize temperature and resistance to screen, obtain the dual-gene deletion mycopremna of a kind of streptococcus suis 2-type Δ Ece1/ Δ Salk/R.
Major advantage of the present invention is:
The present invention is the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R that makes up on the basis of streptococcus suis 2-type Ece1 single-gene deletion mycopremna; its virulence significantly descends after the Salk/R genetically deficient; safe; do not exist virulence to return strong risk; immunogenicity is high; can protect the attack of immune swine opposing streptococcus suis 2-type virulent strain (Local Isolates SC19); and manufacture craft is simple; cost is low, for the generation of effective control China Streptococcus suis and popularly provide useful instrument.
Be that deep evaluation with the application of the dual-gene disappearance living vaccine of streptococcus suis 2-type in the infection of prevention streptococcus suis 2-type of the dual-gene deletion mycopremna preparation of this streptococcus suis 2-type Δ Ece1/ Δ Salk/R, carried out the evaluation of safety evaluation and immune effect with the dual-gene disappearance living vaccine of streptococcus suis 2-type for preparing experimental animal 35-42 age in days piglet.
Description of drawings
Sequence table SEQ ID NO:1 is nucleotide sequence (the GenBank accession number: GeneID:5099191) of missing gene Ece1.
Sequence table SEQ ID NO:2 is the nucleotide sequence (being that Salk and SalR are two two elements of component signal transduction system, its GenBank accession number: GeneID:5099618 and GeneID:5100603) of missing gene Salk/R.
Fig. 1: the structure schema of temperature sensitive type suicide plasmid pSET4s-Δ Salk/R.
Fig. 2: the enzyme of temperature sensitive type suicide plasmid pSET4s-Δ Salk/R is cut qualification result.
M1 is DNA marker (DL 15000) among the figure; M2 is DNA marker (DL 2000); 1,2 is Hind III and EcoR I double digestion pSET4s-Δ Salk/R.
Fig. 3: the PCR qualification result of the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R.
Mark among the figure: M1:DNA marker (DL 15000); M2:DNA marker (DL 2000); 1-4 increases with the p5/p6 primer; 5-8 increases with the p7/p8 primer; 9-12 increases with the p1/p2 primer; 13-16 increases with the p3/p4 primer; 1,5,9,13: template is SS2 virulent strain (streptococcus suis 2-type Local Isolates SC19); 2,6,10,14: template is streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna; 3,7,11,15: template is the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R; 4,8,12,16: blank.
Fig. 4: the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R is in the genetic stability detected result of subculture in vitro separately.
Mark among the figure: M1 is DNA marker (DL 15000); M2 is DNA marker (DL 2000); 1-15 is that the dual-gene deletion mycopremna 1-15 of primer p1/p2 amplification streptococcus suis 2-type Δ Ece1/ Δ Salk/R is for bacterium.
Fig. 5: the growth curve of the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R, streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna and SS2 Local Isolates SC19.
Fig. 6: the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R, streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna and SS2 Local Isolates SC19 are to the ratio that sticks of Hep2 cell.
Fig. 7: the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R, streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna and SS2 Local Isolates SC19 are to the toxicity ratio of Hep2 cell
Fig. 8: the antibody horizontal behind the dual-gene deletion mycopremna immune swine of streptococcus suis 2-type Δ Ece1/ Δ Salk/R.
Fig. 9: strong virus attack pig survival condition after the dual-gene deletion mycopremna immunity of streptococcus suis 2-type Δ Ece1/ Δ Salk/R.
Figure 10: the body temperature that is strong virus attack pig after the dual-gene deletion mycopremna immunity of streptococcus suis 2-type Δ Ece1/ Δ Salk/R changes.
Table 1: SS2 virulent strain (streptococcus suis 2-type Local Isolates SC19) is respectively organized the content of molds detected result after attacking pig after the dual-gene deletion mycopremna immunity of streptococcus suis 2-type Δ Ece1/ Δ Salk/R.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description, but do not limit protection scope of the present invention.
One, design of primers (being used for gene clone and Molecular Detection)
According to upper full genome GB|CP000407.1| (the Chen C of streptococcus suis 2-type 05ZYH33 strain that announces of GenBank, et al.A glimpse of streptococcal toxic shock syndrome from comparative genomics of S.suis 2 Chineseisolates.PLoS ONE, 2007,2:312-315) design primer pSu1/pSu2 and pSd1/pSd2 (sequence sees below described), from streptococcus suis 2-type wild mushroom (Local Isolates) SC19 (the sick pig of this bacterial strain in May, 2005 separation from porcine streptococcosis in Sichuan Province, be virulent strain, have the MRP+EF+SLY+ phenotype; Referring to document Li W, Liu L, Qiu D, Chen H and Zhou R.Identification of Streptococcus suis serotype 2 genes preferential expressed in the natural host.Int JMed Microb, 2010,300:482-488) increase respectively in the genome upstream homology arm Salk/R-up (sequence length is 996bp) and the downstream homology arm Salk/R-down (sequence length is 986bp) of Salk/R gene.The primer that simultaneously design is positioned at Salk/R gene both sides to p3/p4, is used for the dual-gene deletion mutantion strain of Screening and Identification to p1/p2 and the primer that is positioned at Salk/R gene inside.
According to Tan Chen (Tan Chen, the immunogenicity research of the pathogenic and 6PGD albumen of streptococcus suis 2-type Ece1, middle National IP Network, 2010, Ph D dissertation; Http:// epub.cnki.net/grid2008/detail.aspx? QueryID=3﹠amp; CurRec=1) the primers designed sequence synthesized primer thing of the Ece1 gene of report identifies for pcr amplification whether streptococcus suis 2-type Ece1 gene lacks, and reaction conditions is: 94 ℃ of denaturation 10min to p5/p6 and p7/p8; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 2.5min, after 30 circulations, 72 ℃ are extended 10min.The PCR product detects through 0.8% agarose gel electrophoresis, if streptococcus suis 2-type Δ Ece1 deletion mutantion strain, then amplification should obtain the dna segment of a 479bp to primer to p5/p6, if SS2 Local Isolates SC19 then obtains the dna segment of a 2309bp.Another purpose fragment to primer p7/p8 amplification is positioned at the inside of Ece1 gene, and the amplification of streptococcus suis 2-type Δ Ece1 mutant strain should be negative, can the increase fragment of 776bp of wild strain.Above-mentioned primer is synthetic by Nanjing Jin Sirui company limited.The nucleotide sequence of primer is as follows:
PSu1:5 '--CGCAAGCTTGTAGGAACAATCTATACAGAGGC--3 ' HindIII restriction enzyme site
PSu2:5 '--CGCGGATCCTTACCCTTTTTACTCATGATTTT--3 ' BamHI restriction enzyme site
PSd1:5 '--CGCGGATCCGCTGAGCGATATAGATATTAACG--3 ' BamHI restriction enzyme site
PSd2:5 '--CGCGAATTCATAAGAGAACCTATTTAGCTCCC--3 ' EcoRI restriction enzyme site
p1：5′--TGTGCCAAAAACGCTCTATC--3′
p2：5′--CAAACTCCGCTCCCCTAAG--3′
p3：5′--ACAAAGATAAGCCTTTTAGCAAT--3′
p4：5′--TAGACAATCCAAGCGTATCAGTT--3′
p5：5′--GAATTACTTTGTTTGGGAATG--3′
p6：5′--GTTATGACTGGTTAAATAC--3′
p7：5′--ACGTTACTCAAGATATAAAAGACAG--3′
p8：5′--GATTTTAACAACCAATGTATCTAGT--3′
Two, the structure (see figure 1) of pSET4s-SalK/R recombinant transfer plasmid
To pSu1/pSu2 and pSd1/pSd2 increase from SS2 Local Isolates SC19 genome Salk/R gene upstream and downstream homology arm Salk/R-up (996bp) and Salk/R-down (986bp), response procedures is: 94 ℃ of denaturation 10min with primer; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min30sec, 35 circulations, last 72 ℃ are extended 10min.Detect amplification with 0.8% agarose gel electrophoresis.Purifying reclaims the purpose fragment, the Salk/R upstream region of gene homology arm amplified production of purifying is directly connected to HindIII and the BamHI site of plasmid pSET4s (Daisuke doctor Takamatsu of Japanese state-run animal health institute is so kind as to give) after with HindIII and BamHI double digestion, the recombinant plasmid pSET4s-SU that obtains cuts the confirmation structure correctly through HindIII and BamHI enzyme.Subsequently the Salk/R gene downstream homology arm amplified production of purifying is connected to BamHI and the EcoRI site of constructed plasmid pSET4s-SU after with BamHI and EcoRI double digestion, confirm to make up correct through HindIII and EcoRI double digestion, order-checking confirms to mismatch without base, with the plasmid called after pSET4s-Salk/R (seeing Fig. 2) that obtains.The restructuring of plasmid, preparation, restriction analysis all carry out (J. Pehanorm Brooker, EF is the Ritchie not, T Manny A Disi work, Huang Peitang, Wang Jiaxi etc. translate, molecular cloning experiment guide (third edition), Science Press, 2002 years) according to a conventional method.
Three, screening and the evaluation of the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R
Method (Daisuke T et al with reference to Daisuke doctor Takamatsu report, Construction and characterizationof Streptococcus suis-Escherichia coli shuttle cloning vectors.Plasmid, 2001,45:101-113), with the suicide recombinant transfer plasmid pSET4s-Salk/R that obtains at electric Transformation Parameters 2.5KV/cm, electricity is converted in the Electroporation-competent cells that streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna makes under the condition of 500 Ω and 25 μ F, be coated with 100 μ g/ml spectinomycin resistance plates after electricity transforms and put 28 ℃ of lower cultivations, put again 37 ℃ of lower cultivations after growing single bacterium colony.In this process, a homologous recombination acquisition single cross occurs and changes in recombinant transfer plasmid pSET4s-Salk/R and streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna genome, single cross is changed and is identified that we go down to posterity to screen mutant strain the single cross swap-in is capable after correct, and homologous recombination obtains dual-gene deletion mutant thereby it can occur for the second time.Homologous recombination might produce two kinds of different bacterial strains for the second time: a kind of is to produce the dual-gene deletion mutantion strain of needed streptococcus suis 2-type Δ Ece1 Δ Salk/R; Another kind is to produce the parent strain of replying.
The applicant is by the dual-gene deletion mycopremna of PCR method magnanimity screening streptococcus suis 2-type Δ Ece1 Δ Salk/R.Each changes picking cultivation mono-clonal out for single cross and inoculates simultaneously the TSA plate of non-resistant and spectinomycin resistance and cultivate, then picking growing under the spectinomycin resistance condition single bacterium colony well-grown on the non-resistant plate after 3ml contains in the TSB liquid nutrient medium (available from U.S. company BD) of 10% new-born calf serum 37 ℃ of shaking culture as template, utilize primer that p1/p2 and p3/p4 are carried out pcr amplification, reaction conditions is: 94 ℃ of denaturation 10min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 2min30sec, 30 circulations, last 72 ℃ are extended 10min.The PCR product detects through 0.8% agarose gel electrophoresis, if the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1 Δ Salk/R, then amplification should obtain the dna segment of a 500bp to primer to p1/p2, if revert back to parent strain, then obtains the dna segment of a 2268bp.Another purpose fragment to primer p3/p4 amplification is positioned at the inside of missing gene, should be negative to the dual-gene deletion mycopremna amplification of streptococcus suis 2-type Δ Ece1/ Δ Salk/R, streptococcus suis 2-type Local Isolates SC19 and streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna then can obtain the fragment of 117bp.With primer p1/p2 is increased after screening mutant strain by pcr amplification again, and the fragment of amplification is carried out sequencing analysis, the result confirms that the dual-gene deletion mutantion strain of streptococcus suis 2-type Δ Ece1/ Δ Salk/R successfully constructs.
Four, the dual-gene deletion mycopremna biological property analysis of streptococcus suis 2-type Δ Ece1/ Δ Salk/R
4.1 genetic stability analysis
With the continuous passage on the TSA that contains 10% new-born calf serum (TrypticSoy Agar purchases to U.S. company BD) plate of the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R, passed altogether for 15 generations, every generation picking list bacterium colony contains 37 ℃ of shaking culture in the TSB liquid nutrient medium of 10% new-born calf serum in 3ml, with primer p1/p2 every generation bacterium is carried out pcr amplification, the result all can only obtain the fragment (Fig. 4) of 500bp, illustrates that the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R connects in in-vitro culture medium that to pass for 15 generations be stable.
4.2 growth characteristics
The local isolated strains SC19 of streptococcus suis 2-type, streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna and the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R of the present invention are inoculated in respectively middle cultivation of TSB substratum (Tryptic Soy Broth is available from U.S. company BD) that 5ml contains 10% new-born calf serum (available from Hangzhou folium ilicis chinensis Materials Co., Ltd), and 100 μ L bacterium liquid mensuration OD600 value was got respectively at every interval in one hour after cultivating, and drew growth curve (accompanying drawing 5).The result shows the growth curve of the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R, streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna and streptococcus suis 2-type Local Isolates SC19 without significant difference, shows that the disappearance of Ece1 and two genes of Salk/R does not affect the growth of the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R.
4.3 cell adhesion experiment
The bacterial adhesion cell is that the bacterium cells infected causes that the pathogenic the first step also is a critical step, and the ability of bacterial adhesion cell is directly related with its virulence.The method that cell intrusion experiment main reference Charland and Gottschalk (2000) report is carried out (Charland N, et al.Streptococcus suis serotype 2 interactions with human brainmicrovascular endothelial cells.Infect Immunity, 2000,68:637-643; Segura M et al.Streptococcussuis interactions with the murine macrophage cell line J774:adhesion and cytotoxicity.InfectImmunity, 2002,70:4312-4322), concrete operations are: get the streptococcus suis 2-type Local Isolates SC19 that 1ml grows into exponential phase of growth, streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna and the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R are collected bacterium through the centrifugal 5min of 6000rpm, with phosphate buffer soln PBS (prescription: NaCl 137mmol/L, KCl 2.7mmol/L, Na
2HPO
44.3mmol/L, KH
2PO
41.4mmol/L) after the washing, be resuspended in and do not contain antibiotic cell culture medium DMEM (available from Gibco
TMCompany) in, the rear infection multiplicity by 10m.o.i. of dilution is inoculated into Hep2 cell (available from Chinese Typical Representative culture collection center) and has grown up in the 24 porocyte culture plates of individual layer.Then with cell plate in the centrifugal 10min of 800g, to strengthen contacting of bacterium and monolayer cell surface.Afterwards, cell plate in 37 ℃, are cultivated 2h in the 5%CO2 incubator, make bacterium invade cell.Wash cell monolayer 3 times with phosphate buffer soln PBS, then add 1ml in each hole and contain 100 μ g ml-1 gentamicins and 5 μ g ml
-1In the DMEM cell culture medium of penicillin G, contain 5%CO in 37 ℃
2Incubator in cultivate 2h so that extracellular bacterium with attaching to cell surface is killed.Then wash cell monolayer 3 times with phosphate buffer soln PBS, add subsequently the deionized water of 500ul with the destruction cytolemma, and repeatedly blow and beat with pipettor.It is dull and stereotyped that 100ul bacterial suspension coating TSA is got in every hole, at 37 ℃, and 5%CO
2Be incubated overnight in the incubator, observe the formational situation of bacterium colony, and calculate bacterium c.f.u..Invasion efficiency represents with the c.f.u. of bacterium.Experimental result shows, the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R of the present invention is compared the ability of sticking the Hep2 cell with streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna with streptococcus suis 2-type Local Isolates SC19 and is significantly weakened, but the streptococcus suis 2-type Δ Ece1/ dual-gene deletion mycopremna of Δ Salk/R and streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna to the ability of sticking of Hep2 cell without significant difference (Fig. 6).
4.4 cytotoxicity experiment
Bacterium is destroyed an important ring of host's normal cell structure often to the toxicity of cell, method (the Charland N of this laboratory reference Charland and Gottschalk (2000) report, et al.Streptococcus suis serotype 2 interactions withhuman brain microvascular endothelial cells.Infect Immunity, 2000,68:637-643), estimate bacterium to the toxicity of cell by detecting the amount that serum lactic dehydrogenase (lactate dehydrogenase, LDH) outwards discharges in the born of the same parents.Concrete operations are undertaken by test kit specification sheets (the cytotoxicity detection kit is Promega company product): the local isolated strains SC19 of the streptococcus suis 2-type of the centrifugal collection logarithmic phase of difference, streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna and the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R (about 3 * 10
8Individual bacterium), after the phosphoric acid buffer PBS washing, being suspended in respectively 1ml does not contain among serum and the antibiotic DMEM, and carry out suitable dilution with serum-free and antibiotic DMEM, bacterial suspension is added the Hep2 cell have been grown in the 96 porocyte plates of individual layer, the infection multiplicity that makes bacterium is every cell 10m.o.i., and each does 4 repetitions.The spontaneous serum lactic dehydrogenase of effector cell (LDH) is set simultaneously discharges that contrast, the spontaneous LDH of target cell discharge contrast, the maximum LDH of target cell discharges contrast, volume correction contrast, the contrast of substratum background.Tissue Culture Plate is cultivated 4h at 37 ℃ in the 5%CO2 incubator, collects the front 45min of supernatant and adds 5 μ L cell pyrolysis liquids (Lysis solution) to the maximum LDH release of target cell control wells.The centrifugal 5min of 280g, in volley of rifle fire transferase 45 0 μ L supernatant to one 96 a new porocyte culture plate, every hole adds the substrate that 50 μ L prepare, lucifuge incubated at room 30min, add again 50 μ L stop buffers (stopsolution), measure light absorption value under OD value 490nm, by calculation formula: % cytotoxicity=[(experiment-effector cell spontaneous-target cell is spontaneous)/(target cell maximum-target cell is spontaneous)] * 100 calculating different strains are to the toxicity of Hep2 cell.The result shows that the streptococcus suis 2-type Δ Ece1/ dual-gene deletion mycopremna of Δ Salk/R and streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna all significantly are lower than streptococcus suis 2-type Local Isolates SC19 to the toxicity of Hep2 cell, but two mutant to the toxicity of Hep2 cell without significant difference (Fig. 7).
Five, the preparation of the dual-gene deletion of vaccine of streptococcus suis 2-type Δ Ece1/ Δ Salk/R
To identify correct; growth characteristics are normal; inheritance stability; and have good biological and learn the active dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R to be inoculated in the TSB substratum that be added with 10% new-born calf serum (available from Hangzhou folium ilicis chinensis Materials Co., Ltd) overnight incubation according to volume ratio as 1: 100 amount with the TSB substratum; the centrifugal 5min of 5000rpm collects thalline; with behind the physiological saline suspension thalline the more centrifugal 5min of 5000rpm collect thalline; after physiological saline suspends; by bacterium liquid: the gelatin protective material is that 7: 1 volume ratio adds the gelatin protective material; fully be sub-packed in the sterilization penicillin bottle by the 2.0mL/ bottle behind the mixing; put freeze-drying in the freeze drier, rearmounted 4 ℃ of gland saves backup.
Six, experimentation on animals
6.1 safety testing
With the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R of the resuspended freeze-drying of physiological saline, inoculate the negative sodium selenite of 35 age in days suis through musculi colli, divide 10
9, 10
8, 10
7C.f.u.3 dosage group, establishes 10 simultaneously by 5 every group
6C.f.u. the streptococcus suis 2-type SC19 Local Isolates of dosage infects piglet control group and 10
7C.f.u. the streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna of dosage infects the piglet control group, and in the clinical manifestation of infection rear every day of observation experiment pig, observes altogether 21 days.Any untoward reaction does not appear in result's dual-gene deletion mycopremna Pigs Inoculated of streptococcus suis 2-type Δ Ece1/ Δ Salk/R within the whole observation period, and 10
6C.f.u. wild mushroom and 10
7C.f.u. the streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna of dosage infects second day behind the piglet and namely shows clearly Streptococcus suis clinical symptom: high continuous fever, poor appetite, drowsiness, weak, ataxia, arthroncus, limping, opisthotonus, red and swollen, the sleeping ground of whole body do not rise etc., it is all dead in infecting in rear 5 days that wild mushroom infects the control group pig, and it is dead in infecting in rear 5 days that streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna infects 4 of control group pigs.The dual-gene deletion mycopremna of experimental result explanation streptococcus suis 2-type Δ Ece1/ Δ Salk/R will descend much 10 than streptococcus suis 2-type Δ Ece1 single-gene deletion mycopremna virulence
9, 10
8, 10
7The dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R of c.f.u.3 dosage all is safe to piglet.
6.2 the immunity of piglet and antibody test
16 negative sodium selenites of 35 age in days swine streptococcus are divided into 3 groups, wherein Immunization group and nonimmune each 7 of poison groups, 2 of the blank groups of attacking.With the dual-gene disappearance of the streptococcus suis 2-type Δ Ece1/ Δ Salk/R bacterium of the resuspended freeze-drying of physiological saline, attack poison group piglet through the musculi colli immunoprophylaxis, every inoculation 1ml, the bacterium amount is 108c.f.u.; Nonimmune poison group and the blank group of attacking is through musculi colli inoculation 1ml physiological saline, immune 1 time altogether.All experiment pig after immunity 2 the week and took a blood sample by jugular plexus respectively in 3 weeks, the swine streptococcus CPSELISA antibody assay kit of producing with animal biological product limited liability company before the section of Wuhan behind the separation of serum detects the antibody horizontal for streptococcus suis 2-type, the result as shown in Figure 8, the immune group pig has all produced the specific antibody of higher level, and nonimmune control group and blank group are all negative.
6.3 protection test
Rear the 28th day of immunity, Immunization group and the nonimmune poison of attacking are organized piglet through auricular vein injection 10
6C.f.u. streptococcus suis 2-type Local Isolates SC19, blank does not deal with.Cutd open extremely the Immunization group on the 3rd day and attack poison group respectively 2 pigs and blank group piglet with nonimmune after attacking poison, to observe the pathology situation of its histoorgan, the test set of taking a sample is simultaneously knitted content of molds.Immunization group and the nonimmune poison of attacking are organized the observation that 5 other pigs are used for incidence and death condition.As a result Immunization group pig attack the poison rear two days in body temperature slightly raise; return to later on normal level; do not show other untoward reaction; but not all pigs of Immunization group show high continuous fever after attacking poison; poor appetite; drowsiness; weak; ataxia; arthroncus; walk lamely; opisthotonus; whole body is red and swollen; sleeping ground such as does not play at the serious swine streptococcus disease symptoms; and whole Mortality (Fig. 9 in the Yu Yizhou; 10), experimental result illustrates that the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R of the present invention can protect the attack of immune swine opposing streptococcus suis 2-type virulent strain SC19.
It is substantially the same with the pig of blank group to cut open each histoorgan of pig that kills pig discovery Immunization group, finds no obvious pathological change.But not the pig of Immunization group finds that its each histoorgan has pathology to a certain degree, is mainly manifested in after dissecting: alimentary canal mucous membrane, upper respiratory tract mucosa hyperemia, hemorrhage, and lung swelling, congested hemorrhage; Meninx is congested, hemorrhage, hydrops under the meninx, and the cerebral tissue tangent plane has petechial hemorrhage; Endocardium is hemorrhage, and a large amount of faint yellow hydrops are arranged in the pericardium; Liver, kidney and spleen enlargement, hemorrhage, the dark red or royal purple of spleen; Yellow gel-shaped or fiber disposition, purulent exudate are arranged in the joint cavity.
Brain, lungs, liver, heart, spleen, kidney, lymphoglandula that takes by weighing collection etc. organized each 0.2g, adds 1ml phosphoric acid buffer PBS homogenate.Coat the TSA plate that contains 10% new-born calf serum after respectively getting 100 μ L homogenates and doing suitable dilution, the same coating of each 100 μ L of anticoagulation of getting simultaneously experiment pig contains the TSA plate of 10% new-born calf serum, calculate the content of molds (c.f.u./0.1g tissue) that 0.1g organizes after in 37 ℃ of incubators, cultivating 24h, the result is as shown in table 1, Immunization group pig can detect the minute quantity bacterium in heart, kidney, lymphoglandula and blood, remaining tissue does not all carry disease germs, but not all can detect a large amount of bacteriums in each tissue of Immunization control group pig.(reaction conditions is: 94 ℃ of denaturation 10min simultaneously the bacterium that separates acquisition to be carried out pcr amplification with the p1/p2 primer; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, after 30 circulations, 72 ℃ are extended 10min) confirm that the bacterium that separates is streptococcus suis 2-type wild mushroom SC19.Experimental result further specifies the attack that the dual-gene deletion mycopremna of streptococcus suis 2-type Δ Ece1/ Δ Salk/R can be protected immune swine opposing streptococcus suis 2-type virulent strain SC19.
The distribution situation (unit: c.f.u./0.1g tissue) of table .1 swine streptococcus virulent strain SC19 in each tissue
The specification sheets explanation: in order to express easily, the streptococcus suis 2-type bacterial strain that occurs among the present invention and swine streptococcus II type bacterial strain are same bacterial strain.
Claims (3)
1. dual-gene deletion mycopremna of streptococcus suis 2-type, it is characterized in that: the dual-gene deletion mycopremna of described streptococcus suis 2-type is swine streptococcus II type (streptococcus suisII) Δ Ecel/ Δ Salk/R, it is deposited in Chinese Typical Representative culture collection center, deposit number is: CCTCC NO:M2010360, this bacterial strain is on the basis that has lacked single-gene Ecel, continue another Salk/R gene of disappearance, consist of dual-gene gene-deleted strain, the nucleotide sequence of first gene Ecel of its disappearance is shown in sequence table SEQ ID NO:1, and the nucleotide sequence of second gene Salk/R of its disappearance is shown in sequence table SEQ ID NO:2.
2. the dual-gene disappearance living vaccine of streptococcus suis 2-type that is prepared by the dual-gene deletion mycopremna of streptococcus suis 2-type claimed in claim 1.
3. the application of bacterial strain claimed in claim 1 in the dual-gene disappearance living vaccine of preparation streptococcus suis 2-type.
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| CN108410784B (en) * | 2018-01-26 | 2021-07-27 | 华中农业大学 | Streptococcus suis delta CPS/SsnA-mSly (P353L) -SC19 engineering bacteria and application thereof in vaccines |
| CN111662854B (en) * | 2020-07-27 | 2022-02-11 | 中国农业科学院兰州兽医研究所 | Cell culture method and application of Chlamydia abortus |
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