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CN102719389A - Capsule deficiency type Streptococcus equi subsp. zooepidemicus attenuated vaccine strain and preparation method thereof - Google Patents

Capsule deficiency type Streptococcus equi subsp. zooepidemicus attenuated vaccine strain and preparation method thereof Download PDF

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Publication number
CN102719389A
CN102719389A CN2012101730854A CN201210173085A CN102719389A CN 102719389 A CN102719389 A CN 102719389A CN 2012101730854 A CN2012101730854 A CN 2012101730854A CN 201210173085 A CN201210173085 A CN 201210173085A CN 102719389 A CN102719389 A CN 102719389A
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streptococcus equi
zooepidemicus
hasb
preparation
attenuated vaccine
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陈瑶生
魏子贡
付强
刘小红
莫德林
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Sun Yat Sen University
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Sun Yat Sen University
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Abstract

本发明公开了一种荚膜缺失型马链球菌兽疫亚种弱毒疫苗株及其制备方法，属于动物疫苗制备技术领域。本发明的荚膜缺失型马链球菌兽疫亚种弱毒疫苗株命名为马链球菌兽疫亚种C55138∆ hasB（Streptococcusequisubsp.zooepidemicusC55138∆ hasB），于2012年5月14日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2012164。该菌株是通过等位基因置换导致hasB基因失活构建而成，毒力减弱，可用于制备马链球菌疾病的疫苗。疫苗制作工艺简单，疫苗菌株可大量培养，成本低，周期短；既表现细胞免疫又有体液免疫；用量小，安全性高，毒力小，使用方便，能在全世界推广使用。

Figure 201210173085

The invention discloses a attenuated vaccine strain of capsular-deleted Streptococcus equi subspecies zooepidemicus and a preparation method thereof, belonging to the technical field of animal vaccine preparation. The capsule-deleted Streptococcus equi subsp . zooepidemicus attenuated vaccine strain of the present invention is named Streptococcus equi subsp. zooepidemicus C55138 ∆ hasB (Streptococcusequi subsp. The depository center, the deposit number is CCTCC NO:M 2012164. The bacterial strain is constructed by inactivating the hasB gene through allele replacement, has weakened virulence, and can be used for preparing vaccines against equine streptococcal diseases. The vaccine production process is simple, the vaccine strains can be cultivated in large quantities, the cost is low, and the cycle is short; both cellular immunity and humoral immunity are exhibited; the dosage is small, the safety is high, the toxicity is small, the use is convenient, and it can be promoted and used all over the world.

Figure 201210173085

Description

A kind of pod membrane absence type streptococcus equi epizootic disease subspecies attenuated vaccine strain and preparation method thereof
Technical field
The present invention relates to the animal vaccine preparing technical field, be specifically related to a kind of pod membrane absence type bacterial strain streptococcus equi epizootic disease subspecies less toxic vaccine and preparation method thereof.
Background technology
Streptococcus equi epizootic disease subspecies are and mucous membrane of animal and the symbiotic a kind of bacterium of skin, and are especially more common on length, possibly cause septicemia, endocarditis and sacroiliitis to many other kinds animals, like ox, pig, sheep, dog.In China, streptococcus equi epizootic disease subspecies are important pathogenic agent that cause the swine streptococcus disease, and 1975, outburst swine streptococcus epidemic disease caused 300000 pig death in Sichuan, causes enormous economic loss.Up to the present, China's industry of raising pigs still receives the puzzlement of this disease.
Streptococcus equi epizootic disease subspecies constitute a serious threat to the All Around The World industry of raising pigs, but also do not have the effectively preventing method at present, therefore develop effective vaccine prevention streptococcus equi epizootic disease subspecies and infect and be very important.Yet, study very little about the virulence factor of this cause of disease, thus the research and development of obstruction less toxic vaccine, more type of the having only M protein (SzP) of research in the prior art.Recently, there is the investigator successfully to make up a streptococcus equi attenuated vaccine strain through the gene that knocks out coding SzP Δ ATCC35246, and find to make that mouse avoids lethal hit.This achievement in research provides important clue for suis vaccine research direction, as the less toxic vaccine target research direction is provided for exploring other potential virulence factors of suis.
Streptococcus equi can synthesize by operon HasABCThe mucinase pod membrane of coding is through to suis C55138Bacterial strain HasBGene makes this gene inactivation through allelic replacement, and this directly is convenient to study mucinase pod membrane role in the pathogenesis of this bacterium, simultaneously the relevant less toxic vaccine of research and development.
Summary of the invention
The objective of the invention is to provides a kind of pod membrane absence type bacterial strain streptococcus equi epizootic disease subspecies less toxic vaccine to above-mentioned deficiency of the prior art.
Another object of the present invention provides the preparation method of above-mentioned pod membrane absence type bacterial strain streptococcus equi epizootic disease subspecies less toxic vaccine and preparation method thereof.
The present invention realizes above-mentioned purpose through following technical scheme:
A kind of pod membrane absence type bacterial strain streptococcus equi epizootic disease subspecies attenuated vaccine strain, name is called streptococcus equi epizootic disease subspecies C55138 HasB( Streptococcus equiSubsp. ZooepidemicusC55138 HasB), abbreviate mutant bacteria C55138 among the present invention as HasB, being preserved in Chinese typical culture collection center on May 14th, 2012, deposit number is CCTCC NO:M 2012164, preservation address: China. Wuhan. Wuhan University.
This mutant bacteria C55138 HasBBecause the hasB gene function lacks and can not normally synthesize pod membrane, streptococcus equi can synthesize by operon HasABCThe mucinase pod membrane of coding, and discover that the mucinase pod membrane plays an important role in the pathogenesis of swine streptococcus disease, therefore this pod membrane absence type mutant bacteria can reduce the toxicity of bacterial strain itself greatly.
The preparation method of above-mentioned pod membrane absence type streptococcus equi epizootic disease subspecies attenuated vaccine strain respectively gets portion gene with hasB gene upstream and downstream and is connected to plasmid pG +Host5 is last, obtains carrier pG HasB, transform streptococcus equi epizootic disease subspecies C55138, utilize the homologous recombination principle with the hasB genetically deficient in the streptococcus equi epizootic disease subspecies C55138 genome, be built into pod membrane absence type mutant bacteria C55138 HasB
As a kind of preferred version, preparing method's concrete steps of above-mentioned pod membrane absence type streptococcus equi epizootic disease subspecies attenuated vaccine strain are following:
(1) genomic dna with streptococcus equi epizootic disease subspecies C55138 is a template, and nucleotides sequence is classified primer as shown in SEQ ID NO:1 ~ 2, pcr amplification hasBL gene fragment;
(2) genomic dna with streptococcus equi epizootic disease subspecies C55138 is a template, and nucleotides sequence is classified primer as shown in SEQ ID NO:3 ~ 4, pcr amplification hasBR gene fragment;
(3) hasBL gene fragment and the hasBR gene fragment with gained connects into plasmid pG +Among the host5, transform streptococcus equi epizootic disease subspecies C55138, be built into pod membrane absence type streptococcus equi epizootic disease subspecies attenuated vaccine strain.
The application of above-mentioned pod membrane absence type streptococcus equi epizootic disease subspecies attenuated vaccine strain in the preparation vaccine.
A kind of preparation method of streptococcus equi epizootic disease subspecies less toxic vaccine, concrete steps are following:
(1) streptococcus equi epizootic disease subspecies C55138 HasBRecover in TSB 37 ℃ of shaking table overnight cultures;
(2) bacterial classification of getting overnight cultures in new TSB, 37 ℃ of shaking table enlarged culturing 6 ~ 8h;
(3) through bacterium liquid centrifugal 3min under the 8000rpm condition of enlarged culturing, remove supernatant;
(4) bacterium in the deposition mixes with the 20wt.% skimming milk of sterilization, and lyophilize is prepared into freeze-dried live vaccine.This freeze-dried live vaccine can be preserved in-20 ℃ of cryogenic refrigerators.
Above-mentioned TSB is a soybean casein digest medium, can buy existing procucts (OXOID, CM0129); Prescription is: Tryptones 1.5% (g/100mL); Soy peptone 0.5% (g/100mL), sodium-chlor 0.5% (g/100mL), adding distil water is formulated; Regulating the pH value is 7.2 ± 0.2, behind 121 ℃ of pressuresteam sterilizations, uses.
Compared with prior art, the present invention has following beneficial effect:
This streptococcus equi epizootic disease subspecies less toxic vaccine has the following advantages: one, manufacture craft is simple, and vaccine strains can be cultivated in a large number, and cost is low, and the cycle is short, can promote the use of in the whole world; Two, not only show cellular immunization but also humoral immunization is arranged; Three, consumption is little, and is safe, and virulence is little, easy to use.
The present invention adopts genetic engineering technique to make up pod membrane disappearance strains of streptococcus C55138 Δ hasB, this bacterial strain virulence attenuation of.Experiment has proved that this mutant strain is avirulent to mouse, uses its immune mouse, and mouse 100% is protected.
Description of drawings
Fig. 1. gene HasBIn the site and the gene fragment of streptococcus equi epizootic disease subspecies C55138 bacterial strain, arrow is represented to synthesize HasBThe length of gene fragment and transcriptional orientation.
Fig. 2. make up the method and the principle schematic of absence type genophore.
Fig. 3. the method with PCR and RT-PCR is identified mutant bacteria C55138 HasBStructure electrophorogram as a result, WT is a wild-type, hasB is a mutant bacteria.
Fig. 4. the figure as a result of transmission electron microscope observation wild strain and mutant strain, WT is a wild type strain, and pod membrane is thick and fine and close, and hasB is for lacking mutant strain C55138 HasB, the pod membrane disappearance.
Fig. 5. BALB/c mouse is through wild bacterium of abdominal injection and mutant bacteria C55138 HasBSurvival curve afterwards.
Fig. 6. use C55138 HasBThe wild bacteria vaccine (positive control) of mutant bacteria, deactivation, adjuvant (negative control), PBS (blank) be immune mouse respectively; Use wild bacterium that four groups of mouse are attacked poison respectively then; The mouse of attacking poison continues to observe every group of mouse survival rate 12 days.
Fig. 7. use mutant bacteria C55138 HasBPass through the detected immune mouse spleen cell of the method IFN-γ of quantitative PCR and the mRNA level of IL-4 behind the immune mouse.
Embodiment
Embodiment 1 mutant strain C55138 HasBStructure
1. material: streptococcus equi epizootic disease subspecies C55138, available from China Veterinary Drugs Supervisory Inst..Plasmid pG +Host5 available from Appligene company (Illkirch, France).
2. primer design is synthetic
The genome structure of streptococcus equi epizootic disease subspecies C55138 (being called for short bacterial strain C55138) is seen shown in the accompanying drawing 1, is template with bacterial strain C55138 genomic dna, uses Primer Premier5.0 design amplification purpose fragment HasBThe primer of gene, wherein the hasBL primer is to containing restriction enzyme SalI with BamThe restriction enzyme site of HI, the hasBR primer is to containing BamHI with EcoThe restriction enzyme site of RI.It is synthetic that primer is given birth to the worker by Shanghai.
hasBL1：5’-ATTTCTGTCGACGGCTCAGGATA-3’（SEQ?ID?NO:1）；
hasBL2：5’-AATGGATCCTGACGCATTTAGGT-3’（SEQ?ID?NO:2）；
hasBR1：5’-AACCATTACAATAACGGATCCTTTG-3’（SEQ?ID?NO:3）；
hasBR2：5’-ACAACCCTGTAGCGAATTCCCTC-3’（SEQ?ID?NO:4）；
3. pcr amplification goal gene
HasBL gene fragment amplification system (30 μ L):
ddH 2O 17.8μL
10×buffer 3.0μL
Mg 2+? 1.0μL
dNTP 3.0μL
hasBL1 1.5μL
hasBL2 1.5μL
Dna profiling 2.0 μ L
RTaq enzyme 0.2 μ L
The pcr amplification reaction condition: 95 ℃ of 5min, once; 94 ℃ of 1min, 55 ℃ of 45s, 72 ℃ of 45s, totally 30 circulations; 72 ℃ of 5min, once.Obtain hasBL gene fragment amplification product.
The amplification system of hasBR gene fragment (30 μ L):
ddH 2O 17.8μL
10×buffer 3.0μL
Mg 2+? 1.0μL
dNTP 3.0μL
hasBR1 1.5μL
hasBR2 1.5μL
Dna profiling 2.0 μ L
RTaq enzyme 0.2 μ L
Condition: 95 ℃ of 5min, once; 94 ℃ of 1min, 55 ℃ of 45s, 72 ℃ of 45s, totally 30 circulations; 72 ℃ of 5min, once.Obtain hasBR gene fragment amplification product.
4. pod membrane lacks the structure of bacterium
Use bacterial strain C55138 genome as template, hasBL gene fragment amplification product and plasmid pG that the amplification of step 3 method obtains +Host5 uses restriction enzyme SalI with BamHI digestion, digestion product connects with the T4 ligase enzyme, recombinant plasmid in the middle of obtaining, this plasmid and hasBR gene fragment amplification product re-use restriction enzyme BamHI with EcoRI digestion, digestion product connects with the T4 ligase enzyme, and the end product that obtains is recombinant plasmid pG HasB(seeing accompanying drawing 2).
PG HasBTransform entering streptococcus equi epizootic disease subspecies C55138 through electricity, (clone of anti-Oxacyclotetradecane,erythromycin deriv is because pG for screening positive clone on 28 ℃ of Oxacyclotetradecane,erythromycin deriv flat boards +Host5 has the Oxacyclotetradecane,erythromycin deriv resistance).Grow into the logarithm initial stage with the TSB substratum dilution back of not containing Oxacyclotetradecane,erythromycin deriv at 28 ℃ when growing into logarithmic phase behind the positive colony switching TSB substratum.Be transferred to 37 ℃ to culturing bottle and hatch 4h.Subsequently, be uniformly coated on the dull and stereotyped last 37 ℃ of growths of TSA to bacterium, select the erythromycin-sensitive clone and identify with PCR.Identify that correct recombinant clone is labeled as mutant bacteria HasB, 37 ℃ of shaking tables are cultivated in TSB, obtain finite concentration bacterium liquid after ,-80 ℃ of refrigerators are preserved.
5. the evaluation of pod membrane absence type mutant bacteria
The mutant bacteria that makes up with wild-type streptococcus equi epizootic disease subspecies C55138 (representing) and step 4 respectively with WT HasB(use HasBExpression) be template, hasB1, hasB2 are primer (hasB1:ATACGATAACCTTTACCCAAGTCG, SEQ ID NO:5; HasB2:AGGTATTCGCAAATAGCTTGACC, SEQ ID NO:6), the method for conventional RT-PCR obtains purpose fragment, electrophoresis result (seeing accompanying drawing 3).The WT swimming lane amplifies the purpose fragment about 200bp, and mutant bacteria HasBDo not amplify the purpose fragment with the swimming lane of negative control.Then, respectively with wild bacterium C55138 and mutant bacteria HasBGenomic dna be template, hasBL1, hasBR2 are that primer carries out pcr amplification, the electrophoresis result of amplified production is seen (seeing accompanying drawing 3).The WT swimming lane amplifies the purpose fragment about 1200bp, mutant bacteria HasBSwimming lane amplify the purpose fragment about 800bp.
The experimental result of comprehensive RT-PCR and PCR can show mutant strain HasB HasBGenetically deficient the gene fragment about 400bp, mutant strain HasBMake up successfully called after C55138 HasB, be preserved in Chinese typical culture collection center.
6. the outer pod membrane of transmission electron microscope observation bacterial strain born of the same parents
Bacterial strain C55138 and mutant strain HasBSample process negative staining, through transmission electron microscope, amplification 15500 *, acceleration voltage is that 80kV observes bacterial capsule.Observations (seeing accompanying drawing 4): compare mutant bacteria C55138 with wild bacterium HasBPod membrane disappearance.
Embodiment 2 mouse virulence experiments
20 4 ~ 6 age in week BALB/c mouse, be divided into 2 groups at random, inject wild bacterium C55138 for one group, another group injection mutant bacteria C55138 HasB, be used to contrast the virulence size of two kinds of bacterial strains.
After bacterium used PBS resuspended, bacterium liquid suitably was diluted to 1 * 10 5CFU/mL, mouse peritoneal inject 500 μ L, observe the mouse existing state, write down the death time of mouse, and the result is analyzed, and mouse continues to observe 12 days.
Experimental result is seen Fig. 5: mortality ratio reaches 80% in the mouse that wild bacterium is a group 9 days, and shows serious clinical symptom, mutant bacteria C55138 HasB100% survival in one group of mouse 12 days, and have no clinical symptom.
Experimental result shows: mutant bacteria HasBVirulence significantly descend.
Embodiment 3 mutant bacterias HasBActive immunity protection experiment
40 4 ~ 6 the week age female BALB/c mouse be divided into 4 groups at random, 10 every group.The 1st group of mouse, abdominal injection 500 μ L mutant bacteria C55138 for the first time HasBCarry out immunity, bacterial concentration is 2 * 10 6CFU/mL, immune once more after 14 days with same dosage; The 2nd group of mouse carries out immunity as positive control with the vaccine of same method injection formalin-inactivated, and this deactivation vaccine is the wild bacterium of deactivation and freund's adjuvant emulsive inactivated vaccine, and abdominal injection 500 μ L for the first time, follow-up immunization are per 2 all for 1 time.The 3rd group of mouse uses identical freund's adjuvant emulsive PBS to inject mouse as negative control with same method, and the 4th group of mouse uses PBS to inject mouse as blank with same method.
After four groups of all immune completion of mouse, the wild bacterium C55138 of every group of mouse peritoneal injection lethal dose, the survival rate of observing every group of mouse, and record result.
Experimental result is seen Fig. 6: owing to there is not the immunoprotection of vaccine, the 3rd group with the 4th group of mouse behind the wild bacterium of inserting lethal dose, all death in 5 days.The 2nd group of mouse is under the immunoprotection of wild bacterium inactivated vaccine, and survival rate reaches 80%.The 1st group of mouse obtains mutant strain C55138 HasBImmunoprotection, finishing the mouse survival rate up to research is 100%.Phenomenons such as in addition, the mouse of all negative controls and blank all shows tangible clinical symptom, and is for example coarse messy by hair, and IR is slow, however mutant bacteria C55138 used HasBMice immunized does not show any clinical symptom.
Experimental result shows: mutant bacteria C55138 HasBImmune effect than the good immune effect of wild bacterium inactivated vaccine, through mutant bacteria C55138 HasBThe mouse resistibility that immunity is crossed is strong, receive wild virus infection once more after protection ratio reach 100%.
Embodiment 4 induction of immunity reaction types
The T cell mass is made up of two subgroups, is respectively Th1 subgroup and Th2 subgroup.Th1 Expression of Subsets IFN-γ can activate the CDCC of K cell, causes delayed type hypersensitivity, the mediated cell immunoreation.Th2 Expression of Subsets IL-4, the generation of enhancing antibody causes humoral immunization.Mouse is through mutant bacteria C55138 HasBAfter the immunity, can be through the activity of these two cell subsets of the horizontal Indirect evaluation of mRNA of IFN-γ and IL-4 in the detection mouse spleen cell.
6 BALB/c mouses are divided into two groups at random, one group of abdominal injection 1 * 10 6CFU/mL mutant bacteria C55138 HasB, another group injection PBS, dosage all is 0.5mL.Behind the injection 96h, the spleen cell of every mouse is extracted RNA respectively.Use the method evaluation IFN-γ of quantitative PCR and the transcriptional level of IL-4.
Experimental result: the quantitative PCR analysis result shows that IL-4 and the IFN-γ mRNA level in the back 96 hours mouse spleen cell of immunity significantly increases.In addition, the mRNA level of IFN-γ is significantly higher than the mRNA level (seeing accompanying drawing 7) of IL-4.
Experimental result shows: mutant strain HasBCan activate the immunoreation of the interior Th1 of mouse body and two cell masses of Th2, show cellular immunization and humoral immunization simultaneously, and cellular immune level be significantly higher than humoral immunity level.
SEQUENCE?LISTING
 
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Claims (5)

1.一种荚膜缺失型马链球菌兽疫亚种弱毒疫苗株，名称为马链球菌兽疫亚种C55138∆hasB（Streptococcus equi subsp. zooepidemicus C55138∆hasB），2012年5月14日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO: M 2012164。 1. A capsule-deleted attenuated vaccine strain of Streptococcus equi subsp. zooepidemicus C55138 ∆hasB ( Streptococcus equi subsp . Culture Collection Center, the deposit number is CCTCC NO: M 2012164. 2.权利要求1所述荚膜缺失型马链球菌兽疫亚种弱毒疫苗株的制备方法，其特征在于将hasB基因上下游各取部分基因连接到质粒pG+host5上，得到载体pG∆ hasB，转化马链球菌兽疫亚种C55138，利用同源重组原理将马链球菌兽疫亚种C55138基因组中的hasB基因缺失，构建成荚膜缺失型突变菌C55138∆ hasB。 2. The preparation method of the capsular deletion type Streptococcus equi subspecies zooepidemicus attenuated vaccine strain according to claim 1, characterized in that the upper and lower reaches of the hasB gene are connected to the plasmid pG + host5 to obtain the carrier pG ∆ hasB , Streptococcus equi subsp. zooepidemicus C55138 was transformed, and the hasB gene in the genome of Streptococcus equi subsp. zooepidemicus C55138 was deleted using the principle of homologous recombination to construct a capsule deletion mutant strain C55138 ∆ hasB . 3.根据权利要求2所述荚膜缺失型马链球菌兽疫亚种弱毒疫苗株的制备方法，其特征在于步骤如下： 3. according to the preparation method of the capsular deletion type Streptococcus equi subspecies zooepidemicus attenuated vaccine strain according to claim 2, it is characterized in that the steps are as follows: （1）以马链球菌兽疫亚种C55138的基因组DNA为模板，SEQ ID NO:1~2所示核苷酸序列为引物，PCR扩增hasBL基因片段； (1) Using the genomic DNA of Streptococcus equi subsp. zooepidemicus C55138 as a template and the nucleotide sequences shown in SEQ ID NO: 1~2 as primers, PCR amplifies the hasBL gene fragment; （2）以马链球菌兽疫亚种C55138的基因组DNA为模板，SEQ ID NO:3~4所示核苷酸序列为引物，PCR扩增hasBR基因片段； (2) Using the genomic DNA of Streptococcus equi subsp. zooepidemicus C55138 as a template and the nucleotide sequences shown in SEQ ID NO: 3-4 as primers, PCR amplifies the hasBR gene fragment; （3）将所得的hasBL基因片段和hasBR基因片段连接入质粒pG+host5中，转化马链球菌兽疫亚种C55138，构建成荚膜缺失型马链球菌兽疫亚种弱毒疫苗株。 (3) The obtained hasBL gene fragment and hasBR gene fragment were ligated into plasmid pG + host5, and transformed into Streptococcus equi subsp. zooepidemicus C55138 to construct a capsular-deleted Streptococcus equi subsp. zooepidemicus attenuated vaccine strain. 4.权利要求1所述荚膜缺失型马链球菌兽疫亚种弱毒疫苗株在制备疫苗中的应用。 4. The application of the attenuated vaccine strain of capsular-deleted Streptococcus equi subspecies zooepidemicus described in claim 1 in the preparation of vaccines. 5.一种马链球菌兽疫亚种弱毒疫苗的制备方法，其特征在于步骤如下： 5. A preparation method of Streptococcus equi subspecies zooepidemic attenuated vaccine, characterized in that the steps are as follows: （1）马链球菌兽疫亚种C55138∆hasB复苏于TSB中，37℃摇床培养过夜； (1) Streptococcus equi subsp. zooepidemicus C55138 ∆hasB was resuscitated in TSB, and cultured on a shaker at 37°C overnight; （2）取培养过夜的菌种于新的TSB中，37℃摇床扩大培养6~8h； (2) Take the cultured strain overnight and place it in a new TSB, and expand the culture on a shaker at 37°C for 6-8 hours; （3）经扩大培养的菌液在8000rpm条件下离心3min，去上清； (3) Centrifuge the cultured bacterial solution at 8000rpm for 3min, and remove the supernatant; （4）沉淀中的细菌与灭菌的20wt.%脱脂乳混合，冷冻干燥，制备成冻干活疫苗。 (4) The bacteria in the precipitate were mixed with sterilized 20wt.% skim milk, and freeze-dried to prepare a freeze-dried live vaccine.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220182A (en) * 2016-12-22 2018-06-29 新疆农业大学 One plant of horse source streptococcus zooepidemicus strain X JMSY16-1 and its application in streptococcus equi disease vaccine
CN113005072A (en) * 2021-04-09 2021-06-22 佛山科学技术学院 Streptococcus equi subsp zooepidemicus gene deletion strain and preparation method and application thereof
CN115418369A (en) * 2022-04-07 2022-12-02 佛山科学技术学院 Recombinant streptococcus and its construction method and application
WO2024002331A1 (en) * 2022-06-30 2024-01-04 Shanghai Yuguan Biotech Co., Ltd. A live bacteria strain with reduced capsules

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