CN106995489B - A kind of Streptococcus suis truncated protein Sao and application - Google Patents

A kind of Streptococcus suis truncated protein Sao and application Download PDF

Info

Publication number
CN106995489B
CN106995489B CN201610044176.6A CN201610044176A CN106995489B CN 106995489 B CN106995489 B CN 106995489B CN 201610044176 A CN201610044176 A CN 201610044176A CN 106995489 B CN106995489 B CN 106995489B
Authority
CN
China
Prior art keywords
sao
piglet
glu
lys
truncated protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201610044176.6A
Other languages
Chinese (zh)
Other versions
CN106995489A (en
Inventor
金梅林
李敬涛
王雅
康超
吴超
孙小美
朱伟峰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Huazhong Agricultural University
Original Assignee
Huazhong Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Huazhong Agricultural University filed Critical Huazhong Agricultural University
Priority to CN201610044176.6A priority Critical patent/CN106995489B/en
Publication of CN106995489A publication Critical patent/CN106995489A/en
Application granted granted Critical
Publication of CN106995489B publication Critical patent/CN106995489B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0208—Specific bacteria not otherwise provided for
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Mycology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention discloses a kind of Streptococcus suis truncated protein Sao and application, described its amino acid sequence of truncated protein Sao is shown in SEQ ID NO.2.The protein stability is better than full-length proteins, it is not easy to degrade, after vaccine is made using Sao albumen provided by the invention, effectively can generate protective effect to piglet；After attacking poison using the velogen strain SC19 of large dosage, protection still reaches 60%.Sao albumen is safe and non-toxic, and body temperature, breathing, appetite, the state of mind are all normal between the entire observation period after immune to piglet and pregnant sow, and pregnant pig farrowing is normal, situations such as miscarriage, stillborn foetus does not occur.Sao albumen can also combine with other albumen is prepared into multiple vaccines, is suitable for mass production.

Description

A kind of Streptococcus suis truncated protein Sao and application
Technical field
The present invention relates to domestic animal recombinant vaccine fields, and in particular to a kind of Streptococcus suis truncated protein Sao and application.
Technical background
Streptococcus suis (Streptococcus suis, S.suis) is the main pathogen for causing Streptococcus suis, according to thin S.suis points can be 33 serotypes, the respectively type of 1/2 type, 1~31 and 33 by the capsular polysaccharide (CPS) on bacterium surface.Wherein Streptococcus suis 2 (SS2) type is that virulence is most strong, prevalence is wide, is clinically separated the highest serotype of frequency.SS2 is a kind of important people The total illness pathogen of poultry, can cause pig meningitis, endocarditis, septicemia, arthritis, scrositis, pneumonia etc., often cause to wean Piglet or growing and fattening pigs die by visitation of God；The human infection bacterium can lead to meningitis, cause permanent deafness, septicemia, the internal membrane of heart Inflammation, in addition it is dead.It is reported within 1991 Guangdong Province for the first time and finds 2 type streptococcus suspected case of pig, portion of the Jiangsu Province 1998-1999 Season breaks out the disease to continuous 2 years of region-by-region in full summer.Having enough epidemiologic datas at present confirms that SS2 is to China Aquaculture and people's health constitute serious threat.Therefore, effective vaccine is developed to prevent the infection of Streptococcus suis It is extremely urgent with control Streptococcus suis.
Subunit seedling has many advantages, such as that the safety of the immune efficacy height of live vaccine and inactivated vaccine is good, Yuanyi Li et al. Testimony of a witness reality Sao is the surface membrane protein of Streptococcus suis, and the specific antibody of Sao can be with 28 serotypes in 33 serotypes 25 plants in bacterial strain lysate and 26 plants of SS2 have good immunogenic response, and it is high conservative to illustrate that Sao has in Streptococcus suis Property.
In addition, the immune protective effect and adjuvant of Sao albumen have much relations.Yuanyi Li et al. people have found Sao and adjuvant Although Emulsigen-Plus can cause higher antibody level after being used in mixed way, piglet cannot be protected to resist Streptococcus suis Infection.Adjuvant Emulsigen-Plus was substituted for adjuvant Quil A by the researcher later, can but provide 80% or more piglet Protection.K.-J.Hsueh et al. confirms Sao and is commercialized adjuvant w/o/w adjuvants also can be to the protection of mouse offer 80%.
Sao is divided into 3 types according to the difference of gene order length：Sao-S(1.5kb),Sao-M(1.7kb),Sao-L (2.0kb), wherein Sao-M are distributed most wide in bacterial strain.Sao-M full-length proteins have 110KD, Wang Jing et al. to find overall length sao eggs White purifying difficulty is big, and protein stability is poor, degradation speed block, these factors hinder the further research and development of Sao protein vaccines.
Invention content
The object of the present invention is to provide a kind of Streptococcus suis truncated protein Sao, sequence is SEQ ID NO.2 institutes Show.Truncated protein Sao provided by the invention is stability albumen, is dissolved in PBS buffer solutions, is stored in -80 DEG C one month After take out and do not find that albumen has signs of degradation.
It is another object of the present invention to provide Streptococcus suis truncated protein Sao to prepare streptococcus suis infection vaccine In application, truncated protein Sao and adjuvant are prepared into vaccine, intramuscular immunisation piglet attacks poison after booster immunization, can give piglet Higher protection is provided.
The last one has been designed to provide applications of the truncated protein Sao in preparing multiple vaccines to the present invention, this is sent out The truncated protein Sao of bright offer is prepared into multiple vaccines with other protein vaccines, can play the effect of a needle multiple-effect.
In order to achieve the above object, the present invention takes following technical measures：
Full-length proteins Sao is unstable, is easy degradation, applicant is effectively truncated overall length Sao albumen, after truncation Protein sequence is shown in SEQ ID NO.2, and truncated protein Sao becomes stability albumen, is dissolved in PBS buffer solutions, is preserved It is taken out after one month at -80 DEG C and does not find that albumen has signs of degradation.
Applications of the Streptococcus suis truncated protein Sao in preparing streptococcus suis infection vaccine, including by truncated protein Sao and Adjuvant is prepared by mixing into vaccine, intramuscular immunisation piglet in proportion, and energy effective protection piglet resists the infection of Streptococcus suis.
Applications of the truncated protein Sao in preparing multiple vaccines, by truncated protein Sao provided by the invention and other albumen Vaccine preparation is at multiple vaccines, preferably by truncated protein Sao and erysipelothrix ruhsiopathiae surface truncated protein SpaA (SEQID Shown in NO.3) it is prepared into combined vaccine, intramuscular immunisation piglet can effectively improve piglet survival rate after attacking poison, and two are independent of each other Each other to the immune protective effect of piglet.
Specific method refers to specific implementation mode.
Compared with prior art, the present invention has the following advantages：
1. Sao property of protein provided by the invention is stablized, it is not easy to degrade, be better than full-length proteins, be prepared into after vaccine surely It is qualitative good, it is suitable for mass production.
2. after vaccine is made using Sao albumen provided by the invention, effectively can generate protective effect to piglet, utilize big agent After the velogen strain SC19 of amount attacks poison, protection still reaches 60%.
3. Sao albumen provided by the invention is safe and non-toxic, body temperature between the entire observation period after immune to piglet and pregnant sow, Breathing, appetite, the state of mind are all normal, and pregnant pig farrowing is normal, situations such as miscarriage, stillborn foetus does not occur.
4. Sao albumen provided by the invention can also combine with other albumen is prepared into multiple vaccines, a needle multiple-effect can be played Effect.
Description of the drawings
Fig. 1 is the antibody level schematic diagram that mouse is immunized in truncated protein Sao.
Fig. 2 is to attack malicious protection effect diagram after mouse is immunized in truncated protein Sao.
Fig. 3 is that Temperature changing schematic diagram after piglet is immunized in truncated protein Sao.
Fig. 4 is that antibody level schematic diagram after piglet is immunized in truncated protein Sao.
Fig. 5 is to attack malicious protection effect diagram after piglet is immunized in truncated protein Sao.
Fig. 6 is that Temperature changing schematic diagram after piglet is immunized in joint seedling.
Fig. 7 is that truncated protein Sao antibody level schematic diagrames after piglet are immunized in joint seedling.
Fig. 8 is that truncated protein SpaA antibody level schematic diagrames after piglet are immunized in joint seedling
Specific implementation mode
Reagent of the present invention if not otherwise specified, is purchased from commercial channel or the general configuration reagent for this field.Institute It states technical solution if not otherwise specified, is the conventional scheme of this field.
Embodiment 1：
The synthesis of Streptococcus suis truncated protein Sao and vaccine preparation：
1.1 the synthesis of truncated protein Sao
The sequence of truncated protein Sao is closed as shown in SEQ ID NO.2 by Sangon Biotech (Shanghai) Co., Ltd. At.Truncated protein Sao Purities are 99%, every 5mg after synthesis, are white powder, are kept in dark place in -20 degree, completely It is water-soluble.By the protein dissolution in PBS buffer solutions, it is stored in -80 DEG C and takes out after one month and do not find that albumen has degradation Phenomenon.
Before use, with sterile PBS (NaCl 8.0g, KCl 0.2g, KH2PO40.24g, Na2HPO4×12H2O 3.628g, PH7.4 it) is diluted by prescribed concentration.It is preheated in 37 degree of incubators using preposition.
The preparation of 1.2 vaccines
Dosage form：Water-in-oil type (W/O)
Adjuvant forms：White Mineral Oil (Marcol52), Tween 80 (CRILLET4), sorbester p17 (CRILL4).
Oil phase：White Mineral Oil be heated to it is transparent after and sorbester p17 by volume 94：6 prepare.
Water phase：Albumen is formed with Tween 80, and Tween 80 accounts for 0.4% (volume ratio) of water phase.
Vaccine emulsifies：Water phase presses 1 with oil phase：1 volume mixture emulsifies.Make the final concentration of 1mg/ml of truncated protein Sao, uses In following experiment.
1.3 routine inspection
Vaccine character detects：Vaccine instills in clear water after emulsification, and the first drop is scattered, the second oil dripping pearl, shakes liquid level oil Pearl is not demulsified.
Sterility testing：A little vaccine coating TSA plates, 37 DEG C of incubator cultures is taken to be grown without bacterium colony for 18 hours.
Stability test：Whether 3000rpm/min is centrifuged 15 minutes, stablized according to separation situation judgement emulsification, after centrifugation Emulsification vaccine is not layered.
By protein dissolution in PBS buffer solutions, -80 DEG C preserve 1 month, 3 months, 6 months respectively, do not find Proteinaceous Qualitative change.
The good vaccine of above-mentioned emulsification is placed in 4 DEG C to preserve 1 month, 3 months, 6 months respectively, the change of vaccine character is not detected Change.
Embodiment 2：
Applications of the truncated protein vaccine Sao in preparing streptococcus suis infection vaccine
1, the immune protective effect of vaccine is evaluated on mouse model
1.1 experimental program
Experimental animal：4 week old female BAl BIcs/c small white mouses are purchased from Disease Prevention Control Center, Hubei Prov.
Attack toxic bacterial strain：Streptococcus suis 2-type SC19.
Experiment packet：Weight 16g female BAl BIcs/c small white mouses 20 are taken, are randomly divided into 2 groups:Vaccine I groups (Sao), control Group (adjuvant and PBS), every group 10.Docking takes blood weekly between the immune later stage, is used for the monitoring of serum specific antibody.Immune side Method：The dose subcutaneous immunization experiment mouse of vaccine prepared by embodiment 1 by 100 μ l/ only；Booster immunization 1 time is (immune after 2 weeks Dosage and position are with immune for the first time).
The detection of 1.2 serum specific antibodies
It is coated with elisa plate overnight in 4 DEG C using the holes truncated protein Sao100ng/, separation is detected with the method for indirect ELISA The antibody titer of serum；The result is shown in Figure 1.
Discovery is detected to the antibody level of mouse after immune：It first week after first immunisation can inducing mouse generation Higher antibody level, there is 27.When two exempt from 14 days, mouse internal antibody level reaches a higher level, positive serum Mean antibody titer can reach 215。
1.3 immunized mices protect situation after attacking poison
Two exempt from challenge test after 14 days, and it is 3.3 × 10 to attack toxic dose8CFU/ is only.It is observed continuously one week, records facing for mouse Bed feature and death condition.
As a result as follows：
SC19 plants of absolute lethal bacterium amount LD100 is 3.0 × 108CFU/ only, attack malicious result and see Fig. 2, mouse by immune mouse Survival rate is 70%, and 7 mouse of survival are without clinical symptoms；And control group mice is all dead, survival rate is 0.By antibody water Gentle immune group mouse survival rate adds vaccine on mouse made of adjuvant to have higher protection to imitate after can be seen that Sao protein truncations The immunogenicity of fruit, the albumen does not disappear.
2, the immune protective effect of vaccine is evaluated on piglet model
2.1 experimental program
Experimental animal：The negative weanling pig of 4 week old Streptococcus suis detection, purchased from Wuhan economic cycle green grass lake day boar ?.
Attack toxic bacterial strain：Streptococcus suis 2-type SC19.
Experiment packet：4 week old weanling pigs are divided into 3 groups, be immunized 1 group (Sao), be immunized 2 groups (Sao), control group (adjuvant and PBS), 1 group and control group every group 10 is immunized, is immunized 2 groups 5,1 group immune, adjuvant and PBS mixing control groups are used to attack poison, Immune 2 groups, for detecting antibody level, are continuously detected 8 months.
Immunization ways：Buttocks muscles multi-point injection, every 14 days booster immunizations, once (immunizing dose and position were the same as first after head exempts from Exempt from), immunizing dose is 2ml/ heads.
The detection of 2.2 serum specific antibodies
Ear vein takes blood weekly for 1 group immune, adjuvant and PBS mixing control group, is used for the detection of serum specific antibody；Exempt from 2 groups of blood samplings every month of epidemic disease are primary, are used for the detection of serum specific antibody.It is wrapped using the holes truncated protein Sao100ng/ of purifying By elisa plate, 4 DEG C overnight.With the antibody level in indirect ELISA detection piglet body.
2.3 immune rear piglet Temperature changing detections
Body temperature is monitored after first immunisation daily, feeding and the mental condition of immune rear piglet are observed in continuous detection 7 days.
2.4 immune swines protect situation after attacking poison
Two exempt from 14 days after carry out challenge test, attack toxic dose be 1.8 × 106Cfu/ SC19 are piglet lethal doses 1.5 times of LD100.It is observed continuously 14 days, records Clinical symptoms and death condition.
2.5 immune effects are assessed
Piglet does not have clinical onset symptom after immune, and 1 group immune, the immune rear second day body temperature of control group can rise, and rise Amplitude is smaller, has no that symptoms, the third body temperature such as piglet has apocleisis, greedy sleeping, spirit is depressed return to normal, during which piglet is adopted Appetite has no reduction, and the of short duration rising of body temperature is smaller to the growth effect of piglet, and Temperature changing is shown in Fig. 3.
Antibody level detection after piglet immunological Sao is shown：The antibody level of Sao induction piglets gradually rises, in head Exempt from rear 4th week up to 220Left and right, sees Fig. 4；After attacking poison, immune 1 group has 4 piglets dead in 7 days, survives 6, sees Fig. 5. Bacterium, kidney,spleen,liver, the heart will be divided all not to be separated to SC19 with the progress dissect that is euthanized at health survival piglet.Control group piglet land Supervention disease, back leg arthroncus, spiritual depressed, happiness is crouched, conjunctivitis, is struck in swimming shape, there is piglet death successively, last whole It is dead complete.The truncated protein Sao that the experimental result of ontology animal further illustrates the present invention preparation can be provided preferably for piglet Protecting effect.
The antibody level of immune 2 groups of carry out 8 months is detected, starts within 2 weeks to detect antibody, testing result table after booster immunization It is bright：Head exempt from after the 28th day~90 days antibody titer average values 220Left and right；Exempt from latter 4th month to 6th month antibody effect from head Valence is gradually reduced, and antibody level is still higher, and the 6th month antibody titer average value is 210, still there is protection in left and right to piglet Power illustrates that antibody level can at least maintain 6 months.Though 210 days can detect antibody, antibody level is relatively low, and antibody titer is flat Mean value only has 25。
Embodiment 3：Safety examination
In order to examine safety effects of the prepared vaccine to piglet and pregnant sow, to 35 age in days piglets and gestation 70 days Age sow carries out safety examination.
3.1 experimental program：
1) vaccine of preparation is passed through into 35 age in days sodium selenite of buttocks muscles branch injection inoculation, every batch of vaccine injection respectively 5, every inoculation 2ml observes its clinical manifestation and measures body temperature.
2) vaccine prepared passes through buttocks muscles branch injection inoculation gestation healthy sow on the 70th, every batch of vaccine note respectively 5 are penetrated, every inoculation 2ml observes its clinical manifestation and farrowing situation.
2. test result
3.2.1 the safety that subunit vaccine of the invention is inoculated with weanling pig
After the 35 age in days health weanling pig of vaccine inoculation of preparation, every batch of vaccine injection 5, piglet breathes situation, essence Refreshing situation, appetite etc. are normal, and only second day body temperature of vaccine inoculation slightly rises, and third body temperature declines restore just rapidly Often.Illustrate that the vaccine product is fine to weanling pig safety, Temperature changing is shown in (table 1).
Mean body temperature variation after 35 age in days weanling pig of vaccine inoculation prepared by 1 present invention of table
3.2.2 the safety (produce surviving of son situation) that subunit vaccine of the invention is inoculated with pregnant animal (pregnant sow)
As a result the pregnant sow on the 70th of health of 3 batches of subunit vaccines inoculations of the invention between the entire observation period body temperature, exhale Suction, appetite, the state of mind are all normal (table 2), and farrowing is normal, situations such as miscarriage, stillborn foetus (table 3) do not occur.
Mean body temperature variation after the vaccine inoculation health pregnant sow of 2 present invention of table
The healthy pregnant sow inoculation of table 3 prepares the farrowing situation after vaccine
Embodiment 4：
Applications of the truncated protein Sao in preparing multiple vaccines
1. the preparation of combined vaccine
The joint of truncated protein Sao and erysipelothrix ruhsiopathiae surface truncated protein SpaA are prepared by the method in embodiment 1 The content of seedling, Sao and SpaA are respectively 1mg/ml.
The amino acid sequence of the erysipelothrix ruhsiopathiae surface truncated protein SpaA is this hair shown in SEQ ID NO.3 Bright truncated protein SpaA used is genetic engineering bacterium Escherichia coli (Escherichia coli) BL21/pET-28a-spaA, Deposit number is CCTCC NO：M2015076 secretes to obtain.
2. combining seedling immune effect on piglet
1) experimental program
Experimental animal：The negative weanling pig of 4 week old Streptococcus suis detection, purchased from Wuhan economic cycle green grass lake day boar ?.
Attack toxic bacterial strain：(abbreviation SE38, Li Jingtao, Hubei is partly by streptococcus suis 2-type SC19, erysipelothrix ruhsiopathiae SE38 Area's erysipelothrix ruhsiopathiae separation strains drug resistance analysis, cultivation and feed [J] .2015.2,11-14), it is 1.8 that SC19, which attacks toxic dose, ×106CFU/ heads are 1.5 times of lethal dose ID100；It is 9.0 × 10 that SE38, which attacks toxic dose,8CFU/ heads are lethal doses 3 times of LD100.
Experiment packet：4 week old weanling pigs are divided into 4 groups, and 1 group (Sao+SpaA) is immunized, 2 groups (Sao+SpaA) are immunized, is right According to 2 groups, every group each 10 of group (adjuvant+PBS).
Immunization ways：Buttocks muscles multi-point injection, head is primary every 14 days booster immunizations after exempting from, and immunizing dose is 2ml/ heads.
2) detection of serum specific antibody
Randomly selecting piglet in 4 experimental groups, ear vein takes blood weekly, is used for the detection of serum specific antibody.Using pure The holes truncated protein Sao100ng/ of change are coated with elisa plate, and 4 DEG C overnight, anti-with Sao in indirect ELISA detection piglet separation serum Body is horizontal.It is coated with elisa plate using the holes truncated protein SpaA100ng/ of purifying, 4 DEG C overnight, with indirect ELISA detection piglet point From SpaA antibody levels in serum.
3) piglet clinical observation after being immunized
Body temperature is monitored after first immunisation daily, feeding and the mental condition of immune rear piglet are observed in continuous detection 7 days.
4) immune swine protects situation after attacking poison
Two exempt from 14 days after carry out challenge viral dosage, be observed continuously 14 days, record Clinical symptoms and death condition.
5) immune effect is assessed
Piglet does not have clinical onset symptom after immune, and second day body temperature of piglet can slightly rise after being immunized, and have no that piglet has Apocleisis, greedy sleeping, spirit is depressed etc., and symptoms, third body temperature return to normal, and during which piglet feed intake has no reduction, body temperature Of short duration rising is smaller to the growth effect of piglet, and Temperature changing is shown in Fig. 6.
Sao and SpaA antibody levels detection after piglet immunological is shown：After immune piglet, antibody titer gradually rises, The antibody titer of piglet can reach 2 at the end of exempting from 2 weeks to two20Left and right.It is compared with the growth of the antibody level of monomer vaccine immunity, The antibody growth that piglet is immunized in joint seedling has no abnormal changes, and explanation can be used in combination, and be not in what antibody was offset Phenomenon is shown in Fig. 7 and Fig. 8.
It attacks poison and the results are shown in Table 4, truncated protein Sao seedlings can provide the protection of piglet 60%, and SpaA can be carried to piglet For 90% protection.This experimental result illustrates that the joint seedling further prepared does not interfere with the protecting effect to piglet each other.
Table 4 combines seedling and attacks the result after poison
For truncated protein Sao prepared by the present invention relative to full-length proteins, being can be to young after stability albumen and adjuvant mixing Pig provides higher protecting effect, illustrates that its immunogenicity does not reduce.With erysipelothrix ruhsiopathiae truncated protein SpaA mixing systems It is standby that piglet is immunized at combined vaccine, be not in the phenomenon that antibody is offset.It is described above：Individually truncating Sao albumen seedling can protect Piglet resists streptococcus suis 2-type, and the infection of poison SC19 and truncated protein SpaA composition joint seedlings can also protect piglet to resist pig by force The infection of streptococcus or erysipelothrix ruhsiopathiae meets the preventative strategies of current " the anti-more diseases of a needle ", has very important reality Border application value.
SEQUENCE LISTING
<110>Hua Zhong Agriculture University
<120>A kind of Streptococcus suis truncated protein Sao and application
<130>A kind of Streptococcus suis truncated protein Sao and application
<160> 3
<170> PatentIn version 3.1
<210> 1
<211> 777
<212> DNA
<213>Artificial sequence
<400> 1
atgacaacct gatgggggac aggctacttc aaaggcggtt aatgtcaaaa taccagcagt 60
agtacgacta tttggtcgtg agcttctaga aaatgaattt aaatttgagc ttagagaagc 120
gaatggcgag gaactccctg tccttgatac agctcaaaat acaaaagagg gtcaagttag 180
atttaaaaat ctatcattcg ataagcctgg caaatactgg tatacaattt cagaagtaaa 240
agatgagctt ggtggtattg agtatgattc gaaatatatt gtagcaaaaa taactgtaga 300
agatcgaaac gggcaattac aggcaatgat cgaatttatt gataatgaca atgtctttaa 360
caatttctat acacctgctc cagctgctgc tagtctttcg ataaaaaaag tcctcgaggg 420
acgtacctta aacaccggtg aattcgaatt tgttttaaaa aatgaaaaag gcgatgaaat 480
cgaaaaggta agcaatcaag cagatggttc tgtaaacttt agtgccctaa catttacaaa 540
agagggaacc tatacctaca ctgtttcaga agttgatggt ggacttggcg atattatcta 600
tgacaaatca gatattaagg ccactgttac tgtgaaagat aacaatcacg gacaactagt 660
ctcaacagtg acttatgaaa atagcgatca aatcttcgag aatattttga atcctgggaa 720
gttaatagcg ccaaccacgg atagcgttat tactgataat gaagtctcta aggaagc 777
<210> 2
<211> 258
<212> PRT
<213>Artificial sequence
<400> 2
Met Asn Thr Lys Lys Trp Arg Thr Ser Leu Leu Ile Pro Gly Ile Val
1 5 10 15
Leu Phe Gly Thr Val Ala Leu Val Asn Asn Val Ser Ala Gln Glu Val
20 25 30
Lys Asn Thr Ile Ile Ser Ala Lys Gln Pro Asp Gly Gly Gln Ala Thr
35 40 45
Ser Lys Ala Val Asn Val Lys Ile Pro Ala Val Val Arg Leu Phe Gly
50 55 60
Arg Glu Leu Leu Glu Asn Glu Phe Lys Phe Glu Leu Arg Glu Ala Asn
65 70 75 80
Gly Glu Glu Leu Pro Val Leu Asp Thr Ala Gln Asn Thr Lys Glu Gly
85 90 95
Gln Val Arg Phe Lys Asn Leu Ser Phe Asp Lys Pro Gly Lys Tyr Trp
100 105 110
Tyr Thr Ile Ser Glu Val Lys Asp Glu Leu Gly Gly Ile Glu Tyr Asp
115 120 125
Ser Lys Tyr Ile Val Ala Lys Ile Thr Val Glu Asp Arg Asn Gly Gln
130 135 140
Leu Gln Ala Met Ile Glu Phe Ile Asp Asn Asp Asn Val Phe Asn Asn
145 150 155 160
Phe Tyr Thr Pro Ala Pro Ala Ala Ala Ser Leu Ser Ile Lys Lys Val
165 170 175
Leu Glu Gly Arg Thr Leu Asn Thr Gly Glu Phe Glu Phe Val Leu Lys
180 185 190
Asn Glu Lys Gly Asp Glu Ile Glu Lys Val Ser Asn Gln Ala Asp Gly
195 200 205
Ser Val Asn Phe Ser Ala Leu Thr Phe Thr Lys Glu Gly Thr Tyr Thr
210 215 220
Tyr Thr Val Ser Glu Val Asp Gly Gly Leu Gly Asp Ile Ile Tyr Asp
225 230 235 240
Lys Ser Asp Ile Lys Ala Thr Val Thr Val Lys Asp Asn Asn His Gly
245 250 255
Gln Leu
<210> 3
<211> 450
<212> PRT
<213>Artificial sequence
<400> 3
Met Asp Ser Thr Asp Ile Ser Val Ile Pro Leu Ile Gly Glu Gln Val
1 5 10 15
Gly Leu Leu Pro Val Leu Pro Gly Thr Gly Val His Ala Gln Glu Tyr
20 25 30
Asn Lys Met Thr Asp Ala Tyr Ile Glu Lys Leu Val Ser Leu Ile Asn
35 40 45
Gln Lys Val Lys Pro Phe Leu Ile Asn Glu Pro Lys Gly Tyr Gln Ser
50 55 60
Phe Glu Ala Val Asn Glu Glu Ile Asn Ser Ile Val Ser Glu Leu Lys
65 70 75 80
Asn Glu Gly Met Ser Leu Gln Asn Ile His His Met Phe Lys Gln Ser
85 90 95
Ile Gln Asn Leu Ala Thr Arg Ile Gly Tyr Arg Ser Phe Met Gln Asp
100 105 110
Ala Met Tyr Leu Glu Asn Phe Glu Arg Leu Thr Ile Pro Glu Leu Asp
115 120 125
Glu Ala Tyr Val Asp Leu Leu Val Asn Tyr Glu Val Lys His Arg Ile
130 135 140
Leu Val Lys Tyr Glu Gly Lys Val Lys Gly Arg Ala Pro Leu Glu Ala
145 150 155 160
Phe Ile Val Pro Leu Arg Asp Arg Ile Arg Ser Met Asn Glu Met Ala
165 170 175
Ala Glu Val Asn Gly Leu Pro Glu Ala His Glu Asp Phe Leu Val Ser
180 185 190
Asp Ser Ser Glu Tyr Asn Asp Lys Leu Asn Asn Ile Asn Phe Ala Leu
195 200 205
Gly Leu Gly Val Ser Glu Phe Ile Asp Tyr Asn Arg Leu Glu Asn Met
210 215 220
Met Glu Lys Glu Ile His Pro Leu Tyr Leu Glu Leu Tyr Ala Met Arg
225 230 235 240
Arg Asn Arg Gln Ile Gln Val Val Arg Asp Val Tyr Pro Asn Leu Glu
245 250 255
Arg Ala Asn Ala Val Val Glu Ser Leu Lys Thr Ile Lys Asp Ile Lys
260 265 270
Gln Arg Gly Lys Lys Leu Gln Glu Leu Leu Glu Ile Tyr Ile Gln Arg
275 280 285
Ser Gly Asp Val Arg Lys Pro Asp Val Leu Gln Arg Phe Ile Gly Lys
290 295 300
Tyr Gln Ser Val Val Asp Glu Glu Lys Asn Lys Leu Gln Asp Tyr Leu
305 310 315 320
Glu Ser Asp Ile Phe Asp Ser Tyr Ser Val Asp Gly Glu Lys Ile Arg
325 330 335
Asn Lys Glu Ile Thr Leu Ile Asn Arg Asp Ala Tyr Leu Ser Met Ile
340 345 350
Tyr Arg Ala Gln Ser Ile Ser Glu Ile Lys Thr Ile Arg Ala Asp Leu
355 360 365
Glu Ser Leu Val Lys Ser Phe Gln Asn Glu Glu Ser Asp Ser Lys Val
370 375 380
Glu Pro Glu Ser Pro Val Lys Val Glu Lys Pro Val Asp Glu Glu Lys
385 390 395 400
Pro Lys Asp Gln Lys Lys Leu Val Asp Gln Ser Lys Pro Glu Ser Asn
405 410 415
Ser Lys Glu Gly Trp Ile Lys Lys Asp Asn Lys Trp Phe Tyr Ile Glu
420 425 430
Lys Ser Gly Gly Met Ala Thr Gly Trp Lys Lys Val Ala Asp Lys Trp
435 440 445
Pro Tyr
450

Claims (3)

1. a kind of applications of Streptococcus suis truncated protein Sao in preparing streptococcus suis infection vaccine, the Streptococcus suis cuts The polypeptide of short Protein S ao is shown in SEQ ID NO.2.
Application of the polypeptide in preparing multiple vaccines shown in 2.SEQ ID NO.2.
3. application according to claim 2, the multiple vaccines be include polypeptide and SEQ shown in SEQ ID NO.2 The multiple vaccines of polypeptide shown in ID NO.3.
CN201610044176.6A 2016-01-22 2016-01-22 A kind of Streptococcus suis truncated protein Sao and application Active CN106995489B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610044176.6A CN106995489B (en) 2016-01-22 2016-01-22 A kind of Streptococcus suis truncated protein Sao and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610044176.6A CN106995489B (en) 2016-01-22 2016-01-22 A kind of Streptococcus suis truncated protein Sao and application

Publications (2)

Publication Number Publication Date
CN106995489A CN106995489A (en) 2017-08-01
CN106995489B true CN106995489B (en) 2018-11-06

Family

ID=59428811

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610044176.6A Active CN106995489B (en) 2016-01-22 2016-01-22 A kind of Streptococcus suis truncated protein Sao and application

Country Status (1)

Country Link
CN (1) CN106995489B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111378015B (en) * 2019-12-27 2021-01-01 无锡市妇幼保健院 Synthetic peptide for detecting S.suis 2 infection and application thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4755977B2 (en) * 2004-02-27 2011-08-24 一般財団法人化学及血清療法研究所 Method for producing surface antigens of swine erysipelas in Escherichia coli
DK1943342T3 (en) * 2005-09-02 2016-07-25 Valorisation-Recherche Ltd Partnership STREPTOCOCCUS SUIS POLYPEPTIDES AND POLYNUCLEOTIDES ENCODING SAME AND THEIR USE IN DIAGNOSTIC USES vaccination and

Also Published As

Publication number Publication date
CN106995489A (en) 2017-08-01

Similar Documents

Publication Publication Date Title
CN103071151B (en) Special diluent for swine mycoplasmal pneumonia vaccines and preparation method of special diluent
CN102512674B (en) Fish broad-spectrum vibrio subunit vaccine and preparation method
CN104784686B (en) TGEV, PEDV bigeminal live vaccine and preparation method thereof
CN105462936B (en) A kind of Porcine epidemic diarrhea virus MY01 plants and its application
CN104470538B (en) For the chimeric protein vaccine by the microbial pneumonia of pneumonia streptococcus
CN108823218A (en) Chicken infectivity bursa of Fabricius virus VP 2 gene, its expression product, its subunit vaccine and application
CN102847146A (en) Vaccine used for preventing tilapia streptococcal disease
CN109055412A (en) A kind of pig circular ring virus-mycoplasma pneumoniae bigeminy subunit vaccine and preparation method thereof
CN106146626B (en) A kind of erysipelothrix ruhsiopathiae subunit vaccine and preparation method and application
CN104087559B (en) A kind of infectious bursa of Fabricius virus, inactivated vaccine and preparation method thereof
CN106995489B (en) A kind of Streptococcus suis truncated protein Sao and application
CN101157907A (en) Recombinant salmonella choleraesuis strain for expression of pig origin bordetella bronchisepatica fhaB and prn gene segment, bacterin and uses thereof
CN102127554A (en) Japanese encephalitis particle vaccine and preparation method and application thereof
CN101629178A (en) Manually-combined Newcastle diseases virus F gene and recombining expression vector and application thereof
CN104096222A (en) Vaccine composition, and preparation method and application thereof
CN108410784A (en) It Streptococcus suis △ CPS/SsnA-mSly (P353L)-SC19 engineering bacterias and is applied in vaccine
CN102847168B (en) The design of a kind of nucleic acid vaccine PV-Fn preventing bovine mastitis and structure thereof
CN109402035A (en) A kind of parenteral enteropathogenic E. Coli recombinant bacterial strain and its application
CN105497885B (en) A kind of subunit vaccine and its preparation method and application
CN106520623B (en) A kind of serum 7-type haemophilus parasuis low virulent strain and its application
CN101781632A (en) Brucella melilitensis bp26 gene-deleted M5-90 vaccine strain
TW200936759A (en) Fish vaccine
US11717566B2 (en) Brucella canis vaccine for dogs
CN104248761B (en) A kind of vaccine combination and its preparation method and application
CN107982527A (en) Applications of the outer membrane protein V P1243 in vibrio infection is prevented

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant