CN102212120A - Salmonella paratyphi A PagC subunit vaccine and preparation method thereof - Google Patents
Salmonella paratyphi A PagC subunit vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN102212120A CN102212120A CN2011100786203A CN201110078620A CN102212120A CN 102212120 A CN102212120 A CN 102212120A CN 2011100786203 A CN2011100786203 A CN 2011100786203A CN 201110078620 A CN201110078620 A CN 201110078620A CN 102212120 A CN102212120 A CN 102212120A
- Authority
- CN
- China
- Prior art keywords
- pagc
- outer membrane
- vaccine
- membrane protein
- salmonella paratyphi
- 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.)
- Pending
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a salmonella paratyphi A PagC subunit vaccine and a preparation method thereof. The vaccine comprises a salmonella paratyphi A outer membrane protein PagC and a vaccine adjuvant with effective dosages. The amino acid sequence of the outer membrane protein PagC is shown as SEQ ID NO.2. The preparation method of the salmonella paratyphi A PagC subunit vaccine comprises the following steps of: performing in-vitro expression to obtain the outer membrane protein PagC; and mixing the outer membrane protein PagC and the vaccine adjuvant. The preparation method is low in production cost and high in yield of the target protein and has important significance for mass production of the PagC subunit vaccine and the prevention of salmonella paratyphi A infection.
Description
Technical field
This paper belongs to biological technical field, relates to Salmonella paratyphi A subunit vaccine and preparation method thereof.
Background technology
Typhoid fever is a kind of acute infectious disease, comprises the paratyphoid that typhoid fever that salmonella typhi (Salmonella Typhi) causes and first, second, moscow' paratyphi C (Salmonellaparatyphi A, Band C) cause.Typical clinical manifestation comprises that lasting high heat, apathetic expression, abdominal discomfort, hepatosplenomegaly and surrounding hemogram white corpuscle are low, and part patient has roserash and relative infrequent pulse.Typhoid fever also can cause Gao Re and intestinal bleeding, has very high infectivity.The crowd is to the general susceptible of Salmonella paratyphi A, but children and between twenty and fifty sickness rate are the highest.The annual number of suffering from typhoid fever (comprising paratyphoid) in the whole world is about 2,200 ten thousand according to estimates, and mortality ratio surpasses 0.9%.And in recent years,, cause the Pparatyphoid A outbreak of epidemic because strain of Salmonella paratyphi A clinical drug-resistant and multidrug resistant strain extensively exist.
Salmonella paratyphi A is acapsular Gram-negative bacteria, and its outer membrane protein is having very important effect aspect pathogenic and the stimulation immune response.Studies show that the Salmonellas outer membrane protein has good immunogenicity and immune protective.PagC albumen, its genome annotation is the adventitia invasin protein, be the outer membrane protein by a 18kDa of pagC genes encoding, ubiquity in several antityphoid sera type of Salmonellas bacterium is that Salmonellas survives in scavenger cell and show the principal element of virulence in mouse.This albumen also can increase the immunoreactivity of animal body to bacterium in the cholera Salmonellas.Choose the component of PagC, can cause efficient immune, the infection of opposing Salmonella paratyphi A as subunit vaccine.But less to this research both at home and abroad, known PagC kind is less.Up to the present, also PagC albumen is not used to prepare the report of Salmonella paratyphi A vaccine.
Summary of the invention
The objective of the invention is to, a kind of new Salmonella paratyphi A outer membrane protein PagC is provided;
Second purpose of the present invention is to provide the application of this albumen in preparation Salmonella paratyphi A subunit vaccine.
The 3rd purpose of the present invention is to provide a kind of Salmonella paratyphi A subunit vaccine and preparation method thereof.
The aminoacid sequence of paratyphosus A bacillus outer membrane protein PagC of the present invention is shown in SEQID No.2, and experiment shows that this albumen has good immune protection, can be used to prepare medicine, the especially vaccine of prevention or treatment Pparatyphoid A.In addition, because PagC has immunity neutralization activity, can also prepare antibody drug by the specific antibody of preparation PagC.
The present invention's also be provided for encoding gene of PagC, its nucleotide sequence is shown in SEQ IDNo.1.Consider the degeneracy of codon, for example can be in its coding region, under the condition that does not change aminoacid sequence, or at its non-coding region under the condition that does not influence protein expression, above-mentioned proteic gene order is made amendment to encoding.Therefore, the present invention also comprises replacement, the interpolation that the above-mentioned proteic gene order of encoding is carried out and/or lacks one or more Nucleotide, has the nucleotide sequence that has identical function with above-mentioned encoding gene.The present invention also comprises just sequence or the antisense sequences based on described gene, comprises the host cell that contains described nucleotide sequence or its segmental cloning vector or expression vector, contains described carrier.
The present invention also provides preparation to reach the method for Salmonella paratyphi A outer membrane protein PagC, comprising: the gene clone of the PagC that will encode transforms the host bacterium to expression vector, cultivates transformant, obtains the target protein of reorganization.
Described expression vector is selected from plasmid pET30a and is applicable to all pET series expression vectors of intestinal bacteria system.
Described escherichia coli expression bacterial strain is selected from E.coli BL21 (DE3), E.coliBL21 (DE3) pLys, Origami or Rosetta.
The conversion of stating transforms with heat-shocked, and electricity transforms or protoplast transformation.
Described PagC gene clone to plasmid pET30a, is obtained recombinant expression plasmid, and heat-shocked method Transformed E .coli BL21 (DE3) then is through fermentation culture, broken bacterium, centrifugal, PagC recombinant protein that column chromatography purification obtains recombinating.
In a minute embodiment of invention, adopt following method to prepare outer membrane protein PagC:
(1) in Bacillus coli cells, carries out expressing PagC in the born of the same parents
The PagC gene is passed through pcr amplification, and introduce Nde I and Xho I restriction enzyme site and 6 * HIS label, on the expression vector pET30a of the Bacillus coli cells of recombinating behind the double digestion, form recombinant expression vector PagC/pET30a.By heat-shocked method conversion CaCl
2The E.coli DH5 α of preparation, after the checking of extracting plasmid and double digestion is correct, again with recombinant plasmid transformed E.coliBL21 (DE3) to be used for abduction delivering PagC albumen.Contain the further liquid fermenting of intestinal bacteria transformant of goal gene and use the IPTG abduction delivering.Collect and broken Bacillus coli cells, SDS-PAGE and Western blot identify the molecular weight and the immunogenicity of recombinant protein.(2) purifying of the bulk fermentation of recombinant bacterial strain PagC/pET30a/BL21 and target protein thereof
In the 2L triangular flask, recombinant bacterial strain is carried out a large amount of abduction deliverings.Determine the expression-form of target protein, and calculate the expression rate and the productive rate of target protein; Adopt ultransonic mode to break bacterium, make inclusion body through the method for gradient centrifugation; Select the affine prepacked column of Ni of GE company to carry out purifying.Under 4 ℃, the method that adopts gradient dialysis renaturation is to the good metaprotein renaturation of purifying;
In addition; also further carry out the immune effect that the proteic animal immune of PagC is determined PagC: with PagC albumen associating aluminium adjuvant immunity BALB/c mouse; and adopt the wild-type Salmonella paratyphi A to attack malicious mouse; the ELISA method is surveyed the protection ratio of serum total Ig G antibody and statistics candidate vaccine, estimates the immune effect of this vaccine.
Compared with prior art, the present invention has following beneficial effect:
(1) in the clinical trial at the Salmonella paratyphi A vaccine, do not use the proteic relevant report of PagC both at home and abroad basically separately.The present invention uses the main ingredient of PagC as subunit vaccine, utilizes the intestinal bacteria system to obtain to efficiently express.
(2) the reorganization PagC albumen of genetic engineering means structure does not contain bigger fusion tag, only contains 6 * HIS label and is convenient to the later stage purification process.This label can not influence the immunogenicity of PagC albumen itself basically.
(3), can improve the expression level of protein vaccine at the PagC of expression in escherichia coli Salmonella paratyphi A albumen.Because intestinal bacteria are simple in structure, growth fast, be easy to cultivation and fermentation, low, the target protein output height of production cost, this has important practical significance for large-scale production and clinical application thereof.
Description of drawings
Fig. 1 is that PCR obtains purpose fragment (M:DNA Marker DL2000; 1: negative control; The 2:PagC gene fragment);
Fig. 2 is that recombinant plasmid PagC/pET30a double digestion is identified (M:DNA MarkerDL2000; The Nde I of 1:PagC/pET30a and Xho I double digestion);
Fig. 3 is the expression (M: molecular weight of albumen standard that SDS-PAGE detects reorganization PagC; 1: the pET30a/BL21 after inducing; 2: inductive PagC/pET30a/BL21 not; 3: the PagC/pET30a/BL21 after inducing);
Fig. 4 is that Western blot detects reorganization PagC (M: molecular weight of albumen standard; 1: reorganization PagC albumen);
Fig. 5 is that SDS-PAGE detects the proteic purification result of PagC (M: molecular weight of albumen standard; 1: purification of samples not; 2: stream is worn the peak; 3:100mM imidazoles elution peak; 4:1M imidazoles elution peak);
Fig. 6 is a PagC protein-specific IgG change curve in the different time mice serum after the aluminium adjuvant immunity.
Embodiment
Below in conjunction with concrete accompanying drawing, further set forth the present invention with embodiments of the invention.These embodiment only are used to illustrate the present invention, and scope of the present invention are not constituted any restriction.The main genetically engineered molecular biology cloning process that adopts routine among the embodiment, these methods are well known to those of ordinary skill in the art.According to following examples, the slightly modified and conversion be not difficult as the case may be and successfully implement the present invention, these are revised and conversion all drops in the scope of the application's claim.
Expression vector is selected from plasmid pET30a and is applicable to all pET series expression vectors of intestinal bacteria system.The escherichia coli expression bacterial strain is selected from E.coli BL21 (DE3), E.coliBL21 (DE3) pLys, Origami or Rosetta.
In the present embodiment, be example only with the aluminium adjuvant, the immunologic process of recombinant protein of the present invention is described, but is not limited to this adjuvant that freund's adjuvant is also within the scope of embodiment.
Embodiment 1: Salmonella paratyphi A outer membrane protein PagC vivoexpression
(1) acquisition of Salmonella paratyphi A membranin PagC gene
With Salmonella paratyphi A 50973 bacterial strains (derive from: genome Chinese medicine bacterium preservation administrative center) is as template, entrusts the synthetic upstream and downstream primer of DNA Synesis Company:
PagC-f：5-GGGAATTC
CATATGGATACTAACGCCTTTTCCGTGG-3′；
PagC-r：5′-CCG
CTCGAGTTAGTGGTGGTGGTGGTGGTGGAAACGGTATCCAACTCCG-3′。
Wherein, PagC-f, the PagC-r PagC protein gene that is used to increase, and introduce Nde I and Xho I restriction enzyme site and 6 * HIS label.Use Taq plus enzyme to carry out pcr amplification: 94 ℃ of pre-sex change of 10min; 30 circulations (94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of lmin); 72 ℃ of 30min.Identify amplified production with 1% agarose gel electrophoresis.
(2) structure of recombinant expressed bacterium PagC/pET30a/BL21
Cut 2 hours in 37 ℃ of enzymes with Nde I and Xho I respectively after the PagC isogeneity, reclaim the purifying fragment, be connected in 16 ℃ with the T4DNA ligase enzyme with the expression vector pET30a double digestion product of purifying and spend the night, connect product by heat-shocked method conversion CaCl
2The E.coli DH5 α of preparation, coating Kan
+-LB flat board was cultivated several single colony inoculation Kan of picking 16 hours for 37 ℃
+-LB nutrient solution, 37 ℃ of shaking culture 12 hours, alkaline lysis extracting plasmid, Nde I and Xho I double digestion plasmid are identified the PagC gene that contains the expection size, and correct by professional dna sequencing company sequencing result sequence.Make up correct recombinant plasmid adopt aforesaid method once more Transformed E .coli BL21 (DE3) be used for abduction delivering PagC albumen.
(3) PagC induction expression of protein
The single colony inoculation of picking recombinant bacterial strain PagC/pET30a/BL21 is to Kan respectively
+In-LB the nutrient solution, 37 ℃ of concussion overnight incubation were inoculated into fresh Kan respectively by 1% in second day
+In-LB the nutrient solution, 37 ℃ of concussions are cultured to OD600 and are about at 1.0 o'clock, add 0.5mmolIPTG respectively 37 ℃ of abduction deliverings 4 hours.Induce and get an amount of sample after the end and carry out SDS-PAGE and detect, and with anti-his as an anti-Western blot evaluation.The result shows: the proteic apparent molecular weight of PagC is 18.6KDa, with the calculated value basically identical.
In the present embodiment, only with pET30a, E.coli BL21 (DE3) and heat-shocked conversion method are example, the building process of recombinant protein of the present invention is described, but be not limited to this carrier, host bacterium and method for transformation, other pET serial carrier, host bacterium E.coli BL21 (DE3) pLys, Origami, Rosetta and other electrotransformation or protoplast transformation method are all within the scope of embodiment.
Embodiment 2: the purifying of recombinant bacterial strain PagC/pET30a/BL21 bulk fermentation and target protein thereof
(1) recombinant bacterial strain bulk fermentation
Control bacterium liquid OD600 value is about at 0.8 o'clock, adds 1mmol/L IPTG, and 37 ℃ of temperature were fermented 6 hours.The expression rate of target protein is about 40.5%, and productive rate is 153mg/L, the inclusion body expression-form.
(2) preparation of PagC inclusion body
The centrifugal 10min of bacterium liquid 10000g after inducing, the bacterium precipitation is resuspended with Tris damping fluid (0.05mol/L NaCl, 0.02mol/L Tris-Cl), ice-water bath carrying out ultrasonic bacteria breaking (work 5s suspends 10s for broken bacterium time 15min, power 100W).Ultrasonic back liquid is after 800g and 18000g two times centrifugal, and gained 18000g centrifugation is the PagC inclusion body.
(3) proteic purifying of PagC and renaturation
Select the affine prepacked column of Ni of GE company that inclusion body is carried out purifying.Inclusion body sex change liquid (0.1mol/L NaCl, 0.05mol/L Tris-Cl, 8M urea, 0.01mol/L imidazoles, lavation buffer solution (0.1mol/L NaCl is used in pH9.0) sex change then, 0.05mol/L Tris-Cl, 8M urea, 0.05mol/L imidazoles, pH9.0) the washing foreign protein re-uses elution buffer (0.1mol/L NaCl, 0.05mol/L Tris-Cl at last, 8M urea, 0.1mol/L imidazoles, pH9.0) wash-out target protein, SDS-PAGE detects, and purifying protein purity is 93%; Albumen behind the purifying adopts the method for gradient dialysis to carry out renaturation, under 4 ℃, respectively at urea dialyzate (the 0.05mol/L Tris-Cl of different concns, 10% glycerine, 8M-6M-4M-2M-0M urea, 1mMEDTA, pH9.0) leave standstill dialysis in, each gradient keeps 12h, arrive thoroughly more at last damping fluid (0.05mol/L Tris-Cl, pH9.0) in.Albumen is not separated out in the pilot process.
In the present embodiment, be example only with the Ni affinity chromatography, the purge process of recombinant protein of the present invention is described, but be not limited to this purification process, other metal ion-chelant chromatography purification method, molecular sieve purification method, ion exchange chromatography method of purification or hydrophobic chromatography method of purification are all within the scope of embodiment.
The proteic experimentation on animals of embodiment 3:PagC
(1) the proteic animal immune of PagC
BALB/c mouse is divided into 3 groups, and every group is 20 mouse.Every group is divided into two groups again, 10 of every groups, and blood is got by a group, and an other group attacks poison.Adopt the mode of intramuscular injection, respectively 0,2,4 weeks were carried out immunity.The A group is used PBS; The B group is used aluminium adjuvant; The C group is used PagC albumen+aluminium adjuvant.Immunizing dose is every mouse 0.2ml, contains antigen 10 μ g aluminium adjuvant 0.2mg.Back 10 days of each immunity, carry out the serum collection.The first, secondary adopts the blood sampling of angular oculi vein clump, adopts for the third time and plucks the eyeball blood sampling.Use the ELISA method to survey serum total Ig G antibody, and respectively organize the group difference of data with the SPSS software analysis.The result shows: the proteic IgG antibody titer of mouse anti PagC is very high.
(2) the poison protection of attacking of animal is tested
To be divided into 4 groups with 40 of the BALB/c mouse of batch raising, 10 every group.With physiological saline cultured Salmonella paratyphi A 50973 is washed, behind the mixing, than turbid definite bacteria concentration.Then be respectively 60,30 with concentration, the bacterium liquid of four concentration of 15,7.5 hundred million/ml is attacked poison, every mouse 0.5ml, abdominal injection.The death condition of record mouse is determined minimum absolute lethal dose after three days.
Table 1 pathogenic bacteria the determining of minimum absolute lethal dose in Balb/C mouse infection model
Annotate: reaching 100% minimum bacteria concentration with mortality ratio is minimum absolute lethal dose
As shown in Table 1, mouse is all dead when attacking poison with 3,000,000,000/ml bacterial concentration, determines that therefore 3,000,000,000/ml bacterial concentration is minimum absolute lethal dose.Immune the last time back 10 days, with minimum absolute lethal dose dosage, experimental group is attacked the poison experiment, write down the death condition of mouse after three days, determine the protection ratio of albumen PagC.
Table 2 pathogenic bacteria is attacked malicious experimental result to the Balb/C mouse of immunity
Annotate: (3,000,000,000/ml) attack poison to the Balb/C mouse to pathogenic bacteria with minimum absolute lethal dose
As can be seen from Table 2, attack poison after, blank and adjuvant group mouse are all dead, the proteic immune protective rate of PagC is 80%.
Experimentation on animals is the result show, the PagC recombination fusion protein has good immunogenicity.Later stage attacks the poison experiment and proves that then this fusion rotein has good immune protection power.
Claims (10)
1. Salmonella paratyphi A outer membrane protein PagC, its aminoacid sequence is shown in SEQ ID No.2.
2. the gene of coding claim 1 described outer membrane protein PagC.
3. gene as claimed in claim 2, its nucleotide sequence is shown in SEQ ID No.1.
4. the application of the described outer membrane protein PagC of claim 1 in preparation prevention or treatment Pparatyphoid A vaccine.
5. Salmonella paratyphi A PagC subunit vaccine, it comprises the described outer membrane protein PagC of claim 1 and the vaccine adjuvant of effective dose.
6. vaccine as claimed in claim 5 is characterized in that, described vaccine adjuvant is aluminium adjuvant or Fei Shi adjuvant.
7. method for preparing the described outer membrane protein PagC of claim 1, it comprises step: the described gene clone of claim 2 to expression vector, is transformed the host bacterium, cultivate transformant, obtain the target protein of reorganization.
8. method as claimed in claim 7, it is characterized in that, described host bacterium is intestinal bacteria E.coli BL21 (DE3), E.coli BL21 (DE3) pLys, Origami or Rosetta, and described expression vector is the expression vector that is applicable in described host bacterium expression system.
9. method as claimed in claim 7, it is characterized in that comprising the steps: with described PagC gene clone to plasmid pET30a, obtain recombinant expression plasmid, heat-shocked method Transformed E .coli BL21 (DE3) then is through fermentation culture, broken bacterium, centrifugal, PagC recombinant protein that column chromatography purification obtains recombinating.
10. method for preparing the described vaccine of claim 5, it comprises the described method of employing claim 7～9 is prepared outer membrane protein PagC, and it is mixed with an amount of immunological adjuvant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100786203A CN102212120A (en) | 2011-03-30 | 2011-03-30 | Salmonella paratyphi A PagC subunit vaccine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100786203A CN102212120A (en) | 2011-03-30 | 2011-03-30 | Salmonella paratyphi A PagC subunit vaccine and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102212120A true CN102212120A (en) | 2011-10-12 |
Family
ID=44743684
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011100786203A Pending CN102212120A (en) | 2011-03-30 | 2011-03-30 | Salmonella paratyphi A PagC subunit vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102212120A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290918A (en) * | 2016-08-24 | 2017-01-04 | 南京农业大学 | A kind of ELISA kit detecting antibodies toward salmonella |
| CN108359682A (en) * | 2018-02-09 | 2018-08-03 | 河北科技师范学院 | A kind of Wdwardsiella tarda outer membrane protein PagC with immanoprotection action |
| CN112546210A (en) * | 2020-12-15 | 2021-03-26 | 南京农业大学 | Preparation method and application of salmonella inactivated vaccine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318646A (en) * | 2000-02-28 | 2001-10-24 | 圣斯马来西亚大学 | Coded salmonella typhi specific and antigenic outer membrane protein DNA sequence |
-
2011
- 2011-03-30 CN CN2011100786203A patent/CN102212120A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318646A (en) * | 2000-02-28 | 2001-10-24 | 圣斯马来西亚大学 | Coded salmonella typhi specific and antigenic outer membrane protein DNA sequence |
Non-Patent Citations (3)
| Title |
|---|
| HAMID N ET AL: "Characterization of an outer membrane protein of S almonel la enterica serovar typhimurium that confers protection against Typhoid", 《CLINICAL AND VACCINE IMMUNOLOGY》 * |
| MCCLELLAND M ET AL: "outer membrane invasion protein [Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150]，GenBank: AAV77526，185 aa linear", 《NCBI GENBANK》 * |
| 刘春梅: "甲型副伤寒沙门氏菌外膜蛋白的表达及其免疫保护性研究", 《《中国优秀硕士学位论文全文数据库，医药卫生科技辑，南昌大学硕士学位论文》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290918A (en) * | 2016-08-24 | 2017-01-04 | 南京农业大学 | A kind of ELISA kit detecting antibodies toward salmonella |
| CN108359682A (en) * | 2018-02-09 | 2018-08-03 | 河北科技师范学院 | A kind of Wdwardsiella tarda outer membrane protein PagC with immanoprotection action |
| CN108359682B (en) * | 2018-02-09 | 2021-08-20 | 河北科技师范学院 | Edwardsiella tarda outer membrane protein PagC with immune protection effect |
| CN112546210A (en) * | 2020-12-15 | 2021-03-26 | 南京农业大学 | Preparation method and application of salmonella inactivated vaccine |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103172749B (en) | Preparation of African swine fever protein engineering vaccine | |
| CN103585625B (en) | A kind of porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine and preparation method thereof and application | |
| CN107899008B (en) | Sick three subunit vaccines of a kind of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus | |
| CN107337718A (en) | A kind of gene for encoding carrying Cap gene of porcine circovirus type 2 and its application | |
| CN102816246B (en) | Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof | |
| CN114807060B (en) | Coxsackie virus A6 type strain and immunogenic composition and application thereof | |
| CN104628871B (en) | A kind of preparation for recombinating bursal disease protein engineering vaccine | |
| CN102210860B (en) | Mycobacterium tuberculosis TB10.4-F1 fusion protein vaccine and preparation method thereof | |
| CN109136198A (en) | A kind of expression Chicken Infectious Anemia Virus VP1, VP2 genetic recombination bird pox virus live vector vaccine | |
| CN102580076A (en) | Synthetic peptide vaccine for O-type foot and mouth disease of swine and preparation method thereof | |
| CN102127554B (en) | Japanese encephalitis particle vaccine and preparation method and application thereof | |
| CN109021115A (en) | A kind of pig circular ring virus trivalent subunit vaccine | |
| CN102212120A (en) | Salmonella paratyphi A PagC subunit vaccine and preparation method thereof | |
| CN110124025A (en) | A kind of bird flu and 4 type bigeminy genetic engineering subunit vaccine of aviadenovirus and preparation method thereof | |
| CN104250304B (en) | The vaccine combination of a kind of fusion protein and its coding and application | |
| CN113603754A (en) | Waterfowl H5N8 subtype influenza virus HA recombinant protein and preparation method and application thereof | |
| CN104894045A (en) | Recombinant lactobacillus for coexpression of foot and mouth disease virus VP1 gene and immunoadjuvant cattle IL-6 gene, and preparation method and application of recombinant lactobacillus | |
| CN104804099B (en) | A kind of reinforced polyepitope vaccines of recombination H9N2 subtype avian influenza | |
| CN110128545A (en) | A kind of fusion, recombinant expression carrier, antigen and its preparation method and application | |
| CN102311957A (en) | Hydatidovis soluble antigen preparation method and product thereof | |
| CN102206258A (en) | NmpC subunit vaccine of salmonella paratyphi A and preparation method thereof | |
| CN107974458A (en) | Express gene gapdh and its recombination bacillus coli and the application of bacillus rhusiopathiae suis recombinant protein GAPDH | |
| CN108410784B (en) | Streptococcus suis delta CPS/SsnA-mSly (P353L) -SC19 engineering bacteria and application thereof in vaccines | |
| CN102604898B (en) | VP60 protein recombinant baculovirus carrying type O foot and mouth disease virus B cell epitope | |
| CN109266710A (en) | Production method of pig foot-and-mouth disease O-type genetic engineering composite epitope protein vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20111012 |