CN108641993A - A kind of recombinant lactic acid bacteria oral vaccine strain of food-grade expression bovine viral diarrhea virus E2 albumen - Google Patents
A kind of recombinant lactic acid bacteria oral vaccine strain of food-grade expression bovine viral diarrhea virus E2 albumen Download PDFInfo
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- CN108641993A CN108641993A CN201810474026.8A CN201810474026A CN108641993A CN 108641993 A CN108641993 A CN 108641993A CN 201810474026 A CN201810474026 A CN 201810474026A CN 108641993 A CN108641993 A CN 108641993A
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61K2039/541—Mucosal route
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- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention discloses the recombinant lactic acid bacteria oral vaccine strains that a kind of food-grade expresses bovine viral diarrhea virus E2 albumen.The present invention provides a kind of recombinant bacteriums, and specific DNA is imported in Lactococcus lactis and is obtained；The specific DNA includes the encoding gene of bovine viral diarrhea virus E2 albumen.The present invention is directed to the importance of BVDV mucosa-immunes, constructs the Recombinant Lactococcus lactis expression system of cell surface expression BVDV main protection antigen E2 albumen, and the development that live vector vaccine is taken orally for BVDV provides basis.
Description
Technical field
The present invention relates to the recombinant lactic acid bacteria oral vaccine strains that a kind of food-grade expresses bovine viral diarrhea virus E2 albumen.
Background technology
Bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV) belongs to flaviviridae
(Flavividae) pestivirus (Pestivirus), can cause ruminant virus diarrhea/mucosal disease (Bovine
Virus diarrhea-mucosal disease, BVD-MD), clinically with fever, mucosal erosion, ulcer, Neuroleptic Leukocytopenia,
Diarrhea, cow in calf miscarriage or production deformity fetus are main feature.This is reported for the first time after being ill from nineteen forty-six, it is effective due to lacking
Control Technology, it is generally popular in the whole world, great economic loss is caused to cattle-raising.
Vaccine immunization is to prevent the major measure of the disease, but inactivated vaccine effect is unstable, it is difficult to reach prevention
Purpose, there are dissipation virus, virus virulences to return many insecurity such as strong for attenuated live vaccines.Therefore, from BVDV through mucous membrane sense
The pathogenic angle of dye is set out, and the oral vaccine that developing can effectively stimulate mucosal immune system to generate immune response prevents the disease
Jig is significant.
In recent years, mucosal vaccine antigen delivery systems become the new technology hot spot in infectious disease defence field, study at present
Carrier has particulate carrier, bacteria carrier, viral vectors and plant vector, but is concentrated mainly in the bacterium bacterial strain of attenuation, i.e., living
Bacterium carrier.Viable bacteria carrier vaccine is the important component of mucosa-immune vaccine, and the hot spot studied now.Bacillus acidi lactici is made
It is the normal flora in animal and human body for the microorganism of aliment security level, can be long-term be colonized in body plays its promotion
Nutriment molecular degradation and absorption maintain bacterium colony balance in enteron aisle, adjust the prebiotic effects such as immune system effect.Therefore, with
Lactic acid bacteria is that oral vaccine is developed in transmission and expression system of the carrier as foreign gene, has important development prospect.
Invention content
The object of the present invention is to provide the recombinant lactic acid bacteria mouths that a kind of food-grade expresses bovine viral diarrhea virus E2 albumen
Take vaccine strain.
The present invention provides a kind of recombinant bacteriums, and specific DNA is imported in Lactococcus lactis and is obtained；The specific DNA
Include the encoding gene of bovine viral diarrhea virus E2 albumen.
The specific DNA includes the coding base of the coded sequence and bovine viral diarrhea virus E2 albumen of Usp45 signal peptides
Cause.
The sequence 1 of the encoding gene such as sequence table of the bovine viral diarrhea virus E2 albumen holds 106-1140 from 5 '
It is shown.
The specific DNA is by the coded sequence of Usp45 signal peptides, the coded sequence and bovine viral diarrhoea of 6 × His labels
The encoding gene of viral E2 albumen forms.
The sequence 1 of the coded sequence of the Usp45 signal peptides such as sequence table is from shown in 5 ' 7-87, ends.
The specific DNA is as shown in the sequence 1 of sequence table.
Protein shown in the sequence 2 of the specific DNA polynucleotide.
The specific DNA imports Lactococcus lactis by the expression vector containing specific DNA.
The expression vector is that will be inserted into the multiple cloning sites of pNZ8148 expression vectors to obtain containing the segment of specific DNA
It arrives.
The expression vector be using sequence table sequence 1 from 5 ' end 5-1140 shown in double chain DNA molecule replacement
Segment between I restriction enzyme site of Xba I and Nco of pNZ8148 expression vectors obtains recombinant expression carrier.
Any description above Lactococcus lactis concretely Lactococcus lactis NZ9000.
The present invention also protects the recombinant bacterium of any description above as the application in bovine viral diarrhea virus vaccines.
The immunization ways of the bovine viral diarrhea virus vaccines are administered by oral gavage.
The present invention also protects application of the recombinant bacterium of any description above in preparing bovine viral diarrhea virus vaccines.
The present invention also protects application of the recombinant bacterium of any description above in preparing product；The purposes of the product is pre-
Anti- bovine viral diarrhea virus infection.
It is the recombination of any description above that the present invention, which also protects a kind of bovine viral diarrhea virus vaccines, active constituent,
Bacterium.
It is the recombinant bacterium of any description above that the present invention, which also protects a kind of product, active constituent,；The purposes of the product
To prevent bovine viral diarrhea virus infection.
The present invention is directed to the importance of BVDV mucosa-immunes, constructs cell surface expression BVDV main protection antigens E2
The Recombinant Lactococcus lactis expression system of albumen, the development that live vector vaccine is taken orally for BVDV provide basis.
Description of the drawings
Fig. 1 is expression of the SDS-PAGE electrophoresis detection raq genes in lactic acid bacteria.
Fig. 2 is the Western-blot analyses of recombinant bacterium pNZ8148-E2/NZ9000 expression products.
Fig. 3 is the dynamic rule of BVDV E2 specific antibody titres in serum.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
PNZ8148 expression vectors：The Shanghai bio tech ltd Bei Nuo.
Lactococcus lactis NZ9000：The Shanghai bio tech ltd Bei Nuo.
Embodiment 1, express bovine viral diarrhea virus E2 albumen recombinant lactic acid bacteria structure
One, the structure of recombinant expression carrier
Using sequence table sequence 1 from 5 ' end 5-1140 shown in double chain DNA molecule replacement pNZ8148 expression vectors
I restriction enzyme site of Xba I and Nco between segment, obtain recombinant expression carrier (sequence verification).
Insetion sequence merges formation sequence 1 with carrier sequence.In the sequence 1 of sequence table, it is from 5 ' the 7th to 87, ends
The coded sequence of Usp45 signal peptides, the 88th to 105 coded sequence for 6 × His labels, after the 106th to 1140 is optimization
Raq gene sequence.Raq gene after optimization is more suitable for expressing in lactic acid bacteria.
Protein shown in DNA molecular coded sequence 2 shown in the sequence 1 of sequence table.In the sequence 2 of sequence table, the 3rd to
29 are Usp45 signal peptides, and the 30th to 35 is 6 × His, and the 36th to 379 is E2 albumen.
Two, the structure of recombinant lactic acid bacteria and protein expression identification
1, the recombinant expression carrier for obtaining step 1 imports Lactococcus lactis NZ9000, obtains recombinant bacterium pNZ8148-
E2/NZ9000。
2, the recombinant bacterium that step 1 obtains is inoculated in 5ml and contains 0.5% (mass percentage) glucose and 10g/ml chlorine
In the M17 fluid nutrient mediums of mycin, 30 DEG C, 150rpm is incubated overnight.
3, after completing step 2, recombinant bacterium bacterium solution is pressed 1:25 be inoculated in containing 0.5% (mass percentage) glucose and
In the M17 fluid nutrient mediums of 10g/ml chloramphenicol, 30 DEG C, 150rpm cultivates to bacterium solution OD600nmAbout 0.4.
4, after completing step 3,1ml cultures are taken out, 10000r/min room temperatures centrifuge 2min, and taking supernatant, (recombinant bacterium lures
Lead 0h supernatants), 100 μ L 1 × SDS-PAGE albumen sample-loading buffers of precipitation are resuspended (recombinant bacterium induces the full bacterium of 0h).It is remaining
Streptococcus lactis peptide (Nisin) (a concentration of 1ng/ml in cultivating system), 30 DEG C of culture different times are added in cultivating system
(2h, 4h or 8h), induced fusion protein expression take out 1ml cultures (recombinant bacterium induces 2,4 and 8h supernatants), 10000r/min
Room temperature centrifuges 2min, takes supernatant, 100 μ L1 × SDS-PAGE albumen sample-loading buffers of precipitation that (recombinant bacterium induction 2,4 is resuspended
With the full bacterium of 8h).
5, recombinant bacterium is substituted using Lactococcus lactis NZ9000, is operated according to step 2-4, obtains NZ9000 bacterial strains not
Induction supernatant, NZ9000 bacterial strains do not induce full bacterium and the full bacterium of NZ9000 bacterial strain inducings (induction 4h).
6, recombinant expression carrier is substituted using pNZ8148 expression vectors, is operated according to step 1-5, obtains recombinant bacterium
pNZ8148/NZ9000。
Sample in the above process is subjected to SDS-PAGE detection and analysis, coomassie brilliant blue staining shows band.
The results are shown in Figure 1.In Fig. 1, M:Protein standard；Swimming lane 1：NZ9000 bacterial strains do not induce supernatant to train
Nutrient solution；Swimming lane 2：NZ9000 bacterial strains do not induce full bacterium；Swimming lane 3：The full bacterium of NZ9000 bacterial strain inducings；4-7：PNZ8148-E2 positive bacterias
0,2,4 and 8h supernatant culture solutions after strain induction；8-11:The full bacterium of 0,2,4 and 8h after the induction of pNZ8148-E2 positive strains.
The result shows that recombinant protein is expressed in cellular lysate object, expressed for non-secretory, albumen size is about
41.69KD expressed albumen is consistent with expected size.
7, the sample in the above process is subjected to western detection and analysis.
Primary antibody is BVDV E2 rabbit polyclonal antibody (Biorbyt companies, article No.；Orb312169), dilution ratio 1；
2000。
Secondary antibody is goat anti-rabbit igg H＆L (HRP) (Abcam companies, article No.：Ab6721), dilution ratio 1:5000.
As a result see Fig. 2.In Fig. 2, swimming lane 1 is the full bacterium of pNZ8148/NZ9000 that 4h is induced through Nisin；Swimming lane 2 is warp
Nisin induces the full bacterium of recombinant bacterium pNZ8148-E2/NZ9000 of 4h；M is protein standard；The result shows that induction
Recombination pNZ8148-E2/NZ9000 bacterial strains have apparent band at 41.69KD afterwards, the size phase with expected recombination E2 albumen
Symbol illustrates that the albumen of expression has good reactionogenicity as native protein.
Embodiment 2, animal immune experiment
Experimental animal：Screen the healthy calf that 6-12 monthly age BVDV antibody tests are negative in Shihezi of Xinjiang cattle farm.
BVDV antibody uses bovine viral diarrhea virus (BVDV) detection kit (IDEXX, product identification:99-44000).
1, experimental animal is randomly divided into two groups (every group 6), point circle isolated rearing.
I (immune group) of group：Recombinant bacterium pNZ8148-E2/NZ9000 prepared by administered by oral gavage embodiment 1, dosage is each
20ml/ heads, a concentration of the 1 × 10 of recombinant bacterium pNZ8148-E2/NZ900010CFU/mL.It is immunized 2 times altogether, each continuous immunity 2
It, is spaced 15 days.
II (control group) of group：Recombinant bacterium pNZ8148/NZ9000 prepared by administered by oral gavage embodiment 1, dosage is each
20ml/ heads, a concentration of the 1 × 10 of recombinant bacterium pNZ8148/NZ900010CFU/mL.It is immunized 2 times altogether, each continuous immunity 2 days,
Every 15 days.
2,0th, 7,21,36,60, the 90 progress venous blood collection after immune, detaches serum, using using bovine viral
Diarrhea virus (BVDV) detection kit (IDEXX) detects the antibody in serum (detection method is with reference to kit specification).
The results are shown in Figure 3.The result shows that head exempts from the 7th day afterwards, recombinant bacterium pNZ8148-E2/NZ9000 immune groups have anti-
The serum antibody of the serum antibody of BVDV, then antibody titer gradually rise, the 21st day with non-immunized controls group significant difference (P<
0.05), the anti-BVDV E2 protein antibodies potency of the 36th day serum reaches highest, difference extremely significantly (P<0.01).It is opened after the 36th day
Begin to decline.
Sequence table
<110>Xinjiang Academy of Land Reclamation ＆. Cultivation
<120>A kind of recombinant lactic acid bacteria oral vaccine strain of food-grade expression bovine viral diarrhea virus E2 albumen
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1140
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atgggcaaaa aaaagattat ctcagctatt ttaatgtcta cagtgatact ttctgctgca 60
gccccgttgt caggtgttta cgctatgcat caccatcatc atcatcattt ggattgtaag 120
ccagaatttt catatgctat tgctaaagat gaacgtattg gtcaattagg tgctgaaggt 180
ttaactacta cttggaagga atattcacca ggtatgaaat tagaagatac tatggttatt 240
gcttggtgtg aagatggtaa attaatgtat ttgcaacgtt gtactcgtga aactcgttat 300
ttggctattt tacatactcg tgctttacca acttcagttg tttttaaaaa gttgttcgac 360
ggtcgtaaac aagaagatgt tgttgaaatg aatgacaatt tcgaattcgg tttatgtcca 420
tgtgatgcta agccaattgt tcgtggtaaa ttcaatacta ctttgttaaa cggtccagct 480
tttcaaatgg tttgtccaat tggttggact ggtactgttt catgtacttc attcaatatg 540
gatactttgg ctactactgt tgttcgtact tatcgtcgtt caaagccatt tccacatcgt 600
caaggttgta ttactcaaaa gaatttgggt gaagatttgc ataattgtat tttgggtggt 660
aattggactt gtgttccagg tgaccaatta ttatataaag gtggttcaat tgaaagctgt 720
aaatggtgtg gttatcaatt caaagaatca gaaggtttac cacattatcc aattggtaaa 780
tgtaagttag aaaacgaaac tggttaccgt ttagttgatt caacttcatg taatcgtgaa 840
ggtgttgcta ttgttccaca aggtacttta aaatgtaaga ttggtaaaac tactgttcaa 900
gttattgcta tggatactaa gttaggtcca atgccatgtc gtccatatga aattatttca 960
tcagaaggtc cagttgaaaa aactgcttgt acttttaatt acactaagac tttgaagaac 1020
aagtactttg aaccacgtga ttcatatttt caacaatata tgttgaaggg tgaataccaa 1080
tattggtttg atttggaagt tactgatcat catcgtgatt attttgctga atcaatttaa 1140
<210> 2
<211> 379
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 2
Met Gly Lys Lys Lys Ile Ile Ser Ala Ile Leu Met Ser Thr Val Ile
1 5 10 15
Leu Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala Met His His His
20 25 30
His His His His Leu Asp Cys Lys Pro Glu Phe Ser Tyr Ala Ile Ala
35 40 45
Lys Asp Glu Arg Ile Gly Gln Leu Gly Ala Glu Gly Leu Thr Thr Thr
50 55 60
Trp Lys Glu Tyr Ser Pro Gly Met Lys Leu Glu Asp Thr Met Val Ile
65 70 75 80
Ala Trp Cys Glu Asp Gly Lys Leu Met Tyr Leu Gln Arg Cys Thr Arg
85 90 95
Glu Thr Arg Tyr Leu Ala Ile Leu His Thr Arg Ala Leu Pro Thr Ser
100 105 110
Val Val Phe Lys Lys Leu Phe Asp Gly Arg Lys Gln Glu Asp Val Val
115 120 125
Glu Met Asn Asp Asn Phe Glu Phe Gly Leu Cys Pro Cys Asp Ala Lys
130 135 140
Pro Ile Val Arg Gly Lys Phe Asn Thr Thr Leu Leu Asn Gly Pro Ala
145 150 155 160
Phe Gln Met Val Cys Pro Ile Gly Trp Thr Gly Thr Val Ser Cys Thr
165 170 175
Ser Phe Asn Met Asp Thr Leu Ala Thr Thr Val Val Arg Thr Tyr Arg
180 185 190
Arg Ser Lys Pro Phe Pro His Arg Gln Gly Cys Ile Thr Gln Lys Asn
195 200 205
Leu Gly Glu Asp Leu His Asn Cys Ile Leu Gly Gly Asn Trp Thr Cys
210 215 220
Val Pro Gly Asp Gln Leu Leu Tyr Lys Gly Gly Ser Ile Glu Ser Cys
225 230 235 240
Lys Trp Cys Gly Tyr Gln Phe Lys Glu Ser Glu Gly Leu Pro His Tyr
245 250 255
Pro Ile Gly Lys Cys Lys Leu Glu Asn Glu Thr Gly Tyr Arg Leu Val
260 265 270
Asp Ser Thr Ser Cys Asn Arg Glu Gly Val Ala Ile Val Pro Gln Gly
275 280 285
Thr Leu Lys Cys Lys Ile Gly Lys Thr Thr Val Gln Val Ile Ala Met
290 295 300
Asp Thr Lys Leu Gly Pro Met Pro Cys Arg Pro Tyr Glu Ile Ile Ser
305 310 315 320
Ser Glu Gly Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Thr Lys
325 330 335
Thr Leu Lys Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Gln Gln
340 345 350
Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Glu Val Thr
355 360 365
Asp His His Arg Asp Tyr Phe Ala Glu Ser Ile
370 375
Claims (10)
1. specific DNA is imported in Lactococcus lactis and is obtained by recombinant bacterium；The specific DNA includes bovine viral diarrhea
The encoding gene of malicious E2 albumen.
2. recombinant bacterium as described in claim 1, it is characterised in that：The specific DNA includes the coded sequence of Usp45 signal peptides
With the encoding gene of bovine viral diarrhea virus E2 albumen.
3. recombinant bacterium as claimed in claim 1 or 2, it is characterised in that：The coding of the bovine viral diarrhea virus E2 albumen
The sequence 1 of gene such as sequence table is from shown in 5 ' end 106-1140.
4. recombinant bacterium as claimed in claim 3, it is characterised in that：The specific DNA is as shown in the sequence 1 of sequence table.
5. the recombinant bacterium as described in claims 1 to 3 is any, it is characterised in that：The specific DNA is by containing specific DNA
Expression vector imports Lactococcus lactis, and the expression vector is that will be inserted into pNZ8148 expression vectors containing the segment of specific DNA
It is obtained in multiple cloning sites.
6. any recombinant bacterium of claim 1 to 5 is as the application in bovine viral diarrhea virus vaccines.
7. application of any recombinant bacterium of claim 1 to 5 in preparing bovine viral diarrhea virus vaccines.
8. application of any recombinant bacterium of claim 1 to 5 in preparing product；The purposes of the product is to prevent cattle disease
Viral diarrhea virus infects.
9. a kind of bovine viral diarrhea virus vaccines, active constituent is any recombinant bacterium of claim 1 to 5.
10. a kind of product, active constituent is any recombinant bacterium of claim 1 to 5；The purposes of the product is to prevent
Bovine viral diarrhea virus infects.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109718369A (en) * | 2019-02-26 | 2019-05-07 | 海南大学 | The preparation and application of the fish Vibrio harveyi oral DNA vaccine of probiotics transmitting |
| CN110746495A (en) * | 2019-10-31 | 2020-02-04 | 石河子大学 | Recombinant protein E2 and application thereof |
| CN112961224A (en) * | 2021-03-26 | 2021-06-15 | 中国农业大学 | Preparation and application of bovine viral diarrhea virus type 1 virus-like particles |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001000234A3 (en) * | 1999-06-25 | 2001-08-02 | Maxygen Inc | Methods and compositions for engineering of attenuated vaccines |
| CN103656633A (en) * | 2012-09-10 | 2014-03-26 | 刘占良 | Food grade lactic acid bacteria active carrier Group A rotavirus vaccine and preparation method thereof |
| CN103881956A (en) * | 2014-03-12 | 2014-06-25 | 中国药科大学 | Helicobacter pylori-resistant vaccine by taking lactococcus lactis as carrier |
| CN105924506A (en) * | 2016-07-04 | 2016-09-07 | 中国兽医药品监察所 | Preparation method of bovine viral diarrhea virus E2 protein subunit vaccine |
| CN107973841A (en) * | 2016-12-23 | 2018-05-01 | 浙江海隆生物科技有限公司 | Preparation method and application of recombinant bovine viral diarrhea virus E2 protein expressed by CHO (Chinese hamster ovary) cell and subunit vaccine |
-
2018
- 2018-05-17 CN CN201810474026.8A patent/CN108641993A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001000234A3 (en) * | 1999-06-25 | 2001-08-02 | Maxygen Inc | Methods and compositions for engineering of attenuated vaccines |
| CN103656633A (en) * | 2012-09-10 | 2014-03-26 | 刘占良 | Food grade lactic acid bacteria active carrier Group A rotavirus vaccine and preparation method thereof |
| CN103881956A (en) * | 2014-03-12 | 2014-06-25 | 中国药科大学 | Helicobacter pylori-resistant vaccine by taking lactococcus lactis as carrier |
| CN105924506A (en) * | 2016-07-04 | 2016-09-07 | 中国兽医药品监察所 | Preparation method of bovine viral diarrhea virus E2 protein subunit vaccine |
| CN107973841A (en) * | 2016-12-23 | 2018-05-01 | 浙江海隆生物科技有限公司 | Preparation method and application of recombinant bovine viral diarrhea virus E2 protein expressed by CHO (Chinese hamster ovary) cell and subunit vaccine |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: "登录号：M60178.1", 《GENBANK》 * |
| GENPEPT: "登录号：NP_776263.1", 《GENPEPT》 * |
| 齐琳琳: "口蹄疫乳酸乳球菌活载体疫苗的构建及黏膜免疫效果", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109718369A (en) * | 2019-02-26 | 2019-05-07 | 海南大学 | The preparation and application of the fish Vibrio harveyi oral DNA vaccine of probiotics transmitting |
| CN110746495A (en) * | 2019-10-31 | 2020-02-04 | 石河子大学 | Recombinant protein E2 and application thereof |
| CN112961224A (en) * | 2021-03-26 | 2021-06-15 | 中国农业大学 | Preparation and application of bovine viral diarrhea virus type 1 virus-like particles |
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