CN105348376A - Trichinella spiralis recombinant antigen and application thereof - Google Patents
Trichinella spiralis recombinant antigen and application thereof Download PDFInfo
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- CN105348376A CN105348376A CN201510630076.7A CN201510630076A CN105348376A CN 105348376 A CN105348376 A CN 105348376A CN 201510630076 A CN201510630076 A CN 201510630076A CN 105348376 A CN105348376 A CN 105348376A
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- trichinella spiralis
- recombinant antigen
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
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- 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
- A61K39/0003—Invertebrate antigens
Abstract
The present invention provides a trichinella spiralis recombinant antigen which comprises amino-acid sequences encoded by trichinella spiralis aquaporin (TsAQP) gene opening reading frame gene sequences. The present invention also provides a corresponding vaccine and an application thereof. The recombinant antigen provided by the present invention has excellent reactogenicity and immunogenicity, can be used for preparing vaccines used for preventing trichinella spiralis infection, and has excellent immune effects.
Description
Technical field
The present invention relates to a kind of Trichinella spiralis recombinant antigen and application thereof.
Background technology
Trichinella spiralis (Trichinellaspiralis) a kind ofly endangers serious food source property Zoonosis parasitic nematode, can infect more than the 150 kind of Mammals comprising the mankind, in worldwide distribution.Trichonematosis is listed in (all the other are cysticercosis and echinococcosis) first of three large food source property Amphixenosises in China.Since 1964, successively in Tibet, there is the trichinous outbreak of epidemic of people in Yunnan, Jilin, Heilungkiang, Guangxi, 15 provinces (city, autonomous region) such as Sichuan and Hubei, case reaches more than 38700 and rises, dead more than 330 people.In Animal husbandry production, pigs trichina disease not only can cause serious financial loss, and is an important indicator of meat product safety.China every year for the expense of pigs trichina disease inspection up to 1,500,000,000 yuan, but still have a large amount of Pork For Export to be return due to the undetected of this disease, the grievous injury international image of China's meat product security.
At present, for the parasitosis that this harm is serious, the imidazoles such as Top Form Wormer, Vermox is only had to treat, but can toxic side effect be caused, show as appetite stimulator that severity differs, heating, Nausea and vomiting, urticaria, dizzy, tremble, slightly have a sleepless night, alopecia and reversibility hepatic injury etc.At present, also not effective for preventing the method for the vaccine of Trichinella spiralis and the special of DIAGNOSIS OF TRICHINOSIS WITH.Therefore, the main research that new vaccine candidate antigen and drug target are this diseases of prevention and control is found.
Aquaporin (Aquaporins, AQPs) is called again hydrophilic pores albumen, is a kind ofly to be positioned on cytolemma, can the aquaporin of selectivity efficient transmembrane transport water molecules, belongs to major intrinsic protein (MajorIntrinsicProtein, MIP) family member.In erythrocytal plasmatic membranes first people in 1991 since clone, qualification AQP1, there is aquaporin in discovery in virus, bacterium, fungi, protozoon, worm, plant and Mammals at present.Aquaporin is in parasite except regulating Water permeability, and also participate in the processes such as the adaptation of parasite osmotic pressure, nutritive substance transhipment and metabolic waste discharge, research finds that parasite aquaporin can also participate in the transport of anti-parasite medicine.Aquaporin because playing an important role in parasite existence, and is expected to become parasiticide vaccine candidate molecule and drug target, and is subject to extensive concern.But, not yet have at present the research of Trichinella spiralis aquaporin (TsAQP) as vaccine candidate molecule and drug target.
Summary of the invention
The invention provides a kind of Trichinella spiralis recombinant antigen, it has good immunogenicity and reactionogenicity, can be used in the vaccine preparing prevention trichinzation.
The invention provides a kind of Trichinella spiralis recombinant antigen, it comprises the aminoacid sequence by Trichinella spiralis aquaporin (TsAQP) gene open reading frame gene sequences encode.
As preferably, it comprises the aminoacid sequence of sequence table SEQ IDNo.4.
The present invention also provides immunogenic composition, and it comprises the Trichinella spiralis recombinant antigen described in claim 1 or 2.
As preferably, described immunogenic composition also comprises adjuvant or medically acceptable carrier or vehicle; More preferably, described adjuvant is not formula Freund's complete adjuvant and Freund's incomplete adjuvant.
3rd object of the present invention is to provide a kind of vaccine, and the effective constituent of described vaccine is the Trichinella spiralis recombinant antigen described in claim 1 or 2 or the arbitrary described immunogenic composition of claim 3-6.
4th object of the present invention is the preparation method of above-mentioned vaccine, and step is as follows:
(1) Trichinella spiralis recombinant antigen is prepared; As preferably, with preparing Trichinella spiralis recombinant antigen in File of Newborn Larvae of Trichinella spiralis;
(2) by Trichinella spiralis recombinant antigen and adjuvant mixed preparation, vaccine is obtained.
5th object of the present invention is to provide the application in the medicine of preparation prevention trichinzation of above-mentioned Trichinella spiralis recombinant antigen, immunogenic composition.
6th object of the present invention is to provide the nucleic acid of Trichinella spiralis recombinant antigen.
7th object of the present invention is to provide the expression vector comprising nucleic acid described in claim 8.
8th object of the present invention is to provide the host cell comprising expression vector described in claim 9.
Recombinant antigen of the present invention has good reactionogenicity and immunogenicity, can be used in the vaccine preparing prevention trichinzation, has good immune effect.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, together with embodiments of the present invention for explaining the present invention, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the double digestion qualification of recombinant expression plasmid pET-30-NS1.Wherein, M:DNA molecular mass standard; 1:pET-30-NS1 recombinant expression plasmid; 2:pET-30-NS1 recombinant expression plasmid
ecorI and
xhoi double digestion is identified;
Fig. 2 is the TsAQPP1 recombinant protein electrophorogram after purifying; Wherein, M: protein molecular quality standard; 1-4: the TsAQPP1 recombinant protein after purifying;
Fig. 3 is the West-blotting qualification result of TsAQPP1 recombinant protein.Wherein, M: protein molecular quality standard; 1,3: vehicle control group mice serum; 2,4:TsAQPP1 recombinant protein immune serum;
Fig. 4 is that the indirect ELISA of immune serum antibody titers detects;
Fig. 5 is TsAQP gene and the reference gene expression at Trichinella spiralis different growing periods, and wherein, Fig. 5 A is TsAQP gene and the reference gene amplification curve in the cultivation of larvae of Trichinella spiralis from muscle phase; Fig. 5 B is TsAQP gene and the reference gene amplification curve in the Trichinella spiralis three age in days adult stage; Fig. 5 C is TsAQP gene and the reference gene amplification curve in the File of Newborn Larvae of Trichinella spiralis phase;
Fig. 6 is that TsAQP gene is at Trichinella spiralis different development stage differential expression situation histogram; Threshold value: 360.382.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is commercially available.
embodiment 1
1, the clone of Trichinella spiralis TsAQP gene open reading frame (OFR)
The extraction of 1.1 Trichinella spiralis RNA
The preparation of cultivation of larvae of Trichinella spiralis from muscle (ML): muscle larvae (ML) is preserved by Mice Body, by mouse trunk stomach en-and the digestion of 1% hydrochloric acid, then places pear shape bottle and makes its natural sedimentation; Be collected into by throw out in 50mL centrifuge tube, with normal saline flushing 3 times, abandon supernatant, precipitation is cultivation of larvae of Trichinella spiralis from muscle.
The preparation of Trichinella spiralis 3 age in days adult (Ad3): 3 age in days adults (Ad3) take from mouse small intestine.To infect the mouse lethal of muscle larvae 3d, vertical profile mouse small intestine, after cleaning 3 times, cuts off small intestine, invade in physiological saline with physiological saline, considers sieve, be positioned in plate through 50 orders.Place 6h at 37 DEG C, collect the physiological saline in plate, staticly settle, abandon supernatant, throw out is Ad3.
The preparation of File of Newborn Larvae of Trichinella spiralis (NBL): continued to be cultured to 7d in 37 DEG C of incubators by Ad3, filtered through 300 order screens by physiological saline, filtrate is newborn larvae (NBL).
Trizol method is utilized to extract Trichinella spiralis RNA.Step is: add 1mlTrizol after cultivation of larvae of Trichinella spiralis from muscle, 3 age in days adults and newborn larvae is centrifugal respectively, homogenizer is homogenate 5min on ice.Moved to by homogenate supernatant in new EP pipe, room temperature adds 0.2ml chloroform after placing 5min.By Trizol-chloroform mixture concuss 15s, after room temperature places 5min, in 4 DEG C, the centrifugal 15min of 12000g.After centrifugal, mixture is divided into three layers, is transferred in new EP pipe by upper strata water sample, adds isopyknic Virahol, after incubated at room 10min, in 4 DEG C, and the centrifugal 20min of 12000g.Abandon supernatant, add 1ml75% ethanol, vortex mixes, in 4 DEG C, and the centrifugal 5min of 7500g.Abandon supernatant, be placed in the air-dry about 10-30min of super clean bench room temperature, add the ddH of 30 μ l without RNA enzyme
2o ,-70 DEG C save backup.
The acquisition of 1.2 Trichinella spiralis cDNA
Utilize universal primer (poly thymidylic acid) respectively under 42 DEG C of constant temperature the total serum IgE of the cultivation of larvae of Trichinella spiralis from muscle (ML) extracted, 3 age in days adults (Ad3) and newborn larvae (NBL), reverse transcription 30min, acquisition Trichinella spiralis cDNA.
The amplification of 1.3TsAQP gene
The cultivation of larvae of Trichinella spiralis from muscle cDNA obtained with foregoing teachings, for template, carries out with upstream primer sequence AQPPF (5 ¢-GTGAGCAGAGCATATTCACA-3 ¢) and downstream primer sequence A QPPR (5 ¢-GGGGCTTTAGAAATGTGAGA-3 ¢) the open reading frame sequence ORF that pcr amplification obtains TsAQP gene.PCR reaction system: 10 × PCR damping fluid 5 μ L, dNTPmixture(2.5mmol/L) 4 μ L, primer (10 μm of ol/L) each 1 μ L, LATaqDNA polysaccharase (5U/ μ L) 0.5 μ L, templet gene group DNA1 μ L, adds ddH
2o to 50 μ L.Reaction conditions: 94 DEG C of 5min; 94 DEG C of 60s, 56 DEG C of 60s, 72 DEG C of 60s, 35 circulations; 72 DEG C of 10min.
The clone of 1.4TsAQP gene, sequential analysis
(1) the PCR primer DNA purification kit of above-mentioned 1.3 is carried out purifying (Beijing Tian Gen company DNA purification kit), concrete steps are shown in test kit specification sheets.
(2) connection of TsAQP gene and pMD-18T cloning vector
Linked system is: pMD18T cloning vector 1 μ L (50ng/ μ L), TsAQP gene PCR purified product 4 μ L, SolutionI5 μ L, mix rearmounted 16 DEG C and connect Transformed E .coliDH5 α competent cell after 4h, then coat on amicillin resistance LB flat board, 37 DEG C of overnight incubation.Select the single bacterium colony after conversion, carry out PCR positive identification.
(3) sequential analysis of TsAQP gene
Get positive recombinant bacterium respectively and serve the order-checking of Hai Shenggong order-checking company, sequencing result is see SEQ ID No .1, and aminoacid sequence of its coding is see SEQ ID No .2.Sequencing result is utilized Blast comparison.Online software MotifScansoftware sequence correct for comparison is utilized to carry out hydrophilicity analysis.By the hydrophilic region doped, after series connection (AAS1), add restriction enzyme site respectively at 5 ' end and 3 ' end
ecoRi/
xhosynthetic this section of sequence after I, is numbered NS1, and concrete sequence is see SEQ ID No .3, and the aminoacid sequence of its coding is see SEQ ID No .4.
2, the expression of TsAQP protein part fragment
By the NS1 gene product of above-mentioned synthetic and pET-30a plasmid, use respectively
ecoRi/
xhoi carries out double digestion, reclaim product to connect with T4DNA ligase enzyme, transform DH5 α competent cell, obtain recombinant expression plasmid pET-30-NS1, through double digestion and sequence verification correct after (Fig. 1), be transformed in e. coli bl21 competent cell, the single bacterium colony of picking, after 37 DEG C of incubated overnight, be forwarded to (containing penbritin 100 μ g/mL) in new LB substratum by 1:50 and carry out enlarged culturing, work as OD
600when being 0.6, getting 1mL and do not induce bacterium liquid as negative control, in residue bacterium liquid, add IPTG, make its final concentration be 1mmol/L, 37 DEG C, 200rpm, 4h, collect thalline, gets the bacterium liquid after 1mL induction.Residue bacterium liquid carries out collected by centrifugation thalline, 4 DEG C, 5500rpm, 15min, adds phosphate buffered saline buffer (PBS) (1g/5mL), mixing, multigelation 3 times in bacterial sediment.After ultrasonication, centrifugal (4 DEG C, 12000rpm, 15min), collect supernatant, wash precipitation 2 times successively, finally use 8M urea dissolution precipitation with 2M urea.The deposit sample that bacterium liquid, Supernatant samples, 2M urea washes liquid and 8M urea dissolve of not inducing and induce of collecting is processed, carries out SDS-PAGE analysis, obtain recombinant protein TsAQPP1 and mainly express with the form of solubility.Specifically see Fig. 1.
3, the purifying of TsAQPP1 recombinant protein
Used by TsAQPP1 recombinant expression protein Beijing Webster Bo Hui chromatogram Science and Technology Ltd. Glutathione Sepharose FF resin to carry out purifying respectively, method is see working instructions (summary).Carry out SDS-PAGE protein electrophoresis detection display after TsAQPP1 recombinant protein purification, the TsAQPP1 recombinant protein size of expression is about 29kDa(Fig. 2).80 DEG C save backup.
4, the immune protective test of TsAQPP1 recombinant protein
The collection of 4.1 cultivation of larvae of Trichinella spiralis from muscles
The neck that broken by the mouse infecting Trichinella spiralis 35d is lethal, utilizes tissue pulverizer to smash mouse trunk, is collected in 1000ml beaker.In beaker, add 500ml water, 2ml concentrated hydrochloric acid and 2.5g stomach en-, 42 DEG C are stirred digestion 1.5h.Postdigestive mixed solution is transferred in pear shape bottle, leaves standstill 2.5h, bottom liquid is received in 50ml centrifuge tube.Leave standstill 10min, abandon supernatant, add 20ml0.9% physiological saline, after leaving standstill 10min, abandon supernatant.Bottom solution is transferred in 1.5mlEP pipe.Carry out Trichinella spiralis counting under the microscope, carry out packing, packing 200 cultivation of larvae of Trichinella spiralis from muscles, totally 30 μ L in each EP pipe.
4.2 immune programme for children
50 Kunming mouses, dividing two groups: one group is TsAQPP1 recombinant protein immune group (25), the vehicle control group (25) that another group is not formula Freund's complete adjuvant and Freund's incomplete adjuvant immunity.The operating method of recombinant protein immune group: inguinal region, neck dorsal sc injection mouse 200 μ L, first immunisation 50 μ gTsAQPP1 recombinant protein+100 μ L Freund's complete adjuvants, second and third immunity 50 μ gTsAQPP1 recombinant protein+100 μ L Freund's incomplete adjuvants.Immunity three times altogether, each immunization interval two weeks.Vehicle control group immune programme for children is consistent with protein immunization group, unlike: first immunisation is: 100 μ LPBS+100 μ L Freund's complete adjuvants, second and third immunity 100 μ LPBS+100 μ L Freund's incomplete adjuvants.
4.3TsAQPP1 recombinant protein West-blotting identifies and antibody titer detects
The serum of exempting from rear mouse by three utilizes afterbody blood-collecting method to take a blood sample, and collects serum.
TsAQPP1 recombinant protein is carried out SDS-PAGE, then turns 15min to NC film with the half-dried transfer printing instrument of Bio-Rad at 15V voltage electricity.After closing 1h with 5% skimmed milk, add TsAQPP1 recombinant protein immune serum or vehicle control group mice serum (negative control) that 1:500 doubly dilutes, incubated at room 1h.TBST cleans 3 times repeatedly, each 10min.Then add the anti-pig IgG of rabbit of the AP mark doubly diluted with 1:4000, incubated at room 1h, finally add NBT/BCIP mixing nitrite ion lucifuge and to develop the color 5-10min, after colour developing, discard nitrite ion water termination reaction (Fig. 3).Application western-blot test confirms, TsAQPP1 recombinant protein can be combined with the specific antisera of immune mouse, but can not react with the serum of adjuvant group mouse, illustrates that TsAQPP1 recombinant protein reactionogenicity is good.
Immune serum antibody titers routinely indirect ELISA method detects, and the TsAQPP1 recombinant protein after application of purified is envelope antigen, and mouse immune serum is that starting point concentration carries out doubling dilution to 1:102400 with 1:100.Vehicle control group mice serum dilutes by the same way.Result display (Fig. 4), the antibody TsAQPP1 protein immunization mice serum of TsAQPP1 recombinant protein immune serum is tired as 1:51200.
The Protective efficacy of 4.4TsAQPP1 recombinant protein
After final immunization 2 weeks, TsAQPP1 protein immunization group and vehicle control group mouse peroral infection 200 cultivation of larvae of Trichinella spiralis from muscles simultaneously, after 35 days, often group killed 10 mouse, and the method according to 4.1 collects muscle larvae, and counts.Result shows, and an average year worm amount for TsAQPP1 protein immunization group mouse is 2.54 ten thousand ± 0.29, and an average year worm amount for vehicle control group mouse is 3.32 ten thousand ± 0.08.After TsAQPP1 protein immunization, the worm reduction rate of mouse is 23.5%.Illustrating that TsAQPP1 recombinant protein immunogenicity is good, is good Trichinella spiralis candidate vaccine.
5, TsAQP gene is at the expression of Trichinella spiralis different growing periods
The cDNA of 1.2 cultivation of larvae of Trichinella spiralis from muscles obtained, 3 age in days adults and newborn larvae is utilized real-time quantitative PCR (QT-PCR) method, quantitative examination is carried out to the expression of TsAQP gene.Select GAPDH as reference gene.Fluorescent primer sequence used is: AQPQTF (5 ¢-GTATCGGTGCTTGTAGTTG-3 ¢) and AQPQTR (5 ¢-TGCGTTAGGTCCTGTTAC-3 ¢); GADPHF(5 ¢-AGATGCTCCTATGTTGGTTATGGG-3 ¢) and GADPHR(5 ¢-ACTGTCTTTTGGGTTGCCGTT-3 ¢) QT-PCR application of sample system is: each 0.5 μ L(10 μm ol/L of cDNA1 μ L, PCR2 × mix12.5 μ L, AQPQTF and AQPQTR), ddH
2o is added into 25 μ L.QT-PCR reaction system is: 94 DEG C of 5min; 94 DEG C of 60s, 57 DEG C of 60s, 72 DEG C of 60s, 35 circulations; 72 DEG C of 10min.Result shows, TsAQP gene all can be expressed (Fig. 5) in cultivation of larvae of Trichinella spiralis from muscle, 3 age in days adults and newborn larvae, but expression amount is different, and in newborn larvae (NBL), expression amount is the highest, next is at muscle larvae (ML), expression amount minimum (Fig. 6) in 3 age in days adults (Ad3).
6, the virtual screening of TsAQP protein drug target spot
The applicant utilizes information biology Molecular Simulation Technique, and the medicine that prediction may be target spot with TsAQP albumen has: lunarine, isotheaflavin, rutin, Calciumlevofolinate, naringin, pimozide, cefoperazone, 9-hydroxy-risperidone, diosmin, the anti-high I blood pressure medicine of olmesartan medoxomill, Tadalafei, astemizole, cedrelone, parathion-methyl, Sisalgenin, NVP-XAA 723, Septochol methyl esters, Betamethasone Valerate, Sodium Dehydrocholate, Estradiol Cypionate, dpn, Vulkamycin. PA-93, hecogenin, vauqueline, Methyl cholate, dehydrocholic acid, Pitavastatin Calcium, ethinylestradiol, irbesartan, glimepiride, fluorescein, Glipizide, parathion-methyl, diosgenin, dehydrorotenone, dibenzyl ether, pomiferin, osajin, Shui Calusena lansium element, adapalene, Rhoifoloside, 10-hydroxycamptothecine, candesartan cilexetil, telmisartan, cefonicid sodium, penfluridol, dihydrotanshinone I, shellolic acid A, motilium, Glyburide, 17-M-nitro benzoic acid dihydrotestosterone, Berberine chloride has good anti-microbial effect, zhengdingmeisu, Etoposide, Sulcephalosporin and teniposide.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a Trichinella spiralis recombinant antigen, is characterized in that: it comprises the aminoacid sequence by Trichinella spiralis aquaporin (TsAQP) gene open reading frame gene sequences encode.
2. Trichinella spiralis recombinant antigen according to claim 1, is characterized in that: it comprises the aminoacid sequence of sequence table SEQ IDNo.4.
3. immunogenic composition, is characterized in that: it comprises the Trichinella spiralis recombinant antigen described in claim 1 or 2.
4. immunogenic composition according to claim 3, is characterized in that: also comprise adjuvant or medically acceptable carrier or vehicle; As preferably, described adjuvant is not formula Freund's complete adjuvant and Freund's incomplete adjuvant.
5. a vaccine, is characterized in that: the effective constituent of described vaccine is the Trichinella spiralis recombinant antigen described in claim 1 or 2 or the arbitrary described immunogenic composition of claim 3-6.
6. the preparation method of vaccine according to claim 5, is characterized in that: step is as follows:
(1) Trichinella spiralis recombinant antigen is prepared; As preferably, with preparing Trichinella spiralis recombinant antigen in File of Newborn Larvae of Trichinella spiralis;
(2) by Trichinella spiralis recombinant antigen and adjuvant mixed preparation, vaccine is obtained.
7. the application in the medicine of preparation prevention trichinzation of the Trichinella spiralis recombinant antigen described in claim 1 or 2, the immunogenic composition described in claim 3 or 4.
8. the nucleic acid of the Trichinella spiralis recombinant antigen of coding described in claim 1 or 2.
9. comprise the expression vector of nucleic acid described in claim 8.
10. comprise the host cell of expression vector described in claim 9.
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Cited By (1)
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CN110898216A (en) * | 2019-12-31 | 2020-03-24 | 中国农业科学院兰州兽医研究所 | Application of recombinant trichina thioredoxin peroxidase 2 in preparation of vaccine for resisting trichina infection |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110898216A (en) * | 2019-12-31 | 2020-03-24 | 中国农业科学院兰州兽医研究所 | Application of recombinant trichina thioredoxin peroxidase 2 in preparation of vaccine for resisting trichina infection |
CN110898216B (en) * | 2019-12-31 | 2021-12-03 | 中国农业科学院兰州兽医研究所 | Application of recombinant trichina thioredoxin peroxidase 2 in preparation of vaccine for resisting trichina infection |
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