CN114540378B - Expression method of recombinant duck type 3 adenovirus fiber-2 gene - Google Patents
Expression method of recombinant duck type 3 adenovirus fiber-2 gene Download PDFInfo
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- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract
The invention provides an expression method of a recombinant duck 3-type adenovirus fiber-2 gene in a Kluyveromyces marxianus expression system, which comprises the steps of firstly connecting the optimized duck 3-type adenovirus fiber-2 gene into an expression plasmid pUKD-N115, then converting a connection product into uracil deletion type Kluyveromyces marxianus TOP1 for expression, and finally obtaining recombinant yeast for expressing duck 3-type adenovirus fiber-2 protein. The invention successfully realizes the high-efficiency expression of the recombinant plasmid in Kluyveromyces marxianus, and the vaccine containing the expression product has a certain protection effect on the experiment of adenovirus infection of the muscovy ducks.
Description
Technical Field
The invention belongs to the field of biological products for livestock, and particularly relates to an expression method of duck type 3 adenovirus fiber-2 genes.
Background
Adenovirus is a linear double-stranded DNA virus without envelope, the diameter is 70-90 nm, and the adenovirus is symmetric in regular dodecahedron. The capsid is composed of 252 capsomeres arranged in a twenty-face shape, and the diameter of each capsomere is 7-9 nm. Each end has an inverted repeat sequence of about 100bp in length. They are designated as adenoviruses because they tend to infect epithelial cells. Adenovirus was isolated from many vertebrates in succession since the last fifty years of the last century, when adenovirus was isolated from the first strain of Adenovirus (ADV). Adenoviruses are currently classified into five genera, mammalian ADV (maltadenous), avian ADV (aviadeenous), AT-rich ADV (atatenovirus), sialidase ADV (sialadenovus) and american white sturgeon adenovirus (ichtadenous), depending on the host range. Adenoviruses that have been reported to be pathogenic to ducks include: duck adenovirus type 1 (Dadv-1), duck adenovirus type 2 (Dadv 2) and Duck adenovirus type 3 (Dadv-3), wherein Dadv-3 was isolated in 2014 from a group of disease muscovy ducks in Guangdong, and its clinical pathology was manifested as liver fibrosis, swelling, pericardial effusion. Dadv-3 is severely pathogenic to young ducks within forty days of age, and presents a serious hazard to poultry farming, and because of the lack of an effective commercial vaccine against Dadv-3, it is urgent to develop an economical, safe and effective vaccine.
The method adopts the means of exogenous protein expression, and can provide technical support for the large-scale production of biological products such as commercial vaccines, wherein the yeast expression system has the advantages of quick growth, low cost of culture medium, easy maintenance, easy large-scale production and the like of a prokaryotic expression system, and simultaneously has the advantages of a eukaryotic expression system such as protein processing, protein folding and the like, and is suitable for large-scale production. However, on the other hand, yeast expression systems also have the disadvantage of transient glycosylation, which is inefficient in secreting proteins into the growth medium, resulting in retention of proteins in the cell, difficulty in purification, etc.
Disclosure of Invention
The invention aims to provide an expression method of recombinant duck type 3 adenovirus fiber-2 genes.
The invention also aims to overcome the technical defects of low expression quantity, unstable expression strain, easy loss of plasmid, excessive glycosylation and the like of the traditional expression method.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a method for expressing recombinant duck type 3 adenovirus fiber-2 gene, wherein the gene sequence of recombinant duck type 3 adenovirus fiber-2 is shown in SEQ NO 1.
Furthermore, the method is expressed by an expression system constructed by uracil-deficient Kluyveromyces marxianus engineering bacteria.
The expression vector adopted by the expression method is pUKD-N115.
Further, the expression method comprises the following steps:
1) The duck 3-type adenovirus fiber-2 gene is subjected to sequence optimization according to uracil-deficient Kluyveromyces marxianus preference codons;
2) Connecting the recombinant duck 3-type adenovirus fiber-2 gene with a vector pUKD-N115;
3) Transforming the ligation product into Kluyveromyces marxianus TOP1 for expression;
4) Obtaining the expressed product TOP-DAdV-3 and identifying;
5) Fermenting TOP-DAdV-3 at high density to obtain recombinant bacterial liquid;
6) Purifying the recombinant bacterial liquid to prepare the recombinant protein.
Further, the vector pUKD-N115 of the step 2) is subjected to double cleavage with SmaI and NotI and then ligated.
Further, the purification process of step 6) is as follows: firstly, mixing recombinant bacterial liquid with PBS buffer solution containing 0.75M NaCl; then crushing for 2 times by a high-pressure homogenizer under the pressure of 1300 bar; centrifuge at 8000rpm for 5min and filter with 0.2 μm,100K ultrafiltration membrane pack concentrate microporous filter.
Further, the final concentration of the purified protein in step 6) was 453. Mu.g/ml.
A vaccine comprising the recombinant protein of step 6).
Compared with the prior art, the invention has the following advantages:
the invention overcomes the technical defects of low expression quantity, unstable expression strain, easy loss of plasmid, excessive glycosylation and the like of the expression method.
The invention utilizes the Kluyveromyces marxianus expression system to express the optimized duck 3-type adenovirus fiber2 gene, has high expressed protein content, and is suitable for large-scale production.
Kluyveromyces marxianus is food safety-grade yeast, has the advantages of being fast in growth, high in density, resistant to high temperature, capable of utilizing various carbon sources and the like, is an industrial protein expression strain with great potential, has the characteristics of stable genetic property, simplicity in operation, easiness in high-density culture, high protein yield, low production cost, suitability for industrial mass production and the like, has the advantages of exogenous protein post-translational processing and the like which are not possessed by a prokaryotic expression system, and simultaneously avoids the phenomena of instability of other yeast expression strains, easiness in losing plasmids, excessive glycosylation and the like. The invention is found after comparing the conventional expression systems such as pichia pastoris expression system, escherichia coli expression system, baculovirus expression system and the like, and the expression quantity of the Kluyveromyces marxianus expression system is the highest.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 is a PCR identification chart of duck type 3 adenovirus fiber-2 gene;
wherein 1-4 is pUKD-N115-fiber-2 non-optimized gene; 5-8 is pUKD-N115-fiber-2 optimized gene; 9-12 is pUKD-N112-fiber-2 non-optimized gene; 13-15 is pUKD-N112-fiber-2 optimized gene.
FIG. 2 shows PCR identification of Kluyveromyces marxianus transformants containing the fiber-2 gene;
wherein 1-3 is pUKD-N115-fiber-2 non-optimized gene; 4-5 is pUKD-N115-fiber-2 optimized gene; 6-8 is pUKD-N112-fiber-2 non-optimized gene; 9-10 is pUKD-N112-fiber-2 optimized gene
FIG. 3 is a SDS-PAGE detection of duck 3-type adenovirus fiber-2 gene protein expression:
wherein 1 is pUKD-N115 fiber-2 protein after gene optimization; 2 is pUKD-N115 fiber-2 protein with non-optimized gene; 3 is pUKD-N112 fiber-2 protein after gene optimization; 4 is pUKD-N112 fiber-2 protein with non-optimized gene; and 5 is a negative control.
FIG. 4 is a Western blot detection chart of the expression results of duck type 3 adenovirus fiber2 protein;
wherein 1 is pUKD-N115 fiber-2 protein after gene optimization; 2 is a saccharomycete lysate containing pUKD-N115 plasmid; 3 is pUKD-N112 fiber-2 protein after gene optimization; 4 is a saccharomycete lysate containing pUKD-N112 plasmid;
FIG. 5 is a SDS-PAGE detection of the expression level of duck 3-type adenovirus fiber2 protein;
wherein 1-6 are respectively 750 mug/mL, 500 mug/mL, 250 mug/mL, 125 mug/mL, 25 mug/mL and 0 mug/mL of BSA protein standard substance in sequence; m is a protein marker;7 is the target protein after purification (8-fold dilution).
Detailed Description
The following detailed description is exemplary and is intended to provide further explanation of the invention. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.
The reagents, plasmids, media, host bacteria and other biological materials used in the examples below were all from commercial products.
EXAMPLE 1 preparation of recombinant Duck 3-type adenovirus fiber-2 Gene
The disease materials selected in the research are derived from a Muscovy duck farm in Baoding City of Hebei province, the disease ducks with the characteristics of suspected duck type 3 adenovirus infectious diseases are selected as the disease materials, the genome of the disease materials is extracted, primers are designed for PCR detection and sequencing analysis, the fiber2 gene sequence is obtained, the sequence is shown as SEQ NO 2, then the sequence optimization is carried out by Kluyveromyces marxianus preference codons by Beijing biological science and technology Co., ltd, and the optimized gene sequence is shown as SEQ NO 1.
EXAMPLE 2 expression of recombinant Duck 3 adenovirus fiber-2 Gene in Kluyveromyces marxianus
1. The preparation method of the recombinant plasmid in the Kluyveromyces marxianus expression system comprises the following steps:
(1) strain activation: kluyveromyces marxianus TOP1 single colony was inoculated into 3mL YPD liquid medium, cultured at 30℃overnight at 200rpm, and OD value was measured to be 12-15.
(2) Yeast transformation: taking 1mL of the bacterial liquid after overnight culture, centrifuging for 1min at 8000rpm in a 1.5mL EP tube, discarding the supernatant, adding 1mL of sterile water for washing, centrifuging at 8000rpm for 1min, and discarding the supernatant; 1mL of 1 XLiAc/TE is added for washing, centrifugation is carried out at 8000rpm for 1min, the supernatant is discarded, washing is carried out twice, and 3-tube thalli are prepared again according to the washing mode;
respectively adding two vectors pUKD-N115 and pUKD-N112 into 4-tube thalli, respectively connecting the two vectors with an unoptimized 3-duck adenovirus fiber-2 gene and a recombinant vector (1 mug) with a nucleotide sequence optimized according to a Kluyveromyces marxianus preferred codon, 5 mug carrier DNA,600 mug PEG solution and 0.2 mug DTT with a concentration of 1M; after fully mixing, water bath is carried out for 15min at 30 ℃ and water bath is carried out for 15min at 47 ℃; centrifuging at 8000rpm for 1min, removing supernatant, adding 200 μl of sterile water to suspend thallus, sucking 75 μl of bacterial liquid, coating sc-ura plate, and culturing at 30deg.C for two days.
Expression reaction system
(3) And (3) verifying transformants: obvious colonies were picked on sc-ura plates, dissolved in 10. Mu.L of sterile water, and boiled for 20min, and PCR was performed with forward primer PINU-F (5 'CAGCAATTAA ATCCGGGGTA AG 3') and reverse primer PINU-R (5 'TATAAAATGT CGCTGTGACC AGGC 3'), the experimental results of which are shown in FIG. 1.
PCR reaction system:
PCR amplification procedure
(4) Colonies of the correct size expected were selected, inoculated in 3mL YPD liquid medium, cultured overnight at 30℃and 200rpm, blotted on PS plates 150. Mu.L and incubated in a 30℃incubator for two days. Obvious colonies were picked, dissolved in 10. Mu.L of sterile water, and after 20min of boiling water bath, PCR was performed using primers PINU-F/PINU-R, and the results are shown in FIG. 2.
PCR reaction system
PCR amplification procedure
Randomly selecting strains with the positions and sizes of the bands consistent with the expected positions, inoculating 10mL of YPD liquid culture medium to a single colony, culturing at 30 ℃ at 200rpm overnight, transferring the seed liquid cultured overnight into fresh 1L of YD liquid culture medium at an inoculum size of 1%, centrifuging after culturing for 3d, collecting thalli, re-suspending the bacterial liquid after PBS is washed once, and crushing cells for 1-3 times under the pressure of 1100-1400 bar.
Preferably 1300bar is used with a high pressure homogenizer, broken 2 times, centrifuged at 8000rpm for 5min and the supernatant is collected.
Cross experiments of different time and crushing strength show that the cell is crushed by 1300Bar for 1 time, and the crushing rate can reach 50-70%; the crushing is carried out for 2 times, the crushing rate can reach 85% -95%, the crushing rate can reach 95% -98% after 3 times of crushing, the crushing time cost and the proportion of target protein and foreign protein after crushing are combined, and the crushing rate for 2 times is more than 85% and is optimized.
2. Verification of expression results
Recombinant proteins were detected by 12% SDS-PAGE and the staining results are shown in FIG. 3. Transferring the protein separation gel to a PVDF membrane with the same size by a wet transfer method, wherein the transfer condition is 70V for 100min; after the membrane transfer is completed, washing with TBS-T, taking out PVDF membrane, sealing with 5% skimmed milk powder for 1 hour, and washing with TBS-T; the membrane was placed in a hybridization bag, diluted (1:400) primary antibody 2ml was added, and after heat sealing, the bag was closed overnight at 4 ℃. The next day the hybridization bag was cut off and the membrane was placed on a plate and repeatedly washed 2-3 times, diluted (1:1000) secondary anti-goat anti-chicken IgG-HRP 4ml was added, the secondary antibody was poured off and washed with TBS-T, chemiluminescent chromogenic substrate was added dropwise to the front side of PVDF membrane in the absence of light, and the membrane was exposed by Bio-rad chemiluminescent imager, and Western blot results showed (FIGS. 4a and b): after sequence optimization is carried out on the Kluyveromyces marxianus preferred codons, the pUKD-N115 intracellular expression plasmid expression product is connected, so that the pUKD-N115 intracellular expression plasmid expression product can be specifically combined with an antibody, and has obvious reaction bands at a 55KD position, so that the exogenous protein can be well expressed in yeast cells.
High-density fermentation is carried out in a 15L fermentation tank, glucose and ammonia water are continuously supplemented in the fermentation process, the stirring rotation speed is set to 600rpm, and the air flow rate is 2.0m 3 Controlling dissolved oxygen at 1-10%, fermenting for 52 hr, using high pressure homogenizer 1300bar, crushing for 2 times, centrifuging at 8000rpm for 5min, collecting supernatant, and identifying titer as 1 by agar gel diffusion experiment: 256.
to reduce turbidity and purify proteins, PBS buffer solution containing 0.5M NaCl, PBS buffer solution containing 0.67M NaCl and PBS buffer solution containing 0.75M NaCl are respectively used for mixing bacterial solutions to increase the solubility of protein molecules, bacterial cells are crushed by the same condition strain by using the bacterial solution mixed bacterial solutions with different salt concentrations, and after 8000rpm and 5min of centrifugation, the bacterial cells are concentrated by a 100K ultrafiltration membrane bag and filtered by a 0.2 mu M microporous membrane to reduce turbidity. The method for crushing the thalli comprises the following steps: crushing cells for 1-3 times under the pressure of 1100-1400 bar; preferably, the engineering bacteria are disrupted 2 times with PBS buffer containing 0.75M NaCl at a pressure of 1300 bar. Gray analysis of the target protein by BSA standards of different concentrations gave purified protein at a final concentration of 453. Mu.g/ml, turbidity of 385NTU and titer of 1 as shown in FIG. 5: 128.
example 3 evaluation of vaccine Effect
Selecting 50 SPF-grade muscovy ducks of 2 days old, dividing the SPF-grade muscovy ducks into 5 groups, diluting the purified protein obtained in the example 2 by multiple ratio, preparing a vaccine by taking Marcol-52 white oil as an adjuvant according to the titers of 1:32, 1:64 and 1:128, injecting 10 muscovy ducks into each group, injecting 0.3ml into each group by subcutaneous injection, and injecting no toxicity attack control group and no toxicity attack control group; after 21 days of immunization, 1mL of liver supernatant of duck adenovirus 3 was injected subcutaneously into each of the experimental group and the challenge control group, and after 7 days, anus swabs were taken, and after 1mL of PBS was mixed evenly and centrifuged, whether or not to expel toxin was detected by PCR. The forward primer for the PCR reaction was 22K-F (5 'ACCCAAAAGCAAGTGGACGC 3'), and the reverse primer was 22K-R (5 'CGGAATCACCACCTTCCTCG 3'), with the following system:
PCR reaction system
PCR reaction procedure
The results are shown in the following table, titer 1: no detoxification was measured in the 128 vaccine group tested muscovy ducks, titer 1:64 vaccine groups measured 1 tested muscovy duck with detoxification, titer 1: the vaccine group of 32 has 2 tested muscovy ducks with toxin expelling, all muscovy ducks in the toxin attacking control group have toxin expelling, and the blank control group has no toxin expelling.
The invention uses a Marx Kluyveromyces expression system to induce and express recombinant duck 3-type adenovirus fiber-2 gene, and prepares the recombinant duck 3-type adenovirus fiber-2 gene into a vaccine. Animal experiments prove that the vaccine has a certain protection effect on duck type 3 adenovirus. Provides a good basis for further researching duck adenovirus infection.
Sequence listing
<110> Tianjin Reep Biotechnology Co., ltd
<120> method for expressing recombinant duck 3-type adenovirus fiber-2 gene
<141> 2022-03-18
<160> 2
<170> SIPOSequenceListing 1.0
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<400> 1
atgaagagaa ccaacagatc cgacaacggt gaaggtttca ccaagagagc taagatccaa 60
gcttccgctt ccgctaccat cgacttgacc tacccattct gggaacaagc ttcctccgct 120
aacccaatca acccaccatt cgtcactggt ccattgtacg acgacaacgg ttacttgaac 180
gttagaacct ccgacccaat tagaaccgtt ggtaactctt tgtctttgtt gtacgatgac 240
tccttggctg ttacctccgg taagttgggt gttaagattg acccaaacgg tccattggat 300
gaatctccag ctggtttgtc cttggctttg ggtgacggtt tggaagaaga cgaattcacc 360
ggtttgtctg ttaagccaga cccaagaggt ccaatcgaag tttctgatga aggtgttggt 420
atcgcttacg acactgaaac catgtctatt acctctatgc aaccaaactc tcaaatgact 480
ttgggtgtta gattgaaccc agacggtgct ttgcactcta acaacggttt ggatgttaag 540
attgatgacg acgctttgat cgtttctgac gaaggtttga ccgttttggt ttccgaatct 600
ggtccattga ctatcgaccc aggtaagggt ttggacatcg acattgacca atctttgtct 660
gttagaacca acccacaagg tgaaaaggaa ttgggtatca acatcatccc aacctcctgt 720
atcaccttgg acaacggtgg tttggacttg aaggttgatc caggttcctt ggctgtcacc 780
gacaacacct tgcacttgac ctcttcttac tctacctacg ttttcacctc tggttctgac 840
accttgcaaa agacccaagc tcaagtttgt tgtggtgctg gttctgttac tttcccatgt 900
gcttactacg ctaagatcgt ttgttctaac aacgtttctt ctggttacat taccttgaag 960
gtttccgctg aagacgcttc tcacgctgtt gaccaaagat tcgctaccat ccaaccagtt 1020
ttcaccttct ggttgtgtag agacatcggt aacgaaaaca ccgttaactt ctctcactgt 1080
accaacaact cttacaagcc agaagaaacc gctgttgtca aggcttgtat caccccagct 1140
tacaagaact tcgacgtttc taacgtttct gaacacgact tcatcttgta caactactcc 1200
ggtgtcaaca cctccggtgt tccaatcggt accggtaact tgaagaacgt ttacgactac 1260
gtttccagat tctctatcgc tcaagtttct ggttctccaa cctctccaca attgactttc 1320
accatcgaat tgaacaagat cgaccaatct tcttactact tgtacaagca aggtaccacc 1380
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aacccgataa atcctccgtt tgtcaccgga ccactgtatg atgacaacgg gtatcttaac 180
gtaagaacat ccgacccgat aagaaccgtt ggtaacagcc tcagcctact gtacgatgat 240
tcgctagctg tgacaagtgg caaactaggc gttaaaatcg acccaaacgg gcctctagat 300
gagtcgcccg ctggactcag tttagctcta ggagacggtc ttgaagaaga cgagttcaca 360
ggtttatctg taaaaccaga tccacgtggc cctatcgaag tgtcagatga aggagtgggt 420
atagcctatg atacagaaac catgtcaata acatcaatgc aaccaaattc acagatgact 480
ctcggtgtac gactaaaccc cgatggtgca ctacacagta acaacggatt agatgtaaaa 540
attgatgatg atgcgttgat agtcagcgac gaaggcctaa cagtactcgt tagtgaaagc 600
gggccactta caatcgaccc tggcaaagga ctggatattg atatcgatca atcactctcc 660
gttagaacta atcctcaagg agagaaagag ttaggcatca acatcatccc cacatcgtgc 720
ataacactag acaacggagg cctcgacctc aaggtggacc caggaagcct ggccgtcact 780
gacaataccc tacacctcac aagctcatac tcaacctatg tctttacgag tggatctgac 840
acacttcaga agacacaagc ccaagtgtgc tgcggagcag ggtctgttac gtttccgtgc 900
gcatactatg ccaagatcgt atgctcaaac aatgtgtctt caggatatat aacactgaag 960
gtgagcgctg aggatgcatc acatgctgta gatcaacgct tcgcgactat tcaaccagta 1020
ttcacattct ggttatgtcg agacataggt aatgaaaaca ccgtcaattt ttcccactgt 1080
accaacaaca gttataagcc agaggaaacc gcagtcgtta aggcatgcat cacaccagca 1140
tacaaaaatt ttgatgtcag caatgtttca gaacatgact ttattctgta taactatagt 1200
ggggtaaaca catcaggagt tccaataggc acagggaacc taaagaatgt gtatgattac 1260
gtatcaagat tctccatagc ccaagtatca ggaagcccca catcaccaca attaactttt 1320
acaatagaac ttaataagat agatcaatct tcgtactatc tgtacaaaca aggcacaact 1380
ggtgatatta caattggtcc aattccattt tcgtacgtta gcaacccatc aaatgttaat 1440
tag 1443
Claims (3)
1. The expression method of the recombinant duck 3-type adenovirus fiber-2 gene is characterized in that the gene sequence of the recombinant duck 3-type adenovirus fiber-2 is shown as SEQ NO 1;
the expression system adopted by the expression method is constructed by uracil-deficient Kluyveromyces marxianus engineering bacteria;
the expression vector adopted by the expression method is pUKD-N115;
the expression method comprises the following steps:
1) The duck 3 type adenovirus fiber-2 gene is prepared into a recombinant duck 3 type adenovirus fiber-2 gene by optimizing the sequence according to uracil deficiency type Kluyveromyces marxianus preferred codons;
2) Connecting the recombinant duck 3-type adenovirus fiber-2 gene with a vector pUKD-N115;
3) Converting the connection product into uracil-deficient Kluyveromyces marxianus TOP1 for expression;
4) Obtaining the expressed product TOP-DAdV-3 and identifying;
5) Fermenting TOP-DAdV-3 at high density to obtain recombinant bacterial liquid;
6) Purifying the recombinant bacterial liquid to prepare recombinant protein;
the final concentration of the purified protein in step 6) was 453. Mu.g/ml.
2. The method for expressing recombinant duck type 3 adenovirus fiber-2 gene according to claim 1, wherein the vector pUKD-N115 of step 2) is subjected to SmaI and NotI double digestion and then is connected.
3. The method for expressing recombinant duck type 3 adenovirus fiber-2 gene according to claim 1, wherein the purification process of step 6) comprises the steps of: firstly, mixing recombinant bacterial liquid with PBS buffer solution containing 0.75M NaCl; then crushing for 2 times by a high-pressure homogenizer under the pressure of 1300 bar; centrifuge at 8000rpm for 5min and filter with 0.2 μm,100K ultrafiltration membrane pack concentrate microporous filter.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106399350A (en) * | 2016-09-07 | 2017-02-15 | 复旦大学 | Porcine circovirus type II virus-like particle vaccine and preparation method thereof |
| CN113846112A (en) * | 2021-08-23 | 2021-12-28 | 武汉科前生物股份有限公司 | Nucleotide sequence, fiber2 protein and expression method, duck type 3 adenovirus and duck tembusu virus bigeminal inactivated vaccine |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399350A (en) * | 2016-09-07 | 2017-02-15 | 复旦大学 | Porcine circovirus type II virus-like particle vaccine and preparation method thereof |
| CN113846112A (en) * | 2021-08-23 | 2021-12-28 | 武汉科前生物股份有限公司 | Nucleotide sequence, fiber2 protein and expression method, duck type 3 adenovirus and duck tembusu virus bigeminal inactivated vaccine |
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| 严专强 ; 曾凡桂 ; 招丽婵 ; 王占新 ; 鲁俊鹏 ; .1株鸭3型腺病毒的分离鉴定及fiber基因序列分析.养殖与饲料.2020,(第02期),29-32. * |
| 史琪雯等.鸭腺病毒 3 型 Fiber - 2 蛋白在酵母细胞中的 表达及免疫原性研究.南京农业大学学报.2023,第46卷(第1期),99-105. * |
| 袁枫等.禽腺病毒血清４型fiber-2蛋白在毕赤酵母中的优化表达及其免疫原性分析.中国农业大学学报.2022,第27卷(第2期),132-142. * |
| 魏常青等.鸭腺病毒3型Fiber-2蛋白单克隆抗体的制备及初步应用.中国家禽.2023,第45卷(第2期),46-51. * |
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