CN107446029A - A kind of Caprine arthritis encephalitis virus serum antibody diagnostic flag albumen p28 and preparation method thereof - Google Patents

A kind of Caprine arthritis encephalitis virus serum antibody diagnostic flag albumen p28 and preparation method thereof Download PDF

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CN107446029A
CN107446029A CN201710667641.6A CN201710667641A CN107446029A CN 107446029 A CN107446029 A CN 107446029A CN 201710667641 A CN201710667641 A CN 201710667641A CN 107446029 A CN107446029 A CN 107446029A
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albumen
caev
encephalitis virus
caprine arthritis
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王登峰
吴建勇
李建军
古努尔·吐尔逊
杨学云
刘志强
金映红
王文奇
班万里
刘丽娅
刘艳丰
叶锋
马晓菁
蒋晓梅
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Xinjiang Academy Of Animal Sciences
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Xinjiang Academy Of Animal Sciences
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    • 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 belongs to biological technical field, and in particular to a kind of Caprine arthritis encephalitis virus serum antibody diagnostic flag albumen p28, include the nucleotide sequence of the p28 albumen after optimized, as shown in SEQ ID NO.1, uniformity is not less than 95% other sequences；And the amino acid sequence of the p28 albumen after optimized is included, as shown in SEQ ID NO.2, uniformity is not less than 95% other sequences.The CAEV serodiagnosis labelled protein p28 reactionogenicities of preparation are good, are easy to industrialized production, and antigen is provided for exploitation CAEV high flux serological diagnostic methods.

Description

A kind of caprine arthritis-encephalitis virus serum antibody diagnostic flag albumen p28 and its system Preparation Method
Technical field
The invention belongs to biological technical field, and in particular to a kind of caprine arthritis-encephalitis virus serum antibody diagnosis mark Remember albumen p28 and preparation method thereof.
Background technology
Caprine arthritis-encephalitis（Caprine arthritis-encephalitis, CAE）It is by caprine arthritis-brain A kind of chronic viral of goat passes caused by scorching virus (caprine arthritis-encephalitis virus, CAEV) Catch an illness.The adult goat that is mainly characterized by of the disease is in the arthritis that slowly develops, or with interstitial pneumonia and chromic fibrous breast Fang Yan；2-6 monthly age lambs show as the nervous symptoms of ascending paralysis.This sick infection rate is high, incubation period length, and infection goat is lifelong Band poison, and without special treatment method, the production performance particularly milk yield of drove is had a major impact, while also hampers The kind normal trade of animal, two class animal epidemics are classified as by China, and in international trade, relevant animal and product must when entering a country Must quarantine.
Caprine arthritis-encephalitis virus is since nineteen eighty-two is found by Crowford et al., many states of throughout world Family, wherein, the country such as the U.S., Norway, Britain, Japan, Mexico, Brazil is particularly acute, and serosurvey shows its positive rate Exceed 30%.China has separated the encephalitis viruses of caprine arthritis one in the Sa energy milch goat from Britain's import in 1987 and ordered Entitled CAEV89-GB1026.At present, the areas such as Gansu, Shaanxi, Guizhou, Shandong, Heilungkiang, Liaoning, Sichuan are distributed mainly on. Separate and identified 5 different caprine arthritis-encephalitis virus strains, respectively Gansu, Shaanxi, Sichuan, Guizhou and Shandong Strain, the gag-pol gene orders of these strains are similar between 99.2%~99.9%, and genotype is B1 types, with Switzerland, Si Nuo The European countries such as Wen Niya epidemic strain genotype is identical.
Caprine arthritis-encephalitis is in endemicity, and morbidity goat and carriers person are the infection sources.It can infect any Age, the goat of sex.CAEV infection has specificity to host, and goat is this sick major risk animal, wherein milch goat It is most susceptible, infected more in chronic sustained, incubation period length, when the disease is found, whole flock of sheep may all be infected.There is state Outer flock of sheep and the area for introducing a fine variety history, the risk of CAEV infection are higher.Major transmission path is lamb by sucking containing virus Colostrum and often breast and propagated, can also be propagated by susceptible sheep and the adult sheep long-term close contact of infection.
Ago-Gel diffusion test（AGID）It is the most frequently used method for detecting CAEV antibody in serum, Ye Shiguo Border animal doctor organizes the detection method that (OIE) recommends.AGID conventional antigen is totivirus antigen, and capsid protein p28 and cyst membrane Albumen gp135 can also be used as AGID antigen, be serodiagnosis labelled protein.Simultaneously as ELISA is more sensitive, more passes through Ji, and can be used in large-scale serological screening, it was set to the standard method of CAEV detections by OIE in 2004.At present, commonly use Include totivirus antigen and recombinant antigen in ELISA antigens, p28 albumen is important serodiagnosis mark egg in recombinant antigen In vain, the p28 recombinant proteins such as Archambault ELISA progress lowlenthal serum is detected, and shows that its Sensitivity and Specificity is distinguished For 96.9% and 100%.
Mainly the prokaryotic expression using Bacillus coli expression as representative, CAEV are viral for the recombinant expression method of p28 albumen at present Mammalian epithelial cell is infected, is translated, expressed using the protein expression system of host, and the albumen table of prokaryotic Differed greatly up to system and eukaryotic, the difference of environment is formed due to the preference difference and protein structure of codon to be made to express To albumen larger difference be present in amino acid sequence, two level and tertiary structure；Forgive in addition, Bacillus coli expression has been formed Body, it is unfavorable for industrialized production.
The content of the invention
The purpose of the present invention：The application is based on CAEV viral genetics RNA reverse transcription product DNA sequence dna, root According to the preference of codon, the stability of Optimal Expression albumen, formation secreting, expressing etc. in Pichia pastoris GS115 expressing protein The encoding gene of p28 albumen is optimized, the gene cloning of optimization is entered into pPIC9K plasmid construction recombinant plasmids, and will be linear The recombinant plasmid Electroporation of change enters in Pichia pastoris GS115, the monoclonal bacterium colony of screening expression p28 albumen, through induced expression It is defined as secreting, expressing, and antibody generation antigen-antibody reaction caused by non-target animal stimulation is infected with CAEV.
Technical solution of the present invention is as follows：A kind of caprine arthritis-encephalitis virus serum antibody diagnostic flag albumen p28, bag Nucleotide sequence containing the p28 albumen after optimized, as shown in SEQ ID NO.1, uniformity is not less than 95% other sequences； And the amino acid sequence of the p28 albumen after optimized is included, as shown in SEQ ID NO.2, uniformity is other not less than 95% Sequence.
Further limit, the nucleotide sequence of the p28 albumen after optimization, as shown in SEQ ID NO.1, the p28 after optimization The amino acid sequence of albumen, as shown in SEQ ID NO.2.
A kind of caprine arthritis-encephalitis virus serum antibody diagnostic flag albumen p28 preparation method, including following steps Suddenly：
（1）The codon optimization and synthesis of CAEV p28 genes
Codon in Pichia pastoris GS115 expressing protein is optimized and closed to the encoding gene of CAEV p28 albumen Into acquisition pUC57-p28 plasmids；
（2）The structure of recombinant vector
Above-mentioned pUC57-p28 plasmids are passed throughNotⅠWithEcoRⅠIt is connected after double digestion with pPIC9K, constructs recombinant expression plasmid pPIC9K-p28；
（3）The screening of Pichia pastoris GS115 positive recombinant
The recombinant expression plasmid pPIC9K- p28 of linearisation are imported in Pichia pastoris GS115 by Electroporation；
（4）The induced expression of p28/GS115 engineering bacterias, obtain p28 albumen.
Beneficial effect：With this method prepare CAEV serodiagnosis labelled protein p28 reactionogenicities it is good, be easy to industrialize Production, antigen is provided for exploitation CAEV high flux serological diagnostic methods.
Brief description of the drawings
Accompanying drawing 1 is CAEVp28The similitude and genetic divergence of gene order；Accompanying drawing 2 is the coding p28 albumen after optimization Gene order（5’-3’）And the amino acid sequence of albumen（C-N）；Accompanying drawing 3 reflects for the double digestion of pPIC9K-p28 recombinant plasmids Determine result, wherein, 5：Blank control；6：pPIC9KNotI HeEcoThe double digestion results of R I；7：pPIC9K-p28NotI HeEcoR I double digestion result；8：D15000 marker；Accompanying drawing 4 converts GS115 results for linearisation pPIC9K-p28；Accompanying drawing 5 is p28/ GS115 bacterial strain PCR qualification results, wherein, M：marker C（Raw work）；PCR testing results are the positive；Accompanying drawing 6 is p28/ The optimization of GS115 engineering bacteria induced expression times, wherein, M：Pre-dyed albumen Marker；1：Supernatant before expression；2-6：p28/GS11 1-5 days results of engineering bacteria induced expression；Accompanying drawing 7 is that p28 and gp135 protein expressions are quantitatively schemed, wherein, 1-2：P28/GS115 works Journey bacterium Fiber differentiation supernatant（10 times of concentrations）；M molecular weight of albumen marker (low)（TAKARA）；Accompanying drawing 8 recombinates for p28/GS115 Bacterial strain inducing is expressed and western blotting qualification results, wherein, M：Pre-dyed albumen Marker；1：Supernatant before expression；2- 3：P28/GS115 recombinant bacterial strain expressions of results；Accompanying drawing 9 is that WB methods diagnose CAEV infection sheep results M：Pre-dyed albumen Marker； 1：Negative serum sample；2、3：CAEV infects goat positive serum sample；Accompanying drawing 10 is that AGID methods diagnose CAEV infection sheep knots Fruit, wherein, in the width figure of left, center, right three：Left -1, -2, right side -3 is that CAEV infects goat positive serum sample in；A left side -3 and a left side -4, In -3 and in -4, the right side -1 and the right side -2 be CAEV negative serum samples；Left -2, -1, right side -4 is blank control in.
Embodiment
The optimization and synthesis of embodiment, 1.CAEV p28 genes
（1）The homology analysis of CAEV p28 genes
To three plants of caprine arthritis-encephalitis viruses of published isolated in China in GeneBank（CAEV）（GeneBank： KT749879.1, KT749880.1, KT749881.1）The gene for encoding p28 albumen carries out homology analysis.Through sequence alignment Analysis shows, the p28 gene order differences of three plants of CAEV codings are smaller, and homology is more than 98.5%, with disclosed base in the world Because the homology of sequence is more than 91%, the similitude and genetic divergence of Fig. 1 CAEV p28 gene orders are seen.
（2）The codon optimization of CAEV p28 genes
According to the preference of codon, the stability of induction expression protein, formation secreting, expressing in Pichia pastoris GS115 expressing protein Etc. the encoding gene of p28 albumen is optimized, commission Shanghai biotechnology Co., Ltd combining unit carries out full base Because of synthesis, pUC57-p28 plasmids are finally obtained, the sequence of the coding p28 GFPs after optimization（5’-3’）And expressing protein Amino acid sequence（C-N）, see Fig. 2, the gene order of the coding p28 albumen after optimization（5’-3’）And the amino acid sequence of albumen Row（C-N）.
2. the structure of recombinant vector
PUC57-p28 plasmids are passed throughNotⅠWithEcoRⅠIt is connected after double digestion with pPIC9K, constructs recombinant expression plasmid PPIC9K-p28, verify expected band occur through double digestion, see the double digestion identification knot of Fig. 3 pPIC9K-p28 recombinant plasmids Fruit；5：Blank control；6：pPIC9KNotI HeEcoThe double digestion results of R I；7：pPIC9K-p28NotI HeEcoThe double digestion knots of R I Fruit；8：D15000 marker
3. the screening of GS115 positive recombinants
Pichia pastoris GS115 electricity transformed competence colibacillus, a large amount of extracts kits extraction milligram level plasmid are prepared by DTT-LiAc methods, Pass throughSac IGlue reclaim is carried out after linearization for enzyme restriction.Pass through Electroporation（Biorad fungal transformation default parameters）Succeed line Property recombinant plasmid import in Pichia pastoris GS115, be coated with MD flat boards, expected single bacterium colony occur, see Fig. 4, linearize PPIC9K-p28 converts GS115 results.The screening of Pichia pastoris positive recombinant passes through the screening of G418 multicopy recons, choosing Select and be resistant to 1mg/mL respectively, 2mg/mL, 3mg/mL G418 single bacterium colony enter performing PCR identification, purpose band expected from appearance, table Bright target gene realizes the homologous recombination with yeast, sees Fig. 5 p28/GS115 bacterial strain PCR qualification results, M：marker C（It is raw Work）；PCR testing results are the positive.
4. the induced expression of p28/GS115 engineering bacterias
Methanol induction expression is carried out to the positive Pichia pastoris p28/GS115 engineering bacterias of screening（1% methanol）, through SDS-PAGE points There is expected purpose band near 26 KDa in analysis, destination protein；Methanol induction further is carried out to p28/GS115 recombinant bacterial strains The optimization of time（0、1d、2 d、3 d、4 d、5 d）, as a result show that p28/GS115 recombinant bacterial strains expression quantity after the 4th day obtains Peak, sees Fig. 6, the optimization of p28/GS115 engineering bacteria induced expression times, M：Pre-dyed albumen Marker；1：On before expression Clearly；2-6：P28/GS11 engineering bacterias 1-5 days results of induced expression.
Using 30% concentration of alcohol（v/v）10 times of concentrations are carried out to expression supernatant, use TAKARA protein quantification Marker SDS-PAGE analyses are carried out, the gray value of purpose band are calculated using BandScan5.0 softwares, results expression p28 eggs White expression quantity is up to 145mg/L, sees that Fig. 7, p28 and gp135 protein expression are quantitatively schemed；1-2：P28/GS115 engineering bacterias induce Culture supernatant（10 times of concentrations）；M molecular weight of albumen marker (low)（TAKARA）.
5. p28/GS115 recombinant proteins reactionogenicity is identified
P28/GS115 engineering bacterias carry out methanol induction expression（1% methanol）, reactionogenicity is carried out using Western Blotting Identification and analysis, primary antibody are positive serum caused by natural infection CAEV goats, and secondary antibody is the rabbit-anti goat IgG of HRP marks, as a result Display p28 recombinant expression proteins have consistent reactionogenicity with the CAEV albumen synthesized using host protein synthesis system, see figure 8, p28/GS115 recombinant bacterial strain induced expressions and western blotting qualification results；M：Pre-dyed albumen Marker；1：Table Up to preceding supernatant；2-3：P28/GS115 recombinant bacterial strain expressions of results.
Practical application is illustrated
Example 1. uses western blotting method diagnosis CAEV infection sheep
Caprine arthritis-encephalitis virus（CAEV）After infecting goat, stimulate and produce antibody, p28/GS115 engineering bacterias are induced into table The p28 recombinant proteins reach, purified use western blotting method as antigen（Western Blotting）The tested goat of detection Serum antibody, it can be used for CAE serum antibody diagnosis.
1. MATERIALS METHODS
1.1 material
Serum：3 parts of CAEV infection lowlenthal serums sample, wherein 2 parts of positive sample, it is negative 1 part；
Antigen：P28/GS115 engineering bacterias induced expression, purifying p28 albumen；
Secondary antibody：The rabbit-anti goat IgG of HRP marks（Tiangeng Bioisystech Co., Ltd）.
1.2 test method
According to the third edition of Science Press《Molecular Cloning-A Laboratory guide》In " common technology in the molecular cloning of annex 8-exempt from Epidemic disease trace "（p 1723-1726）Carry out：
（1）The p28 recombinant proteins of purifying carry out SDS-PAGE electrophoresis, the μ L of applied sample amount 10 (ng of p28 protein 15s 0), voltage 150 V, the min of electrophoresis 40；
（2）Polyacrylamide gel is transferred on filter membrane；
（3）Closing, it is detected serum（50 times of dilutions）60 min are incubated, are cleaned；
（4）Secondary antibody is incubated（1,000 times of dilutions）, cleaning；
（5）Colour developing
2. result
Primary antibody is positive serum caused by natural infection CAEV goats, and secondary antibody is the rabbit-anti goat IgG of HRP marks, is as a result shown P28 recombinant expression proteins have consistent reactionogenicity with the CAEV albumen synthesized using host protein synthesis system, see Fig. 9, WB Method diagnosis CAEV infection sheep results；M：Pre-dyed albumen Marker；1：Negative serum sample；2、3：CAEV infects goat positive blood Final proof sheet.
The Ago-Gel method of diffusion of example 2. diagnosis CAEV infection sheep
Caprine arthritis-encephalitis virus（CAEV）After infecting goat, stimulate and produce antibody, p28/GS115 engineering bacterias are induced into table The p28 recombinant proteins reach, purified use Ago-Gel method of diffusion as antigen（AGID）The tested lowlenthal serum of detection resists Body, it can be used for CAE serodiagnosis.
1.1 material
Serum：10 parts of CAEV infection lowlenthal serums sample, wherein 3 parts of positive sample, it is negative 6 parts；
Antigen：P28/GS115 engineering bacterias induced expression, purification of Recombinant p28 albumen；
1.2 test method
With reference to《SNT 1171.2-2003 Caprine arthritis-encephalitis antibody detection method-agar immunodiffusion test》Carry out, P28/GS115 engineering bacterias induced expression, the μ g/ holes of purification of Recombinant p28 albumen 5.
2. result
AGID results are shown:P28 recombinant expression proteins produce immuning lines with CAEV infection goat positive serums, with negative blood Clear reactionless line, see Figure 10, AGID methods diagnosis CAEV infection sheep results；In the width figure of left, center, right three：Left -1, -2, right side -3 in Goat positive serum sample is infected for CAEV；A left side -3 and a left side -4, in -3 and in -4, the right side -1 and the right side -2 be CAEV negative serum samples This；Left -2, -1, right side -4 is blank control in.
The indirect ELISA method of example 3. diagnosis CAEV infection sheep
Caprine arthritis-encephalitis virus（CAEV）After infecting goat, stimulate and produce antibody, p28/GS115 engineering bacterias are induced into table The p28 recombinant proteins reach, purified detect tested lowlenthal serum antibody using indirect ELISA method, can be used for as antigen CAE serodiagnosis.
1.1 material
Serum：Through IDEXX MVV/CAEV p28 Antibody screening kits（Production code member: P00303-10）Examination, goat are positive 30 parts of serum sample, it is negative 60 parts；Antigen：P28/GS115 engineering bacterias induced expression, the restructuring p28 albumen of purifying；
1.2 test method
Establish using p28/GS115 engineering bacterias induced expression, purification of Recombinant p28 albumen between the CAEV Serologic detections of antigen ELISA method is connect, negative control and positive control use the standard that IDEXX MVV/CAEV p28 Antibody screenings kit is equipped with Thing.
1）P28/GS115 engineering bacterias induced expression, the restructuring p28 albumen of purifying（10 ng/mL）, 100 μ L/ holes, 4 DEG C overnight.
2）5% skimmed milk（PBS-T）Confining liquid is incubated 30 min, 200 μ L/ holes, cleaning.
3）100 μ L/ holes add tested serum（500 times of dilutions）, it is incubated 1 h, clear plate.
4）The HRP mark rabbit-anti goat secondary antibodies that 100 μ L/ holes add 3000 times of dilutions are incubated 1 h, cleaning.
5）100 μ L/ holes add OPD, and develop the color 10 min
6）50 μ L/ holes add H2SO4Terminate liquid terminating reaction.
7）Serum hole OD values to be checked are judged to the positive with negative control ratio >=2.1.
2. result
After IDEXX MVV/CAEV p28 Antibody screening kit examinations, the iELISA methods being set up detect, 30 portions of goats 26 parts are the positive in positive serum sample, and 4 parts are feminine gender；60 parts of all feminine genders of negative sample.With IDEXX MVV/CAEV P28 Antibody screening kits are compared：Coincidence rate is 95.6%, and specificity is 100%, sensitiveness 86.7%.
SEQUENCE LISTING
<110>The Xinjiang herding academy of sciences
<120>A kind of caprine arthritis-encephalitis virus serum antibody diagnostic flag albumen p28 and preparation method thereof
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 684
<212> DNA
<213>Artificial sequence
<400> 1
gaattcccaa ttgttgttca agccgctggt ggaaggtctt ggaaagctgt tgactctgtc 60
atgttccagc agttgcagac agtcgcaatg caacacgggc tagtatcaga agattttgaa 120
agacaattgg catattacgc tactacttgg acctccaaag atattttgga agttttggca 180
atgatgcctg gaaatagagc acaaaaggaa cttattcaag gtaaattaaa cgaagaggcc 240
gagagatgga ggcgaaataa cccacctccc ccagctggtg gcggattgac agtagaccag 300
atcatgggtg taggtcaaac gaaccaagct gccgcccaag caaatatgga tcaagctaga 360
caaatctgtc tacaatgggt tatcaccgct ttaagggctg tgcgtcatat ggctcataga 420
cccggcaatc caatgttggt gagacaaaag gccaacgaat cctatgaaga atttgctgct 480
aagcttctgg aagccataga tgcagagcct gttactcagc ctattaagga ctacctgaag 540
ttaactctgt cctacactaa cgcttctagt gactgccaaa aacaaatgga tcgagttctt 600
ggacagcgtg tgcagcaagc tagtgtcgag gagaagatgc aggcctgtag agatgtcggt 660
tcagagggtt ttaaaatgca gtaa 684
<210> 1
<211> 227
<212> PRT
<213>Artificial sequence
<400> 1
Glu Phe Pro Ile Val Val Gln Ala Ala Gly Gly Arg Ser Trp Lys Ala
1 5 10 15
Val Asp Ser Val Met Phe Gln Gln Leu Gln Thr Val Ala Met Gln His
20 25 30
Gly Leu Val Ser Glu Asp Phe Glu Arg Gln Leu Ala Tyr Tyr Ala Thr
35 40 45
Thr Trp Thr Ser Lys Asp Ile Leu Glu Val Leu Ala Met Met Pro Gly
50 55 60
Asn Arg Ala Gln Lys Glu Leu Ile Gln Gly Lys Leu Asn Glu Glu Ala
65 70 75 80
Glu Arg Trp Arg Arg Asn Asn Pro Pro Pro Pro Ala Gly Gly Gly Leu
85 90 95
Thr Val Asp Gln Ile Met Gly Val Gly Gln Thr Asn Gln Ala Ala Ala
100 105 110
Gln Ala Asn Met Asp Gln Ala Arg Gln Ile Cys Leu Gln Trp Val Ile
115 120 125
Thr Ala Leu Arg Ala Val Arg His Met Ala His Arg Pro Gly Asn Pro
130 135 140
Met Leu Val Arg Gln Lys Ala Asn Glu Ser Tyr Glu Glu Phe Ala Ala
145 150 155 160
Lys Leu Leu Glu Ala Ile Asp Ala Glu Pro Val Thr Gln Pro Ile Lys
165 170 175
Asp Tyr Leu Lys Leu Thr Leu Ser Tyr Thr Asn Ala Ser Ser Asp Cys
180 185 190
Gln Lys Gln Met Asp Arg Val Leu Gly Gln Arg Val Gln Gln Ala Ser
195 200 205
Val Glu Glu Lys Met Gln Ala Cys Arg Asp Val Gly Ser Glu Gly Phe
210 215 220
Lys Met Gln
225

Claims (3)

  1. A kind of 1. caprine arthritis-encephalitis virus serum antibody diagnostic flag albumen p28, it is characterised in that：After optimized P28 albumen nucleotide sequence, as shown in SEQ ID NO.1, uniformity be not less than 95% other sequences；And include warp The amino acid sequence of p28 albumen after optimization, as shown in SEQ ID NO.2, uniformity is not less than 95% other sequences.
  2. 2. a kind of caprine arthritis-encephalitis virus serum antibody diagnostic flag albumen p28 as claimed in claim 1, its feature It is：The nucleotide sequence of p28 albumen after optimization, as shown in SEQ ID NO.1, the amino acid sequence of the p28 albumen after optimization Row, as shown in SEQ ID NO.2.
  3. 3. a kind of caprine arthritis-encephalitis virus serum antibody diagnostic flag albumen p28 preparation method, comprises the steps：
    （1）The codon optimization and synthesis of CAEV p28 genes
    Codon in Pichia pastoris GS115 expressing protein is optimized and closed to the encoding gene of CAEV p28 albumen Into acquisition pUC57-p28 plasmids；
    （2）The structure of recombinant vector
    Above-mentioned pUC57-p28 plasmids are passed throughNotⅠWithEcoRⅠIt is connected after double digestion with pPIC9K, constructs recombinant expression plasmid pPIC9K-p28；
    （3）The screening of Pichia pastoris GS115 positive recombinant
    The recombinant expression plasmid pPIC9K- p28 of linearisation are imported in Pichia pastoris GS115 by Electroporation；
    （4）The induced expression of p28/GS115 engineering bacterias, obtain p28 albumen.
CN201710667641.6A 2017-08-07 2017-08-07 A kind of Caprine arthritis encephalitis virus serum antibody diagnostic flag albumen p28 and preparation method thereof Pending CN107446029A (en)

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Application publication date: 20171208