CN107056898A - 3 type of carp herpesviral, 1301 plants of ORF136 DNA recombinant expressions albumen, antibody and its application - Google Patents
3 type of carp herpesviral, 1301 plants of ORF136 DNA recombinant expressions albumen, antibody and its application Download PDFInfo
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Abstract
The invention discloses a kind of 3 type of carp herpesviral, 1301 plants of ORF136 DNA recombinant expressions albumen, polyclonal antibody and its application.3 type of carp herpesviral, 1301 plants of ORF136 DNA recombinant expression albumen that the present invention is provided, its full length amino acid sequence such as SEQ ID NO:Shown in 2, the full length nucleotide sequence such as SEQ ID NO of the albumen are encoded:Shown in 1.The present invention chooses ORF136 partial gene sequences and builds pET32a ORF136 recombinant prokaryotic expression vectors, recombinant expression protein is obtained after IPTG induced expressions, polyclonal antibody is obtained through immune rabbit, gained polyclonal antibody is subjected to western blot analyses with the viruses of CyHV 3 of purifying and infection CyHV 3 KS cells, and the detection application of antibody is further verified using indirect immunofluorescence experiment.The be prepared as research of ORF136 protein functions and the foundation of the serological diagnostic methods of CyHV 3 of ORF136 DNA recombinant expression protein polyclone antibodies provide important materials.
Description
Technical field
The invention belongs to biological gene engineering field, and in particular to by 3 type of carp herpesviral, 1301 plants of ORF136 gene weights
Group expressing protein, the polyclonal antibody prepared by ORF136 recombinant expression proteins and its application, further to polyclonal antibody
Preparation method.
Background technology
The type of carp herpesviral 3 (Cyprinid Herpesvirus 3, CyHV-3), also known as Koi herpesvirus (Koi
Herpesvirus, KHV), it is to cause carp, fancy carp and its mutation highly infectious Koi herpesvirus disease (Koi
Herpesvirus disease, KHVD) cause of disease.The disease is since 1997 since Europe is found, multiple countries and regions are all
Report as carp caused by the disease or the fancy carp fulminant phenomena of mortality, brought to carp and fancy carp aquaculture industry serious
Economic loss.CyHV-3 belongs to herpesviral mesh, different herpetoviridae, carp Herpesvirus, is the double-stranded DNA disease with cyst membrane
Poison, a diameter of 167~200nm of virion, full-length genome 295kbp, two ends are 22kbp direct repetitive sequence, altogether coding
164 ORFs (Open reading frame, ORF), including 8 repetition ORF, are the maximum blebs of known group
Virus.
At present, 46 kinds of virus structural proteins have been identified with the method for mass-spectrometry.Wherein, Michel etc. uses liquid phase string
It is unknown that connection chromatographic technique identifies 13 kinds of envelope proteins, 3 kinds of nucleocapsid proteins, 2 seed coat layer albumen and 22 kinds in KHV-FL plants
Albumen totally 40 kinds of structural proteins.Then, Yi etc. is to Asian type KHV-GZ10 and Europe class KHV-GZ11 by carrying out Mass Spectrometric Identification,
The structural proteins of identification are increased to 46 kinds.Certain recognize although identifying there are CyHV-3 virus proteins by proteomic image
Know, but most of albumen is accurately positioned with functional study still in blank, and only a small number of albumen have carried out more deep identification,
Wherein, ORF81 encoding proteins are one of more structural proteins of research.In addition, Fuchs etc. also to part envelope protein ORF25,
ORF65, ORF99, ORF136, ORF149 and major capsid protein ORF92 have carried out Preliminary Identification.
There is nucleotide sequence difference in the ORF136 that the present invention is selected, it is completely opened in Asian type and Europe class separation strains
It is respectively 474bp and 462bp to put reading frame length, the gene can as CyHV-3 molecule partings effective tool.Although albumen
Mass spectral analysis shows that ORF136 encoding proteins are structural proteins, but has no specific checking, and its deep functional localization is also unclear
Chu, more correlative studys still need to be carried out.
Although existing many CyHV-3 checkout and diagnosis method at present, main in it is also based on point actual
The detection technique of son, serodiagnosis is not widely used.For the host of latent infection, it is based on
The detection of molecule can not be judged with the presence or absence of virus in host, and serodiagnosis can overcome the disadvantages that this defect.It is based on
The foundation of immunologic antigen detection method, it is desirable to have energy specific recognition CyHV-3 antibody.
The content of the invention
In view of the shortcomings of the prior art, the present invention provides a kind of 3 type of lithium herpesviral, 1301 plants of ORF136 genetic recombination tables
Up to albumen and its polyclonal antibody of preparation, it is further provided the polyclonal antibody is used to detect answering for ORF136 gene viruses
With, and polyclonal antibody preparation method.
To solve the above problems, on the one hand, the invention reside in provide a kind of 1301 plants of ORF136 genes of 3 type of carp herpesviral
Recombinant expression protein, its full length amino acid sequence such as SEQ ID NO:Shown in 2.
On the other hand, the invention reside in provide a kind of coding carp herpesviral 3 type, 1301 plants of ORF136 DNA recombinant expressions
The nucleotides of albumen, its full length nucleotide base sequence such as SEQ ID NO:Shown in 1.
On the one hand, the present invention provides a kind of polyclonal antibody, and it is with amino acid sequence such as SEQ ID NO:Weight shown in 5
Group expressing protein is obtained by antigen is prepared in immune animal.
Further, it is of the present invention such as SEQ ID NO:Amino acid sequence shown in 5 is SEQ ID NO:Ammonia shown in 2
A part for base acid sequence；Its specific preparation process is that carp herpesviral 3 type, 1301 plants of ORF136 genes are entered into performing PCR amplification
Afterwards, recombinant cloning vector pMD18-T-ORF136 is obtained；By recombinant cloning vector pMD18-T-ORF136, prokaryotic expression carrier
PET-32a (+) is attached after carrying out double digestion respectively, builds recombinant prokaryotic expression vector pET-32a-ORF136；By pET-
32a-ORF136 is converted to Escherichia coli Rosetta and obtained after IPTG induced expressions such as SEQ ID NO:Restructuring table shown in 5
Up to albumen.
Further, Anti-TNF-α production procedure of the present invention is as follows：Will be such as SEQ ID NO:Restructuring shown in 5
Expressing protein carries out subcutaneous multi-point injection to rabbit and is immunized, and is taken a blood sample after being immunized；After blood is stood, serum is collected in centrifugation,
Purified with the mode antagonistic Serum of affinity purification, obtain polyclonal antibody.
On the other hand, the invention reside in provide a kind of polyclonal antibody of the present invention to be used to prepare detection carp blister sore
The application of the immune instrument of malicious 3 types.Immune instrument of the present invention includes but is not limited to, reagent, kit, chip or examination
Paper.
Further, polyclonal antibody of the present invention draws in detection, prevention or the type infection for the treatment of carp herpesviral 3
Application in the relevant disease risen.
On the other hand, the invention reside in a kind of kit for detecting the type of carp herpesviral 3 is provided, containing of the present invention
The antibody of carp herpesviral 3 type, 1301 plants of ORF136 DNA recombinant expression albumen.
On the other hand, the present invention provides a kind of method for preparing polyclonal antibody of the present invention, specific preparation process
It is as follows：
1) expand：Using 3 type of carp herpesviral, 1301 plants of DNA as template, design specific primer is to ORF136 gene codes
Performing PCR amplification is entered in the 91st~471 nucleotide region of albumen, obtains recombinant cloning vector pMD18-T-ORF136；
2) build：Recombinant cloning vector pMD18-T-ORF136, prokaryotic expression carrier pET-32a (+) are carried out respectively double
Connected after digestion, build recombinant prokaryotic expression vector pET-32a-ORF136；
3) express：PET-32a-ORF136 is converted to Escherichia coli Rosetta and obtained after IPTG induced expressions such as SEQ
ID NO:Recombinant expression protein shown in 5；
4) prepare：Will be such as SEQ ID NO:Recombinant expression protein shown in 5 is taken a blood sample after being immunized in immune animal；It is right
Blood obtains polyclonal antibody after being handled.
Further, a kind of method for preparing polyclonal antibody of the present invention of the present invention, the step 2)
The system of middle digestion is：10 × buffer 2ul, EcoR I and Xho I each 1ul, plasmid 1ug, mend ddH2O water is to 20ul；37℃
Water-bath digestion 2h.
Further, a kind of method for preparing polyclonal antibody of the present invention of the present invention, the step 2)
The system of middle connection is：T4Ligase 1.5uL, 10 × T4Ligase buffer 1.5uL, pET-32a (+) 3uL, after digestion
Fragment 9uL；16 DEG C of connections are stayed overnight.
Further, a kind of method for preparing polyclonal antibody of the present invention of the present invention, the step 3)
Middle pET32a-ORF136 is converted into Escherichia coli Rosetta, screening positive clone, is inoculated in the LB culture mediums containing ammonia benzyl
In, culture to OD600 is 0.4, adds IPTG and carries out after induced expression, thalline is collected by centrifugation, thalline is cleaned, crushed,
Collected after centrifugation inclusion body protein, inclusion body protein obtains recombinant expression protein after purification.
Further, a kind of method for preparing polyclonal antibody of the present invention of the present invention, the step 3)
Middle bacterial cell disruption is using ultrasonic disruption on ice.
Further, a kind of method for preparing polyclonal antibody of the present invention of the present invention, the step 4)
Middle recombinant expression protein carries out subcutaneous multi-point injection in immune animal and is immunized, and is taken a blood sample after being immunized；After blood is stood, from
The heart, collects serum, is purified with the mode antagonistic Serum of affinity purification, obtain polyclonal antibody.
Further, specific primer of the present invention is
F:CCGGAATTCGGCACAACAACCATGAACTCTACC(SEQ ID NO:3) (restriction enzyme sites of I containing EcoR, are shown in
Underscore part)
R:CCGCTCGAGTTAGATTTTTCTAAAGTGCACGACGTC(SEQ ID NO:4) (restriction enzyme sites of I containing Xho,
See below dashed part).
The present invention passes through bioinformatics software, the amino acids of screening KHV ORF136 gene coded proteins the 31st~157
Region is more complete potential B cell antigen epi-position, builds pET-32a-ORF136 recombinant prokaryotic expression vectors；Lured by IPTG
Purification of recombinant proteins after expression is led, polyclonal antibody, and CyHV-3 viruses and infection CyHV- with purifying are obtained through immune rabbit
3 KS cells carry out immunoblotting assay (Western blot), and further verify antibody using indirect immunofluorescence experiment
Detection application.The ORF136 protein functions that are prepared as of ORF136 DNA recombinant expression protein polyclone antibodies are studied and CyHV-3
The foundation of serological diagnostic method provides important materials.
The preparation method of antibody of the present invention has the higher rate of recovery and preferably keeps the bioactivity of albumen, is adapted to big
Scale purification and preparation.The purity of polyclonal antibody is high in the present invention, it is easy to industrialization.
Compared with prior art, the present invention has the advantages that：The present invention provides a kind of type 1301 of carp herpesviral 3
Strain ORF136 DNA recombinant expression albumen, and the albumen is produced into polyclonal antibody.The recombinant protein can be used for carp herpesviral 3
The detection application of type.The present invention provides a kind of energy specific recognition CyHV-3 polyclonal antibody, and provides the ORF136 of preparation
The method of DNA recombinant expression protein polyclone antibody, the virus of the Anti-TNF-α physical efficiency specific recognition purifying and infection
CyHV-3 cell, has certain application value in the foundation of follow-up CyHV-3 serological diagnostic methods, available for it is clinical,
Test in laboratory and epidemiological study.The preparation method that the present invention is provided is simple, easy to operate.
Brief description of the drawings
Fig. 1 is CyHV-3ORF136 genes (the 91st~471 nucleotides) pcr amplification product electrophoretogram；M is DL in Fig. 1
marker 2000；1 is the pcr amplification product using KHV DNA as template；
Fig. 2 is recombinant plasmid pET32a-ORF136 double digestion qualification results；M is DL marker 10000 in Fig. 2；1 is
Recombinant plasmid pET32a-ORF136 double digestion products；2 be empty carrier pET32a (+) double digestion product；
Fig. 3 is that SDS-PAGE analyzes recombinant plasmid pET32a-ORF136 expression products；M is low molecule quality protein in Fig. 3
Matter marker；1 is pET32a-ORF136 induced expression supernatants；2~4 be 2 times of pET32a-ORF136 induced expressions inclusion body, 5
Diluted again with 10 times；
Fig. 4 is that western blot analyze ORF136 encoding proteins antiserum characteristics；M Low molecular weight proteins in Fig. 4
marker；1 1301 plants of the CyHV-3 to purify；2 be infection CyHV-3 KS cells；3 be to be uninfected by CyHV-3 KS cells
Clearly；The 4 KS cells to be uninfected by；
Fig. 5 is the cell that virus infection is detected using polyclonal antibody；A is thin for 1301 plants of CyHV-3 of inoculation KS in Fig. 5
Born of the same parents；B is the normal KS cell controls of non-virus inoculation.
Embodiment
With reference to embodiment, the present invention is further illustrated, but is not limited thereto.
Molecular biology experiment technology employed in following examples include PCR amplifications, plasmid extraction, plasmid convert,
DNA fragmentation connection, digestion, gel electrophoresis etc., unless otherwise specified, generally conventionally operate, for details, reference can be made to《Molecule
Cloning experimentation guide》(third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitangs etc. are translated,
2002, Beijing：Science Press), or according to the condition proposed by manufacturer.
Embodiment 1
Step 1: the amplification of 1301 plants of ORF136 genes of KHV
According to CyHV-3ORF136 gene orders in GenBank, with bioinformatics software ABCpred (http://
www.imtech.res.in/raghava/abcpred/)、BepiPred 1.0(http://www.cbs.dtu.dk/
Services/BepiPred/) and DNAstar submodules Protean prediction the potential B cell antigen epi-position of ORF136 genes.
With the designs of Primer Premier 5.0 specific primer, (specific primer is：
F:CCGGAATTCGGCACAACAACCATGAACTCTACC(SEQ ID NO:3) (restriction enzyme sites of I containing EcoR, are shown in
Underscore part),
R:CCGCTCGAGTTAGATTTTTCTAAAGTGCACGACGTC(SEQ ID NO:4) (restriction enzyme sites of I containing Xho,
See below dashed part),
And synthesized by Sheng Gong biotech firms.
Viral genome, which is extracted, presses blood/cell/tissue DNA extraction kit (Tiangeng biochemical technology Co., Ltd, north
Capital) in specification carry out.
PCR reaction systems：12.5ul rTaq Mix, each 1ul of primer (10umol/L) and 1ul DNA profilings, mend aqua sterilisa
To 25ul.
Response procedures are as follows：94 DEG C, 5min；94 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 45s, 30 circulations；72 DEG C, 7min.
Using F/R as primer, for the potential B cell antigen epi-position region of ORF136 genes (i.e. ORF136 gene coded proteins
91st~471 nucleotide region, i.e. the 31st~157 amino acids region), with 3 type of carp herpesviral, 1301 plants are entered for template
Performing PCR is expanded, and 1.5% agarose electrophoretic analysis display obtains a band being consistent with expected size, and clip size is
381bp (specific as shown in Figure 1).
Step 2: recombinant prokaryotic expression vector pET32a-ORF136 structure
Recombinant cloning vector pMD18-T-ORF136, the prokaryotic expression carrier pET-32a (+) that step one is expanded are entered respectively
Row double digestion (enzyme is EcoR I and Xho I enzymes), and the prokaryotic expression after recombinant cloning vector Insert Fragment and double digestion is carried
Body is attached, and obtains recombinant prokaryotic expression vector.Experiment condition is as follows：
Digestion system is：10 × buffer 2ul, EcoR I and Xho I each 1ul, plasmid 1ug, mend dd H2O water is extremely
20ul.37 DEG C of water-bath digestion 2h.After digestion, electroresis appraisal digestion effect, and to vector insert 0RF136 in recombinant clone
Rubber tapping recovery is carried out with the prokaryotic expression carrier pET-32a (+) after double digestion.
Linked system：T4Ligase 1.5uL, 10 × T4Ligase buffer 1.5uL, pET-32a (+) 3uL, digestion
Fragment 9uL afterwards.16 DEG C of connections, overnight.
Connection product is converted to DH5 α competent cells, screening positive clone carries out bacterium solution PCR.
The recombinant prokaryotic expression vector pET32a-ORF136 of structure is subjected to double digestion identification, obtained and expected size phase
Two band of symbol, show that recombinant prokaryotic expression vector is successfully constructed (specific as shown in Figure 2).
Step 3: protein expression
Step 2 is identified that correct recombinant prokaryotic expression vector pET32a-ORF136 is converted to Escherichia coli Rosetta
(DE3) in, screening positive clone is inoculated in the LB culture mediums containing ammonia benzyl, at 37 DEG C shaking table culture to OD600 be 0.4,
The IPTG for adding final concentration of 0.8mM carries out 10000rpm/min centrifugations 5min collection thalline after induced expression, 4h, carries out a small amount of
Expression identification.
By the thalline of collection with after PBS 2 times, it is resuspended, in ultrasonic disruption on ice, 10000rpm/min centrifugations
5min, collects supernatant inclusion body precipitation, with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) respectively
Identified.
SDS-PAGE is analyzed, and as a result shows the protein band for obtaining and being consistent with purpose size, size is about 35kDa (carriers
The amino acids of about 20kDa, ORF136 the 31st~157 prediction molecular weight of albumen is about 14kDa), and destination protein is mainly distributed
In inclusion body (Fig. 3).
Inclusion body protein is purified with reference to the method for Ni- gel purification kits, the recombination expression egg purified
In vain, its amino acid sequence such as SEQ ID NO:Shown in 5.
Step 4: polyclonal antibody preparation and purification
The recombinant expression protein that step 3 is purified is immune to the subcutaneous multi-point injection of rabbit progress, and (first immunisation uses Freund
Freund's complete adjuvant, latter three times immune to use incomplete Freund's adjuvant, and adjuvant and recombinant expression protein volume ratio are 1:1), it is immunized 66 days
Afterwards, arteria carotis blood sampling is carried out.Blood is stood after 1h in 37 DEG C, 3000rpm/min centrifugation 10min collect serum, with affine pure
The mode antagonistic Serum of change is purified, and obtains polyclonal antibody, in preservation at -20 DEG C.
Embodiment 2
SDS-PAGE and immunoblotting assay (Western blot)
Polyacrylamide gel is made up of 12% separation gel and 4% concentration glue, by 1301 plants of diseases of CyHV-3 of purifying
Poison, RIPA lysates cracking postoperative infection CyHV-3 KS cells and be uninfected by CyHV-3 KS cells respectively with 5 × albumen loading
Buffer solution heats point sample after 5min, cooling in boiling water and carries out electrophoretic analysis (200V, 35min) after mixing in proportion.By SDS-
Gel after PAGE separation is transferred to nitrocellulose filter, 5% closing of skimmed milk power room temperature 1h, Ran Houyong with wet turn of method
Sealer 1:The polyclonal antibody that the step 4 of embodiment 1 of 1000 dilutions is obtained is incubated 1h at room temperature, then with 0.05%
PBST is cleaned 3 times, each 10min；Finally use sealer 1:The HRP mark goat anti-rabbit igg incubation at room temperature 1h of 10000 dilutions, are used
0.05% PBST carries out DAB colour developings after cleaning 3 times.
Western blot analysis results show occur a specific band (as shown in Figure 4) between 15~25kDa,
ORF136 encoding proteins predicted molecular weights are about 17kDa, in the same size with expection, illustrate Anti-TNF-α physical efficiency prepared by the present invention
Specific recognition CyHV-3.
Embodiment 3
Indirect immunofluorescence assay detects infection cell
Fancy carp kiss teloblast (KS cells) is passaged in 96 porocyte culture plates, treats that cell length to individual layer is paved with 80% left
Behind the right side, the strain virus of CyHV-3 1301 is infected；Infect after virus 5 d, the culture medium in Tissue Culture Plate is exhausted, 0.01mol/ is used
L PBS is washed 3 times, is added 80% acetone soln incubation at room temperature 30min, is sucked acetone, drying at room temperature 1h；Add 100 μ l per hole
1：The rabbit-anti ORF136 protein polyclone antibodies of 1000 dilutions, 37 DEG C are incubated 1h, and PBST is washed 3 times, adds 1:The sheep of 50 dilutions
Fluorescence secondary antibody 100 the μ l, 37 DEG C of incubation 1h of anti-rabbit IgG-FITC marks；PBST is washed 3 times, is eventually adding nucleus dyestuff iodate
Third pyridine (PI) acts on and observes result under 5min, fluorescence inverted microscope (Nikon, Eclipse Ti-S).Negative control is not feel
Catch an illness poison normal KS cells.
The result of indirect immunofluorescence experiment shows that the Anti-TNF-α physical efficiency of preparation makes the strains of infection CyHV-3 1301
KS cells can produce specific fluorescence (see Fig. 5 A), and not observe fluorescence signal in the normal KS cells of uninfecting virus
(Fig. 5 B).
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
SEQUENCE LISTING
<110>China's Pearl River Fishery Research Institute of Aquatic Science Research Institute
<120>3 type of carp herpesviral, 1301 plants of ORF136 DNA recombinant expressions albumen, antibody and its application
<130> 2017
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 474
<212> DNA
<213>It is unknown
<400> 1
atgaaggcct ctaaactgct gctggttaca tgggtggctt tcgcggccgg ccagaacgcc 60
accaccctgg ctccgctcgc cgccactaac ggcacaacaa ccatgaactc tacctggtcc 120
tctaccgctt cagtcatgaa ctctactacc tggtcctcta ctatgaactc tacctggtcc 180
accaccaccg cggcgagcgg cgactcttgg tggcgcccag aagaggtgct gtctaggtgt 240
agggaccagg ccggactgaa ctggctgggg tgcgctcagg gtatggtggt ggtgatggtc 300
atcttcgcta tcatctttgc catcatcgtg atcgtcgtgg tgtgggtgct gatgcacgtc 360
gtgatcgccc gtcgcgaccc agccggcgcg gcagggccag gagccatgtc tgcatcctac 420
aacaggggct acgtcgagga tgaagacgac gtcgtgcact ttagaaaaat ctaa 474
<210> 2
<211> 157
<212> PRT
<213>It is unknown
<400> 2
Met Lys Ala Ser Lys Leu Leu Leu Val Thr Trp Val Ala Phe Ala Ala
1 5 10 15
Gly Gln Asn Ala Thr Thr Leu Ala Pro Leu Ala Ala Thr Asn Gly Thr
20 25 30
Thr Thr Met Asn Ser Thr Trp Ser Ser Thr Ala Ser Val Met Asn Ser
35 40 45
Thr Thr Trp Ser Ser Thr Met Asn Ser Thr Trp Ser Thr Thr Thr Ala
50 55 60
Ala Ser Gly Asp Ser Trp Trp Arg Pro Glu Glu Val Leu Ser Arg Cys
65 70 75 80
Arg Asp Gln Ala Gly Leu Asn Trp Leu Gly Cys Ala Gln Gly Met Val
85 90 95
Val Val Met Val Ile Phe Ala Ile Ile Phe Ala Ile Ile Val Ile Val
100 105 110
Val Val Trp Val Leu Met His Val Val Ile Ala Arg Arg Asp Pro Ala
115 120 125
Gly Ala Ala Gly Pro Gly Ala Met Ser Ala Ser Tyr Asn Arg Gly Tyr
130 135 140
Val Glu Asp Glu Asp Asp Val Val His Phe Arg Lys Ile
145 150 155
<210> 3
<211> 33
<212> DNA
<213>It is unknown
<400> 3
ccggaattcg gcacaacaac catgaactct acc 33
<210> 4
<211> 36
<212> DNA
<213>It is unknown
<400> 4
ccgctcgagt tagatttttc taaagtgcac gacgtc 36
<210> 5
<211> 127
<212> PRT
<213>It is unknown
<400> 5
Gly Thr Thr Thr Met Asn Ser Thr Trp Ser Ser Thr Ala Ser Val Met
1 5 10 15
Asn Ser Thr Thr Trp Ser Ser Thr Met Asn Ser Thr Trp Ser Thr Thr
20 25 30
Thr Ala Ala Ser Gly Asp Ser Trp Trp Arg Pro Glu Glu Val Leu Ser
35 40 45
Arg Cys Arg Asp Gln Ala Gly Leu Asn Trp Leu Gly Cys Ala Gln Gly
50 55 60
Met Val Val Val Met Val Ile Phe Ala Ile Ile Phe Ala Ile Ile Val
65 70 75 80
Ile Val Val Val Trp Val Leu Met His Val Val Ile Ala Arg Arg Asp
85 90 95
Pro Ala Gly Ala Ala Gly Pro Gly Ala Met Ser Ala Ser Tyr Asn Arg
100 105 110
Gly Tyr Val Glu Asp Glu Asp Asp Val Val His Phe Arg Lys Ile
115 120 125
Claims (9)
1. a kind of 3 type of carp herpesviral, 1301 plants of ORF136 DNA recombinant expression albumen, it is characterised in that its full length amino acid sequence
Row such as SEQ ID NO:Shown in 2.
2. a kind of nucleotides for encoding the recombinant expression protein described in claim 1, it is characterised in that its full length nucleotide base
Sequence such as SEQ ID NO:Shown in 1.
3. a kind of polyclonal antibody, it is characterised in that with amino acid sequence such as SEQ ID NO:Recombinant expression protein shown in 5 is
Obtained by antigen is prepared in immune animal.
4. the Antibody preparation described in claim 3 is used for the application for detecting the immune instrument of the type of lithium herpesviral 3.
5. the antibody described in claim 3 is in relevant disease caused by detection, prevention or the type infection for the treatment of carp herpesviral 3
Using.
6. a kind of method of the polyclonal antibody prepared described in claim 3, it is characterised in that specific preparation process is as follows：
1) expand：Using 3 type of carp herpesviral, 1301 plants of DNA as template, design specific primer is to ORF136 gene coded proteins
Performing PCR amplification is entered in 31st~157 amino acids region, obtains recombinant cloning vector pMD18-T-ORF136；
2) build：Recombinant cloning vector pMD18-T-ORF136, prokaryotic expression carrier pET-32a (+) are subjected to double digestion respectively
After connect, build recombinant prokaryotic expression vector pET-32a-ORF136；
3) express：PET-32a-ORF136 is converted to Escherichia coli Rosetta and obtained after IPTG induced expressions such as SEQ ID
NO:Recombinant expression protein shown in 5；
4) prepare：Will be such as SEQ ID NO:Recombinant expression protein shown in 5 is taken a blood sample after being immunized in immune animal；To blood
Polyclonal antibody is obtained after being handled.
7. method according to claim 6, it is characterised in that
The step 2) in the system of digestion be：10 × buffer 2ul, EcoR I and Xho I each 1ul, plasmid 1ug, mend ddH2O
Water is to 20ul；37 DEG C of water-bath digestion 2h；
The step 2) in connection system be：T4Ligase 1.5uL, 10 × T4Ligase buffer 1.5uL, pET-32a
(+) 3uL, the fragment 9uL after digestion；16 DEG C of connections are stayed overnight.
8. method according to claim 6, it is characterised in that the step 3) in pET32a-ORF136 convert to large intestine
In bacillus Rosetta, screening positive clone is inoculated in the LB culture mediums containing ammonia benzyl, and culture to OD600 is 0.4, is added
IPTG is carried out after induced expression, and thalline is collected by centrifugation, and thalline is cleaned, crushed, collected after centrifugation inclusion body protein, is forgiven
Body protein obtains recombinant expression protein after purification.
9. method according to claim 6, it is characterised in that the step 4) in recombinant expression protein in immune animal
Carry out subcutaneous multi-point injection to be immunized, taken a blood sample after being immunized；After blood is stood, centrifugation collects serum, with the side of affinity purification
Formula antagonistic Serum is purified, and obtains polyclonal antibody.
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| CN108047317A (en) * | 2018-02-08 | 2018-05-18 | 广东省农业科学院动物卫生研究所 | PORF148 recombinant proteins and its preparation method and application |
| CN110407920A (en) * | 2019-07-29 | 2019-11-05 | 盐城工学院 | A kind of prokaryotic expression, the method for purifying proteins of carp herpesvirusⅡtype capsid protein ORF66 |
| CN114685619A (en) * | 2021-06-23 | 2022-07-01 | 中国检验检疫科学研究院 | Antigen protein, monoclonal antibody or polyclonal antibody thereof and application |
| CN114685619B (en) * | 2021-06-23 | 2024-02-13 | 中国检验检疫科学研究院 | Antigen protein, monoclonal antibody or polyclonal antibody and application thereof |
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