CN112094853A - White spot syndrome virus VP28 gene, recombinant protein, polyclonal antibody, preparation method and application - Google Patents
White spot syndrome virus VP28 gene, recombinant protein, polyclonal antibody, preparation method and application Download PDFInfo
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Abstract
The invention belongs to the field of molecular biological gene engineering, relates to a nucleotide sequence of White Spot Syndrome Virus (WSSV) VP28 gene, an amino acid sequence coded by the nucleotide sequence and a nucleotide sequence after codon optimization, and particularly relates to a nucleotide sequence of White Spot Syndrome Virus (WSSV) VP28 gene, and a preparation method thereofWSSVRecombinant expression preparation method of VP28 protein and anti-rabbitAnd (4) preparing a polyclonal antibody. By recombinant expressionWSSVThe VP28 protein can be further used for analyzing the structure and function of the protein in vitro, and the preparation of the polyclonal antibody can lay a foundation for further detecting the WSSV content in shrimp bodies.
Description
Technical Field
The nucleotide sequence and the coded amino acid sequence of the VP28 gene of WSSV and the nucleotide sequence after codon optimization, in particular to a preparation method of VP28 protein of WSSV by recombinant expression and the preparation of anti-rabbit polyclonal antibody. The VP28 protein of WSSV can be expressed in a recombinant mode to further analyze the structure and the function of the protein in vitro, and in addition, the preparation of the polyclonal antibody can lay a foundation for further detecting the content of WSSV in fishes and shrimps.
Background
VP28 of WSSV is the envelope protein of white spot syndrome virus which is most studied at present, and the protein is one of the most main components of the virus, including structural proteins and non-structural proteins. The structural proteins of WSSV comprise three parts of envelope protein, nucleocapsid protein and envelope protein, which maintain the basic structure of virus, protect the nucleic acid of virus and participate in the recognition, adsorption and invasion of host tissue cells by virus. The majority of non-structural proteins are enzymes and regulatory proteins that catalyze and regulate viral replication.
Disclosure of Invention
In order to solve the technical problems, the invention provides a white spot syndrome virus VP28 gene, a recombinant protein, a polyclonal antibody, a preparation method and application thereof, so as to solve the problems that the WSSV measurement cannot be carried out due to the lack of a procambarus clarkia WSSVVP28 detection antibody and the like.
The technical scheme of the invention is as follows:
a white spot syndrome virus VP28 gene, wherein VP28 gene is designated as WSSVVP28, and has the sequence as SED ID NO: 1 is shown.
A white spot syndrome virus VP28 gene after codon optimization has a sequence shown as SED ID NO: 2, respectively.
A recombinant protein of white spot syndrome virus VP28, the amino acid sequence of which is as defined in SED ID NO: 3, respectively.
A preparation method of a white spot syndrome virus VP28 recombinant protein comprises the following steps: optimizing gene sequence SED ID NO of white spot syndrome virus VP28 codon: 2 is connected into a PET-B2M expression vector to obtain pET-B2M- WSSVVP28 recombinant plasmid;
pET-B2M- WSSVVP28 recombinant plasmid was transformed intoCulturing, inducing, cracking and purifying in escherichia coli to obtain the white spot syndrome virus VP28 recombinant protein.
In some embodiments, the codon-optimized sequence SEQ ID No.2 is synthesized and directly connected to a PET-B2M vector, transformed into escherichia coli BL21 (DE 3) competent cells, expressed by IPTG induction, collected and subjected to SDS-PAGE protein detection, bacteria are cracked by ultrasonic disruption, and recombinant protein is purified.
Further, the conversion steps are as follows: to competent cellsE. coliBL21 is added with pET-B2M-WSSV-VP28 recombinant plasmid, gently mixed, kept stand in ice bath for 5min, placed in 42 ℃ water bath for 90 s of heat shock, and quickly transferred to ice bath. Adding 900 μ L sterile LB culture medium into each centrifuge tube, mixing, shaking and culturing for 45 min at 37 deg.C, recovering thallus, sucking 100 μ L recovered bacteria liquid, spreading on LB solid culture medium containing kanamycin resistance, and culturing at 37 deg.C for 12-16 h.
Further, the induction expression step is as follows: selecting escherichia coli containing recombinant plasmids, adding a liquid culture medium, carrying out shake culture at 37 ℃ until the OD600 of a bacterial liquid is 0.4-0.6, adding a proper amount of IPTG to the final concentration of 1 mM, carrying out induced expression for 4h, collecting thalli, resuspending the thalli with sterilized water, freezing and preserving at-80 ℃, and repeatedly freezing and thawing for 1-2 times; and (3) taking the frozen and thawed resuspended thalli, carrying out ultrasonic crushing under the ice bath condition, centrifugally collecting the crushed supernatant, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection.
Further, the crushing conditions are as follows: crushing for 4 s at intervals of 3 s for 30 min.
Further, the purification steps are as follows: collecting the supernatant after ultrasonication with Ni2+Purifying with affinity chromatography column, washing with 15 times column volume Binding Buffer (20 mM Tris-HCl pH 7.9, 5 mM imidazole, 0.5M NaCl), and washing off foreign protein; eluted with 5 mL of Elution Buffer (20 mM Tris-HCl pH 7.9, 500 mM imidazole, 0.5M NaCl) and the eluted protein was collected.
A polyclonal antibody is prepared by immunizing animals with the recombinant protein VP28 of white spot syndrome virus as antigen.
The preparation method of the polyclonal antibody uses the recombinant protein VP28 of white spot syndrome virus as antigen, adopts the conventional preparation method of the polyclonal antibody to prepare antiserum, and separates and purifies the antiserum to obtain the polyclonal antibody. Further, the method comprises the following steps: injecting the recombinant protein of the white spot syndrome virus VP28 as an antigen into a rabbit body, immunizing for four times, collecting rabbit blood, separating serum, and purifying to obtain the anti-rabbit polyclonal antibody of the white spot syndrome virus VP 28.
In some embodiments, a method for preparing anti-rabbit polyclonal antibodies against white spot syndrome virus VP28 comprises the following steps:
2 Japanese big ear rabbits were used, and the VP28 protein concentration of the recombinant WSSV was adjusted to 1mg/ml for antigen injection, and the adjuvant and the antigen were extracted at a volume ratio of 1: 1. Complete adjuvant is adopted for the first time, and incomplete adjuvant is adopted for the second rabbit. The rabbits were injected subcutaneously in multiple spots of 0.2ml each. The second immunization is carried out 14 days after the first immunization, and the interval between the second immunization and the third immunization is 7 days. And (3) collecting small sample serum from the middle ear artery on the 7 th day after the rabbit three-immunization, detecting the small sample serum to be qualified, adding the rabbit three-immunization after 7 days, and collecting the whole blood after 7 days of adding the rabbit three-immunization. The antibody titer in rabbit sera was determined by ELISA (indirect method). Then, the antibody is purified by using affinity chromatography column proteinG, and is applied to subsequent western blot and other experiments.
The white spot syndrome virus envelope protein VP28 gene is cDNA sequence with length of 615bp, type of double-chain, linear and nucleic acid, the described white spot syndrome virus envelope protein VP28 gene is a cDNA sequenceWSSVThe sequence of VP28 was amplified by PCR and sequenced. The specific sequence is shown as follows, SEQ ID No. 1:
atggatcttt ctttcactct ttcggtcgtg tcggccatcc tcgccatcac tgctgtgatt
gctgtattta ttgtgatttt taggtatcac aacactgtga ccaagaccat cgaaacccac
acaggcaata tcgagacaaa catggatgaa aacctccgca ttcctgtgac tgctgaggtt
ggatcaggct acttcaagat gactgatgtg tcctttgaca gcgacacctt gggcaaaatc
aagatccgca atggaaagtc tgatgcacag atgaaggaag aagatgcgga tcttgtcatc
actcccgtgg agggccgagc actcgaagtg actgtggggc agaatctcac ctttgaggga
acattcaagg tgtggaacaa cacatcaaga aagatcaaca tcactggtat gcagatggtg
ccaaagatta acccatcaaa ggcctttgtc ggtagctcca acacctcctc cttcaccccc
gtctctattg atgaggatga agttggcacc tttgtgtgtg gtaccacctt tggcgcacca
attgcagcta ccgccggtgg aaatcttttc gacatgtacg tgcacgtcac ctactctggc
actgagaccg agtaa。
using DNAworks 2.4 online software, toWSSVThe nucleotide sequence of VP28 is subjected to codon optimization, and an Escherichia coli expression system is selected for comparisonWSSVCodon optimized sequence of the VP28 sequence, labeled SEQ ID No.2:
cataacaccgtgaccaaaaccattgaaacccataccggtaatatcgaaaccaacatggatgaaaatctgcgtattccggttaccgcagaagttggtagcggttatttcaaaatgaccgatgttagctttgatagcgataccctgggcaaaatcaaaattcgcaatggtaaaagtgacgcccagatgaaagaagaggacgcagatctggttattacaccggttgaaggtcgtgcactggaagttaccgttggccagaatctgacctttgaaggcacctttaaagtgtggaataataccagccgcaagattaacattaccggcatgcagatggttccgaaaattaacccgagcaaagcatttgttggtagcagcaataccagcagctttactccggttagcattgatgaagatgaagttggcacctttgtttgtggcaccacctttggtgcaccgattgcagcaaccgcaggcggtaacctgtttgatatgtatgttcatgttacctatagcggcaccgaaaccgaa。
saidWSSVThe molecular type of VP28 is protein, and the sequence characteristics are as follows: length section: 30-204aa in total 175aa, a molecular weight of 19KD, amino acid type, and the following sequence information, labeled as SEQ ID No.3:
HNTVTKTIETHTGNIETNMDENLRIPVTAEVGSGYFKMTDVSFDSDTLGKIKIRNGKSDAQMKEEDADLVITPVEGRALEVTVGQNLTFEGTFKVWNNTSRKINITGMQMVPKINPSKAFVGSSNTSSFTPVSIDEDEVGTFVCGTTFGAPIAATAGGNLFDMYVHVTYSGTETE。
the preparation method of the recombinant protein VP28 and the anti-rabbit polyclonal antibody of the envelope protein of the white spot syndrome virus comprises the following steps:
synthesizing a white spot syndrome virus envelope protein VP28 codon optimization gene;
a white spot syndrome virus envelope protein VP28 recombinant expression step;
a preparation method of a white spot syndrome virus envelope protein VP28 anti-rabbit polyclonal antibody.
The invention also provides application of the white spot syndrome virus VP28 recombinant protein/the polyclonal antibody in white spot syndrome virus detection.
The invention is toWSSVThe VP28 codon optimized sequence SEQ ID No.2 is directly connected with pET-B2M vector, transformed into escherichia coli BL21 competent cell, monoclonal is selected for shake bacteria, IPTG induction expression is carried out, thalli are collected for SDS-PAGE protein detection, bacteria are cracked by ultrasonic disruption, Ni is used for cracking bacteria2+And purifying the target protein by using an affinity chromatography column. Then in the purified formWSSVThe VP28 recombinant protein was used as an antigen, and 5 New Zealand white rabbits were used for immunization, and serum was collected and the antibody titer in the rabbit serum was measured by ELISA (indirect assay). Then, the antibody is purified by using affinity chromatography column proteinG, and is applied to subsequent western blot and other experiments. By recombinant expressionWSSVThe VP28 protein can be further used for analyzing the structure and function of the protein in vitro, and the preparation of the polyclonal antibody can lay a foundation for further detecting the content of VP28 of WSSV in shrimp bodies.
Has the advantages that: the invention not only recombines and expresses WSSVVP28 protein with activity, and provides a basis for further research of the function of the protein, but also provides conditions for the production of polyclonal antibody and the detection of VP28 of WSSV in shrimp bodies, and the produced antibody can be used for further scientific research.
Drawings
In the context of figure 1, it is shown,WSSVSDS-PAGE electrophoresis detection of the VP28 recombinant protein. M represents protein marker, 1 represents a bacterial protein not induced by IPTG, 2 represents a bacterial protein induced by IPTG, and 3 represents Ni2+After purificationVP28A recombinant protein.
FIG. 2, Western blot of the VP28 antibody detects muscle protein samples of WSSV-infected crayfishes.
FIG. 3, the result of the detection of the titer of the anti-rabbit polyclonal VP28 antibody.
Detailed Description
Example 1
1. WSSVInducible expression of the VP28-His recombinant protein:
to competent cellsE. coliBL21 with pET-B2M- WSSVThe VP28 recombinant plasmid was gently mixed, and the mixture was allowed to stand in an ice bath for 5min, then placed in a 42 ℃ water bath for 90 s by heat shock, and rapidly transferred to the ice bath. Adding 900 μ L sterile LB culture medium into each centrifuge tube, mixing, shaking and culturing for 45 min at 37 deg.C, recovering thallus, sucking 100 μ L recovered bacteria liquid, spreading on LB solid culture medium containing kanamycin resistance, and culturing at 37 deg.C for 12-16 h. Selecting escherichia coli containing recombinant plasmids, adding a liquid culture medium, carrying out shake culture at 37 ℃ until the OD600 of a bacterial liquid is 0.4-0.6, adding a proper amount of IPTG to the final concentration of 1 mM, carrying out induced expression for 4h, collecting thalli, resuspending the thalli with sterilized water, freezing and preserving at-80 ℃, and repeatedly freezing and thawing for 1-2 times; and (3) taking the frozen and thawed resuspended thalli, carrying out ultrasonic crushing under the ice bath condition, centrifugally collecting the crushed supernatant, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection. As shown in figure 1 of the drawings, in which,WSSVSDS-PAGE electrophoresis detection of the VP28 recombinant protein. M represents protein marker, 1 represents a bacterial protein not induced by IPTG, 2 represents a bacterial protein induced by IPTG, and 3 represents Ni2+After purificationVP28A recombinant protein.
As can be seen from the results, the recombinant protein VP28 can successfully induce expression in large quantity and passes through Ni2+The purified band is single, no other hybrid protein exists, and the requirement of subsequently preparing the polyclonal antibody can be met.
2. WSSVPurification of VP28-His recombinant protein:
to pairWSSVAnd (3) separating and purifying the VP28-His recombinant protein: loading the supernatant sample after ultrasonication with Ni2+And (3) collecting flow-through liquid by an ion affinity chromatography column at the flow rate of 10 times of the column volume per hour. The column was washed with 15 column volumes of Binding Buffer (20 mM Tris-HCl pH 7.9, 5 mM imidazole, 0.5M NaCl) to wash off contaminating proteins. Eluted protein was collected by eluting with 5 mL of Elution Buffer (20 mM Tris-HCl pH 7.9, 500 mM imidazole, 0.5M NaCl).
3. Anti-rabbitWSSVPreparation of VP28-His polyclonal antibody:
2 Japanese big ear rabbits were used to recombineWSSVThe VP28 protein concentration was adjusted to 1mg/ml for antigen injection, and the adjuvant and antigen were extracted at a 1:1 volume ratio. The first-time immunization adopts a complete adjuvant, and the second-time immunization adopts an incomplete adjuvant. The rabbits were injected subcutaneously in multiple spots of 0.2ml each. The second immunization is carried out 14 days after the first immunization, and the interval between the second immunization and the third immunization is 7 days. And (3) collecting small sample serum from the middle ear artery on the 7 th day after the rabbit three-immunization, detecting the small sample serum to be qualified, adding the rabbit three-immunization after 7 days, and collecting the whole blood after 7 days of adding the rabbit three-immunization. The antibody titer in rabbit sera was determined by ELISA (indirect method). Then, the antibody is purified by using affinity chromatography column proteinG, and is applied to subsequent western blot and other experiments.
4. WSSVWestern blot detection of VP28-His polyclonal antibody:
extracting serum total protein of gobiocypris rarus, performing SDS-PAGE electrophoresis, performing PVDF membrane transfer on the serum total protein under the condition of a wet transfer method, then placing the PVDF membrane in 5% skimmed milk powder for sealing for 2h, and carrying out antibody purification according to the weight ratio of 1: 3000, diluting and incubating for 2h, washing the membrane with TBST for three times, 5min each time, then incubating with goat anti-rabbit HRP labeled IgG secondary antibody and PVDF membrane for 2h, washing the membrane with TBST for three times, finally developing with an enhanced DAB developing kit, and observing.
FIG. 2 is a western blot detection of antibody VP28 on muscle protein samples from WSSV-infected crayfishes.
Through western blot verification, a VP28 protein band can be obviously detected from a muscle sample of the WSSV-infected crayfish, and the specificity is good, so that the VP28 polyclonal antibody produced by the method can be used for detection.
FIG. 3 shows the result of the potency test of the prepared VP28 anti-rabbit polyclonal antibody.
Antibody titers were verified by ELISA experiments, from which it can be seen that the titers of antibodies exceeded 1: 50000, shows that the quality of the antibody is better, and can be used for western blot detection.
Sequence listing
<110> Yangzhou university
<120> white spot syndrome virus VP28 gene, recombinant protein, polyclonal antibody, preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 615
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggatcttt ctttcactct ttcggtcgtg tcggccatcc tcgccatcac tgctgtgatt 60
gctgtattta ttgtgatttt taggtatcac aacactgtga ccaagaccat cgaaacccac 120
acaggcaata tcgagacaaa catggatgaa aacctccgca ttcctgtgac tgctgaggtt 180
ggatcaggct acttcaagat gactgatgtg tcctttgaca gcgacacctt gggcaaaatc 240
aagatccgca atggaaagtc tgatgcacag atgaaggaag aagatgcgga tcttgtcatc 300
actcccgtgg agggccgagc actcgaagtg actgtggggc agaatctcac ctttgaggga 360
acattcaagg tgtggaacaa cacatcaaga aagatcaaca tcactggtat gcagatggtg 420
ccaaagatta acccatcaaa ggcctttgtc ggtagctcca acacctcctc cttcaccccc 480
gtctctattg atgaggatga agttggcacc tttgtgtgtg gtaccacctt tggcgcacca 540
attgcagcta ccgccggtgg aaatcttttc gacatgtacg tgcacgtcac ctactctggc 600
actgagaccg agtaa 615
<210> 2
<211> 525
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cataacaccg tgaccaaaac cattgaaacc cataccggta atatcgaaac caacatggat 60
gaaaatctgc gtattccggt taccgcagaa gttggtagcg gttatttcaa aatgaccgat 120
gttagctttg atagcgatac cctgggcaaa atcaaaattc gcaatggtaa aagtgacgcc 180
cagatgaaag aagaggacgc agatctggtt attacaccgg ttgaaggtcg tgcactggaa 240
gttaccgttg gccagaatct gacctttgaa ggcaccttta aagtgtggaa taataccagc 300
cgcaagatta acattaccgg catgcagatg gttccgaaaa ttaacccgag caaagcattt 360
gttggtagca gcaataccag cagctttact ccggttagca ttgatgaaga tgaagttggc 420
acctttgttt gtggcaccac ctttggtgca ccgattgcag caaccgcagg cggtaacctg 480
tttgatatgt atgttcatgt tacctatagc ggcaccgaaa ccgaa 525
<210> 3
<211> 175
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
His Asn Thr Val Thr Lys Thr Ile Glu Thr His Thr Gly Asn Ile Glu
1 5 10 15
Thr Asn Met Asp Glu Asn Leu Arg Ile Pro Val Thr Ala Glu Val Gly
20 25 30
Ser Gly Tyr Phe Lys Met Thr Asp Val Ser Phe Asp Ser Asp Thr Leu
35 40 45
Gly Lys Ile Lys Ile Arg Asn Gly Lys Ser Asp Ala Gln Met Lys Glu
50 55 60
Glu Asp Ala Asp Leu Val Ile Thr Pro Val Glu Gly Arg Ala Leu Glu
65 70 75 80
Val Thr Val Gly Gln Asn Leu Thr Phe Glu Gly Thr Phe Lys Val Trp
85 90 95
Asn Asn Thr Ser Arg Lys Ile Asn Ile Thr Gly Met Gln Met Val Pro
100 105 110
Lys Ile Asn Pro Ser Lys Ala Phe Val Gly Ser Ser Asn Thr Ser Ser
115 120 125
Phe Thr Pro Val Ser Ile Asp Glu Asp Glu Val Gly Thr Phe Val Cys
130 135 140
Gly Thr Thr Phe Gly Ala Pro Ile Ala Ala Thr Ala Gly Gly Asn Leu
145 150 155 160
Phe Asp Met Tyr Val His Val Thr Tyr Ser Gly Thr Glu Thr Glu
165 170 175
Claims (10)
1. The white spot syndrome virus VP28 gene is namedWSSVVP28, sequence as SED ID NO: 1 is shown.
2. A codon optimized gene of white spot syndrome virus VP28 is characterized in thatWSSVNucleotide sequence of VP28 SED ID NO: 1, such as SED ID NO: 2, respectively.
3. A recombinant protein of white spot syndrome virus VP28, characterized in that its amino acid sequence is as defined in SED ID NO: 3, respectively.
4. A polyclonal antibody, which is prepared by immunizing an animal with the recombinant protein of white spot syndrome virus VP28 according to claim 3 as an antigen.
5. The white spot syndrome virus VP28 recombinant protein of claim 3, for use in white spot syndrome virus detection.
6. Use of the polyclonal antibody of claim 4 in white spot syndrome virus detection.
7. A process for the preparation of recombinant protein white spot syndrome virus VP28 according to claim 3, comprising:
optimizing gene sequence SED ID NO of white spot syndrome virus VP28 codon: 2 is connected into a PET-B2M expression vector to obtain pET-B2M- WSSVVP28 recombinant plasmid;
pET-B2M- WSSVThe VP28 recombinant plasmid is transformed into escherichia coli, cultured, induced, cracked and purified to obtain the white spot syndrome virus VP28 recombinant protein.
8. The method for producing a polyclonal antibody according to claim 4, wherein an antiserum is produced by a conventional method for producing a polyclonal antibody using the white spot syndrome virus VP28 recombinant protein according to claim 3 as an antigen, and the antiserum is isolated and purified to obtain a polyclonal antibody.
9. The method for producing a polyclonal antibody according to claim 8, comprising: injecting the recombinant protein of the white spot syndrome virus VP28 as an antigen into a rabbit body, immunizing for four times, collecting rabbit blood, separating serum, and purifying to obtain the anti-rabbit polyclonal antibody of the white spot syndrome virus VP 28.
10. The method for producing a polyclonal antibody according to claim 9, comprising:
adjusting the concentration of the recombinant protein of the white spot syndrome virus VP28 to 1mg/ml, injecting the recombinant protein as an antigen, and extracting the adjuvant and the antigen in a volume ratio of 1: 1; the first-time immunization adopts a complete adjuvant, and the second-time immunization adopts an incomplete adjuvant.
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Application publication date: 20201218 |