CN101023095B

CN101023095B - Genetic engineering protein P40 and application thereof

Info

Publication number: CN101023095B
Application number: CN200480043768.1A
Authority: CN
Inventors: 孟小林; 徐进平; 李红霞; 王健; 鲁伟; 曹旭
Original assignee: Green Life Laboratory Ltd
Current assignee: Green Life Laboratory Ltd
Priority date: 2004-07-09
Filing date: 2004-07-09
Publication date: 2013-07-03
Anticipated expiration: 2024-07-09
Also published as: WO2006005222A1; CN101023095A

Abstract

The invention relates to gene engineering protein P40 as well as coding amino acid sequence and the nucleotide sequence of this P40. The invention also discloses a immunity vaccine of anti-white spot syndrome virus. The vaccine comprises said genetic engineering protein P40. The invention also discloses the host cell of gene engineering protein P40 and detects the reagent kit to prawns white spot syndrome.

Description

Engineered protein P40 and application thereof

Invention field

The present invention relates to the control of the white spot syndrome of crustaceans biology.More particularly, the present invention relates to prevent and treat engineered protein and the application thereof of prawn white spot syndrome.

Background technology

The Chinese prawn aquaculture enters the peak in nineteen nineties, and cultured area reaches 160,000 hm ², ability more than 1,000 ten thousand tails of growing seedlings in year are produced more than 20 ten thousand tons of prawns per year, and year earns foreign exchange more than 500,000,000 dollars, keeps rank first for successive years.After 1993, because prawn ' s virus disease---breaking out of prawn white spot syndrome, significantly landslide has appearred in the Chinese prawn aquaculture, and shrimp culture industry has stepped into low ebb, and the cultured prawn ultimate production sharply descends.Output in 1993 has only 8.7 ten thousand tons, drops to 5.5 ten thousand tons again in 1994, only is equivalent to 1/4 of the period of great prosperity, and a year direct economic loss reaches billions of yuans.Have to change the line of production and culture other kinds or stopping production in many prawn culturings field, the prawn export volume sharply descends.

At present, global prawn annual requirement is at least about 1,200,000 tons, more than 70 hundred million dollars of the volume of trades, and formed Japan, the U.S., European Union, South East Asia four big markets.In recent years, world market prawn demand constantly increases, and price is steadily increasing, and still, prawn white spot syndrome causes respectively producing shrimp and crosses the decline of prawn output.

Prawn white spot syndrome is the disease that a kind of virus (white spot syndrome virus (WSSV) is called for short WSSV) causes, this virus can form prevailing disease rapidly in the prawn population, cause crushing blow to prawn culturing.Current still do not have the good prevention of WSSV and prevent and treat method, and therefore, early discovery, processing in time are to prevent and treat WSSV at present, the best way that the control epidemic situation spreads.

In the field of seeking the effective scheme that solves anti-prawn white spot syndrome, people have made a lot of trials.For example, WO0109340---Proteins Derived From White Spot Syndrome Virus and Use thereof (international filing date on July 26th, 2000), the applicant of this patent application are Dutch AKZO NOBEL companies.This piece document illustration from white spot syndrome virus (WSSV), separate and determined VP28, VP26, VP24 and four main protein of VP19 and studied and adopted these protein to prepare the WSSV immune vaccine.The document has also disclosed the nucleotide sequence of these protein of encoding, and uses these these protein of nucleotide sequence reorganization preparation.What in fact, the document disclosed only comprises the order-checking of VP28, VP26, VP24 and their nucleotide sequence.The document has pointed out that also VP28 and VP19 are envelope proteins.But, do not have to report the full length amino acid sequence of VP19 in this piece document, the gene order of report coding VP19 yet.The document has also been reported the preparation of WSSV vaccine.Point out to comprise in this vaccine the combination of two or more kind proteinoid among VP28, VP26, VP24, the VP19 in general manner.In its preferred scheme, vaccine comprises VP26 and VP28, also can add or not add VP24.But, do not point out to need to add VP19.

WO0222664---Antigenic Proteins of Shrimp White Spot Syndrome Virus and Use thereof (14 days September calendar year 2001 of international filing date), the applicant of this patent are Dutch AKZO NOBEL companies.This piece document illustration derived from antigen protein VP19 and the VP13 of WSSV, and use these protein as vaccine composition, the antibody of these protein, use these antibody as the composition of vaccine, also disclose the vaccine that uses these protein preparation preventions and treatment white spot syndrome, comprised the test kit of relevant antibody or nucleic acid.In the vaccine that the document discloses, contain VP13 and/or VP19.The VP19 that also touched upon has separately and to delay prawn in the death that suffers after WSSV attacks.But the document is not touched upon the combination of VP28 and VP19.

WO03000900---White Sport Syndrome Virus Vaccine (international filing date on June 18th, 2002), the applicant of this patent are Dutch AKZO NOBEL companies.The document has disclosed a very long WSSV viral genome, and it is cloned and checks order, and names to be ORF167, and it has 667KD, comprises 18234 Nucleotide, and provided its full length sequence.But also point out that this gene be considered to encode protein of the similar shape of tail part of WSSV virus terminal is analyzed this ORF167 and found to have above it several immune sites to be suitable for use in the vaccine.The 10th page the 7th row and the 11st row at document have disclosed the fragment with 180 amino-acid residues and 200 amino-acid residues respectively, also have some other big or small fragments.But this piece document does not disclose the combination of VP28 and VP19.

The exercise question that people such as Jeroen Witteveldt are published on " Journal of Virology " in February, 2004 is " protection of white spot syndrome virus resisting is provided for prawn with oral vaccine " [Jeroen Witteveldt et al.; Protecton of Penaeus monodon against White Spot Syndrome Virus by Oral Vaccination; Journal of Virology; Feb.2004; p.2056-61] in the literary composition; disclosed and given oral VP28 itself only or the VP28 of comprising of prawn in conjunction with the mixture of VP19, thereby the immunizing power of anti-white spot syndrome is provided for prawn.The document points out that the effect of the mixture of VP28 and VP19 is effective not as independent VP28's.But also point out that providing the immunity to white spot syndrome to prawn is to be unable to do without VP28, the protection that namely provides is that VP28 is specific.In the experiment of the document, the result who adopts VP19 to carry out immunity is 83% accumulation lethality rate, and the protection of any reality namely almost is not provided.And the result who adopts VP28 to carry out immunity is that lethality rate has reduced by 30% to 50% relatively.

In sum, also do not find the highly effective phylactic agent of white spot syndrome virus and method so far.Therefore, press for phylactic agent and the method that anti-white spot syndrome can be provided for prawn that find.

Summary of the invention

One of purpose of the present invention provides a kind of engineered protein P40, and the aminoacid sequence of its aminoacid sequence and SEQ ID NO.1 has 70% homology at least, and its molecular weight is about 40 kilodaltons.Described engineered protein P40 has the antigenicity of anti-white spot syndrome virus (WSSV).

One of purpose of the present invention provides a kind of engineered protein P40, its amino acid sequence coded is made of two portions, the i.e. aminoacid sequence that has at least 96% homology with white spot syndrome virus protein VP19, and the aminoacid sequence that has at least 99% homology with white spot syndrome virus protein VP28.

One of purpose of the present invention provides a kind of engineered protein P40, its amino acid sequence coded is made of two portions, the i.e. aminoacid sequence that has at least 96% homology with white spot syndrome virus protein VP19, and with the aminoacid sequence that white spot syndrome virus protein VP28 has at least 99% homology, have two to connect amino-acid residues between two fragments.

One of purpose of the present invention provides the nucleotide sequence of a kind of encoding gene engineered protein P40, and the nucleotide sequence of its nucleotide sequence and SEQ ID NO.2 has 50% homology at least.

One of purpose of the present invention provides the nucleotide sequence of a kind of encoding gene engineered protein P40, and the nucleotide sequence of its nucleotide sequence and SEQ ID NO.2 has 70% homology at least.

One of purpose of the present invention provides the nucleotide sequence of a kind of encoding gene engineered protein P40, and the nucleotide sequence of its nucleotide sequence and SEQ ID NO.2 has 80% homology at least.

One of purpose of the present invention provides the nucleotide sequence of a kind of encoding gene engineered protein P40, this sequence is made of two portions, the i.e. nucleotide sequence that has at least 90% homology with the coding nucleotide of white spot syndrome virus protein VP19, and the nucleotide sequence that has at least 95% homology with the coding nucleotide of white spot syndrome virus protein VP28.

One of purpose of the present invention provides a kind of preparation method of dna fragmentation, and this fragment comprises the nucleotide sequence of encoding gene engineered protein P40 of the present invention.

One of purpose of the present invention provides a kind of host cell, and this host cell comprises the nucleotide sequence of the engineered protein P40 of the present invention that encodes, perhaps comprises the dna fragmentation of the engineered protein P40 of the present invention that encodes.

One of purpose of the present invention provides a kind of construction process of e. coli host cell, and this host cell comprises the nucleotide sequence of engineered protein P40 of the present invention.

One of purpose of the present invention provides a kind of immune vaccine of anti-white spot syndrome, described vaccine comprises described protein P40, or the nucleotide sequence of coded protein P40 or its functional DNA fragment, or the host cell of described nucleotide sequence or its functional DNA fragment or their two or more mixtures have been comprised.

One of purpose of the present invention provides a kind of method for preparing the immune vaccine of white spot syndrome, it is characterized in that described method comprises protein P40, or the nucleotide sequence of the described protein P40 that encodes or its functional DNA fragment, or comprised the host cell of described nucleotide sequence or its functional DNA fragment, or their two or more mixtures, mix with acceptable carrier on the pharmacology.

One of purpose of the present invention provides a kind of prawn feed, it is characterized in that described prawn feed includes described protein P40, or the nucleotide sequence of the described protein P40 that encodes or its functional DNA fragment, or the host cell of described nucleotide sequence or its functional DNA fragment or their two or more mixtures have been comprised.

One of purpose of the present invention provides a kind of antiserum(antisera), it is characterized in that described antiserum(antisera) is antigen prepd with engineered protein P40.

One of purpose of the present invention provides a kind of white spot syndrome detection kit, it is characterized in that described detection kit comprises described antiserum(antisera).

The accompanying drawing summary

Above-mentioned and other purposes of the present invention, characteristics and advantage can obtain the content shown in the detailed description and the accompanying drawings of the preferred embodiment of the present invention from following apparently, and reference symbol identical in the different views represents identical part.Accompanying drawing might not be shown to scale, and it focuses on illustrating principle of the present invention.

The preparation process of Fig. 1 fusion gene;

Fig. 2 construction of recombinant plasmid process;

Fig. 3 recombinant expression vector pET-22b (+)-p40vp (19+28) enzyme is cut the checking result;

Swimming lane 1.PCR Markers;

Swimming lane 2.pET-p40vp (19+28)/BamHI+HindIII enzyme is cut checking;

Swimming lane 3.pET-22b (+)/BamHI+HindIII enzyme is cut the result;

Swimming lane 4.Lambda DNA/HindIII+EcoRI Markers;

Fig. 4 engineering strain is expressed the SDS-PAGE electrophoresis result that merges envelope protein;

Swimming lane 1:E.coli BlL21 (DE3) is (pET22) before the bacterial strain inducing;

Swimming lane 2:E.coli BlL21 (DE3) (pET22) behind the bacterial strain inducing 5 hours;

Swimming lane 3:E.coli BlL21 (DE3) (pET22) does not induce;

Swimming lane M. protein molecular weight standard;

Before swimming lane 5. engineering strains are induced;

Swimming lane 6. engineering strains were induced back 3 hours;

Swimming lane 7. engineering strains were induced back 4 hours;

Swimming lane 8. engineering strains were induced back 5 hours;

The engineering strain that swimming lane 9. is not induced;

Fig. 5 merges the SDS-PAGE electrophoresis result behind the envelope protein purifying;

Albumen substep gleanings behind the swimming lane 1-8:Ni column purification;

Swimming lane M. protein molecular weight standard

Fig. 6 test strip structural representation;

(1) water adsorption glass fiber;

(2) the plain film of golden labeling antibody binding substances-glass fibre;

(3) WSSV antibody solid phase nitrocellulose filter (detection line);

(4) anti-WSSV antibody solid phase nitrocellulose filter (nature controlling line);

(5) absorbent filter;

(6) plastic bottom board;

The result schematic diagram of Fig. 7 test strip rapid detection white spot syndrome virus (WSSV);

(1) positive findings;

(2) negative findings;

(3) null result;

Fig. 8 is aminoacid sequence ID No.1;

Fig. 9 is aminoacid sequence ID No.2.

Detailed Description Of The Invention

The preferred embodiment of the invention is described below.

In one embodiment of the invention, the applicant finds surprisingly, a kind of engineered protein P40 provided by the present invention, the aminoacid sequence of its aminoacid sequence and SEQ ID NO.1 has 70% homology at least, its molecular weight is about 40 kilodaltons, the immunogenicity that this engineered protein P40 has anti-white spot syndrome virus (WSSV).

In one embodiment of the invention, provide a kind of engineered protein P40, the aminoacid sequence of its aminoacid sequence and SEQ ID NO.1 has 80% homology at least.

In one embodiment of the invention, provide a kind of engineered protein P40, the aminoacid sequence of its aminoacid sequence and SEQ ID NO.1 has 90% homology at least.

The aminoacid sequence of engineered protein P40 of the present invention comprises two major portions, the i.e. aminoacid sequence that has at least 96% homology with white spot syndrome virus protein VP19, and the aminoacid sequence that has at least 99% homology with white spot syndrome virus protein VP28.

Research for VP19 and the VP28 of white spot syndrome virus has had multiple report.In gene library storehouse (GENEBANK), there has been the information about the aminoacid sequence of these two kinds of virus components.At present, the report of the relevant white spot syndrome of this area has assert that WSSV has two envelope proteins, i.e. VP19 and VP28.

In one embodiment of the invention, the aminoacid sequence that engineered protein P40 of the present invention has is checked order, and concrete outcome is as follows:

MATTTNTLPFGRTGAQAAGPSYTMEDLEGSMSMARMGLFLIVAISIGVLVLAVMNVWMGPKKDSDSDTDKDTDDDDDTANDNDDEDKYKNRTRDMMPLAGSALLFLVSAATAFMSYPKRRQ? MDLSFTLSVVSAILAITAVIAVFIVIFRYHNTVTKTIETHTDNIETNMDENLRIPVTAEVGSGYFKMTDVSFDSDTLGKIKIRNGKSDAQMKEEDADLVITPVEGRALEVTVGQNLTFEGTFKVWNNTSRKINITGMQMVPKINPSKAFVGSSNTSSFTPVSIDEDEVGTFVCGTTFGAPIAATAGGNLFDMYVHVTYSGTETE?

Wherein, dash area is represented the sequence of carrier, and other is gene order, and box indicating VP19 is connected with restriction enzyme site with VP28 and two amino acid having more.

The aminoacid sequence of engineered protein P40 of the present invention can be modified within the specific limits, change, protein after the modification that obtains or the fragment of protein have identical biological function with engineered protein P40, particularly proteantigen does not change, and can both stimulate body to produce similar immune response.To protein modify and the method that changes for conventional method, by those skilled in the art is familiar with.According to the theory of modern life science, it is to be the protein with identical biological function that the protein of amino acid sequence homology more than 70% is often annotated in biology.The modification of in this scope protein amino acid sequence being carried out and sudden change all are considered to not change the biological characteristics of protein, and these changes and modification comprise:

A) indivedual amino acid are suddenlyd change, particularly NOT-function determines the amino acid in site.These amino acid whose variations are neutral often, and in fact, even under field conditions (factors), this change all is extensively to exist, and is all variant such as the sequence of the WSSV strains that separate from different areas.Now not clear to functional site and the antigenic determinant site of WSSV, but theoretically, the change of same acidic amino acid (structural similitude, iso-electric point close) does not generally influence the function of protein.

B) lack or insert indivedual amino acid, particularly NOT-function determines the amino acid in site.If such change does not change the space conformation of albumen, just can not influence the biological function of protein, also can not change the immunogenicity of protein.

C) insert special amino-acid residue.In order to increase or change the solubility of protein, increase stability, in the genetically engineered production process, N, the C end that tends at protein adds some amino-acid residues, to avoid forming inclusion body, reduces the degraded of host cell proteins matter restriction endonuclease; Perhaps add some special aminoacid functional sites at N, C end and be beneficial to protein expression and purifying.Such as adding 6 continuous Histidines so that carry out protein purification with the Ni affinity chromatography at N end or C end; Perhaps add the enteropeptidase site at the N end, such protein can obtain and the duplicate engineered protein of natural protein through the cutting of enteropeptidase; Perhaps add hemagglutination prime factor Xa, can specific recognition and cut off 4 peptide sequence Ile-Glu-Gly-Arg.

More than to protein modify and the method that changes for conventional method, by those skilled in the art is familiar with.There are the protein of 70% homology or immunity fragment all to have the biological function identical with engineered protein P40 of the present invention by what aforesaid method obtained with engineered protein of the present invention, namely can both stimulate prawn to produce the anti-WSSV immune response of specificity, can both stimulate animal to produce the specific antibody of WSSV cyst membrane fusion rotein, can both be used for realizing one or more purposes of the present invention.

Amino acid according to protein is formed, and in conjunction with the analytical results of SDS-PAGE, the molecular weight of engineered protein P40 involved in the present invention is about 40 kilodaltons.If part or local change take place the aminoacid sequence of protein, the molecular weight of protein can change.Select different the appearance for use? host strain and expression vector because posttranslational modification, the molecular weight of protein also can change.Select different detection methods for use, the molecular weight of protein also has some differences, but these change and difference can not surpass 10% of protein molecular weight in principle.

White spot syndrome virus of the present invention (White Spot Syndrome Virus, WSSV) separation is from the prawn of falling ill in the Chinese North Sea, gene order homology Yin Jiyin between the strain isolated of WSSV different areas is different and have larger difference, and wherein the sequence homology of envelope protein is between 72%-99%.Therefore, the aminoacid sequence that has at least 96% homology with WSSV envelope protein VP19, and can merge by direct or indirect mode with aminoacid sequence that envelope protein VP28 has at least 99% homology, resulting fusion rotein all has the antigenic determinant site of all WSSV envelope protein VP19 and VP28, all have the biological function identical with engineered protein P40 of the present invention, can both be used for realizing one or more purposes of the present invention.

The applicant has carried out amino acid sequencing for protein VP19 and the VP28 of white spot syndrome virus (WSSV).Table 1 as follows has provided the basic condition of the gene sequencing of VP19 and VP28.

Table 1, vp19 vp28 gene and connect basic condition as the gene behind the expression vector

The applicant has also carried out pcr amplification to VP19, and the result who obtains is as follows:

Vp19 gene PCR amplified sequence and deduced amino acid sequenceATGGCCACCACGACTAACACTCTTCCTTTCGGCAGGACCGGAGCCCAGGCCGCTGGCCCTTCTTACACCATGGAAGATCTTGAAGGCTCCATGTCTATGGCTCGCATGGGTCTCTTTTTGATCGTTGCTATCTCAATTGGTGTCCTCGTCCTGGCCGTCATGAATGTATGGATGGGACCAAAGAAGGACAGCGATTCTGACACTGATAAGGACACCGATGATGATGACGATACTGCCAACGATAACGATGATGAGGACAAATATAAGAACAGGACCAGGGATATGATGCCTCTGGCTGGGTCCGCTCTTCTGTTCCTCGTTTCCGCCGCCACCGCTTTTATGTCTTACCCCAAGAGGAGGCAGMATTTNTLPFGRTGAQAAGPSYTMEDLEGSMSMARMGLFLIVAISIGVLVLAVMNVWMGPKKDSDSDTDKDTDDDDDTANDNDDEDKYKNRTRDMMPLAGSALLFLVSAATAFMSYPKRRQ

As previously mentioned, the gene order to VP19 has had multiple report.The applicant compares at the homology that the VP19 that has reported has carried out gene, and concrete outcome is as follows:

The homology of vp19 gene relatively among the GenBank

The applicant has also carried out pcr amplification to VP28, and the result who obtains is as follows:

Vp28 gene sequence by amplified PCR and deduced amino acid sequenceATGGATCTTTCTTTCACTCTTTCGGTCGTGTCGGCCATCCTCGCCATCACTGCTGTGATTGCTGTATTTATTGTGATTTTTAGGTATCACAACACTGTGACCAAGACCATCGAAACCCACACAGACAATATCGAGACAAACATGGATGAAAACCTCCGCATTCCTGTGACTGCTGAGGTTGGATCAGGCTACTTCAAGATGACTGATGTGTCCTTTGACAGCGACACCTTGGGCAAAATCAAGATCCGCAATGGAAAGTCTGATGCACAGATGAAGGAAGAAGATGCGGATCTTGTCATCACTCCCGTGGAGGGCCGAGCACTCGAAGTGACTGTGGGGCAGAATCTCACCTTTGAGGGAACATTCAAGGTGTGGAACAACACATCAAGAAAGATCAACATCACTGGTATGCAGATGGTGCCAAAGATTAACCCATCAAAGGCCTTTGTCGGTAGCTCCAACACCTCCTCCTTCACCCCCGTCTCTATTGATGAGGATGAAGTTGGCACCTTTGTGTGTGGTACCACCTTTGGCGCACCAATTGCAGCTACCGCCGGTGGAAATCTTTTCGACATGTACGTGCACGTCACCTACTCTGGCACTGAGACCGAGTAAMDLSFTLSVVSAILAITAVIAVFIVIFRYHNTVTKTIETHTDNIETNMDENLRIPVTAEVGSGYFKMTDVSFDSDTLGKIKIRNGKSDAQMKEEDADLVITPVEGRALEVTVGQNLTFEGTFKVWNNTSRKINITGMQMVPKINPSKAFVGSSNTSSFTPVSIDEDEVGTFVCGTTFGAPIAATAGGNLFDMYVHVTYSGTETE

As previously mentioned, the gene order to VP28 has had multiple report.The applicant compares at the homology that the VP28 that has reported has carried out gene, and concrete outcome is as follows:

The homology of vp28 gene relatively among the GenBank

Annotate: our sequence is just the same with the sequence of China Report.

Respectively there is an amino acid different with external two sequences.

Reference: Yang, F., He, J., Lin, X., Li, Q., Pan, D., Zhang, X.and Xu, X., Complete genome sequence of the shrimp white spot bacilliform virus, J.Virol.75 (23), 11811-11820 (2001)

Vp19 and vp28 join end to end rear clone to the middle actual coding region sequence of expression vector pET22b (+)

Dash area is represented the sequence of carrier, and other is gene order, and box indicating is used for the restriction enzyme site that gene connects

ATGGCCACCACGACTAACACTCTTCCTTTCGGCAGGACCGGAGCCCAGGCCGCTGGCCCTTCTTACACCATGGAAGATCTTGAAGGCTCCATGTCTATGGCTCGCATGGGTCTCTTTTTGATCGTTGCTATCTCAATTGGTGTCCTCGTCCTGGCCGTCATGAATGTATGGATGGGACCAAAGAAGGACAGCGATTCTGACACTGATAAGGACACCGATGATGATGACGATACTGCCAACGATAACGATGATGAGGACAAATATAAGAACAGGACCAGGGATATGATGCCTCTGGCTGGGTCCGCTCTTCTGTTCCTCGTTTCCGCCGCCACCGCTTTTATGTCTTACCCCAAGAGGAGGCAG?

ATGGATCTTTCTTTCACTCTTTCGGTCGTGTCGGCCATCCTCGCCATCACTGCTGTGATTGCTGTATTTATTGTGATTTTTAGGTATCACAACACTGTGACCAAGACCATCGAAACCCACACAGACAATATCGAGACAAACATGGATGAAAACCTCCGCATTCCTGTGACTGCTGAGGTTGGATCAGGCTACTTCAAGATGACTGATGTGTCCTTTGACAGCGACACCTTGGGCAAAATCAAGATCCGCAATGGAAAGTCTGATGCACAGATGAAGGAAGAAGATGCGGATCTTGTCATCACTCCCGTGGAGGGCCGAGCACTCGAAGTGACTGTGGGGCAGAATCTCACCTTTGAGGGAACATTCAAGGTGTGGAACAACACATCAAGAAAGATCAACATCACTGGTATGCAGATGGTGCCAAAGATTAACCCATCAAAGGCCTTTGTCGGTAGCTCCAACACCTCCTCCTTCACCCCCGTCTCTATTGATGAGGATGAAGTTGGCACCTTTGTGTGTGGTACCACCTTTGGCGCACCAATTGCAGCTACCGCCGGTGGAAATCTTTTCGACATGTACGTGCACGTCACCTACTCTGGCACTGAGACCGAG?

Dash area is represented the sequence of carrier, and other is gene order, and box indicating vp19 is connected with restriction enzyme site with vp28 and two amino acid having more

MATTTNTLPFGRTGAQAAGPSYTMEDLEGSMSMARMGLFLIVAISIGVLVLAVMNVWMGPKKDSDSDTDKDTDDDDDTANDNDDEDKYKNRTRDMMPLAGSALLFLVSAATAFMSYPKRRQ?

MDLSFTLSV?VSAILAITAVIAVFIVIFRYHNTVTKTIETHTDNIETNMDENLRIPVTAEVGSGYFKMTDVSFDSDTLGKIKIRNGKSDAQMKEEDADLVITPVEGRALEVTVGQNLTFEGTFKVWNNTSRKINITGMQMVPKINPSKAFVGSSNTSSFTPVSIDEDEVGTFVCGTTFGAPIAATAGGNLFDMYVHVTYSGTETE?

In one embodiment of the invention, a kind of engineered protein P40 is provided, its amino acid sequence coded is made of two portions, the i.e. aminoacid sequence that has at least 96% homology with white spot syndrome virus protein VP19, and with the aminoacid sequence that white spot syndrome virus protein VP28 has at least 99% homology, have two to connect amino-acid residues between two fragments.

In the preparation process of the engineering strain of expressed fusion protein, can external vp19 and vp28 be coupled together according to the restriction enzyme site of carrier and the sequences Design restriction enzyme site of gene, a kind of method of attachment is provided in one embodiment of the invention, with EcoR I two genes are coupled together, its advantage is, this enzyme is restriction enzyme site commonly used, and does not have this restriction enzyme site two gene inside, is beneficial to the operation of gene.When two gene Fusion are expressed, often between two genes, add some catenation sequences, length is generally about 2-6 amino acid, its objective is and in two sections peptide sequences, add an end " hinge area ", be beneficial to two polypeptide chains and be folded to form correct natural higher structure separately separately, keep original biological function separately.

The preparation method of a kind of fusion rotein P40 is provided in one embodiment of the invention, the aminoacid sequence of fusion rotein such as SEQ ID NO.1, VP19 and the position of VP28 gene in fusion rotein are respectively: 32-152,155-358, its connecting zone (153-154) are two amino acid E (L-glutamic acid) and F (phenylalanine).The advantage of this mode of connection is, do not change isoelectric point of protein, and computer analysis results shows the secondary structure that can not change protein, does not change the antigenicity of protein.

In one embodiment of the invention, a kind of engineered protein P40 is provided, and this albumen is genetic engineering fusion protein, and the amino acid of its N end and C end is vector encoded, the N end is MKYLLPTAAAGLLLLAAQPAMAMDIGINSDP, and C holds KLAAALEHHHHHH.

The present invention selects for use pET22b (+) expression vector to prepare engineered protein P40, and host cell is e. coli bl21 (DE3), and the advantage of this system is:

1) the stringent type control expressed of target protein: goal gene is cloned on the carrier, transcribed by force by phage t7 and translation signals is controlled, and expression need provide t7 rna polymerase by host cell.In lac operon sequence of the downstream of T7 promotor next-door neighbour.Simultaneously, host cell BL21 (DE3) is the lysogen of a kind of phage DE3, and transcribing of its t7 rna polymerase controlled by the lac promotor also.Therefore the lac aporepressor both can suppress the expression of carrier promotor and then control goal gene, also can suppress the leakage expression of transcribing and then suppress the goal gene that any RNA polymerase causes of host's t7 rna polymerase.The tight control of this target protein is expressed, and can reduce target protein to the toxicity of cell.Under non-inductive condition, can make goal gene be in silence state fully and do not transcribe, when the host fully induced, nearly all cell resource all was used for expressing target protein, after inducing several hrs, target protein is the highest can to account for more than 50% of total protein of cell.

Genetic engineered product is excessive in bacterial cell closes and becomes, and will inevitably influence growth and the metabolism of host cell, and the damage of host cell metabolism suppresses the synthetic of exogenous genes products again.In pET22-e. coli bl21 (DE3) expression system, when the host cell raised growth, suppress expression of exogenous gene, when the biomass of host cell reaches capacity, carry out the abduction delivering of engineered protein again, perhaps can also slacken the expression of albumen as required easily by the concentration that reduces inductor.

2) expression amount height, the highest 30-50% that can reach the tropina total amount of the expression amount of engineered protein.

The sequence (30 amino acid) of 3) amalgamation and expression: N end helps the correct folding of protein; increase the solubility of protein; avoid forming inclusion body; can also protect target protein to avoid the effect of the proteolytic enzyme in the host cell; prolong the transformation period of engineered protein, improve the stability of engineered protein.The C end contains 6 continuous Histidines, is beneficial to separation and the purifying of protein.Histidine is rare amino acid in general albumen, introduces 6 continuous Histidines and has very high specificity.His6 tail and metal ion have very strong binding ability, and very little to the influence of the structure and function of target protein, and biologic activity can be kept usually, without removing the directly biologic activity of research purpose albumen of His6 tail.

4) safe: escherichia expression system is a kind of safe expression system, the success give expression to multiple medical protein with escherichia expression system, safety non-toxic, nontoxic to people and animals and environment.

5) be beneficial to industrialization production: in all gene engineering expression systems, colibacillary production cost is minimum, and fermentation equipment and the production method of moulding have been arranged.The expression system that the present invention adopts is conducive to realize industrialization production.Penbritin is the most cheap microbiotic, can further reduce production costs.

Any change of engineered protein aminoacid sequence all is the change of its nucleotide sequence after all.Biological be made up of various protein, and the amino acid of constitutive protein matter has only about 22 kinds, the nucleotide sequence of coded protein but is to be constituted according to different series arrangement by mononucleotide (A, T, G, C) in 4.

Therefore, theoretically, the change of any nucleotide sequence all might change its amino acids coding, and then changes the structure and function of its coded protein.But biological amino acid code exists and annexs property, i.e. the different codon identical amino acid of can encoding, such as, GCA, GCC, GCG, the GCT Ala (L-Ala) that all encodes.The biological significance of this merger is, in the reproduction process of DNA, genetic material can change because of inside or outside conditioned disjunction factor, if these changes cause amino acid whose variation immediately, will directly influence the particularly structure and function of functional protein of protein.The variation of most of nucleotide sequence is the sudden change that is unfavorable for organism, and the merger of codon has just significantly reduced the sudden change of Nucleotide to the influence of protein function.In addition, the merger of codon also is that amino acid whose aberration rate is well below the reason of nucleotide diversity rate.Therefore, the nucleotide sequence of engineered protein P40 of the present invention can suddenly change, but its encoded protein matter or immunity fragment have identical biological function with engineered protein P40, be that proteantigen does not change, can both stimulate body to produce similar immune response.These sudden changes comprise:

A) same sense mutation: the sudden change of indivedual Nucleotide, but its amino acids coding does not change, so all biological properties of its encoded protein matter are with identical originally.

B) missense mutation: the change of indivedual Nucleotide, its amino acids coding also changes, but these amino acid whose changes often NOT-function determine the amino acid in site, these amino acid whose variations are neutral often.In fact, even under field conditions (factors), this change all is extensively to exist, and is all variant such as the sequence of the WSSV strain that separates from different areas.Now not clear to functional site and the antigenic determinant site of WSSV, but theoretically, the replacement between the amino acid of structural similitude does not generally influence the function of protein.

C) phase shift mutation: increase or the disappearance of indivedual Nucleotide, and then cause disappearance or insert indivedual amino acid, if such change does not change the space conformation of albumen, just can not change the biological function of protein.In fact, in the research process of engineered protein, in order to increase or change the solubility of protein, increase stability, N, the C end that tends at protein adds some amino-acid residues, perhaps adds some functional sites at N, C end and is beneficial to protein expression and purifying.Such as adding 6 continuous Histidines so that carry out protein purification with the Ni affinity chromatography, perhaps add the enteropeptidase site, such protein can obtain and the duplicate engineered protein of natural protein through the cutting of enteropeptidase.Perhaps add hemagglutination prime factor Xa, can specific recognition and cut off 4 peptide sequence Ile-Glu-Gly-Arg.

More than Nucleotide is modified and the method that changes is conventional method, such as realizing that these methods are familiar with by those skilled in the art by the method for PCR, need not carry out creative work can obtain.The nucleotide sequence with nucleotides sequence of the present invention is shown 50% homology that obtains by this method all has the biological function identical with nucleotide sequence of the present invention, the WSSV cyst membrane fusion rotein of namely can both encoding, can stimulate prawn to produce the anti-WSSV immune response of specificity, can both stimulate animal to produce the specific antibody of WSSV cyst membrane fusion rotein, can both be used for realizing one or more purposes of the present invention.

In one embodiment of the invention, provide the nucleotide sequence of a kind of encoding gene engineered protein P40, the nucleotide sequence of its nucleotide sequence and SEQ ID NO.2 has 70% homology at least.

In another embodiment of the invention, the nucleotide sequence of a kind of encoding gene engineered protein P40 is provided, the nucleotide sequence of its nucleotide sequence and SEQ ID NO.2 preferably has 80% homology at least.

The nucleotide sequence of encoding gene engineered protein P40 of the present invention can change within the specific limits, the nucleotide sequence that obtains has identical biological function with the nucleotide sequence of encoding gene engineered protein P40, particularly the antigenicity of coded protein does not change, and can both stimulate body to produce similar immune response.

According to the concept of modern biology, the theory of information biology especially, homology can be judged to be at the nucleotide sequence more than 70% has significant similarity, and homology can be judged to be at the nucleotide sequence more than 80% has identical biological function.

More than Nucleotide is modified and the method that changes is conventional method, such as realizing that these methods are familiar with by those skilled in the art by the method for PCR, need not carry out creative work can obtain.The nucleotide sequence of showing 70% homology with nucleotides sequence of the present invention, preferably having 80% homology that obtains all has with nucleotides sequence of the present invention shows identical biological function, the WSSV cyst membrane fusion rotein of namely can both encoding, can stimulate prawn to produce the anti-WSSV immune response of specificity, can both stimulate animal to produce the specific antibody of WSSV cyst membrane fusion rotein, can both be used for realizing one or more purposes of the present invention.

In one embodiment of the invention, the nucleotide sequence of a kind of encoding gene engineered protein P40 is provided, this sequence is made of two portions, the i.e. nucleotide sequence that has at least 90% homology with the coding nucleotide of white spot syndrome virus protein VP19, and the nucleotide sequence that has at least 95% homology with the coding nucleotide of white spot syndrome virus protein VP28.

Envelope protein is the important factor of determination of virus infection and virulence, also is important antigenic determinant region.WSSV has two Envelope Protein Gene vp19 and vp28.Analyze vp19 and the vp28 sequence of existing WSSV among the GenBank, can find that the homology of different regional strain isolated vp28 is higher than vp19, this illustrates during evolution, vp28 is more conservative, and this also may act on more crucial the invasion of virus from another one aspect hint vp28.

WSSV of the present invention separates the prawn of falling ill from the Chinese North Sea, and the gene order homology Yin Jiyin between the strain isolated of WSSV different areas is different and have larger difference, and wherein the nucleotide sequence homology of its envelope protein is between 48%-99%.Therefore, the nucleotide sequence that has at least 90% homology with WSSV Envelope Protein Gene vp19, and can merge by direct or indirect mode with nucleotide sequence that envelope protein vp28 has at least 95% homology, resulting fusion gene has the antigenic determinant site of all WSSV envelope protein vp19 and vp28, can both encode has the protein of identical biological function with engineered protein P40 of the present invention, can both be used for realizing one or more purposes of the present invention.

The preparation method of the nucleotide sequence of a kind of encoding gene engineered protein P40 is provided in one embodiment of the invention, the nucleotide sequence of fusion gene such as SEQ ID NO.2, wherein the position of vp19 and vp28 gene is respectively 94-456,462-1116.

Other gene fusion mode merges at back, vp28 the preceding such as vp19.In this case, vp19 and vp28 be the independent higher structure that forms separately separately, the antigenic determinant of all envelope proteins of still can encoding.Thereby the protein identical with engineered protein P40 biological function of the present invention of still can encoding, can both be used for realizing one or more purposes of the present invention.

In one embodiment of the invention, the nucleotide sequence of a kind of encoding gene engineered protein P40 is provided, its amino acid sequence coded is made of two portions, be white spot syndrome virus envelope protein vp19 and vp28 gene, two genes connect fusion by EcoRI (GAATTC) site, and the gene after the fusion is inserted in the expression vector by BamH I (GGATCC) and two sites of Hind III (AAGCTT).

In the preparation process of the engineering strain of expressed fusion protein, can external vp19 and vp28 be coupled together according to the restriction enzyme site of carrier and the sequences Design restriction enzyme site of gene, a kind of method of attachment is provided in one embodiment of the invention, with EcoR I two genes are coupled together, the gene after the fusion is inserted in the expression vector by BamH I (GGATCC) and two sites of Hind III (AAGCTT).Its advantage is that these enzymes are restriction enzyme site commonly used, and do not have these restriction enzyme sites two gene inside, are beneficial to the operation of gene.

In one embodiment of the invention, provide the nucleotide sequence of a kind of encoding gene engineered protein P40, the upstream and downstream of this sequence also comprises the sequence of carrier:

Upstream ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCT GCCCAGCCGG CGATGGC CATGGAT ATCGGAATTAATTC GGATCCT, downstream AAGCTTGCGGCCGC ACTCGAGCACCACCACCACCACCACTGA, wherein GGATCC is the restriction enzyme site that gene is connected with AAGCTT.

The stringent type control that target protein is expressed is to reduce target protein to the toxicity of cell.

The expression amount height, the highest 30-50% that can reach the tropina total amount of the expression amount of engineered protein.

The sequence of amalgamation and expression: N end helps the correct folding of protein, increases solubility and the stability of protein.The C end contains 6 continuous Histidines, is beneficial to separation and the purifying of protein.

Safe, be beneficial to industrialization production.

In one embodiment of the invention, provide a kind of dna fragmentation and preparation method thereof.This fragment comprises the nucleotide sequence of encoding gene engineered protein P40 of the present invention.

Inventing related dna segment can obtain in the following manner:

Synthetic, can directly use dna synthesizer synthetic dna fragmentation of the present invention, perhaps salvage dna fragmentation of the present invention, these synthetic products have the biological function identical with dna fragmentation of the present invention, can realize one or more purpose of the present invention.

Pcr amplification, be template with dna fragmentation of the present invention, be template with plasmid, carrier, the host cell that contains dna fragmentation of the present invention perhaps, obtain dna fragmentation by pcr amplification, these PCR products have the biological function identical with dna fragmentation of the present invention, can realize one or more purpose of the present invention.

Above method is method commonly used in the molecular biology, by those skilled in the art are familiar with, do not need to obtain by creative work, the dna fragmentation that obtains is regarded as showing identical biological function with nucleotides sequence involved in the present invention, can further realize one or more goal of the invention of the present invention by engineered method.

In one embodiment of the invention, provide a kind of host cell, this host cell comprises the nucleotide sequence of the engineered protein P40 of the present invention that encodes, perhaps comprises the dna fragmentation of the engineered protein P40 of the present invention that encodes.

Dna fragmentation involved in the present invention can change in the host cell by chemistry or biological method, as conversion, transduction, special seat, particle gun injection etc., these host cells include but are not limited to intestinal bacteria, insect cell, mammalian cell, yeast, its assignment of genes gene mapping can be on karyomit(e), also can separately or depend on other genetic material to be present in outside the karyomit(e).These host cells are beneficial to preservation and the amplification of gene on the one hand, on the other hand realization that can be direct or indirect one or more goals of the invention involved in the present invention.

The preparation method of host cell is method commonly used in the molecular biology, by those skilled in the art are familiar with, do not need can obtain by creative work, the host cell that obtains can further be realized all goals of the invention of the present invention by engineered method.

In one embodiment of the invention, a kind of e. coli host cell is provided, this host cell comprises the nucleotide sequence of engineered protein P40 of the present invention, called after (E.coli) BL21 (DE3)-pET-22b (+)-p40vp (19+28) bacterial strain, this bacterial strain is numbered CCTCC M204050 (undetermined) China typical culture collection center.

In one embodiment of the invention, provide a kind of construction process of e. coli host cell, this host cell comprises the nucleotide sequence of engineered protein P40 of the present invention.

Technical scheme

A) the virus genomic preparation of WSSV: the WSSV genome is double chain DNA molecule, and different plant type virus genome sequence have larger difference.Wherein, VP24 and VP26 albumen are the capsid associated protein, and VP19 and VP28 albumen are the cyst membrane associated protein.The virus genomic preparation method of WSSV has many pieces of bibliographical informations, by personnel in this professional skill field are familiar with, does not need creative work.A kind of WSSV is provided virus genomic preparation method in one embodiment of the invention.

B) preparation of preparation vp28 and vp19 gene: vp28 and vp19 gene can obtain by several method, be that the method that template is passed through PCR obtains as the genome with virus, perhaps by genomic digestion with restriction enzyme is obtained corresponding gene segment, can also from genomic library or cDNA library, directly obtain.Provide a kind of method by PCR to prepare the method for vp28 and vp19 gene in one embodiment of the invention, amplimer is according to the WSSV sequences Design of having delivered among the GeneBank.Genome with WSSV is template, adopts conventional PCR method amplifying target genes vp28 and vp19.Reclaim the PCR product and it is cloned in the T carrier, and transformed into escherichia coli (E.coli) JM109, be used for propagation and the preservation of gene.

C) splicing P40 open reading frame: the recon that will contain vp28 and vp19 gene is with suitable digestion with restriction enzyme, reclaim corresponding segment by correct reading frame (ORF) gene fusion construct, the P40 that builds is correct by sequencing checking result.

D) be transformed in the Bacillus coli cells: the P40 open reading frame that splicing is good is cut connection by enzyme and is inserted in the expression vector by correct reading frame, the recombinant expression vector transformed into escherichia coli that builds, method for transformation includes but are not limited to electrotransformation, calcium chloride transformation.A kind of calcium chloride method for transformation is provided in one embodiment of the invention, and the expression vector of use is pET22b (+), and host cell is e. coli bl21 (EDE3).

More than molecular biological working method be familiar with by those skilled in the art, can be with reference to Joe Sambrook, the Molecular Cloning:A Laboratry Manual that David Russell etc. writes, Cold Spring Harbor Lab (CSHL) Press, 2001.

In one embodiment of the invention, a kind of immune vaccine of white spot syndrome virus resisting is provided, described vaccine comprises engineered protein P40 of the present invention, or the nucleotide sequence of the described engineered protein P40 that encodes, or the dna fragmentation of the described engineered protein P40 that encodes, or host cell of the present invention, or their two or more mixtures.

Though do not limited by concrete theoretical mechanism, it has been generally acknowledged that vertebrates has sound immunity system, can produce the invasion and attack that many disease-resistance substances resist virus and other cause of disease, invertebrates lacks by the special resistance of immunity acquisition to certain virus.Although invertebrates lacks similar mammiferous immunity system, the Venegas of Japan etc. discover that to japonicus prawn has a kind of similar immune disease-resistant mechanism, i.e. accurate immunity system.Survivor after the japonicus white spot syndrome breaks out, survival rate is up to 94% when inoculating WSSV.Survivor after the manual injection WSSV virus, at the live virus of injection WSSV, surviving rate is also up to 66-74% after 32 days.The enhancing of anti-virus ability is not because the survivor is natural mutant, but because prawn has the disease-resistant mechanism of a kind of similar immunity system, i.e. accurate immunity system.Behind virus infection, the accurate immunity system of prawn is activated.Behind virus infection, the accurate immunity system of prawn obtains propagation, with WSSV injection prawn after 17 days, during prawn body fluid is found to have and virus activity.

The stimulation of the accurate immune response of prawn and microorganism has this point of relation to be proved to be, after the Vibrio spp (a kind of vibrios) with deactivation is injected in tigar prawn and the japonicus body, prawn self can produce effective immunological effect, resists the Vibrio spp that does not have deactivation of invasion again.

Engineered protein P40 involved in the present invention can also be used for the prevention of white spot syndrome as subunit vaccine.The immunity system that prawn does not have higher animal to have does not have the immune response of high animals such as immunological memory yet.But the invasion of virus needs virus to be combined with the specificity of cell surface receptor, and the related genetically engineered fusion envelope protein of the present invention can emulatively combine with the virocyte acceptor, and then absorption and the intrusion of influence virus.Therefore, the related engineered protein P40 of the present invention can be used as the control that a kind of " accurate vaccine " is used for prawn WSSV virus.

The engineered protein P40 that is used for vaccine production is envelope protein VP19 and the VP28 that merges, fusion rotein has two kinds of amino acid coding that albumen is all, the immune effect of this engineered protein P40 has been improved in antigenic determinant site with two kinds of albumen.

The expression system that the present invention uses is escherichia expression system, and expression vector is pET22b (+), and the host bacterium is BL21 (DE3), and is different with disclosed patent with the document of any report.Compare with other gene engineering expression system, particularly compare with insect cell-baculovirus expression system, this system all have following excellent a bit, (1) expression amount height; (2) production cost is low, and (3) separation and purification is simple, and (4) are convenient to industrialization production.

The main active ingredient of prawn WSSV vaccine involved in the present invention is engineered protein P40, this protein is the microbiological degradation utilization easily in environment, therefore prawn WSSV vaccine involved in the present invention have free from environmental pollution, to person poultry safety's advantage, and can not influence the aquatic ecological environment, be conducive to the sustainable development of water industry.

The immune vaccine of white spot syndrome virus resisting provided by the invention, its main active ingredient is engineered protein P40, in the production use, this protein can use separately, also can directly use the host cell of engineered protein, the plasmid that perhaps comprises fusion gene perhaps comprises the fusion gene dna fragmentation, or their two or more mixtures.

In one embodiment of the invention, a kind of immune vaccine of white spot syndrome virus resisting is provided, the main active ingredient of this vaccine is engineered protein P40, in the production use, this vaccine can also comprise acceptable carrier on the pharmacology, to increase immune effect, improve the stability of vaccine.

The preparation of prawn WSSV vaccine can be carried out according to the ordinary method of aquatic product medication preparation; the main active ingredient of this kind vaccine is engineered protein P40; the validity period of this protein in environment is shorter; therefore can add various auxiliary agents or adjuvant as required; comprise the protein antioxidant; stablizer; sanitas; solvent oil; activity increase agent; synergistic agent; uv-absorbing agent etc.; its objective is the protection engineered protein; increase immune effect; these auxiliarys commonly used in this area include but not limited to; insoluble aluminium salt (as chromium alum etc.); profit emulsion (as freund's adjuvant); the peanut oil emulsion adjuvant, soil temperature 80, liposome; polyose (as lipopolysaccharides, zymosan); propolis etc.

WSSV vaccine involved in the present invention can use separately or mix use with other medicines, feed, and use-pattern includes but not limited to throw something and feed, injects, soaks or sprays.Range of application comprises the prawn kind that all are common, especially the economic prawn of artificial breeding, these prawn kinds include but not limited to: Penaeus vannamei (Penaeus vannamei) Chinese prawn (is commonly called as prawn, Penaeus orientalis), penaeus penicillatus (Penaeus penicillatus), Penaeus merguiensis de man. (Penaeus merguiensis), green tiger prawn (Penaeus semisulcatus), tigar prawn (is commonly called as grass shrimp, Penaeus monodon), japonicus (being commonly called as the ring shrimp, Penaeus japonicus), the new prawn of cutter volume (Metapenseus ensis), must red shrimp (Metapenaeopsis barebata).

In one embodiment of the invention, a kind of prawn feed is provided, the main active ingredient of this feed is the host cell of engineered protein P40 or producer gene engineered protein P40, the plasmid that perhaps comprises engineered protein P40 fusion gene, the dna fragmentation that perhaps comprises engineered protein P40 fusion gene, or their two or more mixtures.

The preparation method of prawn feed of the present invention can adopt method well known to those of ordinary skill in the art, for example, includes but not limited to add engineered protein P40 of the present invention and be prepared into prawn feed in the raw material of the prawn feed of routine.Perhaps, engineered protein P40 of the present invention is mixed mutually with commercially available conventional prawn feed.

In one embodiment of the invention, provide a kind of antiserum(antisera), this antiserum(antisera) is antigen prepd with engineered protein P40.

The engineered protein P40 of purifying can be used as antigen to be mixed back direct immunization animal and prepares antiserum(antisera) with adjuvant, can be used for preparing sero-fast animal and include but are not limited to rabbit, rat, mouse, horse, sero-fast preparation method is conventional method, by those skilled in the art are familiar with.

Antiserum(antisera) with the method for the invention preparation has following advantage: compare with polyclonal antibody, its specificity is strong, can not produce false positive results, prepares as antigen because it is engineered protein P40 with purifying.Compare with the WSSV monoclonal antibody, it has the advantage that method is easy, cost is low because by fermentation can be quick, a large amount of the preparation genetically engineered merge envelope protein.

A kind of method for preparing rabbit anti-serum is provided in one embodiment of the invention, and the antiserum(antisera) that adopts the preparation of this method has reached 100% to the neutralization of WSSV.

In one embodiment of the invention, provide a kind of prawn white spot syndrome detection kit, it is characterized in that described detection kit comprises antiserum(antisera) of the present invention.

The present invention is applied to the detection of WSSV with the immune colloidal gold chromatography technology, and the colloidal gold test of preparation can quick and precisely be diagnosed sample in the open air.

Fast diagnose test paper bar is a novel vitro diagnostic techniques that has grown up on monoclonal antibody technique, colloidal gold immunochromatographimethod technology and new material technology basis since the nineties in 20th century.Development in recent years is rapid, has particularly obtained widespread use in the medical test at biomedical sector.This technology mainly is that specific antigen or antibody are fixed on nitrocellulose (NC) film with ribbon, colloid gold label reagent is adsorbed on the pad, be added on the sample pad of test strip one end when testing sample after, move forward by wicking action, react to each other behind the colloid gold label reagent on the dissolving pad, when moving to fixing antigen or antibody regional again, the specificity combination takes place again with it and is trapped in the mixture of determinand and gold marked reagent, accumulate in to detect and be with, by the colloid gold label thing that can the estimate result that developed the color intuitively.

In one embodiment of the invention, provide a kind of detection kit, this test kit can be used for the detection of prawn WSSV, and the main active ingredient of this detection kit is the antiserum(antisera) of engineered protein P40.

Antiserum(antisera) of the present invention can be used for purchasing all WSSV detection kit based on the antigen antibody reaction principle, comprises ELISA detection kit, gold-immunochromatographyreagent reagent for assay box.The preparation method of these test kits is familiar with by those skilled in the art, advantage with serum pref of the present invention is: (1) is compared with polyclonal antibody, its specificity is strong, can not produce false positive results, prepares as antigen because it is engineered protein P40 with purifying.(2) compare with the WSSV monoclonal antibody, it has the advantage that method is easy, cost is low because by fermentation can be quick, a large amount of the preparation genetically engineered merge envelope protein.

In one embodiment of the invention, provide a kind of gold-immunochromatographyreagent reagent for assay box, this test kit can be used for the detection of prawn WSSV, and the main active ingredient antiserum(antisera) figure cloth of this detection kit is on the solid substrate material.

In colloidal gold strip is purchased process, adjust the size of antibody concentration and colloid gold particle according to actual needs, the size of test strip involved in the present invention, shape, material, assembling mode can be adjusted, and these changes all are familiar with by this professional skill field personnel.In colloidal gold strip involved in the present invention, contain two kinds of key components, gold mark WSSV antibody and anti-WSSV antibody.

The preparation method of a kind of rapid detection white spot syndrome virus (WSSV) (WSSV) colloidal gold strip is provided in an embodiment of the present invention, this test strip can be used for the WSSV of rapid detection sample, this detection method and have fast, easy and simple to handle, the result of reaction and be easy to advantages such as observation, the good diagnostic reagent bar of mark can prolonged preservation, at any time use, and reagent and amount of samples are few, need not plant and instrument, simplified loaded down with trivial details routine operation process, also reduce simultaneously the error that causes because of operation, be particularly suitable for vast grass-roots unit.

Embodiment

The virus genomic preparation of embodiment 1WSSV:

In lung, stomach and the heart of getting the disease shrimp on ice, T N damping fluid (the 50mmol/L Tris-HCl that adds 10 times of volumes, 0.4mmol/L NaCl, pH 7.4) in homogenizer, ice bath homogenate, the centrifugal 5min of 8000r/min gets supernatant liquor, add Proteinase K (100 μ g/ml), DS (50mmol/L KCl; 10mmol/L Tris-Cl PH8.3; Gelatin 0.1mg/ml; NP-40,0.45%; T ween 20,0.45%), in boiling water, boil about 15min, ice bath 5min immediately, the centrifugal 10min of 12,000r/min gets its supernatant liquor and does template DNA, all the other 4 ℃ of preservations.

Embodiment 2PCR amplification vp28 and vp19 gene

Amplimer is synthetic: according to the WSSV sequences Design PCR oligonucleotide amplimer of having delivered among the GenBank.The vp19 upstream primer: Gga tccAtggccaccacgactaac ac (followingization line is BmaH I restriction enzyme site), downstream primer GaattcCtg cct cct ctt ggg gt (followingization line is EcoR I restriction enzyme site), vp28 upstream primer: aga Gaa ttcAtg gat ctt tct ttc ac (followingization line is EcoR I restriction enzyme site), downstream primer cac Aag cttTta ctc ggt ctc agtgc (followingization line is the HindIII restriction enzyme site).Genome with WSSV is masterplate, adopts conventional PCR method amplifying target genes vp28 and vp19, and whether the PCR product conforms to expection by its size of detected through gel electrophoresis.Reclaim the PCR product and it is cloned in the T carrier, and transformed into escherichia coli (E.coli) JM109, picking colony rapid extraction plasmid carries out enzyme and cuts evaluation, and the sub-called after pUCm-T19 of resulting positive colony and pUCm-T28 are used for propagation and the preservation of gene.

The preparation of embodiment 3vp28+vp19 fusion gene

Recon pUCm-T19 and (37 ℃ of the EcoR I single endonuclease digestions of pUCm-T28 that will contain vp28 and vp19 gene, 4hr), 1.0% agarose electrophoresis, big fragment (about 3300bp) after cutting glue and reclaiming small segment (about 450bp) after the pUCm-T19 enzyme is cut and pUCm-T28 enzyme and cut, the two purpose fragments of downcutting are reclaimed test kit (Omega company product) with glue purification respectively to be reclaimed, behind the big fragment dephosphorylation of pUCm-T28, be connected with the pUCm-T19 small segment again, (linked system: T4DNA ligase enzyme damping fluid 5 μ l, small pieces segment DNA 30 μ l, large fragment DNA 6.5 μ l, T4DNA ligase enzyme 3.5 μ l, aseptic double-distilled water 5 μ l), 16 ℃ connect 15hr, get to connect product 30 μ l transformed into escherichia coli competence JM109, choosing the single enzyme of single bacterium colony PstI identifies, extract recombinant plasmid pUCm-T (19+28) dna double deoxidation method and measure nucleotide sequence, sequencing primer is the M13 promoter primer, and sequencing result verification vp28 and vp19 enzyme cut that to be connected the result correct.The preparation process of fusion gene as shown in Figure 1.

The structure of the engineering strain of embodiment 4 expressed fusion proteins (vp28+vp19)

HindIII and BamH I be double digestion pUCm-T (19+28) and expression vector pET-226 (+) respectively, and after fully enzyme was cut, 1.0% agarose gel electrophoresis detected, and the purpose band is reclaimed in rubber tapping, reclaims test kit with glue and reclaims target DNA.The construction of recombinant plasmid process as shown in Figure 2.

Linked system: T4DNA ligase enzyme damping fluid 5 μ l, pUCm-T (19+28) small pieces segment DNA 25 μ l, pET-226 (+) DNA 5 μ l, T4DNA ligase enzyme 4 μ l, aseptic double-distilled water 11 μ l), 16 ℃ connect 15hr.

Connect product transformed into escherichia coli (E.coli) BL21 (DE3) competent cell, HindIII and BamH I double digestion Screening and Identification go out recon (note is made BL21 (DE3)-pET-22b (+)-vp (19+28)) recon, and enzyme is cut the checking result as shown in Figure 3.

The expression of embodiment 5 genetic engineering fusion proteins

To rule at the LB flat board that is added with penbritin with bacterial classification BL21 (DE3)-pET-22b (+)-vp (19+28) p40 that glycerine is preserved, cultivated liquid; Chose single colony inoculation in second day and enrich in the liquid nutrient medium to the fresh 50ml LB that adds penbritin, 230rpm37 ℃ is shaken 8-10hr, deposits for 4 ℃ and spends the night.Being inoculated into new LB by 4% in second day enriches in the liquid nutrient medium, 220rpm, 37 ℃ when shaking OD=0.6, add IPTG (final concentration 0.6mM) and induce, inducing temperature is lowered to 35 ℃ simultaneously, after shaking 4-5hr, 4000rpm is centrifugal, collects thalline, and an amount of 1 * bind buffer of thalline is resuspended, add PMSF (final concentration 1mM) again, 4 ℃ of preservations behind the mixing.The sample SDS-PAGE electrophoresis detection that takes a morsel, result show at IPTG induces the back engineering strain can expressed fusion protein, and the expression product molecular weight conforms to expection, as shown in Figure 4.

The purifying of embodiment 6 genetic engineering fusion proteins

With-20 ℃ of frozen fermentation thalline multigelations several times after, use ultrasonic disruption again, behind the 18000rpm high speed centrifugation 20min, get Ni post on the supernatant, albumen behind the Ni column purification, dialyse repeatedly again, concentrate, finally be concentrated into about (albumen/physiological saline) 380 μ l/ml, namely come immune rabbit as antigen.The sample SDS-PAGE electrophoresis detection that takes a morsel purification result as shown in Figure 5.

The preparation of embodiment 7 prawn WSSV specific antibodies

About the antigen 200 μ g of preparation, behind isopyknic complete Freund's adjuvant mixing, rabbit is carried out the lymph injection.After two weeks, react with 200 μ g purifying antigen booster immunizations again.After one week, the strength arterial blood extracting.Blood places 37 ℃ of 30min, places 4 ℃ of refrigerators then, spends the night.Next day, behind 8, the 5000rpm high speed centrifugation 5min, collect serum.The sodium azide of adding 0.02% is anticorrosion in the serum, mixing, and preceding 56 ℃ of deactivation 30min are used in-70 ℃ of preservations.

The preparation of embodiment 8 colloidal gold strips

The preparation of (1) 20～30nm colloid gold particle:

Adopt the configuration of citrate three sodium reduction method, method is referring to [Shen Guanxin etc., modern immunological experiment technology, Wuhan: Hubei science tech publishing house, 1998].With 1000ml deionized water heated and boiled, when stirring, add 4% gold trichloride 2.5ml and 1% trisodium citrate 15ml, after heated and stirred treats that solution becomes burgundy, to continue to boil to suitable concentration, pH to 7.2-7.5 is transferred with 0.2mol/L salt of wormwood in the cooling back.With 0.22 μ m filter membrane asepsis injector press filtration, 4 ℃ of preservations of filtrate.

(2) preparation of Radioactive colloidal gold-antibody conjugates and purifying

Get a certain amount of Radioactive colloidal gold for preparing, the antibody that adds the WSSV envelope protein of 1mg purifying, stirred 1 hour under the room temperature, adding 10%BSA makes its final concentration be 0.5%, 4 ℃ and places after 3～4 hours centrifugal 30 minutes of 7000r/min, abandon supernatant, precipitation washes twice with 1%BSA, adds 10% polyoxyethylene glycol (PEG2000) 1ml (final concentration is 0.2%), stirs 5 minutes under the room temperature.Centrifugal 50 minutes of 12000～15000r/min, precipitation is dissolved in 5ml and preserves in the liquid.With 0.45 μ m membrane filtration, 4 ℃ of refrigerators are preserved

(3) preparation of the plain film of Radioactive colloidal gold-antibody conjugates glass fibre

Get 3ml Radioactive colloidal gold-antibody conjugates, add 3ml diluent mixing.Put into the plain film of glass fibre and soaked 10 minutes, take out to put in 37 ℃ of baking boxs and dry, heat sealing, it is standby to put 4 ℃ of refrigerators.

(4) preparation of antibody solid phase nitrocellulose filter (NC)

The antibody of getting the WSSV envelope protein becomes 3mg/ml with the solid phase solution dilution.Get goat anti-rabbit antibody and become 1mg/ml with the solid phase solution dilution.Two kinds of antibody are got 1 μ l respectively, and point sample is on nitrocellulose filter.The insolubilized antibody nitrocellulose filter is put in the confining liquid, hatched 1 hour in 37 ℃ of water-baths.Take out the insolubilized antibody nitrocellulose filter and be immersed in and wash 2 times in the washings, air-dry under the room temperature, it is standby to put 4～8 ℃ of preservations.

(5) assembling of test strip

As shown in Figure 6, adhere to water adsorption glass fiber, the plain film of golden labeling antibody binding substances-glass fibre, absorbent filter successively in white plastic plate inboard, 2mm overlaps each other, and cover WSSV antibody solid phase nitrocellulose filter and anti-WSSV antibody solid phase nitrocellulose filter band in the above, fix with adhesive tape, be cut into the wide band of 5mm with paper knife, it is standby to put 4 ℃ of preservations.

Embodiment 9 usefulness colloidal gold strip rapid detection white spot syndrome virus (WSSV)

(1) processing of test sample

Get prawn to be checked, peel off the shrimp shell, get about 1 gram shrimp body homogenate and smash to pieces, add a small amount of pure water mixing, the prawn with health compares simultaneously.

(2) WSSV detects

Take out test strip and return to room temperature.The water adsorption glass fiber end is inserted in the test sample liquid judged result about 10 minutes.If test strip only Quality Control point (adding goat anti-rabbit antibody) pink color dot occurs and test point does not develop the color, the result is negative; If pink color dot all appears in Quality Control point and check point (adding WSSV antibody) on the test strip, the result is positive; If test strip Quality Control point and check point all do not have colour developing, illustrate that then this test strip lost efficacy, the result is invalid, as shown in Figure 7.

(3) detected result

The only Quality Control point colour developing of healthy prawn is with malicious prawn Quality Control point and check point pink color dot all to occur and detect, and illustrates that this test strip can the rapid detection white spot syndrome virus (WSSV).

Embodiment 10 merges the envelope protein antiserum(antisera) to the neutralizing effect of WSSV

Preparation and using method: get the antiserum(antisera) for preparing and mix by 1:1 with oil-emulsion, mix the tigar prawn of throwing something and feeding with feed then, the dosage 100ml/kg shrimp of throwing something and feeding, carry out the WSSV virus attack simultaneously, establish the contrast of virus attack contrast and emulsifying agent, every group of 50 shrimps, do a repetition simultaneously, day by day calculate death toll after 3 days, ended until 20 days, calculate prevention effect.Raising temperature 20-26 ℃.

Prevention effect: as shown in table 2 below, test-results shows that the antiserum(antisera) medicine has good prevention effect to WSSV.

Table 2

The engineering fermentation of embodiment 11 engineering strains

Bacterial classification: engineering strain E.coliBL21 (DE3)-pET-22BL21 (DE3)-pET-22b (+)-p40b (+)-Vp (19+28)

The fermentation initial medium: fermention medium 14L transfers pH7.0, the sterilization in place of 121 ℃, 20 minutes condition bottom fermentation jars by following formulated.

Annotate: MgSO ₄Sterilization separately adds when inoculation, otherwise easily forms precipitation.Supplemented medium:

Supplemented medium 4L is by following formulated, 121 ℃, 20 minutes, is divided in the feed supplement bottle of sterilization before using and adds penbritin to final concentration and be 200mg/L.

Process record:

The sterilization in place after 20 minutes in fermentor tank of 121 ℃ of fermention mediums is lowered the temperature and is stabilized in 36.5 ℃, inoculation 3%LB seed, the MgSO that adds penbritin to final concentration simultaneously and be 200mg/L and sterilized in advance ₄Solution.Initial mixing speed 300RPM observes dissolved oxygen and changes, and guarantees the DO value more than 30%, and DO drops to 30% need improve mixing speed when following.4.7 hour the time, under the constant situation of oxygen supply condition, DO rises rapidly, shows that nutrient exhausts in the initial medium, and mixing speed is brought up to predetermined top speed 500RPM, beginning flow feeding substratum.The supplemented medium arbitrary way is the periodicity automatic feeding, observes the variation of DO value after feed supplement, adjusts feed supplement speed, and assurance DO value is kept the level of 30%-40%.Fermenting began to be cooled to 34 ℃ in 11.7 hours, and disposable adding IPTG is 0.4mmol/L to final concentration, the beginning abduction delivering, and the abduction delivering stage is adjusted feed supplement speed, and DO is about 30% in control, induces and finishes fermentation in 4.8 hours, puts jar.Sampling is centrifugal under 12000RPM, 10 minutes conditions when putting jar, abandoning supernatant, and every liter of substratum contains about 60 grams of wet thallus.

The preparation of embodiment 12 genetically engineered p40 protein purification samples

Ni ²⁺The column chromatography sample preparation is got ferment tank bacterium liquid through the centrifugal wet thallus 30g that obtains, resuspended with 60ml 1 * Binding Buffer (0.5M NaCl+5mM imidazoles+20mMTrisHCl pH7.9), ultrasonic disruption 3 * 30 minutes (the effectively broken time is 30 minutes), albumen suspension after the fragmentation is centrifugal under 15000RPM, 30 minutes conditions, get supernatant liquor, use the membrane filtration of 0.45 μ m again, be settled to 100ml Ni with 1 * Binding Buffer ²⁺The column chromatography sample.

Ni ²⁺The pre-treatment of post is with 10 1 * Binding Buffer, 20 sterile water wash post beds; With 10 1 * Charge Buffer (5mM NiSO4), in conjunction with Ni ²⁺After using 10 1 * Binding Buffer balance columns beds again, standby (1 namely is the volume of medium in the post).

The sample that column chromatography concentrated passes through Ni in a looping fashion through peristaltic pump ²⁺Post makes the abundant and Ni of the protein sample moving phase that has 6 * His ²⁺Ni in the post ²⁺Combine, back Ni spends the night ²⁺Column outlet is collected not and Ni ²⁺In conjunction with sample (leakage liquid).

Clean post and adopt 50 1 * Binding Buffer respectively; 75 60mM imidazoles; 75 100mM imidazoles; Post interior small amount of sample and and Ni are stayed in 30 150mM imidazoles gradient washings ²⁺The non-target protein of post combination.

Wash-out Ni ²⁺Post is with 1 * Elute Buffer (1M imidazoles+0.5MNaCl+20mM TrisHCl pH7.9) wash-out and Ni ²⁺The target protein of post combination, fraction collection, every bed volume are 5 pipes.By the distribution of SDS-PAGE check target protein in elutriant.

Ni ²⁺Post continues wash-out with 1 * Elute Buffer, washes whole albumen in the sample post; Use the Ni on 1 * Strip Buffer (100mM EDTA+0.5M NaCl+20mM TrisHClpH7.9) flush away pillar again ²⁺Ion.

P40 content determines in the embodiment 13p40 purification of samples

With 11ml Ni ²⁺The sample of post wash-out, collection is packed in the dialysis tubing of handling well, and adds PBS to final volume 60ml.PEG is concentrated into shrivelled state, adds that PEG concentrates behind the PBS again.Repeat PEG and concentrate, reach 300ml until the cumulative volume that adds PBS.Good dialysis tubing ddH will be concentrated ₂After O rinses well, put into fill 1000ml PBS beaker anti-saturating, spend the night.In anti-saturating good dialysis tubing, add 50ml PBS, be concentrated into shrivelled state with PEG again.Add 1.6ml PBS the albumen in the dialysis tubing is developed, more than operation is all carried out at 4 ℃.

With Bradford method quantification of protein, the concrete operations step is as follows with the sample before the dialysis treatment: 1. testing sample is moved to-5ml EP pipe in (maximum volume of adding≤100 μ l); 2. add 1 * Einte Buffer to final volume 100 μ l; 3. add the 1mlBradford working fluid, and the concussion mixing; 4. behind 2min, survey OD ₅₉₅5. according to typical curve: Y=21.01X+0.07686, Z=11X (μ g/ μ l) calculates p40 protein content in the purification of samples.

Embodiment 14 genetically engineereds merge the application of envelope protein in the WSSV control---and Procambius clarkii is infected attack through injection engineered protein p40 opposing WSSV injection

1. for the examination material

The preparation of injection WSSV suspension is put-20 ℃ of frozen Procambius clarkiis through the WSSV infection and is removed crust, appendage, hepatopancreas on ice, get cephalothorax (organs such as the cheek, liver, heart), and be cut into small pieces with surgical scissors, put into glass homogenizer, totally 10, and by 1: 5 (W/V) adding PBS.With the homogenate of glass homogenizer ice bath.4 ℃ of centrifugal 20min of homogenate 12000r/min.Supernatant liquor is with 0.45 μ m membrane filtration degerming, and-20 ℃ of packing 1.5ml EP pipes are frozen standby.Obtain 40ml WSSV viral suspension altogether.With above-mentioned WSSV viral suspension, 100 a μ l/ WSSV suspension returns the sense Procambius clarkii, 22-24 ℃ of infection, and 72 hours mortality ratio reach 35.4%, 96 hour mortality ratio and reach 89.6%, 108 hour mortality ratio and reach 100%.

Procambius clarkii (Procambarus clarkii), the long 8cm of average body is bought by the country fair, and it is built-in in the laboratory that (every basin is 10 in the plastics of 50cm * 35cm * 15cm), cultures not have in two weeks and continues dead healthy shrimps, the prawn feed of throwing something and feeding.

The P40 engineered protein of purifying contains 0.68mg/mL, and-4 ℃ of preservations are standby.0.01mol/L PBS pH7.4 sterilization is standby.

Prawn feed (No. 0) Zhanjiang Yuehua Aquatic Feed Co., Ltd.

2. experimental technique

With the healthy Procambius clarkii that culture with prawn feed in the laboratory, the P40 engineered protein 80l/ head of injection purifying; Injection PBS 80 μ l/ heads.In Procambius clarkii uromere voluntary muscle injection third from the bottom, once a day, injected 2 days the normal prawn feed of throwing something and feeding sooner or later every day continuously.The 3rd day, in Procambius clarkii uromere voluntary muscle injection third from the bottom WSSV viral suspension 100 μ l/ heads, the injection back was extracted syringe needle with medical cotton stick flicking injection place 2-3 out after second, puts back in the plastic tub.Afterwards, the normal prawn feed of throwing something and feeding, totally 2 groups of processing are handled 10 shrimps for every group.Other establishes three PBS 100 μ l/ heads of one group of 10 shrimps injection, the control group of the normal bait of throwing something and feeding.Observed and recorded every group of dead shrimp number of handling every day.

3. experimental result

See Table 3

Corrected mortality=(contrast survival rate-processing survival rate)/contrast survival rate * 100%

During 120h, lower mortality ratio appears in injection P40 infected group, is 50% with respect to the survival rate of injecting the PBS infected group.The result shows that Procambius clarkii is through injection engineered protein p40, and injection is infected and can be produced the excellent protection effect to WSSV.

Table 3

Embodiment 15 genetically engineereds merge the application of envelope protein in the WSSV control---and attack is infected in Procambius clarkii oral administration engineered protein p40 opposing WSSV injection

1. for the examination material

Prawn feed (No. 0) Zhanjiang Yuehua Aquatic Feed Co., Ltd.

The prawn feed of P40 engineered protein bag quilt prepares engineering strain E.coliBL21 (DE3)-pET-22b (+)-vp (19+28) p40 through bulk fermentation, collects thalline.100 gram wet thallus are resuspended with 600ml PBS, multigelation, and ultrasonic disruption, centrifugal, get supernatant.According to (V: W=1000: amount 6) adds the abundant mixing of macromolecular material coating agent, dissolving.According to V: W=1: the same prawn feed of solution (No. 0) that 2.5 amount will contain the polymer coating agent mixes (being the prawn feeds that the P40 engineering bacterial cell disruption liquid supernatant of 100ml adds 250 grams) fully, and every gram prawn feed contains the P40 engineering bacterial cell disruption liquid supernatant that weight in wet base is 66.7mg.After drying in the shade ,-20 ℃ frozen standby.

The prawn feed of E.coliBL21 (DE3)-pET-22b (+) bag quilt prepares E.coliBL21 (DE3)-pET-22b (+) through shake flask fermentation, collects thalline.100 gram wet thallus are resuspended with 600ml PBS, multigelation, and ultrasonic disruption, centrifugal, get supernatant.According to (V: W=1000: amount 6) adds the abundant mixing of macromolecular material coating agent, dissolving.According to V: W=1: the same prawn feed of solution (No. 0) that 2.5 amount will contain the polymer coating agent mixes (being the prawn feeds that the P40 engineering bacterial cell disruption liquid supernatant of 100ml adds 250 grams) fully, and it is E.coliBL21 (DE3)-pET-22b (+) bacterial cell disruption liquid supernatant that every gram prawn feed contains weight in wet base.After drying in the shade ,-20 ℃ frozen standby.

3. experimental result

See Table 4

During 120h, the P40 bag lower mortality ratio occurred by bait infectable infection group, is 43.33% with respect to BL21 (DE3) bag by the survival rate of bait infectable infection group and normal bait infectable infection group.The result shows, Procambius clarkii oral administration engineered protein p40, and injection is infected and can be produced the excellent protection effect to WSSV.

Table 4

Embodiment 16 genetically engineereds merge the application of envelope protein in the WSSV control---and Procambius clarkii oral administration engineered protein p40 opposing WSSV is oral to infect attack

1. for the examination material

The preparation of the usefulness of throwing something and feeding WSSV is put-20 ℃ of frozen Procambius clarkiis through the WSSV infection to remove crust, appendage, hepatopancreas on ice, gets cephalothorax (organs such as the cheek, liver, heart), and be cut into small pieces with surgical scissors, put into glass dish, totally 25, it is ℃ standby to put into refrigerator-20.

Procambius clarkii is bought by the country fair that built-in in the laboratory (every basin is 10 in the plastics of 50cm * 35cm * 15cm), cultures not have in two weeks and continues dead healthy shrimps, the prawn feed of throwing something and feeding.

Prawn feed (No. 0) Zhanjiang Yuehua Aquatic Feed Co., Ltd.

The prawn feed of P40 engineered protein bag quilt prepares engineering strain E.coliBL21 (DE3)-pET-22b (+)-vp (19+28) through bulk fermentation, collects thalline.100 gram wet thallus are resuspended with 600ml PBS, multigelation, and ultrasonic disruption, centrifugal, get supernatant.According to (V: W=1000: amount 6) adds the abundant mixing of macromolecular material coating agent, dissolving.According to V: W=1: the same prawn feed of solution (No. 0) that 2.5 amount will contain the polymer coating agent mixes (being the prawn feeds that the P40 engineering bacterial cell disruption liquid supernatant of 100ml adds 250 grams) fully, and every gram prawn feed contains the P40 engineering bacterial cell disruption liquid supernatant that weight in wet base is 66.7mg.After drying in the shade ,-20 ℃ frozen standby.

2. experimental technique

With the healthy Procambius clarkii of laboratory with the prawn feed breed, use the bait of P40 engineered protein bag quilt, the prawn feed of BL21 (DE3) bacterial cell disruption liquid supernatant bag quilt respectively, normal prawn feed is thrown something and fed sooner or later, threw something and fed cheek sheet and the cephalothorax tissue block of the Procambius clarkii that infects WSSV death of throwing something and feeding in the 3rd day continuously 2 days.Afterwards, the prawn feed of the bait of the P40 engineered protein bag quilt of throwing something and feeding respectively, BL21 (DE3) bacterial cell disruption liquid supernatant bag quilt, normal prawn feed, totally 3 groups of processing are handled 30 shrimps for every group.Other establishes the bait that one group of 30 shrimp is used P40 engineered protein bag quilt always, the control group of throwing something and feeding and culturing.Observed and recorded every group of dead shrimp number of handling every day.

3. experimental result

See Table 5.

During 120h, P40 bag lower mortality ratio occurred by the bait feeding infected group, is 36.67% with respect to BL21 (DE3) bag by the survival rate of bait feeding infected group, is 33.33% with respect to the survival rate of normal bait feeding infected group.The result shows that Procambius clarkii oral administration engineered protein p40 can produce the excellent protection effect to oral the infecting of WSSV.

Table 5

Embodiment 1617 genetically engineereds merge the application of envelope protein in the WSSV control---and Penaeus vannamei oral administration engineered protein p40 opposing WSSV is oral to infect attack

1. for the examination material

Penaeus vannamei (Penaeus vannamei) specification: the long 4.0-4.5cm of body.Introduced by thing lake, Wuhan City Bai Quan plant, built-in in the laboratory (plastic box of 65cm * 55cm * 45cm) is cultured the healthy shrimp in a week, the prawn feed of throwing something and feeding.

Prawn feed (No. 0) Zhanjiang Yuehua Aquatic Feed Co., Ltd.

The prawn feed of P40 engineered protein bag quilt prepares engineering strain E.coliBL21 (DE3)-pET-22b (+)-p40vp (19+28) through bulk fermentation, collects thalline.100 gram wet thallus are resuspended with 600ml PBS, multigelation, and ultrasonic disruption, centrifugal, get supernatant.According to (V: W=1000: amount 6) adds the abundant mixing of macromolecular material coating agent, dissolving.According to V: W=1: the same prawn feed of solution (No. 0) that 2.5 amount will contain the polymer coating agent mixes (being the prawn feeds that the P40 engineering bacterial cell disruption liquid supernatant of 100ml adds 250 grams) fully, and every gram prawn feed contains the P40 engineering bacterial cell disruption liquid supernatant that weight in wet base is 66.7mg.After drying in the shade ,-20 ℃ frozen standby.

2. experimental technique

With the healthy Penaeus vannamei that culture with prawn feed in the laboratory, use bait, the BL of P40 engineered protein bag quilt respectively ₂₁DE ₃The prawn feed of bacterial cell disruption liquid supernatant bag quilt, normal prawn feed is thrown something and fed sooner or later, throws something and feeds cheek sheet and the cephalothorax tissue block of the Procambius clarkii of the infection WSSV death of throwing something and feeding in the 3rd day continuously 2 days.Afterwards, the prawn feed of the bait of the P40 engineered protein bag quilt of throwing something and feeding respectively, BL21 (DE3) bacterial cell disruption liquid supernatant bag quilt, normal prawn feed, totally 3 groups of processing, preceding 2 groups 20/every group, the 3rd group 7.Other establishes the bait that one group of 30 shrimp is used P40 engineered protein bag quilt always, the control group of throwing something and feeding and culturing.Observed and recorded every group of dead shrimp number of handling every day.

3. experimental result

See Table 6.

During 120h, P40 bag lower mortality ratio occurred by the bait feeding infected group, is 70% with respect to BL21 (DE3) bag by the survival rate of bait feeding infected group, is 80% with respect to the survival rate of normal bait feeding infected group.The result shows that Penaeus vannamei oral administration engineered protein p40 can produce the excellent protection effect to oral the infecting of WSSV.

Table 6

Embodiment 1718 genetically engineereds merge the application of envelope protein in the WSSV control---and the little long-armed shrimp river prawn oral administration engineered protein p40 opposing WSSV of China is oral to infect attack

1. for the examination material

China little long-armed shrimp (Palaemonetes sinensis), body is long to be 5.2cm, the river prawn spawning time river prawn.Introduced by the Ezhou Liangzi Lake, built-in in the laboratory (plastic box of 65cm * 55cm * 45cm) is cultured the healthy shrimp in a week, the prawn feed of throwing something and feeding.

Prawn feed (No. 0) Zhanjiang Yuehua Aquatic Feed Co., Ltd.

2. experimental technique

With the healthy river prawn of laboratory with the prawn feed breed, use the bait of P40 engineered protein bag quilt, the prawn feed of BL21 (DE3) bacterial cell disruption liquid supernatant bag quilt respectively, normal prawn feed is thrown something and fed sooner or later, threw something and fed cheek sheet and the cephalothorax tissue block of the Procambius clarkii of the infection WSSV death of throwing something and feeding in the 3rd day continuously 2 days.Afterwards, throw something and feed respectively bait, BL21 (DE3) BL of P40 engineered protein bag quilt ₂₁DE ₃The prawn feed of bacterial cell disruption liquid supernatant bag quilt, normal prawn feed, totally 3 groups of processing, 20/every group of the first two group, one group 7 of backs.Other establishes the bait that one group of 20 shrimp is used P40 engineered protein bag quilt always, the control group of throwing something and feeding and culturing.Observed and recorded every group of dead shrimp number of handling every day.

3. experimental result

See Table 7.

During 72h, P40 bag lower mortality ratio occurred by the bait feeding infected group, is 36.84% with respect to BL21 (DE3) bag by the survival rate of bait feeding infected group, is 37.59% with respect to the survival rate of normal bait feeding infected group.The result shows that river prawn oral administration engineered protein p40 can produce the excellent protection effect to oral the infecting of WSSV.

Table 7

Though at length illustrate and introduced the present invention with reference to preferred embodiment, skilled in the art will recognize that various variations formal and that the details aspect is done all do not break away from the spirit and scope of the present invention that appending claims is determined.Those skilled in the art can approve, or only uses conventional experiment just can find various schemes with the specific embodiment equivalence of putting down in writing especially of the present invention here.These schemes are also contained in the scope of claims.

Claims

1. engineered protein P40 has the antigenicity of anti-white spot syndrome virus (WSSV), it is characterized in that described engineered protein P40 is made up of the aminoacid sequence of SEQ ID NO.1.

2. engineered protein P40 according to claim 1, the molecular weight that it is characterized in that described protein P40 is about 40.3 kilodaltons.

3. engineered protein P40 according to claim 1 is characterized in that described protein P40 by the aminoacid sequence of white spot syndrome virus protein VP19, and the aminoacid sequence of white spot syndrome virus protein VP28 is formed.

4. engineered protein P40 according to claim 3 is characterized in that the link position of described white spot syndrome virus protein VP19 aminoacid sequence between the aminoacid sequence of white spot syndrome virus protein VP28 has amino acid EF at least.

5. engineered protein P40 according to claim 1 is characterized in that described engineered protein P40 also comprises carrier sequence MKYLLPTAAAGLLLLAAQPAMAMDIGINSDP and KLAAALEHHHHHH.

6. the nucleotide sequence of an encoding gene engineered protein P40 or its immunogenic fragments is characterized in that described nucleotides sequence classifies the nucleotide sequence of SEQ ID NO.2 as.

7. nucleotide sequence according to claim 6, it is characterized in that the nucleotide sequence of described encoding gene engineered protein P40 or its immunogenic fragments by the coding nucleotide sequence of white spot syndrome virus protein VP19, and the coding nucleotide sequence of white spot syndrome virus protein VP28 is formed.

8. nucleotide sequence according to claim 6 is characterized in that the nucleotide sequence of described encoding gene engineered protein P40 or its immunogenic fragments further comprises restriction enzyme site GGATCC, GAATTC and the AAGCTT that gene connects.

9. nucleotide sequence according to claim 6 is characterized in that the nucleotide sequence of described encoding gene engineered protein P40 or its immunogenic fragments also further comprises carrier sequence A TGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCG GCGATGGCCATGGATATCGGAATTAATTC T and GCGGCCGCACTCGAGCACCACCACCACCACCACTGA, wherein

With

Restriction enzyme site for the gene connection.

10. a dna fragmentation is characterized in that being made up of the described nucleotide sequence of claim 6.

11. a host cell is characterized in that comprising nucleotide sequence as claimed in claim 6, or dna fragmentation as claimed in claim 10.

12. host cell as claimed in claim 11, it is characterized in that described host cell is intestinal bacteria (E.coli) BL21 (DE3)-pET-22b (+)-p40vp (19+28) bacterial strain, this bacterial strain is at the CCTCCM204050 that is numbered at China typical culture collection center.

13. a method that makes up the described host cell bacterial strain of claim 12 is characterized in that described method comprises the following steps:

A. be equipped with the white spot syndrome virus genome;

B.PCR amplification vp28 and vp19 gene;

C. splice the P40 open reading frame and obtain comprising nucleotide sequence as claimed in claim 5; And

D. the nucleotide sequence that step C is obtained is transformed in the Bacillus coli cells.

14. the immune vaccine of a white spot syndrome virus resisting, it is characterized in that described vaccine comprises as claim 1 or 3 described protein P40, or nucleotide sequence as claimed in claim 6, or dna fragmentation as claimed in claim 10, or as claim 11 or 12 described host cells, or their two or more mixtures.

15. immune vaccine according to claim 14 is characterized in that described vaccine also comprises acceptable carrier on the pharmacology.

16. method for preparing the immune vaccine of white spot syndrome virus, it is characterized in that described method comprises as claim 1 or 3 described protein P40, or nucleotide sequence as claimed in claim 6, or dna fragmentation as claimed in claim 10, or as claim 11 or 12 described host cells, or their two or more mixtures, mix with acceptable carrier on the pharmacology.

17. prawn feed, it is characterized in that described prawn feed includes as claim 1 or 3 described protein P40, or nucleotide sequence as claimed in claim 6, or dna fragmentation as claimed in claim 10, or as claim 11 or 12 described host cells, or their two or more mixtures.

18. an antiserum(antisera) is characterized in that described antiserum(antisera) is antigen prepd with engineered protein P40.

19. a white spot syndrome virus detection kit is characterized in that described detection kit comprises antiserum(antisera) as claimed in claim 18.

20. detection kit according to claim 19 is characterized in that described antiserum(antisera) is coated on the substrate material.