CN100371443C - Compositions and methods for enhancing disease resistance in fish - Google Patents

Compositions and methods for enhancing disease resistance in fish Download PDF

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CN100371443C
CN100371443C CNB2003801083571A CN200380108357A CN100371443C CN 100371443 C CN100371443 C CN 100371443C CN B2003801083571 A CNB2003801083571 A CN B2003801083571A CN 200380108357 A CN200380108357 A CN 200380108357A CN 100371443 C CN100371443 C CN 100371443C
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polypeptide
attacin
sequence
fish
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CN1735688A (en
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R·A·顿哈姆
刘占江
G·W·瓦尔
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Auburn University
MUSC Foundation for Research Development
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Abstract

Compositions and methods for conferring enhanced disease resistance in a fish are provided. Compositions include novel recombinant constructs that induce transgenic expression of anti-pathogenic polypeptides, including cecropin proteins or biologically active variants thereof, in a fish. Expression of the anti-pathogenic polypeptide is under the control of novel synthetic promoters, naturally occurring fish promoters, or biologically active variants thereof. Compositions of the invention also include transgenic fish cells, fish eggs, and fish, particularly catfish, having the recombinant constructs of the invention stably integrated within their genome. Methods for expressing a polypeptide of interest within a host cell are also provided, wherein expression is under control of the synthetic promoters of the invention.

Description

Strengthen the composition and the method for disease resistance in fish
Invention field
The present invention relates to the fish biology field, relate in particular to the adjusting of genetic expression in the catfish and the raising of disease resistance.
Background of invention
U.S.'s catfish industry production value surpasses 3,000,000,000 dollars, and catfish output has accounted for more than 70% of U.S.'s aquaculture ultimate production.The catfish of aquaculture is infected easily.Infection has caused output decline, and the expense-every year that has increased the human consumer is above 100,000,000 dollars.
The immune sozins of finding in vertebrates blood and the tissue fluid is divided into two classes: the induced antibody of antigen-specific (or immunoglobulin (Ig)), the defense molecule of antigen non-specific (or natural), comprise antibacterial peptide, they aspect a lot of with similar (Marchalonis (1977) the Immunity in Evolution (Harvard UniversityPress) of Broad spectrum antibiotics; Hancock (1997) Peptide Antibiotics 349:418-42).Bony fish uses this two classes defense molecule opposing to infect.Bony fish can produce antibody (Wilson and Warr (1992) the Ann.Rev.Fis.Dis 2:201-2 of two types of IgM and IgD to infecting the generation response; Wa rr (1995) Dev.Comp.Immunol.19:1-12; Wilson etal. (1997) Proc.Natl.Acadsci.USA..4593-4597).In addition, bony fish can produce and secrete the broad-spectrum antimicrobial composition, antibacterial peptide for example, and be not only enzyme, for example N,O-Diacetylmuramidase (Lemaitre et al. (1996) Eur.JBiochem.240 (1): 143-149; Oren and Shai (1996) Eur.JBiochem.237:303-3).Yet these Alliance Defense mechanism are not enough to protect fully cultured fishes to avoid the invasion and attack of disease, and from the actual foreground of modern commerce fishery, the natural immunity of fish reaction all is not enough.
Channel catfish (channel catfish, Ictalurus punctatus) is a kind of species that economic worth is arranged very much, and may still study the most thorough bony fish immunity animal pattern (Clemetal. (1991), Phylogenesis of Immunological Functions, ed.Warr and Cohen (CRC Press, Boca Raton), pp.1-13; Miller etal. (1994) J.Immunol.152:2180-2189; Ghaffari and Lobb (1991) J.Immunol.146; 1037-1046; Graves et al. (1985) J.Immunol.134:75-85, Warr et al. (1991) Eur.J.Immunogenetics 18:393-379; Wa r r (1991) EurJ.Immunogene tics 18:393-379; Warr (1995) Dev.Comp.Immunol.19:1-2; Wilson et al. (1997) Proc.Natl.Acad.Sci.USA..4593-4597).Catfish suffers that easily various bacteria infects, and such infection can cause very big destruction to the fingerling of fish farm.For example, intestines septicemia ((Edwardsiella ictaluri) causes by the catfish tarda) is the problem that costs a lot of money that present catfish industry faces repeatedly.The researchist has attempted having carried out some inoculation tests, and hope can protect catfish to avoid the invasion and attack of intestines septicemia, but does not up to the present also find real effectively catfish tarda immunization scheme.In fact, though a lot of application of immune programme for children in other fish all successful (atlantic salmon, the cold water vibriosis of salt lake salmon; Referring to Holm andHorgensen (1987) J.Fish Dis.10:85-90), but the immunization strategy is not very effective to many catfish diseases on the whole.
Catfish is cultured the fisherman and attempts to avoid fish disease with three kinds of methods.First method is to attempt immune fish.For example, United States Patent (USP) 4,287,179 have described fish are immersed in and have come immune fish in the water of the Lu Shi Yersinia that contains deactivation, enable the red mouth disease of anti-intestines.Second method be attempt by use can non-specific activating immune system compound, for example the yeast cells wall preparation strengthens the innate immune power (Wang and Wang (1997) Comp.Immunol.Microbiol.Infect.Dis.20:261-270) of aquatic products fish.United States Patent (USP) 5,593,678 have described the use protein phosphatase inhibitor prophylactically protects bony fish to avoid (comprising catfish) infection of microbial pathogen.The third method is to carry out breeding with the genetically modified catfish that can express encoding antimicrobial peptide.The transgenosis peptide can be given non-specific immunity to fish under the situation that does not need external processing.
Every kind of method part of all having any problem.Small fish to embryo and hatching just can't carry out effective immunity, because their immunity system is also enough unripe, and can not be to the vaccine effecting reaction.And immunity is a kind of time-consuming, effort and expensive method, feeds or immunization is carried out in submergence if not employing, and is then especially true.Or even under effective situation, the time that immune non-specific enhancing continued also can be very short.The development transgenic method needs a large amount of energy, and very expensive; But compare with other method, come in the long term, it is best that transgenic method is likely, because genetically engineered fish does not need post-processed in case affect.
In the document antibacterial peptide there is a lot of descriptions.The number of antibacterial peptide family is very huge, and extensively there is (Hancock (1997) PeptideAntibiotics 349:418-42 probably in antibacterial peptide in various species; Hoffmann et al. (1996) Curr.Opin.Immunol.8:8-13; Boman (1996) Scand.J.Immunol.43:475-482; Bevins and Zasloff (1990) Annu.Rev.Biochem.591:395-414; Lehreret al. (1993) Annu.Rev. Immunol.11:105-128).The very deep antibacterial peptide of developing the earliest and studying has attacin (cecropin), and they are the little peptides of cationic (Steineret al. (1981) Nature 292:246-248) that identify in Jin Xing Acer negundum Io moth (Hyalophora cecropia) at first.Attacin had both had germicidal action, had bacteriostatic action again.In fact, the researchist has confirmed that attacin can suppress or kill the pathogenic agent of other type, comprises virus, fungi and protozoon.
Attacin has the important feature that much might be used in aquatic products.These peptide classes have the anti-gram negative bacterium activity of wide spectrum, comprise great majority (Kelly et al. (1990) the J.Fish Dis.13:317-321 in the predominantly bacteria pathogenic agent of catfish; Thune (1993) FishMedicine, and ed.Stoskopf (Saunders Co., Philadelphia), pp.511-520).These peptide classes are avirulent (Jaynes et al. (1989) Pept.Res.2:157-160) to eukaryotic cell.Attacin has been found extensive existence, and its source comprises insect and Mammals, for example pig (people (1989) Proc.Natl.Acad.Sci.USA86:9159-9162 such as Lee).At last, these peptide classes have had very detailed description in the literature, the physicochemical property and the binding mode (Christensen et al. (1988) Proc.Natl.Acad.Sci.USA 85:5072-5076) that comprise them, therefore do not need to do experiment, perhaps the requirement to test decreases.
Experimental evidence can prove the using value of attacin in the aquatic products system.The passive attacin derivative of using can protect fish to avoid the infection (Kellyet al. (1993) J.App.Aqculture.3:25-34) of catfish tarda; this bacterium is main pathogens (Thune (1993) the Fish Medicine that cultures catfish; Stoskopf compiles (Saunders Co.; Philadelphia), pp.511-520).
Compare with the immunization method of routine, cultivating the catfish of expressing antibacterial peptide transgenosis (for example attacin) has a lot of potential advantages.At first, fish reach maturity far away and just the etap was expressed attacin in early days before immunization produced reaction in immunity system.Secondly, the attacin transgenosis can be given the immunizing power that fish is resisted multiple pathogenic agent, thereby need not to prepare multiple pathogen specific inoculation preparation and carry out various processing.Therefore, the attacin genetically engineered fish can avoid two main commerce losses, the one, disease is to the destruction of kind of fish, another be need to continue health kind fish is carried out prophylactic treatment or immunity.
Some genetically engineered fishes have become United States Patent (USP).For example United States Patent (USP) 6,207, and 817 relate to the dna sequence dna and the reorganization IGF-II promotor of plain like growth factor II (IGF-II) promoter region of fish pancreas, and IGF-II promoter region and the expression of reorganization IGF-II promotor in eukaryotic cell and fish embryo.
United States Patent (USP) 6015713 relates to the genetically engineered fish and the application of described transgenosis Regular Insulin in treating diabetes of expressing human Regular Insulin.It is worthy of note that although fish is carried the humanization insulin transgenic, and unmodified drives the fish Regular Insulin adjusting sequence of this transgene expression.
United States Patent (USP) 5545808 has been described the transgenosis salmon of expressing the external source salmon growth hormone.The claimed a kind of method that improves salmon growth speed of this patent; comprise the steps: 1) in the kind of salmon system, import the gene of the coding salmon growth hormone that is connected with 3 type antifreeze protein promotor operability, 2) in the level of salmon expression growth hormone gene its growth velocity is compared under the condition of 4 times of raisings at least to cultivate salmon with non-transgenic fish control group.
According to having separated sockeye 1 type and 2 type growth hormone genes and sockeye histone and metallothionein gene promotor described in the United States Patent (USP) 5998697, and check order.The end sequence of growth hormone gene also comes forth.The carrier that comprises these promotors and end sequence (and intermediate sequence) has been used to transform the fish ovum, and the fish ovum that will transform is then cultivated and is genetically engineered fish.
United States Patent (USP) 5719055 has been described a kind of carrier based on transposon, and it can promote that DNA is incorporated into host genome, especially in the eukaryotic gene group.This carrier has been used to the non-constructive expression's of coding cecropin B transgenosis is transformed in Mammals and the fish cell.
United States Patent (USP) 6,156 discloses transformed animal, the transformed animal cell that can express external source bacteriolyze peptide in 568, by to the gene of eucaryote cell of the exogenous promoter control of the responding property of inductor of acute phase protein with based on the conversion carrier of transposon.The claimed particularly gene in vitro eukaryotic cell that is under the control of wild-type cecropin B promotor that contains of this patent, wherein said promotor is an external source with respect to this cell.The cell of being put down in writing is vertebrates and mammalian cell.
United States Patent (USP) 5998698 claimed transgenosis catfish with the cecropin B encoding gene that is connected with natural cecropin B promotor operability; wherein the cecropin B promotor can be brought into play function and instruct the cecropin B expression of gene, and the resistibility to pathogenetic bacteria has been given in the expression of cecropin B.This patent is claimed transgenosis koi and bony fish (bony fish) with cecropin B encoding gene further.
United States Patent (USP) 6156568 discloses a kind of genetically engineered fish with the cecropin B encoding gene that is connected with natural cecropin B promotor operability.This genetically engineered fish can avoid the infection of catfish tarda.
Therefore, attempted to express coding and killed the problem that the proteic genetically modified fish of disease-resistant originality of pathogenic agent is solved disease infection by raising.But this scheme has caused another problem.Genetically modified expression is subjected to the control of viral promotors usually, but the human consumer thinks that probably the fish that carries virus " fragment " is unhealthy or unsafe, therefore, even this food can be by the approval of FDA, but the human consumer still unlikely can buy.Therefore, breed fish and main be in or be forced to bear the loss that high fish disease incidence causes or select to culture genetically engineered fish but because human consumer's prejudice and can't selling in the dilemma of disease-resistant fish.
Therefore, needing production has improved disease resistance and has met FDA and the method for the genetically engineered fish that the human consumer requires.
Summary of the invention
The invention provides the composition and the method that can be used for giving the enhanced disease resistance to fish.Comprise new recombinant precursor in the composition, this construct can inducing anti-disease originality polypeptide, comprises attacin albumen or the transgene expression of its biologic activity variant in fish.The expression of attacin albumen or its biologic activity variant can be given pathogenic agent, for example the disease resistance of virus, parasite and bacterium provides caused the resistibility of disease by catfish tarda, column Flavobacterium (Flavobacterium columnare), Pseudomonas fluorescens (Psendomonasfluorescens) and Vibrio anguillarum (Vibrio anguillarum).Composition of the present invention also comprises genetically engineered fish cell, fish-egg and fish, the especially catfish with stable integration recombinant precursor of the present invention in its genome.
In some embodiments, described novel recombinant precursor is to comprise the novel synthetic promoter of the present invention and the expression cassette of the nucleotide sequence of the coding desired polypeptides that connects of operability, especially interested disease-resistant originality polypeptide with it.The example of interested disease-resistant originality polypeptide comprises ripe attacin albumen and preceding giant silkworm antimicrobial peptide protein is former, attacin proteinogen, preceding giant silkworm antimicrobial peptide protein form, and these proteinic biologic activity variants.The described recombinant precursor leader sequence that operability connects of can encoding is convenient to the coded attacin albumen or the translation post-treatment of its variant.In the embodiment of accommodation, recombinant precursor of the present invention is to realize the expression cassette that the enhancing of attacin albumen or its biologic activity variant is expressed by the fish promotor.Such fish promotor comprises but not only is confined to the myostatin promotor, the alpha Actinin promotor, β actin promoter (for example FV-1 and FV-2 promotor), the creatine kinase promotor, I type Keratin sulfate promotor, II type Keratin sulfate promotor and metallothionein promoter, and their functional variant.Attacin albumen is well-known in the art, and in fact can derive from any species, comprises the attacin (moth cecropin B) that for example derives from moth, and the attacin that is separated to from pig (pig attacin P1).
Recombinant precursor of the present invention can be used in and drives polypeptide of interest at fish, the intravital non-tissue specific expression of for example catfish.When described polypeptide of interest was disease-resistant originality polypeptide, for example attacin albumen or its biologic activity variant, this constructive expression's pattern provide disease resistance can for full fish.Comprising the fish promotor exclusively also can make the disease resistant transgenic fish approve at commercial easier quilt with the recombinant precursor that drives the expression of attacin albumen or its biologic activity variant.
The present invention also provides the method for expressing target polypeptides in host cell, for example fry cell.This method comprises expression cassette is imported in the target host cell that described expression cassette comprises the novel synthetic promoter of the present invention that is connected with the nucleotide sequence operability of coding target polypeptides.The present invention also provides the method that strengthens the catfish disease resistance, comprise to comprise the synthetic promoter of the present invention that is connected with the nucleotide sequence operability of coding attacin albumen or its biologic activity variant or the expression cassette of fish promotor imports in the catfish ovum, and be suitable for cultivating fish-egg under the condition that the catfish egg development becomes catfish.Like this can be to the different plant species of catfish, comprise spot fork-tail brightness show repentance Hui Bian Channel-catfish (channelcatfish), blue Cha Wei Channel-catfish (blue catfish), spot fork-tail Huang Channel-catfish-blue Cha Wei Channel-catfish hybridization catfish provides disease resistance.The invention also discloses breed and pathogenic agent is had the method for the transgenosis catfish of enhanced disease resistance.
The accompanying drawing summary
Fig. 1 has described and has made up the general approach that the carrier of attacin is expressed in the control be in promotor (for example cmv enhancer, carp β actin promoter, the perhaps synthetic promoter of announcing) herein down.
Fig. 2 has described the associated nucleotide encoding sequence that the cecropin B expression vector that is used for production disease resistance enhanced genetically engineered fish comprises.The corresponding aminoacid sequence of encoded polypeptide be listed in each encoding sequence below.What use is single-letter amino acid symbol.The Working position that oblique line is represented the known of expressed peptide or estimated.The potential cleavage site usefulness of signal peptidase " " expression, the potential cleavage site of dipeptidyl peptidase is represented with "/".Encoding sequence and the aminoacid sequence of the former B of attacin (preprocecropinB) before SEQ ID NO:12 and SEQ ID NO:11 represent respectively.SEQ ID NO:21 and SEQ ID NO:22 represent encoding sequence and the aminoacid sequence of the former B of catfish Ig Vh leader sequence/attacin (procecropin B) respectively.SEQ IDNO:23 and SEQ ID NO:24 have shown the encoding sequence and the aminoacid sequence of catfish Ig Vh leader sequence/cecropin B respectively.
Fig. 3 has described the correlative coding nucleotide sequence (SEQ ID NO:27) that the attacin P1 expression vector that is used for production disease resistance enhanced genetically engineered fish comprises.The corresponding aminoacid sequence of coded polypeptide is listed in (SEQ ID NO:28) below the encoding sequence, with single-letter amino acid representation.The aminoacid sequence that runic shows is the expressed expectation mature amino acid sequence of peptide after being processed by leader peptidase.
Fig. 4 has described the preceding former B of attacin of coding and has driven the nucleotide sequence (SEQ ID NO:13) of the construct of expressing and the encoding amino acid sequence of estimating (SEQ ID NO:11) by the synthetic promoter sequence shown in the SEQ ID NO:3.
Fig. 5 has described the nucleotide sequence (SEQ ID NO:14) of the construct of the preceding former cecropin B of coding under the synthetic promoter sequence control that is in shown in the SEQ ID NO:8 and the encoding amino acid sequence of estimating (SEQ ID NO:11).
Fig. 6 has described the nucleotide sequence (SEQ ID NO:35) and the amino acid sequence coded (SEQ ID NO:11) of the construct of the preceding former cecropin B of coding that is under carp β actin promoter (the SEQ ID NO:29) control.
Detailed Description Of The Invention
Hereinafter with reference to the accompanying drawings the present invention is done more complete description, wherein provided more of the present invention but be not whole embodiments. In fact, the present invention may have a lot of concrete forms, only is confined to the embodiment mentioned herein and should not be construed as. Provide these embodiments just can satisfy the legal requiremnt of patent application for the content that exposes.
This invention relates to the method and composition of not using viral nucleotide sequences in transgenosis and the transgenosis disease resistance being provided to fish. Therefore, the present invention has enhancing fish, especially catfish to the resistance of disease, nor can cause the advantage of consumer's potential conflict. Adopted natural " full fish " promoter in the method for the present invention, " full fish " promoter element of perhaps in synthetic promoter, arranging. These two kinds of promoters can drive the expression of disease-resistant gene in fish, for example catfish. And these promoters can start interested transgenosis, for example composition, the non-tissue-specific expression of disease-resistant originality gene, and do not need viral nucleotide sequences. These promoter characteristics are of great use, because fish at the disease-resistant originality albumen of whole expression in vivo, breed Anywhere thereby pathogen can not enter in the fish tissue or therein easily. Advantageously, this protective effect need not inductivity stimulates and can provide.
Synthetic " full fish " disclosed in this invention promoter also has other advantage except the disease resistance that can be used for strengthening fish. In the time of in being incorporated into expression cassette, the nucleotides sequence interested that these novel synthetic promoters can replace the native species promoter to be used for the connection of driving operability is listed in the expression of fish. Therefore, these Novel promoters can be used in the almost expression of any nucleotide sequence interested of driving, and the render transgenic fish more can be accepted by the consumer simultaneously, thereby has commercial value. In addition, these synthetic promoters kept composition, namely with the advantageous feature of non-tissue-specific mode express transgenic.
" promoter " refers to the dna sequence dna of can promotor gene transcribing. Usually promoter is the DNA control region that comprises the TATA box, can the guide RNA polymerase II synthetic at the initial RNA of suitable transcription initiation site of specific coding sequence. In general, promoter is positioned at 5 ' district of gene, near the transcription initiation site of coded sequence. Promoter can also comprise other promoter and the enhancer recognition sequence that generally is positioned at TATA box 5 ' end or upstream, is called as upstream promoter and enhancer element, and they can affect transcription initiation speed.
Synthetic promoter is the nucleotide sequence of manually making, it is not natural generation, but artificial design, must be directed in the organism or among the organic first ancestor so that control the expression of the nucleotide sequence that operability with it connects." can be operatively connected " and be meant that promotor is connected with the functional of another sequence, wherein this promoter sequence can initial sum mediation transcribing corresponding to the dna sequence dna of this second sequence.In general, can be operatively connected the nucleotide sequence that finger connects and adjoin mutually, and when needs connect two protein coding regions, then refer to them and adjoin mutually and be in the identical reading frame.
Synthetic promoter of the present invention comprises core promoter as described below at least.In addition, described promotor also can comprise at least one upstream element.These elements comprise upstream active region (UAR), and comprise alternatively and can influence other dna sequence dna that structure gene is transcribed, for example synthetic property upstream element or other enhancer element.The upstream active region is position or direction dependent form element normally, mainly instructs tissue, cell type or modulated expression.Enhanser is to improve the DNA controlling element of transcribing efficient with respect to the distance of transcription initiation site or orientation independent ground with enhanser.
" core promoter " or " minimal promoter " contains the required basic nucleotide sequence of encoding sequence of expressing the operability connection, comprises TATA box and transcription initiation site.According to this definition, core promoter can improve activity or give under the situation of the active distinguished sequence of tissue specificity and have or do not have detectable activity lacking.Synthetic promoter among the present invention comprises the goldfish minimal promoter shown in the SEQ ID NO:1 (Wilson et al., Mol.Immuno l.28:449) as its core promoter district, and the function of promotor is provided.The core promoter district can be used in combination with enhanser, upstream element and/or from the activation sequence of expressible gene 5 ' flanking region.
In one embodiment of the invention, described synthetic promoter comprises and has contained in 5 ' end coupling the goldfish minimal promoter of the TATA box primitive of upstream element (SEQ ID NO:1).This upstream element comprises at least three fish Sp1 in conjunction with primitive (CGGGGCGGGG; SEQ ID NO:2; Consult for example Baudler etal.1997.J.Biol.Chem.272:131-137) and novel interleaving property catenation sequence.In such embodiment, described synthetic promoter comprises the sequence shown in the SEQID NO:3.When this synthetic promoter sequence was connected with purpose nucleotide sequence, for example protein coding sequence operability, it can drive coded product the target host cell constructive expression in the catfish cell for example." composition " is meant in whole host, for example in the fish, expresses in most of the time and great majority tissue.The enhanced disease resistance is provided to fish, for example by expressing disease-resistant originality albumen (for example attacin polypeptide) when realizing this purpose, it is very useful that non-tissue-specific height is expressed.These albumen are harmless to the g and D of fish, and restricted tissue expression can make pathogenic agent that some other invasion approach is arranged.For example, driving a transcriptional control element that disease-resistant originality albumen only expresses in skin can not protect the host to avoid being subjected to by stomach and intestine or stab the infection that the position enters intravital bacterium.
In another embodiment, the invention provides to comprise and contain and the upstream element synthetic promoter of the goldfish minimal promoter of link coupled TATA box primitive (SEQ ID NO:1) mutually, this upstream element comprises with the following elements of 5 ' to 3 ' direction assembling and the joint sequence of interleaving property: at least one fish Sp1 is in conjunction with primitive (CGGGGCGGGG; SEQ ID NO:2) may be operably coupled to few fish C/EBP α primitive (ATAATGTTTCATCACACTT; SEQ ID NO:4; Referring to for example Chan et al. (1997) Eur.J.Bi ochem.247:44-51) may be operably coupled to and lack a fish Oct primitive (ATGTAAAT; SEQ ID NO:5; Referring to for example Magoret al. (1997) Immunogenetics 46:192-198) may be operably coupled to and lack a fish NF-κ B primitive (GGGACGTCCC; SEQ ID NO:6) may be operably coupled to few fish AP-1 in conjunction with primitive (ATGACTCAG; SEQ ID NO:7).In such embodiment, this synthetic promoter comprises the sequence shown in the SEQ ID NO:8.Described synthetic promoter also can be used for strengthening nucleotides sequence that operability connects and is listed in transcriptional level in host organisms cell, for example fish cell.
Upstream element described herein can be connected with goldfish minimal promoter and/or other upstream elements (comprising UARs) by any ordinary method well-known in the art, as long as constructed effective element or promotor.These upstream elements generally are connected with 5 ' terminal operability of goldfish minimal promoter.In preferred embodiments, described upstream element connects to be attached thereto near the mode of goldfish minimal promoter very much.Here " very close " refers in about 1～50 Nucleotide scope.Yet, should admit, one upstream element and goldfish minimal promoter may be separated greater than the distance of 50 Nucleotide.
The upstream element of one or more copies can be used with the goldfish minimal promoter.When using multiple copied, they can be the series connection repetitions of a primitive, also can be the combinations of several primitives.Like this, can control the expression level of the nucleotide sequence interested of operability connection by the primitive number that exists in the promoter construct.Therefore, can strengthen the activity of the goldfish minimal promoter of operability connection with multiple copied in conjunction with primitive.
The biologic activity variant of synthetic promoter sequence disclosed herein also can be used in the expression cassette described herein.Such variant sequence will have the subtle change that can not destroy the promoter activity of this synthetic promoter as described below.Preferably, described variation occurs in corresponding to outside the zone of core goldfish minimal promoter and outside the zone corresponding to the particular upstream element that exists in the synthetic promoter of the present invention, for example, this variation occurs in the interleaving property sequence that core promoter and particular upstream promoter element (be Sp1 in conjunction with primitive, C/EBP α primitive, Oct primitive, NF-κ B primitive and AP-1 in conjunction with primitive) are linked together.But, we recognize, as long as the variant promoter sequence is functional promotor, then this small variation also can occur in the core promoter, in one or more upstream element (be Sp1 in conjunction with primitive, C/EBP α primitive, Oct primitive, NF-κ B primitive and AP-1 in conjunction with in the primitive), perhaps not only in core promoter but also in one or more upstream elements.
" functional promotor " means this promotor can be initial or strengthen and transcribe.Whether those of ordinary skills are fully aware of, can be listed in and transcribe under the situation that this promotor exists and easily determine the functional of promotor by measuring nucleotides sequence that operability connects.Determine to transcribe and translate the method that whether takes place and be widely known by the people in this area, these methods comprise mRNA or the proteinic output that generates when detection is under the encoding sequence with target protein places promotor control.Can not inducible transcription or the certain right and wrong of promoter sequence of translation functional.Useful part of the present invention has been wherein to describe and can have driven transcribing and the functional synthetic promoter of coded proteic translation of encoding sequence that operability connects.Those skilled in the art can recognize that the functional variant of synthetic promoter disclosed by the invention also can be used for driving the expression of operability purpose of connecting nucleotide sequence.Here " expression " speech means the biosynthesizing of coded product.Expression comprises that encoding sequence is transcribed into mRNA and mRNA further is translated as one or more functional proteins or polypeptide." functional protein " or " functional polypeptide " refers to that described protein or polypeptide bring into play its predetermined purposes.For example, functional disease-resistant originality albumen or disease-resistant originality polypeptide will kill or suppress the growth of pathogenic agent.
Promoter activity can be by following technology for detection: for example the Northern blot hybridization, transcribe active detection of report of syzygy, like that.For example consult Sambrooke tal (1989) Molecular Cloning:A Laboratory Manual (2 NdEd., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York), this document is incorporated herein by reference.Perhaps, promoter fragment or variant be can be determined at and reporter gene such as green fluorescent protein (GFP), luciferase, the beta-galactosidase enzymes (LacZ) that gives generation under the sequence control, the expression level of E.C. 2.3.1.28 (CAT) etc. started.Referring to for example, Astola et al (2003) Gen.Comp.Endocrinol.134:57-61; Hwanget al (2003) Biochem.Biophys.Acta 1625:11-18; Kim et al. (2000) Gene 252:173-181; Volckaert et al. (1994) Mol.Mar.Biol.Biotechnol.3:57-69; These documents are incorporated herein by reference.
Therefore, the biologic activity variant of synthetic promoter disclosed by the invention is estimated also to can be used in the compositions and methods of the invention.Such biologic activity variant will have functional promoter activity, and with as about at least 70% sequence identity being arranged with reference to the synthetic promoter (being the synthetic promoter of SEQ ID NO:3 or 8) of molecule, general about at least 75%, 80%, 85% sequence identity, preferred about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% sequence identity, more preferably about at least 98%, 99% or higher sequence identity.The variant promotor needn't have with reference to the identical promoter activity of synthetic promoter.As long as it is just enough that the variant promotor has function, in other words, the variant promotor has the ability of the nucleotide sequence expression that drives the operability connection.
Aligned sequences is widely known by the people in this area with the method that compares.The sequence alignment best approach can be undertaken by using following algorithm: the algorithm of Mye r s and Miller (1988) (CABIOS 4:11-17); Local homology's algorithm of Smith et al. (1981) Adv.Appl.Math.2:482; The homology alignment algorithm of Needleman and Wunsch (1970) J.Mol.Biol.48:443-453; The similarity searching algorithm of Pearson and Lipman (1988) Proc.Natl.Acad.Sci.85:2444-2448; The algorithm of Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 87:2264 has been done improvement in Karlin andAltschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5877.Can carry out these mathematical algorithms by computer and come comparative sequences, to determine their sequence identity.These steering routines including, but not limited to: the CLUSTAL in the PC/Gene program (can be available from Intelligenetics, Mountain View, California); ALIGN program (second edition) and GAP, BESTFIT, BLAST, FASTA and TFASTA (GCG Wisconsin genetics software package, the tenth edition, can be available from Accelrys Inc.9685ScrantonRoad, San Diego, California, USA).Can use default parameters to carry out the comparison of these programs.Comparison usually also can be by checking and manually comparing and finish.
For the object of the invention, use GAP (version 10 or upgrade version) and default parameters to determine the sequence identity per-cent of any two nucleotide sequences (for example between the synthetic promoter of SEQ ID NO:3 or SEQ ID NO:8 and the promoter sequence) as the functional variant of arbitrary sequence in them.GAP has used Needleman and Wunsch (1970), and the algorithm of J.Mol.Biol.48:443-453 to determine the comparison between two complete sequence, makes the coupling between them count maximum, and the breach number is minimum.GAP will consider the position of all possible comparison and breach, and sets up the comparison with the minimum breach of maximum coupling alkali cardinal sums.In Wisconsin GeneticsPackage version 10, the default gap of protein sequence is produced point penalty value and breach, and to extend the point penalty value be respectively 8 and 2.For nucleotide sequence, the breach of acquiescence produces the point penalty value and breach extension point penalty value then is respectively 50 and 3.
For the constructive expression of operability purpose of connecting nucleotide sequence is provided, synthetic promoter of the present invention can be structured in the expression cassette.Expression cassette can place plasmid or virus vector, so that transformed host cell, such as fish cell.In preferred embodiments, disclosed synthetic promoter sequence can be used to realize non-tissue specificity, the constructive expression of the product that target nucleotide sequence that operability connects is coded among the present invention.In such embodiment, the synthetic promoter shown in SEQ ID NO:3 or the SEQ ID NO:8 can be inserted in the expression cassette, realize the high level expression of any target protein.
When synthetic promoter sequence of the present invention is assemblied in DNA construct, is in the expression cassette, when promotor was connected with target nucleotide sequence operability, the nucleotides sequence that promptly can drive this operability connection was listed in by the expression in the fish cell of this DNA construct stable conversion.This target nucleotide sequence can with stand-by this construct host transformed fish homology (natural or allos (be external or non-natural exists)).
Expression cassette comprises (by 5 ' to the 3 ' direction of transcribing): synthetic promoter of the present invention (SEQ ID NO:3 for example, SEQ IDNO:8 or its have the variant of promoter activity), the target nucleotide sequence has transcribing and translation termination zone (being the terminator) of function in fish.Generally believe,, then it is worked in the expression cassette, make this encoding sequence comprise initiator codon if described nucleotide sequence is an encoding sequence.If expression product is a fusion rotein, (the immunoglobulin variable heavy chain of catfish immunoglobulin heavy chain variable region hereinafter described for example, IgVh) during leader sequence/cecropin B polypeptide, the sequence that is about to encoding said fusion protein is worked in the expression cassette, makes it to comprise the preceding initiator codon of encoding sequence that is in catfish immunoglobulin heavy chain variable region (Ig Vh) leader sequence N-terminal.
The terminator can be natural for this target nucleotide sequence, perhaps can originate from other.Expression cassette can also comprise 3 ' enhanser and/or the polyadenylation signal sequence that is in the terminator codon downstream.For example, in one embodiment, expression cassette comprises Trobest 3 ' the non-translational region sequence that is in the terminator codon downstream, to strengthen protein expression level.In such embodiment, expression cassette comprises the bovine growth hormone gene 3 ' non-translational region that contains nucleotide sequence shown in the SEQ ID NO:9 that operability connects, and wherein comprises the polyadenylation signal sequence that contains AATAAA primitive (the Nucleotide 145-150 among the SEQ ID NO:9).Referring to for example: U.S.Patent No.5,122,458; Higgs et al. (1983) Nature306:398-400); Woychik et al. (1984) Proc.Natl.Acad.Sci.USA81:3944-3948; These documents are incorporated herein by reference.
A series of restriction site is provided in such expression cassette,, makes it to be in control region, be under the transcriptional control of disclosed synthetic promoter sequence among the present invention to insert the target nucleotide sequence.Can also contain selectable marker gene in the expression cassette, be convenient to screen the cell and/or the organism that contain expression cassette, preferably, such expression cassette stably is incorporated in the genome of described cell or organism.
The target nucleotide sequence can comprise any sequence of coded protein, polypeptide or peptide, and described protein, polypeptide or peptide give useful characteristic can for the fish cell or the host fish of expressing this target code sequence.For the purposes of the present invention, term " polypeptide ", " peptide " and " protein " can exchange use, refer to the polymkeric substance of amino-acid residue.These terms are applicable to that wherein one or more amino-acid residues are aminoacid polymerss of the artificial chemical analog of corresponding natural amino acid, and the polymkeric substance of natural amino acid.The sequence of coding target protein can be used separately, perhaps uses with the combined sequence of giving useful property to fish cell or host fish with coding other albumen or medicament.These useful characteristics comprise, still are not limited to the promotion growth, and improvement local flavor, color and quality are cold-resistant, disease-resistant and sterile.For example, target nucleotide sequence codified fish growth hormone, fish growth hormone releasing hormone, winter flounder (winter flounder) antifreeze protein, disease-resistant originality albumen (comprising antibody and attacin), perhaps coding can change the proteinic sequence of fish sex or ploidy.
Perhaps, the target nucleotide sequence that is connected with one of disclosed synthetic promoter operability among the present invention can be by the antisense sequences of target gene." antisense DNA nucleotide sequence " refers to be in normal 5 ' to 3 ' the side sequence in the opposite direction with this nucleotide sequence.After they were sent fish cell, the expression of antisense dna sequence can stop by the normal expression of the dna nucleotide sequence of target gene.The rna transcription thing of antisense base sequences coding with transcribed endogenous messenger RNA(mRNA) (mRNA) complementation that is produced by the dna nucleotide sequence of target gene, and can hybridize.In this case, be suppressed the phenotypic response that obtains expecting by the generation of the coded native protein of target gene.Like this, disclosed synthetic promoter sequence and antisense dna sequence among the present invention can be able to be operatively connected, to weaken or to suppress the expression of protein in the fish (for example gonadotropin releasing hormone (GnRH), myostatin and viral protein).
In one embodiment, expression cassette uses synthetic promoter of the present invention, driving disease-resistant originality polypeptide expression, thereby provides disease resistance to fish.Known in this area have many disease-resistant originality polypeptide, and they comprise attacin, magainin (magainins), defensin (defensin) and sarcophagid toxin (sarcotoxin).Because these polypeptide all are amphipathic, they can destroy the cytolemma or the cell walls of pathogenic agent, thereby kill or suppress the growth of pathogenic agent.Disease-resistant originality polypeptide can also play a role indirectly by the cytolemma of break virus cells infected.Disease-resistant originality polypeptide does not hereinafter described have toxicity or toxicity very little to the fish cell of expressing them or host fish with such cell.
Disease-resistant originality polypeptide is particularly useful for providing disease resistance, can not damage or postpone the growth of transgenosis fish simultaneously.Those skilled in the art are known to have many proteinoids that disease resistance can be provided, and has described so concrete albumen herein by way of example, but has been not limited thereto.Those skilled in the art also can recognize, can identify disease-resistant originality polypeptide by the disease-resistant originality activity test method shown in hereinafter.
" disease resistance " refers to that disease-resistant originality polypeptide expression has been avoided because the disease symptoms that fish-pathogenic agent interacts and causes.In other words, pathogenic agent can not cause fish disease and produce relevant disease symptoms.If expression cassette of the present invention comprises the synthetic promoter of the present invention that the encoding sequence with disease-resistant originality polypeptide can be operatively connected; can protect resulting genetically engineered fish to avoid disease after importing this expression cassette in the fish, especially those diseases that cause by fish pathogen." disease-resistant originality " polypeptide refer to those therefore have disease-resistant former activity, can suppress, control also/or kill the protein of invading the primary object of venereal disease.With respect to having similar genetic constitution but can not expressing in the wild-type fish of disease-resistant originality polypeptide of comparable level for the observed disease symptoms, disease-resistant originality polypeptide can stimulate the disease symptoms that causes to be reduced by at least about 5%-50% by pathogenic agent, at least about 10%-60%, at least about 30%-70%, at least about 40%-80%, perhaps about at least 50%-90% is even higher.Therefore, method of the present invention can be used for protecting fish to exempt from disease, especially the disease that is caused by fish pathogen.
The method that detects active method of disease-resistant originality and the disease resistance of quantification fish behind pathogenic infection all is widely known by the people in the art.Consult for example U.S. Patent number 5998698, be incorporated herein by reference in its entirety.These technology include, but are not limited to detect the mortality ratio that the fish of inoculation pathogenic agent are taken place in time, and detect when having this disease-resistant originality polypeptide to exist the restraining effect situation in time to pathogenic growth.For example, can express disease-resistant originality polypeptide owing to hereditary change or express the fish of higher levels of disease-resistant originality polypeptide with pathogenic agent inoculation, and mortality ratio was mapped to the time.The mortality ratio of these results and control group can be compared.The fish of described control group promptly be have identical genetic background, but do not carry out hereditary change and the wild-type fish of expressing described disease-resistant originality polypeptide or expressing the inoculation of this disease-resistant originality polypeptide with higher level.Absolute mortality ratio or the relative reduction of comparing with control group lethal mean time have illustrated that this disease-resistant originality polypeptide can give the resistance to this pathogenic agent.Perhaps, can disease-resistant originality polypeptide arranged or external cultivation pathogenic agent under the situation that the cell of expressing this disease-resistant originality polypeptide exists arranged.The viability of pathogen culture thing is compared with undressed control group and is shown that reduction has then shown the antipathogen effect of this disease-resistant originality polypeptide.
Although following embodiment at the synthetic promoter of the present invention that is to use drive the specific disease-resistant originality polypeptide expression of a class, thereby in fish, strengthen disease resistance, will be appreciated that, synthetic promoter of the present invention also can be used to drive the expression of (comprising defensin, magainin and sarcophagid toxin) of the disease-resistant originality polypeptide of any purpose, to realize strengthening the purpose of disease resistance in the fish.About the summary of disease-resistant originality polypeptide, referring to for example Boman (2003) J.Intern.Med.254:197-215; Zhang et al. (2000) Vet.Res.31:277-296, Moore et al. (1996) Antimicrob.Chemother.37:1077-1089; Merrifield et al. (1994) Ciba Found.Symp 186:5-20, and the discussion among the 20-26; And U.S. Patent number: 5166321; At this they are incorporated herein by reference in full.
Therefore, in some embodiments, synthetic promoter of the present invention be used to drive hereinafter described the attacin polypeptide or the expression of its biologic activity variant.Attacin is the amphipathic little peptide that 30 to 40 amino-acid residues are formed, and has broad-spectrum disease resistance originality activity and (for example consults Andra et al. (2001) Med.Microbiol.Immunol (Berl.) 189 (3): 169-173; Baoman (2003) J.I ntern.Med.254 (3): 197-215; Moore etal. (1996) Antimicrob.Chemother.37:1077-1089).Sophisticated attacin polypeptide experiences two stage process in natural insect cell.Attacin is positioned in the endoplasmic reticulum as preceding former attacin (being the preceding former form of described polypeptide) at first, the excision leading peptide produces former attacin (being the former form of described polypeptide) afterwards, remove four n terminal residues by dipeptidyl peptidase, produce ripe attacin polypeptide.Multiple ripe attacin known in the state of the art, their preceding former and former form, and their corresponding encoded sequence comprise, still are not limited to attacin A (Qu et al. (1982) Euro.J.Biochem.I27:219-224; Lidholm et al. (1987) FEBS Lett.226:8-12; From the GenBank typing P01507 of Hyalophora cecropia, X06672 is coded by the GenBank typing; From the GenBank typing BAA04217 of Bombyx mori, D17394 is coded by the GenBank typing) and attacin-melittin T1249 (Boman et al. (1989) FEBS lett.259 (1): 103-106); Cecropin B (Qu et al. (1982) Euro.J.Biochem.127:2l9-224; Van Hofsten eta l. (1985) Proc.Natl.Acad. Sci.USA 82 (8): 2240-2243; Taniai etal (1992) Biochim.Biophys.Acta 1132 (2): 203-206; Kato et al. (1993) Insect.Biochem.Mol.Biol.23 (2): 285-290); Taniai et al. (1995) Gene 163 (2): 215-219; Yamono et al. (1994) Biosci.Biotechnol.Biochem.58:l476-1478; From the GenBank typing P01508 of Hyalophora cecropia, M10309 is coded by the GenBank typing; From the Genbank typing P04142 of Bombyxmori, D11113 is coded by the GenBank typing; GenBank typing P01509 from Antheraeapernyi) and analogue cecropin B 1 and B3 (Wang et al. (1998) J.Biol.Chem.273 (42): 27438-27448), attacin D (Qu et al.Euro.J.Biochem.l27:219-224; Hultmark et al. (1982) Euro.JBiochem. (127:207-217; Lidholm et al. (1987) FEBS lett.226:8-12 is from the GenBank typing P01510 of Hyalophoracecropia, X06673 is coded by the GenBank typing), attacin P1 (Lee et al. (1989) Proc.Natl.Acad.SCi.USA86 (23): 9159-9162); And attacin polypeptide (U.S. Patent number 5166321) with various C-terminal modifications.All mentioned documents all are incorporated herein by reference in full.
These attacins have the common structure, promptly comprise charged N-terminal district (residue 1-21) and one section quite conservative long water repellent region (residue 22-32) (referring to for example U.S. Patent number 5166321) subsequently.These α helical molecules by penetrate microbial pathogen for example the film of bacterium etc. show them activity (referring to for example Steiner et al (1981) Nature 292:246-248; Moore et al (1996) J.Antimicrob.Chemother.37 (6): 1077-1089; Zhang et al. (2000) Vet.Res.31 (3): 277-296; Chen et al. (2003) Eur.J.Biochem.270 (5): 911-920).
According to the compositions and methods of the invention, the nucleotide sequence of interested attacin of encoding can be mounted to DNA construct, be expression cassette, thereby the encoding sequence of this attacin polypeptide and synthetic promoter operability of the present invention couple together, and drive the expression of attacin encoding sequence in fish cell.
In its genome stable integration the fish of this construct with the described interested attacin polypeptide of constructive expression, strengthen their disease resistances thus to many pathogenic agent.Such pathogenic agent comprises, still is not limited to fungi, bacterium, protozoon and virus.In one embodiment, the catfish of expressing attacin by mode described herein will demonstrate disease resistance after being exposed to pathogenic agent, described pathogenic agent comprises for example catfish tarda (Edwardsiellaictaluri), blunt tarda (Edwardsiella tardi), column Flavobacterium (Flavobacterium columnare), Pseudomonas fluorescens (Pseudomonasfluorescens), aeromonas salmonicida (Aeromonas salmonicida), Aeromonas hydrophila (Aeromonas hydrophila) and Vibrio anguillarum (Vibrio anguillarum) etc.Yet, being well known in the art, attacin has the former activity of broad-spectrum disease resistance, and the provide protection at some other disease can be provided, for example Saprolegnia fungi and the thin virus disease of spot fork-tail.
Any functional attacin polypeptide or its biologic activity variant can be applied to the present invention.Therefore, functional attacin polypeptide can be full ripe attacin polypeptide (promptly having removed signal peptide and leading peptide), the attacin polypeptide of precursor forms (being that signal peptide adds ripe attacin peptide sequence), the attacin polypeptide of substance form (being that leading peptide adds ripe attacin peptide sequence), perhaps the attacin polypeptide (being that signal peptide adds leading peptide and adds ripe attacin peptide sequence) of preceding substance form.Processing can be removed some or all precursor or substance aminoacid sequences in the cell, forms ripe attacin polypeptide, and can not cause disadvantageous effect to the function of this polypeptide.The attacin polypeptide can be the natural polypeptides that derives from any species, comprises isolating attacin from pig, moth and fish.In addition, described in hereinafter, expectation can replace the biologic activity variant of the natural attacin polypeptide in the box described herein, obtains identical result, and the disease resistance to many pathogenic agent promptly is provided in fish.
The biologic activity variant of the precursor of known ripe attacin polypeptide and attacin polypeptide, substance, preceding substance form all can be used for the compositions and methods of the invention.Suitable biologic activity variant can be fragment, analogue and the derivative of endogenous or natural attacin polypeptide." fragment " refers to the polypeptide of only being made up of the part of complete attacin peptide sequence.These fragments can be the attacin polypeptide of C-terminal disappearance or N-terminal disappearance." analogue " refers to full-length polypeptide (comprising preceding attacin, the former or ripe attacin polypeptide of former, the preceding attacin of attacin) or its segmental analogue of biologic activity, comprising native sequences and the structure with one or more amino acid replacements, insertion or disappearance)." analogue " also comprises the peptide (consulting International Publication No.WO 91/04282) with one or more plan peptides (peptide mimics)." derivative " refers to natural polypeptides or its segmental any suitable modified forms, perhaps their corresponding analogs, and such as glycation product, phosphorylation product or add other products of ektogenic is as long as kept active.
Preferably, natural or the non-natural variant of attacin polypeptide have with reference to attacin polypeptide (for example, endogenous or natural attacin A, cecropin B, D or P1) or should be with reference to the segmental aminoacid sequence of the weak point of attacin polypeptide at least about 70%, 75% and 80%, preferably at least about 85%, 90%, 91%, 92%, 93%, 94% or 95% identical aminoacid sequence.More preferably, this variant polypeptide and described identical with reference to attacin polypeptide at least 96%, 97%, 98% or at least 99%.
For the purposes of the present invention, the sequence homogeny per-cent of (for example, between endogenous or natural preceding former cecropin B and this variant polypeptides of sequence shown in the SEQ ID NO:11) can use GAP program (the 10th edition or renewal version) to determine by default parameter between any two peptide sequences.As previously mentioned, GAP utilizes the algorithm among Needleman and Wuns ch (1970) J.Mol.Blol.48:443-453 to seek can make between two complete sequence maximization of coupling number and the minimized comparison of breach number.For protein sequence, default gap generation point penalty value and breach extension point penalty value are respectively 8 and 2 in the Wisconsin Genetics software package.For example, variant polypeptide and attacin have the difference of 1-10 amino-acid residue with reference to polypeptide, and be as 6-10, few to 5,4,3, even the difference of 21 amino-acid residues.
As for the comparison of the bests of two aminoacid sequences, the variant aminoacid sequence with compare its continuous section with reference to aminoacid sequence in can have unnecessary amino-acid residue or disappearance amino-acid residue.The sequence section that adjoins of comparing with the reference aminoacid sequence comprises 20 successive amino-acid residues at least, can be 30,40,50 or amino acids residue more.Also can carry out proofreading and correct (consulting the GAP program) with the sequence homogeny that conserved amino acid substitutes or breach is relevant.
As following further discussion, this area is relevant for the preparation of such variant and the substance instruction of use.What the fragment of attacin polypeptide generally comprised total length attacin polypeptide (can be ripe attacin sequence at least about 10 continuous amino acid residues, perhaps experience preceding former attacin, preceding attacin, the former attacin sequence of ripe attacin polypeptide translation post-treatment), perhaps, preferably include about 15-25 continuous amino acid residue of total length attacin polypeptide, most preferably comprise about 20-30 or more a plurality of continuous amino acid residues of total length attacin polypeptide.
For example, can carry out conserved amino acid at nonessential amino-acid residue one or more expectations, preferred place substitutes." nonessential " amino-acid residue refers to attacin (attacin A for example, B, D or P1) wild-type sequence in can change but residue that its biologic activity does not change, and " essential " amino acid is that its biologic activity is necessary." conservative amino acid replacement " is the type that amino-acid residue replaced that amino-acid residue is had similar side chain.Determined to have the amino-acid residue family of similar side chain in the prior art.These families comprise that the amino acid with basic side chain is (as Methionin, arginine and Histidine), amino acid (as aspartic acid and L-glutamic acid) with acid side-chain, amino acid (glycine for example with uncharged polar side chain, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), amino acid (L-Ala for example with non-polar sidechain, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met) and tryptophane), have the amino acid (Threonine for example of β-minute branching side chain, Xie Ansuan and Isoleucine) and have the amino acid (tyrosine for example of aromatic series side chain, phenylalanine, tryptophane and Histidine).Should not carry out such substituting for conservative amino-acid residue or the amino-acid residue that is positioned at conservative primitive.
Perhaps, variant attacin coding nucleotide sequence can prepare by introduce sudden change at random along all or part sequence of attacin encoding sequence, for example prepare by the saturation mutagenesis method, and to the disease-resistant originality biologic activity of the screening mutant attacin of gained, to identify the mutant of retentive activity.After mutagenesis, recombinant expressed coded protein is also by this proteinic disease-resistant originality activity of standard test technical measurement as herein described.
When making up expression cassette of the present invention, the target nucleotide sequence can be the natural encoding sequence of target attacin polypeptide, also can be the variant of natural encoding sequence.For example, when target attacin polypeptide was the former B of preceding attacin of SEQ ID NO:11, the natural or interior source coding sequence shown in the SEQ ID NO:10 can be used in the expression cassette.Select as another kind, the variant of this encoding sequence, sequence shown in SEQ ID NO:12 can be used in the expression cassette.The variant of target nucleotide sequence with the coding attacin with reference to nucleotide sequence, perhaps the shorter part of canonical sequence with the coding attacin has about at least 70%, 75%, 80%, preferred about at least identity of 85%, 90%, 91%, 92%, 93%, 94% or 95%, what encode attacin for example is the endogenous or natural encoding sequence of attacin A, cecropin B, attacin D or attacin P1 with reference to nucleotide sequence.More preferably, the variant nucleotide sequence has 96%, 97%, 98% or at least 99% identity at least with having with reference to nucleotide sequence of attacin of coding.
For the purposes of the present invention, we with GAP program (the 10th edition or upgrade version), by default parameter determine any two target nucleotide sequences (for example, shown in the SEQ ID NO:10 natural or endogenous before the variant of the former B encoding sequence of attacin and this encoding sequence) between sequence identity per-cent.For nucleotide sequence, the default gap in the Wisconsin Gernetics software package the 10th edition forms point penalty value and breach, and to extend the point penalty value be respectively 50 and 3.
Wishing the expression product guiding special cells device such as the plastosome of the nucleotide sequence that operability connects, perhaps secreting that expression cassette can further comprise the encoding sequence of transit peptides on the cell surface or be secreted under the extracellular situation.In these cases, from the leader sequence of heterologous protein, promptly signal peptide can be used to produce location attacin polypeptide to mosaic or the syzygy of specific cell device further to be processed into ripe attacin polypeptide.In some embodiments of this invention, in the fusion rotein of targeted molecular also specifically is encompassed in attacin and the cell.Like this, link to each other with the N-terminal of target attacin polypeptide from immunoglobulin heavy chain variable region (Ig Vh) leader sequence of channel catfish or the fragment of this leader sequence.The expression of encoded polypeptide and translation post-treatment subsequently cause producing sophisticated attacin polypeptide.The leader sequence of targeted cells device is well known in the art.Therefore, using channel catfish Ig Vh leader sequence or its fragment that attacin is targeted to endoplasmic reticulum is an example, rather than is confined to this.
In one embodiment, expression cassette (for example comprises synthetic promoter of the present invention, SEQ IDNO:3, SEQ ID NO:8 or its have the variant of promoter activity), this promotor operability is connected to nucleotide sequence (the cecropia moth of coding from the former B of preceding attacin of golden star Acer negundum Io moth (Hyalophorra cecrropia), Genbank AccessionNo.P01508, shown in the SEQ ID NO:11), for example operability is connected to the nucleotide sequence shown in the SEQ ID NO:10 (Genbank Accession No.M10309) or the variant of this encoding sequence shown in SEQ IDNO:12.In such embodiment, expression cassette comprises sequence listed among Fig. 4 (SEQ ID NO:13).Select as another kind, expression cassette comprises sequence listed among Fig. 5 (SEQ ID NO:14).
In another embodiment, expression cassette (for example comprises synthetic promoter of the present invention, SEQID NO:3, SEQ ID NO:8 or its have the variant of promoter activity), this promotor operability is connected to the nucleotide sequence (SEQ ID NO:16) of coding from the former B of attacin of Jin Xing Acer negundum Io moth (Hyalophora cecropia), and for example operability is connected to the variant of nucleotide sequence shown in the SEQ ID NO:15 or this encoding sequence shown in SEQ ID NO:17.Select as another kind, expression cassette (for example comprises synthetic promoter of the present invention, SEQ ID NO:3, SEQ ID NO:8 or its have the variant of promoter activity), this promotor operability is connected to the nucleotide sequence (SEQ ID NO:19) of coding from the ripe cecropin B of Jin Xing Acer negundum Io moth (Hyalophora cecropia), for example, operability is connected to the variant of sequence shown in the SEQ ID NO:18 or this encoding sequence shown in SEQ ID NO:20.
In selectable embodiment, the encoding sequence of the preceding former B of attacin, the former B of attacin, preceding cecropin B or ripe cecropin B can comprise the change of codon, thereby the biologic activity variant of the former B of attacin, the former B of attacin, preceding cecropin B or ripe cecropin B polypeptide before the expression cassette coding, these biologic activity variants meet function defined above and structure standard (be disease-resistant originality active and with the sequence identity of reference polypeptide at least 70%.)
In other embodiments, expression cassette (for example comprises synthetic promoter of the present invention, SEQID NO:3, SEQ ID NO:8 or its have the variant of promoter activity), this promotor operability is connected to coding and comprises nucleotide sequence with the polypeptide of catfish immunoglobulin heavy chain variable region (Ig Vh) leader sequence that as one man is connected from the former B of attacin of golden star Acer negundum Io moth (Hyalophoracecropia) or cecropin B reading frame.In such embodiment, the polypeptide (the former B of catfish IgVh leader sequence/attacin) shown in the expression cassette coding SEQ ID NO:22, and comprise the nucleotide sequence of this polypeptide of encoding, the nucleotide sequence shown in SEQ ID NO:21.In a selectable embodiment, the polypeptide (catfish Ig leader sequence/cecropin B) shown in the expression cassette coding SEQ ID NO:24, and comprise the nucleotide sequence of this polypeptide of encoding, the nucleotide sequence shown in SEQ ID NO:23.In other embodiments, the encoding sequence of the encoding sequence of the catfish Ig Vh leader sequence part of this fusion polypeptide and/or the former B of attacin of this fusion polypeptide or cecropin B part can comprise the change of codon, thereby the biologic activity variant of the expression cassette coding catfish former B of Ig leader sequence/attacin or catfish Ig leader sequence/cecropin B, these biologic activity variants meet function defined above and structure standard (be disease-resistant originality active and with the sequence identity of reference polypeptide at least 70%).When expressing, the translation post-treatment of the fusion polypeptide of coding causes producing sophisticated cecropin B, is the variant with disease-resistant former active ripe attacin protein B at where applicable perhaps.
In other embodiments of the present invention, expression cassette (for example comprises synthetic promoter of the present invention, SEQ ID NO:3, SEQ ID NO:8 or its have the variant of promoter activity), the nucleotide sequence that this promotor operability is connected to coding attacin P1 (identifies in the chitling tissue; Genbank Accession No.P14661; Shown in the SEQ ID NO:26), for example operability is connected to the nucleotide sequence shown in the sequence SEQ ID NO:25.In selectable embodiment, expression cassette (for example comprises synthetic promoter of the present invention, SEQ ID NO:3, SEQ ID NO:8 or its have the variant of promoter function), this promotor operability is connected to the nucleotide sequence that coding comprises the polypeptide of the catfish Ig Vh leader sequence that as one man is connected with attacin P1 reading frame.In such embodiment, the polypeptide (catfish Ig Vh leader sequence/attacin P1) shown in the expression cassette coding SEQ IDNO:28, and comprise the nucleotide sequence of this polypeptide of encoding, for example nucleotide sequence shown in the SEQ ID NO:27.In other embodiments, the encoding sequence of attacin P1 or catfish Ig Vh leader sequence/attacin P1 polypeptide can change on codon to some extent, thereby the biologic activity variant of this expression cassette coding attacin P1 polypeptide or catfish Ig Vh leader sequence/attacin P1 fusion polypeptide, wherein the biologic activity variant meets the standard (be disease-resistant former activity or with the sequence identity of reference polypeptide at least 70%) of function defined above and structure.When construct coding catfish IgVh leader sequence/attacin P1 polypeptide, its expression and translation post-treatment cause producing sophisticated attacin P1 or its biologic activity varient.
In a word, synthetic promoter of the present invention can be structured among the expression cassette, so that drive the expression of the target nucleotide sequence of any operability connection in non-tissue specificity mode.This box can also contain at least one extra with transcribe the target nucleotide sequence that is connected with translational control zone operability, thereby this additional sequences also is directed in the genome of host living beings, for example in the genome of a kind of fish.Other target sequences comprise the selective marker of the screening that can make things convenient for transgenic organism, but are not limited only to this.Select as another kind, this additional object nucleotide sequence and corresponding regulation and control zone can be provided on one or more extra expression cassettes.
Target nucleotide sequence in the selected host living beings to be imported (for example fish) can contain some and can be increased in the modification of expressing among the specific cells host.This modification comprises the sequence of removing the false polyadenylation signal of coding, exon-intron splice site signal, swivel base increment tumor-necrosis factor glycoproteins, and some other through fine sign may be for the deleterious sequence of genetic expression.In addition, by calculating, the G-C content of sequence can be adjusted to the mean level (ML) of specified cell host with reference to the known of in host cell, expressing.Under possible situation, sequence can be modified, to avoid producing predictable hairpin structure in secondary mRNA structure.
In the building process of expression cassette, can handle various dna fragmentations, so that provide dna sequence dna with correct frame with correct direction and suitably the time.In order to reach this purpose, can utilize adapter or joint to connect dna fragmentation, perhaps adopt restriction site that other operation provides convenience, remove unnecessary DNA, remove restriction site etc.For achieving the above object, can adopt vitro mutagenesis, primer repair, digestion with restriction enzyme, annealing, displacement again (as conversion and transversion).
Synthetic promoter disclosed herein can be used for the fish genetic engineering, to express the nucleotide sequence that any operability connects, the coded product of these nucleotide sequences can be given the phenotype of wanting, and comprises with aforesaid attacin encoding sequence obtaining to have enhanced disease resistance phenotype.Select as another kind, this same target of the disease-resistant fish that acquisition can be accepted by the human consumer can reach by the nucleotide sequence that operability connects native species promoter sequence and aforementioned attacin polypeptide of coding or attacin fusion polypeptide.By this way, the expression cassette that contains the attacin encoding sequence under the fish promoter regulation can be used for improving the resistance against diseases of fish such as catfish, and the acceptance that improves the human consumer.
Therefore, in selectable embodiment of the present invention, adopt native species promotor or its biologic activity variant to replace synthetic promoter, in the target fish, to express the attacin polypeptide." natural " fish promotor is the promoter sequence that exists with natural regulating and controlling sequence in fish.In these embodiments, expression cassette is designed to contain the native species promotor, and its nucleotide sequence operability with above-mentioned coding target attacin polypeptide (comprising aforesaid attacin polypeptide and catfish I g leader sequence-attacin polypeptide) is connected.
Interested native species promotor comprises, but be not limited only to carp beta-actin promotor (promotor as shown in SEQ ID NO:29), channel catfish myostatin promotor (SEQ ID NO:30), channel catfish α-Ji Dongdanbai promotor (SEQ ID NO:31), channel catfish creatine kinase promotor (SEQ ID NO:32), salmon metallothionein promoter (SEQ ID NO:33), salmon histone H 3 promotor (SEQ ID NO:34), channel catfish I type Keratin sulfate promotor and channel catfish II type Keratin sulfate promotor.
One skilled in the art will recognize that behind the nucleotide sequence that discloses native species promoter region as herein described, other controlling elements among the 5 ' non-translational region of the upstream, special promoter zone that separation and evaluation are identified herein belong to prior art.Therefore, when expression cassette comprises natural promoter district disclosed herein or its biologic activity variant, this expression cassette can further comprise the upstream controlling element, and these upstream controlling elements make it possible to any heterologous nucleotide sequence that constitutive expression is connected with one of disclosed promoter sequence operability.One skilled in the art will further recognize that zone disclosed herein, fragment and complete promotor can separately or be united use, to drive the expression of the attacin encoding sequence that operability connects.
The biologic activity variant of native species promoter sequence also can be used in the expression cassette of the present invention, with the expression of the attacin encoding sequence of driving operability connection in fish cell or whole piece fish.Such variant comprises the fragment of these native species promotors." fragment " is meant the part of promotor nucleotide sequence.The fragment of promotor nucleotide sequence can still keep its regulation activity.Therefore, for example, the sequence that is less than whole natural promoter sequence disclosed herein can be used for driving the target nucleotide sequence that operability connects, as the expression of the nucleotide sequence of coding attacin polypeptide.Determine that whether such fragment reduces expression level or change expression characterization is that constitutive expression or inducible expression are within the technical scope of this area.
Nucleic acid molecule as the promotor nucleotide sequence fragment contains at least 15,20,25,30,35,40,45,50,75,100,325,350,375,400,425,450,500,550,600,650,700,800 or 900 Nucleotide, or reaches the Nucleotide number (promptly being respectively 1571,1586,1208,1799,272 and 470 for SEQ ID NO:29,30,31,32,33 or 34) that exists in the total length promotor nucleotide sequence disclosed herein.
The promoter sequence fragment that keeps its regulation activity contains at least 30,35,40 successive Nucleotide of concrete promotor nucleotide sequence disclosed herein, preferred at least 50 successive Nucleotide, more preferably at least 75 successive Nucleotide are more preferably at least 100 successive Nucleotide.Preferred fragment length depends on concrete object, also changes according to specific promoter sequence.
Segmental nucleotide sequence like this contains the TATA recognition sequence of this concrete promoter sequence usually.Such fragment can obtain by natural promoter nucleotide sequence disclosed herein is carried out digestion with restriction enzyme; Obtain by native sequences synthesizing ribonucleotide sequence from the promoter DNA sequence; Or by using round pcr to obtain.Specifically referring to people such as Mullis, (1987), Methods Enzymol.155:335-350 and Erlich chief editor's PCRTechnology (Stockton Press, New York) (1989).
The biologic activity variant of these native species promotors can also comprise those natural variants.The natural variant of the previous native species promotor of identifying can be identified by using well-known Protocols in Molecular Biology, for example uses polymerase chain reaction (PCR) and hybridization technique.The variant promoter sequence also comprises synthetic deutero-nucleotide sequence, for example sequence that obtains by site-directed mutagenesis.Mutagenesis and nucleotide sequence change method are well-known in the art.Referring to for example Kunkel (1985) Proc.Natl.Acad Sci.USA 82:488-492; People such as Kunkel (1987) Methods in Enzymol.154:367-382; U.S.PatentNo.4,873,192; Walker and Gaasta, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York), and other reference literatures of wherein being quoted.
Usually, the variant of native species promotor (for example native species promoter sequence shown in the SEQ ID NO:29,30,31,32,33,34,35), have about at least 70% with native species promotor as the reference molecule, usually at least 75%, 80%, 85%, preferably about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, more preferably about at least 98%, 99% or higher sequence identity.The identity per-cent of nucleotide sequence can be determined the default parameters of correlation parameter for above mentioning for synthetic promoter of the present invention with GAP the 10th edition (or upgrading version).For nucleotide sequence, it is 50 that the breach of acquiescence forms point penalty, and it is 3 that the breach of acquiescence extends point penalty.
The biologic activity variant for example comprises the native species promoter sequence with one or more nucleotide subsitutions, disappearance or insertion.These variant promoter sequences contain the TATA recognition sequence of this concrete native species promoter sequence usually.Promoter activity can be by using above about synthetic promoter of the present invention and definite technology is measured, and comprises the expression level of the reporter gene that the above-mentioned operability of monitoring connects.
In some embodiments of the present invention, expression cassette has closed the native species promotor (as SEQID NO:29,30,31,32,33, promotor shown in 34 or 35), or its biologic activity variant, they are connected with the nucleotide sequence operability that is selected from following coding attacin polypeptide: SEQ ID NO:11 (the former B of preceding attacin), SEQ ID NO:16 (the former B of attacin), SEQ ID NO:19 (ripe cecropin B), SEQ ID NO:22 (the former B fusion polypeptide of catfish Ig leader sequence-attacin), SEQ ID NO:24 (catfish Ig leader sequence-cecropin B fusion polypeptide), SEQ ID NO:26 (attacin P1), with SEQ ID NO:28 (catfish Ig leader sequence-attacin P1 fusion polypeptide), perhaps meet the attacin polypeptide biologic activity variant (be disease-resistant former activity and at least 70% sequence identity is arranged with separately canonical sequence) of aforementioned functional and construction standard.In such embodiments, expression cassette can comprise SEQ ID NO:10 or SEQ ID NO:12 (coding SEQ ID NO:11), SEQ ID NO:15 or SEQ ID NO:17 (coding SEQ ID NO:16), SEQ ID NO:18 or SEQ ID NO:20 (coding SEQ ID NO:19), SEQID NO:21 (coding SEQ ID NO:22), SEQ ID NO:23 (coding SEQ ID NO:24), sequence shown in the SEQ ID NO:25 (coding SEQ ID NO:26), the perhaps sequence shown in the SEQ IDNO:27 (coding SEQ ID NO:28), the attacin encoding sequence that connects as operability.Also can be referring to the construct shown in Fig. 6 and the SEQ ID NO:35.
Comprise the embodiment of top disclosed native species promotor for all, can comprise extra promotor and enhancer element in the expression cassette, to improve the transcriptional level of target attacin polypeptide.These elements can be synthetic, or from fish.Such element comprises the extra promotor and the enhancer element of the core goldfish minimal promoter sequence upstream that is incorporated into synthetic promoter disclosed herein, but is not limited only to this.
Expression cassette described herein can make up or be inserted in any suitable conversion carrier, so that import to subsequently in the target host fish.The method that conversion carrier is imported target host cell is depended in the selection of suitable conversion carrier.For transforming fish cell, can adopt multiple methods known in the art, comprise that the transposon carrier is (referring to for example U.S. Patent number 5,719,055; Quote it in full as a reference at this).In one embodiment, the plasmid vector that carries expression cassette described herein is grown to high copy number in bacterium.These carriers are carried out purifying, linearizing, and inject fish-egg.These fish-eggs are cultivated into fry with the method for suitable aquaculture, and the ripe fish mating of the target nucleotide sequence that high level expression is imported is to stablize the expression that these import sequences.
The controlling element that importing target nucleotide sequence is connected with its operability is well known in the art with the method that produces genetically engineered fish.These methods comprise that microinjection is gone in the zygote, microinjection is gone in the ovocyte and electroporation, but is not limited thereto with linearizing recombinant precursor (expression cassette for example described herein).Referring to for example Inoue (1992) Mol.Mar.Biol.Biotechnol.1 (4-5): 266-270, and Dunham, R.A. wait the people, " Enhanced bacterialdisease resistance of transgenic channel catfish; Ictlauruspunctatus, possessing cecropin genes " .MarineBio technology 4:338-344 (2002).
The expression cassette that contains the synthetic promoter described herein that is connected with target nucleotide sequence operability can be imported in any target fish, to obtain the expression of useful albumen in the gained genetically engineered fish.During when the nucleotide sequence coded disease-resistant originality polypeptide of target, as attacin polypeptide described herein, the expression of coded product can be under the regulation and control of synthetic promoter or native species promotor as the aforementioned.This kind expression cassette has higher market this advantage of acceptance in importing any target fish the time, because controlling element is derived from fish.
The target fish comprise carp, koi, goldfish, salmon, and tilapia and the member of catfish section (channel catfish, blue Cha Wei Channel-catfish and channel catfish-blue Cha Wei Channel-catfish Hybrid), but be not limited only to this.Catfish can be following genus and kind: Ictalurus punctatus, Ictalurusfurcatus, Ictaluru clarias, Ictalurus silurus, Ictaluruspangasius, Ictalurus rafinesque, Ictalurus, balsanus, Ictalurusbrunneus, Ictalurus catus, Ictalurus dugesi, Ictalurus lupus, Ictalurus melas, Ictalurus meridionalis, Ictalurus natalis, Ictalurus natalis erebennus, Ictalurus nebulosus, Pimelodusnebulosus, Ameiurusus nebulos, Ictalurus nebulosus catulus, Ictalurus nebulosus marmoratus, Ictalurus platycephalus, Ictalurus pricei, Ictalurus punctatu, Ameurus punctatus, Ictalurus robustus, Ictalurus simpsoni, Pimelodus argentinus, Pimelodus argystus, Pimelodus caerulescens, Pimeloduscaudafurcatus, Pimelodus furcifer, Pimelodus gracilis, Pimelodus graciosuss, Pimelodus hammondi, Pimelodus houghi, Pimelodus maculates, Pimelodus megalops, Pimelodus nolatus, Pime1odus pallidus, Pimelodus vulpes, Synechoglanis bead1eipunctatus, Silurus punctatus, the filial generation of Ictalurus serracanthus and these fishes.
By instruction and the various technology known in the art and the means of associating present disclosure, can be created in fry cell expressing gene, genetically modified and whole piece fish under the control of controlling element described herein.In most of the cases, all there is alternative way in each stage for whole process.The factor of variation is depended in the selection of way, for example is selected to the plasmid vector system of clone and importing recombinant DNA molecules, fingerling class to be finished, and employed concrete gene, promoter element and upstream element.Those skilled in the art can select and use suitable optional technical scheme to reach expected effect.Also known in this area, many fingerling classes are transformable, thereby can obtain to contain and be expressed in the whole piece fish of the required gene under the regulation and control of synthetic promoter molecule of the present invention.The selection of the gene that the selection of the promotor of brachymemma is connected with operability is other parameter, and they can be optimized to obtain required expression pattern in host fish, and this is well known by persons skilled in the art, and instruction is arranged herein.
Be appreciated that in the nucleotide sequence in mixing expression cassette described herein and may have small sequence variation.Can realize subtle change, and not have negative impact for the function of promoter sequence or the coded proteinic function of nucleotide sequence that connects by operability.Subtle change may occur in the promotor nucleotide sequence, occurs in the nucleotide sequence that operability connects, or occurs in simultaneously in promotor nucleotide sequence and the nucleotide sequence that operability is connected.When the nucleotides sequence of operability connection was classified the encoding sequence of target protein as, the subtle change that exists in encoding sequence owing to the degeneracy of genetic code can cause giving expression to same albumen.Select as another kind, occur in subtle change in the encoding sequence and also may cause giving expression to and compare the protein that has displacement, inserts and/or lack with the reference molecule." subtle change " is meant the variant sequence and is at least 70%, 75%, 80%, 85% with reference to the sequence identity of molecule, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, perhaps even at least 99%, wherein sequence identity is measured by foregoing measuring method.
" with reference to molecule " is meant the sequence of determining as the sequence comparison basis.Canonical sequence can be part or all of particular sequence; For example, the fragment of the synthetic or natural promoter sequence of total length, the perhaps synthetic or natural promoter of total length; The perhaps fragment of complete encoding sequence, or complete encoding sequence.
When these subtle change occur in the synthetic promoter disclosed herein, they can pass through measured by standard techniques, and these methods will make those of ordinary skills can operate and the necessary promoter element functional unit of transcription initiation that uses the nucleotide sequence that is connected for the driving operability.
Thereby as long as the transcribing of the nucleotide sequence that the promoter sequence that changes can initial operability connects, employed synthetic promoter sequence or native species promotor can contain the subtle change with respect to the reference molecule in the expression cassette of the present invention.Similarly, the coding nucleotide sequence of target protein (as the attacin polypeptide) can be natural encoding sequence, also can be different sequence owing to the degeneracy of genetic code, these different sequences can be natural or (for example, using site-directed mutagenesis) that produce through artificial the interference.In addition, the attacin polypeptide of expressing with expression cassette disclosed herein comprises ripe attacin polypeptide, with and the day before yesterday silkworm antibacterial peptide, attacin is former and preceding attacin is former form, and the biologic activity variant of aforementioned polypeptides.Such protein variant can have desirable disease-resistant originality defensive protein activity.Obviously, occur in sudden change in the dna sequence dna of coding misfolded proteins and necessarily sequence can not be placed and read outside the frame, preferably can not form the complementary region that can produce secondary mRNA structure.
Provide following examples to be used for explanation, but limited.
Experiment
On commercial fish market, there are two important problem.First problem is fish disease has caused multi-billion dollar every year to the fish farm loss.For head it off, the investigator attempts cultivating the genetically engineered fish with commercial desirable phenotype.Yet, cultivate genetically engineered fish itself and brought second problem, because transgenosis is all expressed under viral promotors usually.The human consumer thinks that possibly the catfish that has virus " fragment " is unsound or unsafe, therefore unlikely buys.So must find terms of settlement, this predicament of genetically engineered fish that had not only had desirable phenotype but also can have been accepted by the human consumer with Processing of Preparation.
The present invention has solved this predicament by openly having the method and composition that does not use viral nucleotide sequences and carry out the advantage of transgene expression in transgenosis in fish.Therefore, transgenosis described herein system reduced the human consumer for the fish of using composition disclosed herein and method to cultivate may conflict property.Two kinds of selectable methods that are used for cultivating commercial acceptable fish have been described in this invention.The first, by using the synthetic promoter of the present invention that in expression cassette, is connected, can give fish with any valuable phenotype with target nucleotide sequence operability.Because these synthetic promoters only contain the element that derives from fish, so these fishes should be able to be accepted by the human consumer.The second, the disease resistance of catfish can obtain by the nucleotide sequence upstream that natural fish promotor is placed on coding attacin albumen and its biologic activity variant.Because promotor (or functional variant of promotor) derives from fish fully, so these catfish should be able to be accepted by the human consumer.
Synthesized the peptide of ripe cecropin B (SEQ ID NO:18) and ripe attacin P1 (SEQ ID NO:25) in advance, carried out purifying through high pressure liquid chromatography, and their external activity is tested.Test result shows that these polypeptide have the fungicidal activity at catfish Ai Dehuashi bacterium and aeromonas salmonicida (Aeromona salmonicida) of expection, and these bacteriums are the reasons that cause disease in channel catfish.See people such as Kjull (1999) J.FishDis.22:387-394.
Cecropin B and attacin P1 test people (1994) J.Immunol.152:2180-2189s such as () Millers external by polypeptide is carried out titration to clone's catfish category-B lymphoblast clone 1B10 to the toxicity of catfish cell.When 3 μ g/ml cultivate 5 days under the attacin concentration of 100 μ g/ml, these 2 kinds of attacins all do not show any tangible toxic effect (data not shown) for this clone.Because can produce the transgenosis catfish with the construct of expressing these peptides, this has proved that also cecropin B and attacin P1 do not have toxicity (as follows) for catfish.
Embodiment 1: be used for the construct that contains CMV at transgenosis catfish disease resistance enhancemen
Make up the transgenosis catfish, express with the proof attacin and given disease resistance to catfish.Cultivated the genetically engineered fish of 2 strains.The fish of a strain is expressed the ripe cecropin B albumen shown in the SEQ IDNO:19 through engineered one-tenth, and the catfish of another strain is expressed the former B of preceding attacin shown in the SEQ ID NO:11 through engineered one-tenth.
At first, designed a series of construct, these constructs can be expressed ripe cecropin B (SEQ ID NO:19), the former B of preceding attacin (SEQ ID NO:11) or ripe attacin P1 (SEQ ID NO:26) under the regulation and control of cytomegalovirus (CMV) enhanser in fry cell.Like this, cytomegalovirus (CMV) enhanser is connected with the nucleotide sequence operability of coding target attacin.Why select cmv enhancer to be because it can promote transgenosis strong and non-tissue-specific expression in fish cell.In catfish T cell that the construct under the regulation and control of this enhanser is being cultivated and B clone, express well people (1998) J.Immunol.160:3874-3882 such as () Ross.The general design cycle of these constructs and the construct described among other embodiment is below seen Fig. 1.
For the cecropin B construct, have be essential at 2, at first, secondly the attacin guiding Secretory Pathway of expressing, be induced the terminal processing of N-of attacin in the endoplasmic reticulum.Known attacin in natural insect cell through two stage process.At first the former form of attacin is directed in the endoplasmic reticulum before the attacin, produce cutting away leading peptide attacin former after, remove 4 n terminal residues by dipeptidyl peptidase, produce sophisticated attacin.
With first construct (CMV:: the former B of preceding attacin) be designed to express the natural preceding former B polypeptide of attacin shown in the SEQ IDNO:11 (by SEQ ID NO:12 coding).With second construct (CMV:: the former B of catfish Ig Vh leader sequence/attacin) be designed to express the fusion polypeptide (by SEQ ID NO:21 coding) of catfish immunoglobulin heavy chain variable region leader sequence (Ig Vh) shown in the SEQ ID NO:22 and the former B of attacin.With the 3rd construct (CMV:: catfish Ig Vh leader sequence/arbitrary sequence/cecropin B) be designed to express the catfish Ig Vh leader sequence shown in the SEQ ID NO:24 and the fusion polypeptide of ripe cecropin B, insert the arbitrary amino acid sequence by the decision of clone's strategy (by SEQ ID NO:23 coding) therebetween between this fusion polypeptide has.With the 4th construct (CMV:: catfish Ig Vh leader sequence/attacin P1) be designed to express the fusion polypeptide (by SEQ ID NO:27 coding) of catfish Ig Vh leader sequence shown in the SEQ ID NO:28 and attacin P1.Each construct is cloned between 5 ' HindIII and the 3 ' Xbal restriction site.The details of construct and the attacin sequence that occurs in construct are seen Fig. 2 and 3.Expression by each the coded attacin product in these constructs all is under the control of cmv enhancer.The genetically engineered fish that comprises these constructs can avoid the attack of pathogenic agent (face embodiment 4 as follows).
Embodiment 2: drive the design of the synthetic promoter of target gene product expression
Although the construct among the embodiment 1 can protect the transgenosis catfish not to be subjected to the attack of pathogenic agent, the CMV controlling element derives from a kind of human virus, and it is unacceptable using for agricultural owing to the conflict of human consumer's purchase.Therefore, we design complete synthetic promotor from the regulating and controlling sequence of fish.This synthetic promoter comprised 5 ' and end goldfish minimal promoter that be connected with upstream element, that contain TATA box primitive (people (1991) such as SEQ ID NO:1 Wilson, Mol.Immunol.28:449).This upstream element comprises 3 fish Sp1 in conjunction with primitive (CGGGGCGGGG; SEQ ID NO:2; See people such as Baudler (1997) J.Biol.Chem.272:131-137) and novel intervening sequence.This minimal promoter contains the necessary nucleotide sequence of expression of the encoding sequence that connects for operability, comprises TATA box and transcriptional initiation sequence.This synthetic promoter sequence is placed 5 of the preceding former B encoding sequence of attacin (shown in the SEQ ID NO:12) ' end, so that drive the non-tissue-specific constitutive expression of this albumen in fry cell.The synthetic promoter that is connected with the former B encoding sequence of preceding attacin operability is presented in the sequence shown in the SEQ ID NO:13.Also visible Fig. 4.
Then the above-mentioned construct that contains this synthetic promoter that is connected with the former B encoding sequence of preceding attacin operability is cloned into plasmid (pBS, Stratagene, 11011N.TorreyPines Road, La Joll a, CA92037).The restriction site (being Not1 and Kpnl) that is positioned at the both sides, polylinker site of pBS makes it possible to remove the carrier sequence before the use encoding sequence produces genetically engineered fish.Therefore, this expression cassette contains with high level and drives the synthetic controlling element of genetic expression in non-tissue specificity mode.By the construct that design has the synthetic promoter sequence that is connected with the sequence operability of coding specific objective gene product, this synthetic promoter also can be used to drive the high level of any target gene product, non-tissue specific expression.
Embodiment 3: another drives the design of the synthetic promoter of target gene product expression
Another sequence and structure with manual activation that the nucleotide sequence operability of the preceding former B of attacin of coding is connected is seen Fig. 5.Be used to design the controlling element of this promotor from MT gene (people (1994) Nature 371:209-210 such as DevLin) or FV-1 and FV-2 actin promoter (people (1990) Biotechnology S:1268-1272 such as Liu).FV-2 is a kind of strong promotor, FV-1 then be a kind of medium strong promotor (people (1990) such as Liu, as above).
To drive the ability of high level and the expression of non-tissue-specific gene and select a series of transcription factor is placed on the upstream of the goldfish minimal promoter that comprises the TATA box in conjunction with primitive according to their, thereby form another manual activation.Particularly, expression cassette comprises comprising and contains TATA frame primitive (SEQ ID NO:1; People such as Wilson (1991), the synthetic promoter of goldfish minimal promoter Mol.Immunol.28:449), this synthetic promoter is connected with upstream element, and this upstream element comprises the following element that is assembled together with 5 '-3 ' direction and interleaving property joint sequence: fish SP1 combines primitive (CGGGGCGGGG; SEQ ID NO:2), operability is connected to fish C/EBP α primitive (ATAATGTTTCATCACACTT; SEQ ID NO:4 sees for example people (1997) Eur J.Biochem.247:44-51 such as Chan), operability is connected to fish Oct primitive (ATGTAAAT; SEQ ID NO:5; See for example people (1997) Immunogenetics46:192-198 such as Magor), operability is connected to fish NF-κ B primitive (GGGACGTCCC; SEQ IDNO:6), operability is connected to fish AP-1 in conjunction with primitive (ATGACTCAG; SEQ ID NO:7).This promotor (shown in the SEQ ID NO:8) is connected with nucleotide sequence (for example seeing SEQ ID NO:12) 5 ' end operability of the preceding former B of attacin of coding.This promotor and encoding sequence also are shown in (also visible SEQ ID NO:14) among Fig. 5.
Then, the construct (seeing SEQ ID NO:14) that will have this optional synthetic promoter that is connected with preceding attacin original encoding sequence operability as previously mentioned is cloned into plasmid (pBS, Stratagene, 11011 N.Torrey Pines Road, La Jolla, CA92037) in.The restriction site (being Not 1 and Kpn1) that is positioned at the both sides, polylinker site of pBS makes it possible to remove the carrier sequence before the use encoding sequence produces genetically engineered fish.Therefore, this expression cassette contains with high level and drives the synthetic controlling element of genetic expression in non-tissue specificity mode.By the construct that design has the synthetic promoter sequence that is connected with the sequence operability of coding specific objective gene product, this synthetic promoter also can be used to drive the high level of any target gene product, non-tissue specific expression.
Embodiment 4: have the resistance of the genetically modified catfish of attacin of stable integration to the disease attack
The construct importing of embodiment 1 is in the catfish zygote in unicellular stage, cultivate resulting fry, and screening contains genetically modified fry.Contain CMV:: the parent of the former B construct of preceding attacin is (P1) transgenosis catfish and contains CMV:: the P1 transgenosis catfish of cecropin B construct lays eggs, thereby transgenosis is separately given for (F by mating breeding transmission 1).
What particularly point out is, in water temperature was 26～27 ℃ running water fishpond, channel catfish carried out artificial spawning by using inducing of carp hypophysis extract (CPE).Raun is injected the CPE of this body weight of 2mg/, and gives the solution dosage of 8mg/kg after 12 hours, with induced ovulation.Ovum is peeled off from the raun that lays eggs, and put into culture dish.Add milt liquid and water, and gentle agitation, to finish fertilization and dispersive process.
Carry out transgenosis with Baekon 2000 electroporation devices.The fish-egg of 300 channel catfish is primary treatment simultaneously in the culture dish of 50mm, has 1.5ml damping fluid (TE, 0.88mM NaCI) and 50 μ g/ml to comprise the plasmid DNA of attacin sequence in the culture dish.Use the electroporation of non-contact type, its electroporation parameter is provided with as follows: 6kV, 108 burst, 4 circulations, 27 pulses, and 160 microseconds/pulse.
These P1 fishes are put in a suitable place to breed in the mud sump that has the genetically engineered fish limiting device of ratifying through USDA.Each genetically engineered fish to supposition lays eggs in fishpond.Independent fish-egg agglomerate is hatched in incubation tank, by the analysis of PCR dot blotting screen (see people such as Dunham (1992) " Transfer; expression and inheritance of salmonid growthhormone genes in channel catfish; Ictalurus punctatus; andeffects on performance traits; " Mol.Mar.Biol.and Biotech.1:380-389), to guarantee the heredity of attacin gene.Hatch positive ovum, fry is at first supported in incubation tank, puts in a suitable place to breed then in the mud sump of 0.04 hectare band limiting device.The fish of arbitrarily feeding catches, and carries out pathogenic agent and attack when 6 months age of a fish.
Disease is attacked
In incubator, the catfish tarda was grown 24 hours in 26 ℃ in the BHI substratum.Fish is to carry out pathogenic agent in 500 liters the fishpond to attack by the unified volume that is placed on.Pouring into concentration in the pond is 10 8The catfish tarda of cell/ml soaked fish 1 hour under 25 ℃ of temperature still.After this, recover water (25 ℃) stream again.The monitoring fish was subjected to clinical symptom that the catfish tarda infects and mortality ratio meter 14 days.The dead fish of collecting is carried out necrotomy with definite cause of the death and infection, and attempt from these fishes, isolating again the catfish tarda.Carrying out Columnaris by natural epizootic attacks.From mud sump, break out serious Columnaris before the results.Collect live fish and dead fish and (see people such as Dunham (1992), as above) and necrotomy to carry out DNA analysis.Necrotomy has confirmed dead and infection comes from column Flavobacterium (Flavobacterium columnare).
In the first time, pathogenic agent was attacked, have CMV:: the transgenosis catfish of cecropin B construct and non-transgenic compatriot are fully all attacked by the catfish tarda in the pond.Though genetically engineered fish and non-transgenic fish all can have death, the survival rate of transgenosis individuality is 2 times of contrast.(seeing Table 1)
In the second time, pathogenic agent was attacked, have CMV:: the transgenosis catfish of the former B construct of preceding attacin and non-transgenic compatriot's contrast are fully all attacked by the column Flavobacterium.The mortality ratio of contrast is about 63%, and the mortality ratio of genetically engineered fish is 0% (seeing Table 1).Therefore, transgenosis is given genetically engineered fish with resistance completely.In all tests, do not observe manifold effect to growth.
Table 1:
In epizootic and the attack of artificial pond, the transgenosis spot fork-tail catfish that contains the construct of the attacin of encoding has the enhanced resistance for bacteriosis
Transgenosis Disease is attacked Environment Survival rate (%) genetically engineered fish Contrast
CMV:: the former B of preceding attacin F.columnare The pond 100 27.3
CMV: cecropin B E.ictaluri The pond 40.7 14.8
Above data show that the genetically engineered fish with attacin construct has the enhanced resistibility for non-natural and natural pathogenetic bacteria attack.Yet the genetically engineered fish with viral DNA sequence is not accepted commercial, therefore must remove the CMV promotor and use synthetic promoter instead or " full fish promotor ", and is described as the following examples 5.
Embodiment 5: have the resistance of the genetically modified catfish of attacin under the synthetic promoter regulation and control of stable integration to the disease attack
As discussed above, the synthetic fish promotor that is connected with attacin gene operability can be used for forming and not only has disease resistance but also the commercial genetically engineered fish that can be accepted by the human consumer.Validity for the synthetic promoter of measuring SEQ ID NO:3 and 8 is connected these synthetic promoter sequences in expression cassette with 5 of preceding attacin original encoding sequence ' end operability, see above embodiment 2 and 3 for details.Like this, made up the expression cassette that comprises SEQ ID NO:13 (synthetic promoter with SEQ ID NO:3 drives the preceding former expression of attacin) or SEQ ID NO:14 (synthetic promoter with SEQ IDNO:8 drives the preceding former expression of attacin).
Utilize embodiment 4 described methods, transform the catfish ovum with the expression cassette that comprises SEQ ID NO:13.The render transgenic fry is growth and maturity under suitable condition.With P1 is the mating breeding, obtains F1 system.
In second group of test, transform the catfish ovum with the expression cassette that comprises SEQ ID NO:14.The render transgenic fry is growth and maturity under suitable condition.With P1 is the mating breeding, obtains F1 system.
Use embodiment 4 described same procedure, measure F1 for the disease resistance of catfish to the target pathogenic agent.Different with the transgenosis catfish among the embodiment 4, these catfish do not change any viral DNA over to.Therefore, these catfish commercial be acceptable, but also be not subjected to affect.
Embodiment 6: have the resistance of the genetically modified catfish of attacin under natural " full fish promotor " regulation and control of stable integration to the disease attack
As discussed above, also can be used for forming with the native species promotor that is connected of sequence operability of coding attacin and not only have disease resistance but also the commercial transgenosis catfish that can be accepted by the human consumer.Use embodiment 1 described method, the construction expression box, make it to comprise SEQ IDNO:29,30,31,32,33,34 or 35 promoter sequence, this promoter sequence is connected with 5 ' end operability of the nucleotide sequence of coding attacin, described nucleotide sequence for example is (the former B of attacin before the coding of the encoding sequence shown in SEQ IDNO:10 or 12, SEQ IDNO:11), encoding sequence shown in the SEQ ID NO:15 or 17 (the former B of coding attacin, SEQ ID NO:16), encoding sequence shown in the SEQ ID NO:18 or 20 (encoding mature cecropin B, SEQ ID NO:19), perhaps (the encoding mature attacin P1 of the encoding sequence shown in the SEQ ID NO:25, SEQ ID NO:26). another kind of selectable mode is, the construction expression box, make it to comprise SEQ ID NO:29,30,31,32,33,34 or 35 fish promoter sequence, this promoter sequence is connected with the encoding sequence 5 ' end operability of the former fusion polypeptide of catfish Ig Vh leader sequence-attacin, described encoding sequence for example is (the former B fusion polypeptide of coding catfish Ig Vh leader sequence-attacin of the encoding sequence shown in the SEQ ID NO:21, SEQ ID NO:22), perhaps be connected with the encoding sequence 5 ' end operability of catfish Ig Vh leader sequence-attacin fusion polypeptide, described encoding sequence for example is (the coding catfish Ig Vh leader sequence-cecropin B fusion polypeptide of the encoding sequence shown in the SEQ IDNO:23, SEQ ID NO:24) or the encoding sequence shown in the SEQ ID NO:27 (coding catfish Ig Vh leader sequence-attacin P1 fusion polypeptide, SEQ ID NO:28).Referring to construct for example shown in Figure 6 (SEQ ID NO:35), wherein the former B polypeptide expression of the preceding attacin of SEQ ID NO:11 is regulated and control by carp beta-actin promotor (SEQ ID NO:29).
Adopt embodiment 4 described methods, with one of the construct described in present embodiment transfection catfish ovum.The render transgenic fry is growth and maturity under suitable condition.P1 system is carried out outbreeding, to produce F1 system.Use embodiment 4 described same procedure, measure F1 for the disease resistance of catfish to the target pathogenic agent.Owing to do not contain viral DNA, use catfish that these constructs make commercial also be acceptable.
Mentioned all publications and applications for patents are understood one of ordinary skill in the art's of the present invention state of the art in this specification sheets.This sentences same degree and introduces all publications and patent application as a reference, and these publications or patent application are concrete and are incorporated herein by reference in full individually.
Though in order to be expressly understood, comparatively detailed elaboration has been carried out in foregoing invention by the diagram and the mode of giving an example, yet, obviously can make some change and modification within the scope of the invention.
Sequence table
<110>Dunham，Rex A.
Liu，Zhanjiang
Warr，Gregory W.
＜120〉composition and the method for enhancing fish disease resistance
<130>035721/271295
<150>60/424,329
<151>2002-11-06
<160>35
<170>FastSEQ for Windows Version 4.0
<210>1
<211>82
<212>DNA
<213>Caressius auratus
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉goldfish minimal promoter
<400>1
actgtgttat aaactggttc ctcagtcagt gtttgtgttc tgctgctgtg cagtttcttt 60
tcctttgact gtttttggat cc 82
<210>2
<211>10
<212>DNA
＜213〉fish
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉fish Sp1 is in conjunction with primitive
<400>2
cggggcgggg 10
<210>3
<211>161
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic promoter
<400>3
gaattcctgc agaacggggc ggggatctcg agttcggggc ggggataggc gtttcggggc 60
ggggaactgc aggactgtgt tataaactgg ttcctcagtc agtgtttgtg ttctgctgct 120
gtgcagtttc ttttcctttg actgtttttg gatccggcac c 161
<210>4
<211>19
<212>DNA
＜213〉fish
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉fish C/EBP α primitive
<400>4
ataatgtttc atcacactt 19
<210>5
<211>8
<212>DNA
＜213〉fish
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉Oct primitive
<400>5
atgtaaat 8
<210>6
<211>10
<212>DNA
＜213〉fish
<220>
<221>mi6c_feature
<222>(0)...(0)
＜223〉NF-κ B primitive
<400>6
gggacgtccc 10
<210>7
<211>9
<212>DNA
＜213〉fish
<220>
<221>misc_ feature
<222>(0)...(0)
＜223〉AP-1 is in conjunction with primitive
<400>7
atgactcag 9
<210>8
<211>207
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic promoter
<400>8
gaattcctgc agaacggggc ggggatctcg agttataatg tttcatcaca cttatacgcg 60
tttatgtaaa tatctcgagt tgggacgtcc catctcgagt tatgactcag aactgcagga 120
ctgtgttata aactggttcc tcagtcagtg tttgtgttct gctgctgtgc agtttctttt 180
cctttgactg tttttggatc cggcacc 207
<210>9
<211>222
<212>DNA
<213>Bos taurus
<220>
<221>miBc_feature
<222>(0)...(0)
＜223〉Trobest 3 ' non-translational region
<400>9
agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc 60
cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 120
gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 180
ggtgtcattc tattctgggg gactagttct agagcggccg cc 222
<210>10
<211>189
<212>DNA
<213>Hyalophora cecropia
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉cecropin B 1 precursor
<400>10
atgaatttct caaggatatt tttcttcgtg ttcgctttgg ttctggcttt gtcaacagtt 60
tcggctgcac cggagccgaa atggaaagtc ttcaagaaaa ttgaaaaaat gggtcgcaac 120
attcgaaacg gtattgtcaa ggctggacca gcgatcgcgg ttttaggcga agccaaagcg 180
ctaggataa 189
<210>11
<211>62
<212>PRT
<213>Hyalophora cecropia
<400>11
Met Asn Phe Ser Arg Ile Phe Phe Phe Val Phe Ala Leu Val Leu Ala
1 5 10 15
Leu Ser Thr Val Ser Ala Ala Pro Glu Pro Lys Trp Lys Val Phe Lys
20 25 30
Lys Ile Glu Lys Met Gly Arg Asn Ile Arg Asn Gly Ile Val Lys Ala
35 40 45
Gly Pro Ala Ile Ala Val Leu Gly Glu Ala Lys Ala Leu Gly
50 55 60
<210>12
<211>186
<212>DNA
<213>Hyalophora cecropia
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉the former DNA of preceding attacin
<400>12
atgaatttca gcagaatctt cttcttcgtg ttcgccctcg tgctcgccct ctctaccgtg 60
agcgccgccc cagaaccaaa atggaaagtg ttcaaaaaaa tcgagaaaat gggaagaaat 120
atcagaaatg gaatcgtgaa agccggacca gccatcgctg tgctcggaga agccaaagcc 180
ctctag 186
<210>13
<211>569
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic promoter
<400>l3
gaattcctgc agaacggggc ggggatctcg agttcggggc ggggataggc gtttcggggc 60
ggggaactgc aggactgtgt tataaactgg ttcctcagtc agtgtttgtg ttctgctgct 120
gtgcagtttc ttttcctttg actgtttttg gatccggcac catgaatttc agcagaatct 180
tcttcttcgt gttcgccctc gtgctcgccc tctctaccgt gagcgccgcc ccagaaccaa 240
aatggaaagt gttcaaaaaa atcgagaaaa tgggaagaaa tatcagaaat ggaatcgtga 300
aagccggacc agccatcgct gtgctcggag aagccaaagc cctctagagg gccctattct 360
atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 420
gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 480
tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 540
tctgggggac tagttctaga gcggccgcc 569
<210>14
<211>615
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic promoter
<400>14
gaattcctgc agaacggggc ggggatctcg agttataatg tttcatcaca cttatacgcg 60
tttatgtaaa tatctcgagt tgggacgtcc catctcgagt tatgactcag aactgcagga 120
ctgtgttata aactggttcc tcagtcagtg tttgtgttct gctgctgtgc agtttctttt 180
cctttgactg tttttggatc cggcaccatg aatttcagca gaatcttctt cttcgtgttc 240
gccctcgtgc tcgccctctc taccgtgagc gccgccccag aaccaaaatg gaaagtgttc 300
aaaaaaatcg agaaaatggg aagaaatatc agaaatggaa tcgtgaaagc cggaccagcc 360
atcgctgtgc tcggagaagc caaagccctc tagagggccc tattctatag tgtcacctaa 420
atgctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 480
gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 540
aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg ggggactagt 600
tctagagcgg ccgcc 615
<210>15
<211>123
<212>DNA
<213>Hyalophora cecropia
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉the former B DNA of attacin
<400>15
gcaccggagc cgaaatggaa agtcttcaag aaaattgaaa aaatgggtcg caacattcga 60
aacggtattg tcaaggctgg accagcgatc gcggttttag gcgaagccaa agcgctagga 120
taa 123
<210>16
<211>40
<212>PRT
<213>Hyalophora cecropia
<400>16
Ala Pro Glu Pro Lys Trp Lys Val Phe Lys Lys Ile Glu Lys Met Gly
1 5 10 15
Arg Asn Ile Arg Asn Gly Ile Val Lys Ala Gly Pro Ala Ile Ala Val
20 25 30
Leu Gly Glu Ala Lys Ala Leu Gly
35 40
<210>17
<211>120
<212>DNA
<213>Hyalophora cecropia
<220>
<221>misc_f eature
<222>(0)...(0)
＜223〉the former B DNA of attacin variant
<400>17
gccccagaac caaaatggaa agtgttcaaa aaaatcgaga aaatgggaag aaatatcaga 60
aatggaatcg tgaaagccgg accagccatc gctgtgctcg gagaagccaa agccctctag 120
<210>18
<211>111
<212>DNA
<213>Hyalophora cecropia
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉ripe attacin DNA
<400>18
aaatggaaag tcttcaagaa aattgaaaaa atgggtcgca acattcgaaa cggtattgtc 60
aaggctggac cagcgatcgc ggttttaggc gaagccaaag cgctaggata a 111
<210>19
<211>36
<212>PRT
<213>Hyalophora cecropia
<400>19
Lys Trp Lys Val Phe Lys Lys Ile Glu Lys Met Gly Arg Asn Ile Arg
1 5 10 15
Asn Gly Ile Val Lys Ala Gly Pro Ala Ile Ala Val Leu Gly Glu Ala
20 25 30
Lys Ala Leu Gly
35
<210>20
<211>108
<212>DNA
<213>Hyalophora cecropia
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉ripe attacin DNA variant
<400>20
aaatggaaag tgttcaaaaa aatcgagaaa atgggaagaa atatcagaaa tggaatcgtg 60
aaagccggac cagccatcgc tgtgctcgga gaagccaaag ccctctag 108
<210>21
<211>174
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the former B encoding sequence of catfish Ig Vh leader sequence/attacin
<400>21
atgctctcta ccagcctgct cctgctcctc gccctgctct cttatgtgca tggcgcccca 60
gaaccaaaat ggaaagtgtt caaaaaaatc gagaaaatgg gaagaaatat cagaaacgga 120
atcgtgaaag ccggaccagc catcgctgtg ctcggagaag ccaaagccct ctag 174
<210>22
<211>58
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the former B of catfish Ig Vh leader sequence/attacin
<400>22
Met Leu Ser Thr Ser Leu Leu Leu Leu Leu Ala Leu Leu Ser Tyr Val
1 5 10 15
His Gly Ala Pro Glu Pro Glu Lys Trp Lys Val Phe Lys Lys Ile Glu
20 25 30
Lys Met Gly Arg Asn Ile Arg Asn Gly Ile Val Lys Ala Gly Pro Ala
35 40 45
Ile Ala Val Leu Gly Glu Ala LysAla Leu
50 55
<210>23
<211>180
<212>DNA
＜213〉artificial sequence
<220>
＜223〉catfish Ig Vh leader sequence/cecropin B encoding sequence
<400>23
atgttatcta catctctact gctcctgctg gcagctgctt cctatgtgca tggtcaggga 60
ctgactctag agaaatggaa agtgttcaaa aaaatcgaga aaatgggcag aaacatcaga 120
aacggaatcg tgaaagccgg accagccatc gccgtgctcg gagaagccaa agccctctag 180
<210>24
<211>59
<212>PRT
＜213〉artificial sequence
<220>
＜223〉catfish Ig Vh leader sequence/cecropin B
<400>24
Met Leu Ser Thr Ser Leu Leu Leu Leu Leu Ala Ala Ala Ser Tyr Val
1 5 10 15
His Gly Gln Gly Leu Thr Leu Glu Lys Trp Lys Val Phe Lys Lys Ile
20 25 30
Glu Lys Met Gly Arg Asn Ile Arg Asn Gly Ile Val Lys Ala Gly Pro
35 40 45
Ala Ile Ala Val Leu Gly Glu Ala Lys Ala Leu
50 55
<210>25
<211>96
<212>DNA
<2l3>Sus scrofa
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉pig attacin P1 encoding sequence
<400>25
agctggctct ctaaaaccgc caaaaagctg gaaaatagcg ccaaaaaaag aatctctgag 60
ggcatcgcca tcgccatcca gggaggccca agatag 96
<210>26
<211>31
<212>PRT
<213>Sus scrofa
<400>26
Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys
1 5 10 15
Arg Ile Ser Glu Gly Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg
20 25 30
<210>27
<211>150
<212>DNA
＜213〉artificial sequence
<220>
＜223〉catfish Ig Vh leader sequence/attacin P1 encoding sequence
<400>27
atgctctcta ccagcctgct cctgctcctc gccctgctct cttacgtgca tggcagctgg 60
ctctctaaaa ccgccaaaaa gctggaaaat agcgccaaaa aaagaatctc tgagggcatc 120
gccatcgcca tccagggagg cccaagatag 150
<210>28
<211>49
<212>PRT
＜213〉artificial sequence
<220>
＜223〉catfish Ig Vh leader sequence/attacin P1
<400>28
Met Leu Ser Thr Ser Leu Leu Leu Leu Leu Ala Leu Leu Ser Tyr Val
1 5 10 15
His Gly Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser Ala
20 25 30
Lys Lys Arg Ile Ser Glu Gly Ile Ala Ile Ala Ile Gln Gly Gly Pro
35 40 45
Arg
<210>29
<211>1571
<212>DNA
<213>Cyprinos carpio
<220>
<221>misc feature
<222>(0)...(0)
＜223〉carp beta-actin promotor
<400>29
tttgatgaaa atcgcttagg ccttgctctt caaacaatcc agcttctcct tctttcactc 60
tcaagttgca agaagcaagt gtagcaatgt gcacgcgaca gccgggtgtg tgacgctgga 120
ccaatcagag cgcagagctc cgaaagttta ccttttatgg ctagagccgg catctgccgt 180
catataaaag agcgcgccca gcgtctcagc ctcactttga gctcctccac acgcagctag 240
tgcggaatat catctgcctg taacccattc tctaaagtcg acaaaccccc ccaaacctaa 300
ggtgagttga tctttaagct ttttacattt tcagctcgca tatatcaatt cgaacgttta 360
attagaatgt ttaaataaag ctagattaaa tgattaggct cagttaccgg tctttttttt 420
ctcatttacg tgcgaactct gcttaaactc tagttattct ttattaatat gtggttattt 480
ttatatatgt atgttatcat aactgtactg gctatgtcag gtggtaatga ctgtaacgtt 540
acgttactcg ttgtaggcac gacattgaat gggccggtgt tgaaataagt cttcaacccc 600
ttttaacctc aaaatgtgct ctggttaaca aggattttaa cagctatcag tatgactgtg 660
cggttttaaa gccgttagtg aggcacgttg cacacttgat ggatggccgg aatgggaagt 720
tctttatgca ggcagtgctg cagcagggtg tgacctactt tagctaacgt tagccggcta 780
accagcattc atctgccggt aacttgagtc taatattctc tatgtgatat cgaagtgatc 840
aaagacacgt ctgttagctc actttaacca actgtagtga aaaatagcgc agtgtgcagc 900
ccttcaagtc tttcatttag gctgattatt caatcatttt attaactatt aacgcgttac 960
taaacgtaag gtaacgtagt cagtttttaa taactggtga aaagtactgg ttgggtttaa 1020
atggtgactt ataattgtgt tggaggggga aacctttttg ataaaggcta tataatctca 1080
aatgaatggg ctgaggatgg tgttcacagg tgctttagtg aagtccgctc gtgaagagtc 1140
gctgaagtga ctgcagatct gtagcgcatg cgttttggca gacggccgtt gaaattcggt 1200
tgagtaattg ataccaggtg aggctagagg atgtagaaat tcatttgtgt agaatttagg 1260
gagtggcctg gcgtgatgaa tgtcgaaatc cgttcctttt tactgaaccc tatgtctctg 1320
ctgagtgcca caccgccggc acaaagcgtc tcaaaccatt gccttttatg gtaataatga 1380
gaatgcagag ggacttcctt tgtctggcac atctgaggcg cgcattgtca cactagcacc 1440
cactagcggt cagactgcag acaaacagga agctgactcc acatggtcac atgctcactg 1500
aagtgttgac ttccctgaca gctgtgcact ttctaaaccg gttttctcat tcatttacag 1560
ttcagccaag g 1571
<210>30
<211>1586
<212>DNA
<213>Ictalurus punctatus
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉channel catfish myostatin promotor
<400>30
cccaatattc ccagcaggtg atcagcagag agagagacac acaggagaga tagcgagaca 60
gacagagaga aattgagaga cagacaacag agagatatag agacagagac agagagagag 120
agatatagag agacagacag agagagagag atagtagtga gacagagaga gatagagaga 180
cacagagaga gacagagaga gacatagaga gagacagaca gagagagtgt ctctcatatg 240
tcaacatatg tgtagggcat atgttgggtt tttttctgtg tgtgtgtgtg aggtaatgca 300
gaatgccaac agcaggatat attgtgggtt tggattaaac atgctcttta atttctttga 360
atacatgtta actattctat gaaacactgg agcggtagtg tagtggtagt tgcagtgtag 420
gtggtagtgg tagtggagcg gtagttgtat gtagtggcag tgttattggg agtgtagtgg 480
gagtggtagt gtagtggagc tgtagtggag tgtagtggta gtggagtata gtggtagtgt 540
agttggagtg cgtagtgtaa tgtagtggta gtgtaatatg gtgtagtggt agtgtaatgt 600
agtggtagtg gagtggtaat gtagtgtagt ggtagtggag tggtagtgta atgtagtgta 660
gtggtagtgt agtggtagtt gagcggtagt gtaatgtagt gtagtggtag tgtaatgtag 720
tgtagtggag cggtagtgta gtggtagtgg agcggtagtg taatgtagtg gagtggtagt 780
ggagcggtag tgtaatgtag tggagtggta gtggagcggt agtgtagtgg tagtggagcg 840
gtagtgtaat gtagtggagt ggtagtgtag tggtagtgta gtggtagtgg agcggtagtg 900
taatgtagtg tagtggtagt ggagcgggta gtgtagtgga gtggtagtgg agtggtagtg 960
gagtggtagt gtaatgtagt gtagttggta gtggagcggt agtgtaatgt agtggagttg 1020
gtagtggtta cggtggtagt ggagcggtag tgttaatgta gtggagtggt agtgtaatgt 1080
aggtggtagt ggtagtggag tggtagtgta atgtagtgga gtggtagtgg tagtggtagt 1140
ggagcggtag gtgtaatgta gtggagtggt agtgtaatgt agtggtagtg gtagtggagt 1200
ggtagtgtaa tgtagtggag tggtagtgga gtggtagtgt aatgtagtgg agtggtagtg 1260
tagtggtagt gtaatgtagt gtagtggaga aagttgtggg tctgtctctt taaggtttca 1320
gcgctggaaa gggaggaaaa aaatccggac tgaagtccac ctctgattta ttgttgctcc 1380
gagtagccaa tcatagattt cgacgccaga gcctaaataa gagcggcgga ataatttggc 1440
ggtataaaaa ggcttttggg cgaattgaag catgacatct cgcgctacct gtccggtgtg 1500
catggcgcac ggtgttcctg ttactgctgc cacacagaaa acacaaccgc gcgcgcactc 1560
ctctctgaga cctgacctgg ctgatc 1586
<210>31
<211>1208
<212>DNA
<213>Ictalurus punctatus
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉channel catfish α-Ji Dongdanbai promotor
<400>31
ttctttaata aagactcgaa catctataaa atatgtattt acgtatcaat aattaataca 60
taatttaaat accaaaaata gaatatatct ccccttcctc cgcggacgag ccaagcaaac 120
cctatgtatt cctttacatc tacatatgtc aaattttatg atgctactat gactgatacg 180
ctcgcatgat ccttgtggtg tggtgacgtg tctgctctct tcactttgct taactataag 240
ggaaaaccgc ctgcgtgtta acacggtttt cggggtgaaa cttttctaca acggtgcgtc 300
ctccggtttc cttgttgtcc agaaaagctg acaagactcg cgcagccgca gacaggagac 360
gccaaattgt cgtggaaatt agacaacgct cgcagactcg tcctctgaag gtaaaaaagg 420
ttttattaca gaaagccggt ttaatacagg agaggaatta aagcagggag agaataatga 480
gagcgctctg aagtgcctcg tgctgaaccg ctactataga tatgaaacgc agagcacgac 540
atacttctgt atacccataa gaagcggttt gaggcagttc aaacagtttt aggatcttgg 600
aagatgttta gacgaacctc gaacagaaga acatgtttgt gtctgtacgc agataaacat 660
tctgtacgga tctcagtgac atgacatggc cctctcaggc gttatcctca gatgaacatg 720
aacaaacttc tctacacggt gtgcggtctc gggagttttg cagaattgtt cagtcatgtg 780
cactcgtgaa atccaccctg cagtacagac gatgctgagt gctgcccctt cacttataca 840
cacgtaaact gctcgtcgtg tccattagct tctttgcttg catcccattg tctctactaa 900
ctggcgtgat gaacacgtgt aatcgtaaat agaattacga tgagaaaaag tcaaatcgtt 960
gaaacccaac ctttcacgcg tgtgcttaac tatgaatgaa gtgacggtga tgccttactg 1020
agcaccttgt tctttccaca tttcaaaaaa cataacagga ggagaatttt ttttttttat 1080
ggactaaaat atatgcactt ttaatgtagt cccagtaggc ataggttaga atacacactt 1140
aggtgtattg tgtgtgtgtg tgtgtttcat tcctcatcgt gtgcttctat atcaggaacc 1200
cattcaac 1208
<210>32
<211>1799
<212>DNA
<213>Ictalurus punctatus
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉channel catfish creatine kinase promotor
<400>32
tgtgtgtgtg tgtgtaagag tgagagatat atattaacag ctgttttgat ttggtacata 60
ttctagactg ccatggcagc tgccccagta tcagctgacc atctcttagc ctggcagatt 120
gatgcgaatg catccctacc ctgtttttcc atctccatct cccgctctct ttctcaccag 180
cacttagctg aagtcatcat ctccaatagc aagcaaagta aacattcctc tcatatcctg 240
ttcactactt agcactactc agttgagtta aaccagacct tctctttcga tgtaatatca 300
attttaaggt aacaattaac attttgaagg taatataaaa tagtgcaaaa gtgagaaaat 360
tgaaaacggc actgttatat acactagtct atggaataat atacttctcg ctagcagcac 420
tatggtatta atatcaaaaa agctttccaa gcatcctctg tccaagtgtg tctcttcagc 480
caggtagaca aaaacagtct tcccgagctg ccttctttct atttatttat ttatttgaag 540
aaaaaaaaaa atctttatcc ttttttggcc tctgaataaa aactaaatgt tagcaacacg 600
aacaaaccta aaaaaaaaaa aaaaagcagt atcaaggctg gctagttacc gtagctagtt 660
aacatttgtt ttaaaataac aacaacaata aaatcatgaa cagaatccat gagtgtcttc 720
atagtgatgt caactggaga tgctagttga gaagttaaaa ctacagagct ccaaaccttg 780
gcagcctcgt agctagttcg agccattctc ttgtcaccaa ataatgcctc acttccagct 840
attgttccct attttgaatc acataatgtg ctcaattaaa gttttgcatt aaaaatgatc 900
ttgagccaga gcaagatgac tcaggcctat agcatgaatg cccagacgaa tgtctcgaac 960
acatgcagtt tttaagaaaa gtagaaatcc gagttattca atttttttag aagcccttag 1020
gactcgacat tagatttttg cacaaaaaaa acacaaaaaa aacaggaata tggacatttt 1080
ttctcacatt tcagcagaat ctgcttcatc agctttcagt tttaggatct tcaaggatgg 1140
atgaccttac agatttataa ccatatgcct gtgcaatata aatcaagtga aatacaccct 1200
cctcctcctc ctcaaagtac ttgcatacac acacactgtg gttagcacac cttcacaatc 1260
ctatacatct tcagaaatat gctgtttttt ttacagaacg ctatgtttaa tgtattaata 1320
tatgattttt tttttccatc casatgttcc acaatgtaca attcaagagt ttcatttcat 1380
tttaatatac aaaaattcca ttgagaagat aatgcagtga taggctcagt tcattctttt 1440
tcaaggtctt tgctggacgt gagcgctgct gcgttccctg gcacacatgg caaaactctc 1500
actcagcctt tttagccttc ataacccccc accccccgac ccccctaact ttcaatcctc 1560
caggcttata tggagaccta tatatggggg aggaggaggg gtctacgaga gagggccagg 1620
ccacagctgc caccccatct cagatgacgg aaatgtaaat gcaggacctg tttcgtaagc 1680
taaactgggt atcagagatg tgccctgtcc aatcgcagtc catcacgctc taaaatggac 1740
ctctggagta agcagtatat aaagctgaac tgaacccttc tccgctggtg acccctatt 1799
<210>33
<211>272
<212>DNA
<213>Oncohynus nerka
<220>
<221>misc feature
<222>(0)...(0)
＜223〉salmon metallothionein promoter
<400>33
taaataaata taggtgtagc cttaattaat cgatgatcaa cgtggtaatc aggtttatgt 60
aacaggctat ggaatttgga aacaatagga aactcttcct tgattatttt cgcgcagtat 120
aatgaaataa cccgggtgca aaccctgatc gtctgaacgc gagactgttt tgcacacggc 180
acccgtctgt ccctgacgct ataaaaacgg tcttcgccaa agagaaattt aaagcttaca 240
actcacaagt gaaattgagc tgaaatactt ca 272
<210>34
<211>470
<212>DNA
<213>Oncorhynus nerka
<220>
<221>misc_feature
<222>(0)...(0)
＜223〉salmon histone H 3 promotor
<400>34
gtaaaatcgc tggtgcggct gcaacttgac tactcaaccc ccaaaggctc ttttaagagc 60
caaccacctg gctcagccaa aaaagcagtg tcctctctct ctatggctgg ccaactattt 120
ggcgtgtttg ttaaatacac acacatatac acggcacagt atcaagtgcc cacatgaggc 180
ctacatgaag aataacaact actaggctaa aatgaagaga agcgttattg cccgtaaagt 240
gtaacgttgc tcgcggccct aacaaaagaa ccaagcagcg cctcggcgag ggatgggggt 300
tgcattttgg ggcgtcacgg agaggtccga gcctcccgtc caatgggcgg aggaggcctc 360
cgcaacgggc caatcagggc ggtgcggaga tggtgaccaa tcagcagacg ccgctgccgg 420
ctttataaac ttcacatagg catttggagg ctatactccg actgtgaaag 470
<210>35
<211>1997
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the former B encoding sequence of attacin before carp beta-actin promotor connects
<400>35
ggtaccgggc cccccctcga ggtcgacggt atcgataagc ttgatatcga attgggtttg 60
atgaaaatcg cttaggcctt gctcttcaaa caatccagct tctccttctt tcactctcaa 120
gttgcaagaa gcaagtgtag caatgtgcac gcgacagccg ggtgtgtgac gctggaccaa 180
tcagagcgca gagctccgaa agtttacctt ttatggctag agccggcatc tgccgtcata 240
taaaagagcg cgcccagcgt ctcagcctca ctttgagctc ctccacacgc agctagtgcg 300
gaatatcatc tgcctgtaac ccattctcta aagtcgacaa acccccccaa acctaaggtg 360
agttgatctt taagcttttt acattttcag ctcgcatata tcaattcgaa cgtttaatta 420
gaatgtttaa ataaagctag attaaatgat taggctcagt taccggtctt ttttttctca 480
tttacgtgcg aactctgctt aaactctagt tattctttat taatatgtgg ttatttttat 540
atatgtatgt tatcataact gtactggcta tgtcaggtgg taatgactgt aacgttacgt 600
tactcgttgt aggcacgaca ttgaatgggc cggtgttgaa ataagtcttc aacccctttt 660
aacctcaaaa tgtgctctgg ttaacaagga ttttaacagc tatcagtatg actgtgcggt 720
tttaaagccg ttagtgaggc acgttgcaca cttgatggat ggccggaatg ggaagttctt 780
tatgcaggca gtgctgcgca gggtgtgacc tactttagct aacgttagcc ggctaaccag 840
cattcatctg ccggtaactt gagtctaata ttctctatgt gatatcgaag tgatcaaaga 900
cacgtctgtt agctcacttt aaccaactgt agtgaaaaat agcgcagtgt gcagcccttc 960
aagtctttca tttaggcttt attcaatcat tttattaact attaacgcgt tactaaacgt 1020
aaggtaacgt agtcagtttt taataactgg tgaaaagtac tggttgggtt taaatggtga 1080
cttataattg tgttggaggg ggaaaccttt ttgataaagg ctatataatc tcaaatgaat 1140
gggctgagga tggtgttcac aggtgcttta gtgaagtccg ctcgtgaaga gtcgctgaag 1200
tgactgcaga tctgtagcgc atgcgttttg gcagacggcc gttgaaattc ggttgagtaa 1260
ttgataccag gtgaggctag aggatgtaga aattcatttg tgtagaattt agggagtggc 1320
ctggcgtgat gaatgtcgaa atccgttcct ttttactgaa ccctatgtct ctgctgagtg 1380
ccacaccgcc ggcacaaagc gtctcaaacc attgcctttt atggtaataa tgagaatgca 1440
gagggacttc ctttgtctgg cacatctgag gcgcgcattg tcacactagc acccactagc 1500
ggtcagactg cagacaaaca ggaagctgac tccacatggt cacatgctca ctgaagtgtt 1560
gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta cagttcagcc 1620
aaggcccgat ccggcaccat gaatttcagc agaatcttct tcttcgtgtt cgccctcgtg 1680
ctcgccctct ctaccgtgag cgccgcccca gaaccaaaat ggaaagtgtt caaaaaaatc 1740
gagaaaatgg gaagaaatat cagaaatgga atcgtgaaag ccggaccagc catcgctgtg 1800
ctcggagaag ccaaagccct ctagagggcc ctattctata gtgtcaccta aatgctagag 1860
ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 1920
ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 1980
aaattgcatc ggccgcc 1997

Claims (17)

1. the synthetic promoter that in fish cell, has function, wherein said synthetic promoter is made up of the nucleotide sequence that is selected from down group:
A) nucleotide sequence shown in SEQ ID NO:3 or the SEQ ID NO:8;
B) has one or more non-essential amino acid residues place conservative property alternate nucleotide sequence at the nucleotide sequence shown in SEQ ID NO:3 or the SEQ ID NO:8.
2. expression cassette, it comprises the described synthetic promoter of claim 1 that is connected with purpose nucleotide sequence operability with suitable reading frame.
3. a carrier wherein comprises the described expression cassette of claim 2.
4. a host cell is stablized in its genome and has been mixed the described expression cassette of claim 2.
5. an expression cassette wherein is included in the promotor that function is arranged in the fish cell, and described promotor is the described synthetic promoter of claim 1;
Wherein said promotor links together with the nucleotide sequence operability of suitable reading frame with the disease-resistant originality polypeptide of coding purpose, and the disease-resistant originality polypeptide of described purpose is selected from following group:
A) mature form of attacin polypeptide;
B) the preceding former form of described attacin polypeptide;
C) the substance form of described attacin polypeptide;
D) fusion polypeptide of forming by the substance form of catfish immunoglobulin heavy chain variable region (Ig Vh) leader sequence and described attacin polypeptide;
E) fusion polypeptide of forming by the mature form of catfish immunoglobulin heavy chain variable region (Ig Vh) leader sequence and described attacin polypeptide;
F) have disease-resistant originality activity and with a), b), c), d) or e) described in one or more non-essential amino acid residues place of polypeptide carry out the polypeptide of conservative amino acid substitutions.
6. the expression cassette of claim 5, wherein said attacin polypeptide is cecropin B polypeptide or attacin P1 polypeptide.
7. the expression cassette of claim 6, the mature form of wherein said cecropin B polypeptide has the aminoacid sequence shown in the SEQ ID NO:19.
8. the expression cassette of claim 7, wherein said disease-resistant originality polypeptide are coded by the nucleotide sequence that is selected from down group:
A) nucleotide sequence of forming by sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21 or the SEQ ID NO:23; And
B) has one or more non-essential amino acid residues place conservative property alternate nucleotide sequence with nucleotide sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21 or the SEQ ID NO:23.
9. the expression cassette of claim 6, the mature form of wherein said attacin P1 polypeptide has the sequence shown in the SEQ ID NO:26.
10. the expression cassette of claim 5, wherein said disease-resistant originality polypeptide are coded by the nucleotide sequence that is selected from down group:
A) nucleotide sequence of forming by sequence shown in SEQ ID NO:25 or the SEQ ID NO:27; And
B) have one or more non-essential amino acid residues place conservative property alternate nucleotide sequence with nucleotide sequence shown in SEQ ID NO:25 or the SEQ ID NO:27.
11. the expression cassette of claim 5, wherein d) described in fusion polypeptide form by the aminoacid sequence shown in the SEQ IDNO:22.
12. the expression cassette of claim 5, wherein e) described in fusion polypeptide form by the aminoacid sequence shown in SEQ IDNO:24 or the SEQ ID NO:28.
13. a carrier wherein comprises the described expression cassette of claim 5.
14. a host cell, the stable described expression cassette of claim 5 that mixed in its genome.
15. method of in host fish cell, expressing desired polypeptides, described method comprises in this host fish cell and imports expression cassette, described expression cassette comprises the functional synthetic promoter that is connected with the nucleotide sequence operability of the described desired polypeptides of coding with suitable reading frame, and described promotor comprises the nucleotide sequence that is selected from down group:
A) nucleotide sequence shown in SEQ ID NO:3 or the SEQ ID NO:8;
B) has one or more non-essential amino acid residues place conservative property alternate nucleotide sequence with nucleotide sequence shown in SEQ ID NO:3 or the SEQ ID NO:8.
16. the described method of claim 15, wherein said desired polypeptides are disease-resistant originality polypeptide, described host fish cell is the catfish cell.
17. the cultural method of a transgenosis catfish, comprise and to hybridize according to the catfish that described method obtains and produce the step of transgenic progeny, wherein said method comprises in the described expression cassette importing of the claim 5 catfish ovum, cultivate this catfish ovum then under the condition that is suitable for the described catfish maturation of ovum and growth or catfish, wherein said catfish is selected from down in the group: spot fork-tail silver xenocypris, blue fork-tail silver xenocypris or spot fork-tail silver xenocypris-blue fork-tail silver xenocypris hybridization catfish.
CNB2003801083571A 2002-11-06 2003-11-06 Compositions and methods for enhancing disease resistance in fish Expired - Fee Related CN100371443C (en)

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US5998698A (en) * 1995-06-07 1999-12-07 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Transgenic fish capable of expressing exogenous lytic peptides
CN1154970A (en) * 1996-01-16 1997-07-23 中国农业科学院生物技术研究中心 Antibiotic peptide and production method and application thereof

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