CN1774507A - Nucleic acid controlling the expression of a useful polypeptide in the posterior silk glands of a lepidoptera and application thereof - Google Patents
Nucleic acid controlling the expression of a useful polypeptide in the posterior silk glands of a lepidoptera and application thereof Download PDFInfo
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- CN1774507A CN1774507A CN 200480010261 CN200480010261A CN1774507A CN 1774507 A CN1774507 A CN 1774507A CN 200480010261 CN200480010261 CN 200480010261 CN 200480010261 A CN200480010261 A CN 200480010261A CN 1774507 A CN1774507 A CN 1774507A
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Abstract
The invention relates to a nucleic acid controlling the expression of a useful polypeptide, particularly in the posterior silk glands of Bombyx mori . The invention further relates to an expression cassette comprising a useful polynucleotide placed under the control of a nucleic acid such as defined above. The invention also relates to an integration recombinant vector, comprising a nucleic acid or an expression cassette as defined above and a host cell, modified by integration of said nucleic acid or said cassette in the genome thereof.
Description
The present invention relates to the proteinic synthetic and secretion of industrial purposes, as be used for the fibrous protein of textiles, particularly is new silk or the protein with biomedical applications.Relate to polynucleotide of interest and/or polypeptide in some animal tissues and the more properly expression of the special target in the product sericterium of silkworm (Bombyx mori).According to the present invention, the product sericterium of silkworm can be as can producing a large amount of protein and it outputed to " bio-reactor " of a cocoon, can be from the silk cocoon these protein of purifying.
Prior art
Existed multiple generation to be used for the method for the fibrous protein of textiles, for example the mammary gland by transgenic goat produces spider silk fiber method of protein (Nexis Biotechnologies, Inc.1000 St-Charles Avenue, Bloc B, Vaudreuil-Dorion, QC, J7V 8PV, Canada).Yet these methods can not make fibrous protein be organized into the form of line.As for the production of the soluble protein that biomedical applications is arranged, the production of some known protein matter means carries out that microbial culture, clone are cultivated or vertebrates such as milk cow, goat or even the breed of transgene rabbit.This of recombinant protein type production is very expensive.On the other hand, when using the synthetic recombinant protein of vertebrates system, it is not contain to pass to human pathogenic contaminants that their industrial use particularly must relate to the composition that confirms these protein in advance or comprise them in food or purposes pharmaceutically.
Consider the important economic function of recombinant protein production, in technical field, there is demand to novel method, described method promptly is used for the method that the enough remote organism of a large amount of low costs and use and human species produces recombinant protein, described organism can avoid pollution problem, mainly be when protein be when being used for biomedical purpose.Also have the demand to the novel method of producing recombinant protein, when described recombinant protein when being fibrous, described novel method makes can organize described protein with the form of line.In order to obtain high yield, protein is must be in tissue special synthetic and output to animal, the outside of preferred invertebrates.
Yet, according to the knowledge of the applicant, also do not have invertebrates to make a response to the demand.For example, transgenic bombyx mori is well-known (Tamara etc., Germline transformation ofthe silkworm Bombyx mori L.using a piggyBac transposon derived vector.Nature Biotechnology 18 volumes, 2000), however utilize this method can't make the recombinant protein mass production become possibility.
Therefore, but the applicant is devoted to research and develop the expression system that particularly can overcome previously known security of system expensive shortcoming.
Summary of the invention
The invention provides the recombinant protein expression system of the type now.
The present invention relates to instruct target protein matter behind silkworm, to produce the nucleic acid of specifically expressing in the silk gland cell, wherein said nucleic acid from 5 ' terminal to 3 ' end comprise (i) comprise be used for polynucleotide of interest the regulatory region of the conditioning signal of described product silk gland cell specifically expressing and (ii) coding be in the zone of the modified signal peptide under the described regulatory region control, among wherein said nucleic acid and the sequence SEQ ID № 1 Nucleotide 1150 to the polynucleotide of Nucleotide 2026 on Nucleotide, exist at least 90% identity and wherein the Nucleotide 1486 of sequence SEQ ID № 1 encode to the trinucleotide of Nucleotide 1488 and be selected from L-Ala, Isoleucine and a leucic amino acid.
The invention still further relates to and comprise the expression cassette that is in the polynucleotide of interest under the nucleic acid defined above control, polynucleotide of interest is the adopted polynucleotide of having of coded polypeptide.
The invention still further relates to recombination and integration carrier that comprises nucleic acid defined above or expression cassette and the host cell of being modified by described nucleic acid of integration or described expression cassette in its genome.In addition, the objective of the invention is nucleic acid defined above, expression cassette, recombinant vectors or the recombinant host cell purposes in obtaining transgenic nonhuman animal.
The invention still further relates to and obtain by in its genome, integrating the method for the transgenic nonhuman animal that nucleic acid defined above, expression cassette, recombinant vectors or recombinant host cell modified.
The invention still further relates to guidance and can reduce the nucleic acid of fibroin H synthetic purpose RNA specifically expressing in the back product silk gland cell of silkworm, described nucleic acid comprises to contain and is useful on the regulatory region of polynucleotide of interest at the conditioning signal of described product silk gland cell specifically expressing, and the Nucleotide 1150 of described nucleic acid and sequence SEQ ID № 1 to the polynucleotide of Nucleotide 1452 have at least 90% Nucleotide identity.
For the part of " additional nucleic acid of the present invention " nucleic acid such as above-mentioned nucleic acid is described in further detail at title.
Detailed Description Of The Invention
According to the present invention, known nucleic acid comprises at least one copy that (i) comprises the target nucleotide motif of SGF1 " jaw (forkhead) " type transcription factor, with the regulatory region in suitable transcripting promoter site and the zone of the modified signal peptide of (ii) encoding, described zone is placed under the control of described regulatory region, when the nucleotides sequence of the described modified signal peptide of the nucleotide sequence of coding target protein matter and coding was listed in " homophase " and merges, described nucleic acid can instruct target protein matter special synthesizing in back the products silk cell of lepidopterous insects silkworm.
According to the present invention, when 3 ' terminal Nucleotide of last codon of the sequence that is positioned at the modified signal peptide of coding had been positioned at before the 5 ' terminal Nucleotide of first codon of sequence of coding target protein matter, the nucleotide sequence of coding target protein matter was that " homophase " merges with the nucleotide sequence of the described modified signal peptide of coding.
Be astoundingly, the applicant pointed out with the codon of the halfcystine in the former fibrohexamerin signal peptide of the preceding protein that is present in silk yarn fibre with hydrophobic amino acid for example L-Ala, Isoleucine or even leucine replace and can make the protein that merges with described modified signal peptide output to silkworm to produce outside the sericterium as the composition protein of silk thread.
Purpose of the present invention relates to the nucleic acid that instructs target protein matter to produce the silk gland cell specifically expressing behind silkworm, wherein said nucleic acid from 5 ' terminal to 3 ' end comprise (i) comprise be used for polynucleotide of interest the regulatory region of the conditioning signal of described product silk gland cell specifically expressing and (ii) coding be in the zone of the modified signal peptide under the described regulatory region control, the Nucleotide 1150 among wherein said nucleic acid and the sequence SEQ ID № 1 to the polynucleotide of Nucleotide 2026 on Nucleotide, have at least 90% identity and wherein the Nucleotide 1486 of sequence SEQ ID № 1 encode to the trinucleotide of Nucleotide 1488 and be selected from L-Ala, Isoleucine and a leucic amino acid.Preferably, any nucleic acid of the present invention and any polypeptide exist with the form of isolating or purifying.
Term in implication of the present invention " isolating " is defined as the biomaterial that extracts from its initial environment (environment of its natural discovery).For example, the polynucleotide that are present in organism with native state are not isolating.From natural be inserted into the organism genome to close on the identical polynucleotide that nucleic acid separates be isolating.This type polynucleotide can be included in the carrier and/or this kind polynucleotide can be included in the composition and still keep separate stage, and this is because carrier or composition do not constitute its natural surroundings.Identical reasoning also is applicable to the polypeptide that extracts from its natural surroundings.
Term " purifying " does not require that material exists with the absolute pure form that does not have other compound to exist.It is a relative definition.At starting material or at least one order of magnitude of natural materials purifying, behind preferred 2 or 3 and preferred 4 or 5 orders of magnitude, polynucleotide or polypeptide are in purified state.
For the present invention, word " nucleotide sequence " can not refer to polynucleotide or nucleic acid with making any distinction between.Word " nucleotide sequence " comprises genetic material itself and therefore is not limited to the relevant information of its sequence.Term " nucleic acid ", " polynucleotide ", " oligonucleotide " or even " nucleotide sequence " comprise DNA, RNA, cDNA sequence or even the heterozygosis RNA/DNA sequence of more than Nucleotide, no matter they are single stranded form or duplex form.
Term " Nucleotide " refers to natural nucleotide (A, T, G, C and U).
For the present invention, when the complementary base of each base of article one Nucleotide second polynucleotide opposite with direction is matched, think that article one Nucleotide and second Nucleotide are " complementations ".Complementary " base " is A and T (perhaps A and U) and C and G.
According to the present invention, on amino acid levels, have at least 90% with reference to nucleic acid with reference to article one nucleic acid and the described second that nucleic acid has at least 90% identity with second, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3%, 98.6%, 99%, 99.6% identity." identity percentage ratio " between two nucleotide sequences in the implication of the present invention is by at optimum ratio following two sequences being compared definite and can observe by comparison window.
Right for the optimum ratio that obtains between the two sequences, the part of the nucleotide sequence in comparison window can comprise with respect to reference sequences (it does not comprise insertion or disappearance) to be inserted or disappearance (for example " breach ").In order to obtain the identity percentage ratio of the Nucleotide between the two sequences, by determining in the two sequences that is compared, to observe the position number of identical nuclear base, with two sums of examining the number of the identical position of base divided by position in the comparison window, then the result be multiply by 100 and calculate identity percentage ratio then.
It is right that the method that uses a computer can be carried out optimum ratio to the sequence that is compared by well-known algorithm.
In the mode that is more preferably, use CLUSTAL W software (version 1.82) to adopt the following identity percentage ratio that is provided with to determine sequence: (1) cpu model=ClustalW mp; (2) comparison=" entirely "; (3) output format=" aln w/ number "; (4) output order=" alignment "; (5) colored comparison=" no "; (6) KTUP (word size)=" default "; (7) length of window=" default "; (8) score value type=" percentage ratio "; (9) TOPDIAG=" default "; (10) pairing breach=" default "; (11) phylogenetic tree/tree type=" nothing "; (12) matrix=" default "; (13) breach is opened=" default "; (14) finish breach=" default "; (15) breach extends " default "; (16) breach distance=" default "; (17) tree type=" evolutionary tree " and (18) tree RAP distance=" hiding ".
The nucleic acid that has at least 90% Nucleotide identity with nucleic acid of the present invention comprises " variant " of nucleic acid of the present invention.
" variant " of nucleic acid of the present invention is defined as with respect to reference nucleic acid has one or more nucleotide substitutions, adding or disappearance and the nucleic acid different with reference nucleic acid.The variant of nucleic acid of the present invention can be for example naturally occurring allele variant of natural origin.This kind nucleic acid variant also can be the non-natural nucleic acid that for example obtains by mutafacient system.
Usually, has quite similar nucleotide sequence by reference nucleic acid and variant nucleic acid and many identical regional this modes make the difference between reference nucleic acid and the variant nucleic acid reduce.
Regulon nucleic acid variant of the present invention comprise with sequence SEQ ID № 1 in Nucleotide 1379 have the complete nucleotide sequence of 100% Nucleotide identity to the zone of Nucleotide 1390.In addition, the trinucleotide correspondence of adjusting nucleic acid variant of the present invention Nucleotide 1486 among the sequence SEQ ID № 1 and is selected from L-Ala, Isoleucine and a leucic amino acid to the trinucleotide and the coding of Nucleotide 1488.
Therefore, in regulon nucleic acid of the present invention, the trinucleotide below 1488 the trinucleotide from Nucleotide 1486 to Nucleotide of sequence SEQ ID № l can be selected from:
(i) trinucleotide of Xia Mian coded amino acid L-Ala: GCU, GCT, GCC, GCA and GCG;
(i) trinucleotide of Xia Mian coded amino acid Isoleucine: AUU, ATT, AUC, ATC, AUA and ATA; With
The (iii) leucic trinucleotide of Xia Mian coded amino acid: CUU, CTT, CUC, CTC, CUA, CTA, CUG and CTG.
The variant of adjusting nucleic acid of the present invention has been preserved and has been instructed the specific purpose polynucleotide to produce the ability of expressing in the silk gland cell behind lepidopteron.In order to confirm nucleic acid of the present invention " variant ", comprise the functional character that has the nucleic acid of 90% Nucleotide identity with sequence SEQ ID № 1, those skilled in the art can be implemented in the specifically expressing test of report described in the example or marker gene.
" polynucleotide of interest " in implication of the present invention is defined as and can instructs polypeptide fusion synthetic polynucleotide between modified signal peptide and the desired polypeptides.
Nucleic acid of the present invention comprises the sequence with promoter function, and described promotor has constituted the conditioning signal that can instruct polynucleotide of interest to produce specifically expressing in the silk gland cell behind lepidopteron.
For this specification sheets, nucleic acid with " promotor " function also be called " promotor " or even " promoter sequence ", comprise and have RNA polymerase identification motif and the nucleic acid of " TATA " box and " CAAT " box more in particular, their structure is well-known for the expert of the art.
The present invention show nucleic acid sequence SEQ ID № 1 comprises for polynucleotide of interest produce the necessary adjusting sequence of specifically expressing in the silk gland cell behind silkworm.This specific specificity is at Tamura (Tamura etc., Germline transformation of the silkworm Bombyxmori L.using a piggyBac transposon derived vector.Nature Biotechnology18 volume, 2000) do not observe in the adjusting sequence of Miao Shuing, its expression is not limited to produce silk gland cell behind the silkworm.The present invention shows that also the nucleic acid from 5 ' the terminal Nucleotide 1150 that comprises sequence SEQ ID № 1 to 3 ' end to the polynucleotide of Nucleotide 2026 can instruct polynucleotide of interest to produce specifically expressing in the silk gland cell behind silkworm.
Sequence SEQ ID № 1 comprises, from 5 ' terminal to 3 ' end, and the promotor of 1451 fibrohexamerin gene from Nucleotide 1 to Nucleotide.This promotor comprises " TATA " box that is arranged in 1423 Nucleotide of the 1420th Nucleotide to the of sequence SEQ ID № 1.This promotor also comprises and is arranged in the 1252nd Nucleotide of sequence SEQ ID № 1 as far as " CAAT " of the 1365th Nucleotide box.Therefore this promoter sequence comprises the sequence " TATTTATTTAA " that is arranged in 1390 Nucleotide of the 1397th Nucleotide to the of sequence SEQ ID № 1.The TATA box has been formed promoter element itself, and it is usually located at apart from the place of about 30 bases of transcription initiation site.CAAT box is a cis-acting elements of finding in promotor and activation subregion (" enhanser ") usually.Sequence SEQID № 1 comprises the fibrohexamerin exons 1 of the modified signal peptide of coding of the 1452nd Nucleotide to 1525 Nucleotide.
Sequence SEQ ID № 1 comprises the fibrohexamerin introne 1 of the 1526th Nucleotide to 2026 Nucleotide.Sequence SEQ ID № 1 comprises the polynucleotide of the 2027th Nucleotide to 2040 Nucleotide.Back one polynucleotide are unessential for the present invention and only are the structures that the extra coding that helps being included in the 2041st Nucleotide and the 2721st Nucleotide is reported the sequence SEQ ID № 1 of sub-protein " Ds-Red " Nucleotide.
Another object of the present invention be comprise the 1st Nucleotide of (i) sequence SEQ ID № 1 as far as the regulation domain of the 1451st Nucleotide or contain as hereinbefore defined TATA, CAAT box and at least one " CTATTTATTTAA " sequence this nucleic acid bioactive fragment and (ii) coding place the nucleic acid in the zone of the modified signal peptide under the control of this kind nucleic acid.
The present invention has shown that as hereinbefore defined the exons 1 of fibrohexamerin has among the sequence SEQ ID № 1 the 1486th and modifies to 1488 trinucleotide, so that be correct by the selection of polynucleotide of interest encoded polypeptides; And so that can secrete with silk thread by the polynucleotide of interest encoded polypeptides.The the 1486th to 1488 trinucleotide of sequence SEQ ID № 1 encoding aminothiopropionic acid must be modified into coding and be selected from L-Ala, Isoleucine and a leucic amino acid whose trinucleotide.Because to finishing that the DNA that comprise all or most of restricted parts among the sequence SEQ ID № 1 make up, the present invention has shown that nucleic acid of the present invention makes and has instructed polynucleotide of interest to produce behind silkworm that expression becomes possibility in the silk gland cell specifically.Ds-Red report subbase because of existence make that can prove that the modification of fibrohexamerin exons 1 makes can obtain correct secretion by the polynucleotide of interest encoded polypeptides.
Expression cassette comprises coding report subbase because of being the polynucleotide of interest of Ds-Red protein gene, and this expression cassette is placed in the whole of as hereinbefore defined the exons 1 that comprises specific tissue's conditioning signal, modified fibrohexamerin, fibrohexamerin introne 1 and has under the control of adjusting nucleic acid of promoter function.
These expression cassettes are used to transform the cell of silkworm, more accurately are to use the gamete precursor of transgenic animal, and reporter gene is expressed at the different tissues of animal afterwards.The result shows to transcribe with the active necessary conditioning signal of strongly expressed for the specific tissue that produces silk gland cell behind silkworm and is included in particularly sequence SEQ ID № 1 Nucleotide 1150 in the zone of Nucleotide 2026.
Speak by the book more, the present invention has shown that this zone comprises the motif " CTATTTATTTAA " (sequence SEQ ID № 2) of 1390 Nucleotide of the 1379th Nucleotide to that are positioned at sequence SEQ ID № 1.This motif has been formed the binding site (Julien of SGF1 jaw type transcription factor, E-, Bordeaux, M.C., Garel, A. and Couble, P. (2002) Fork head alternativebinding drives stage specific expression in the silk gland of Bombyx mori.Insect Biochem Molecul.Biol.32,377-387.).
Be reluctant to be subjected to any theoretical institute to fetter, the contriver believes that motif " CTATTTATTTAA " formed the important structure feature of nucleic acid of the present invention, and it has transcription sequence activation subfunction (" enhanser ") and specific tissue's expression regulators function.
According to the present invention, the new motif " CTATTTATTTAA " of the natural motif upstream that comprises makes that not changing the expression rate that increases by the polynucleotide of interest encoded polypeptides under the specific situation be possible in the fibrohexamerin promotor.Enter the DNA that makes the piggyBac integrative vector carry in the ovum of animal by microinjection and be incorporated in the domestic silkworm gene group, this makes and shows that in unexpected mode the modification of the exons 1 of fibrohexamerin gene makes that the addressing (addressing) of available target protein matter and this target protein matter are compatible with the silky fibre secretion.In other words, 3 ' terminal when same-phase merges when polynucleotide of interest and fibrohexamerin gene extron 1, the modification of this gene extron 1 of the modified signal peptide of encoding makes target protein matter equally keeping secretion with fibrohexamerin.
According to the present invention, the motif of sequence SEQ ID № 10 or SEQ ID № 11 can be incorporated on the promotor of fibrohexamerin so that Dimerized " CTATTTATTTAA " motif and therefore do not changing the expression rate that improves the polynucleotide of interest encoded polypeptides under the specific condition.Each comprises " CTATTTATTTAA " motif in the natural nucleus glycoside acid environment of being in of above having narrated sequence SEQ ID № 10 and SEQ ID № 11.
Other is expressed and activates subsequence, no matter homology or allos can be incorporated in the fibrohexamerin promotor.As an example, people can quote well known by persons skilled in the art corresponding to yeast Gal4/UAS system sequence and be not described in detail.People also mentioned corresponding to the amplicon of silkworm kytoplasm actin gene (Mange A.Julien E.Prudhomme JC. and Couble P.A strong inhibitory element down-regulates BRE-stimulatedtranscription of the A3 cytoplasmic actin genes f Bombyx mori.J Mol Biol.1997 January 24; 265 (3): 266-74.Fatyol K., Illes K. and Szalay A.A.Analternative intronic promoter of the Bombyx A3 cytoplasmic actin geneexhibits a high level of transcriptional activity in mammalian cells; MolGen Genet.19993 month .261 (2): 337-45).At last, people can mention amplicon system corresponding to baculovirus IE1/Hr5 or Hr3 (Lu M., Farrell P.J., Johnson R. and latrou K.J Biol Chem.1997 December 5; 272 (49): 30724-8).
Adjusting nucleic acid of the present invention
Purpose of the present invention relates to the nucleic acid that instructs target protein matter to produce the silk gland cell specifically expressing behind silkworm, wherein said nucleic acid from 5 ' terminal to 3 ' end comprise (i) comprise be used for polynucleotide of interest the regulatory region of the conditioning signal of described product silk gland cell specifically expressing and (ii) coding be in the zone of the modified signal peptide of described regulatory region under controlling, the Nucleotide 1150 among wherein said nucleic acid and the sequence SEQ ID № 1 to the polynucleotide of Nucleotide 2026 on Nucleotide, exist at least 90% identity and wherein the Nucleotide 1486 of sequence SEQ ID № 1 can encode to the trinucleotide of Nucleotide 1488 and be selected from L-Ala, Isoleucine and a leucic amino acid.
According to first embodiment, particularly be according to nucleic acid above-mentioned, described nucleic acid be characterised in that it from 5 ' terminal to 3 ' end comprise (i) comprise be used for polynucleotide of interest the regulatory region of the conditioning signal of described product silk gland cell specifically expressing and (ii) coding be in the zone of the modified signal peptide under the described regulatory region control, described nucleic acid comprises sequence SEQ ID № 1 from 5 ' end to 3 ' end Nucleotide 1150 to the polynucleotide of Nucleotide 2026 and wherein the Nucleotide 1486 of sequence SEQ ID № 1 can encode to the trinucleotide of Nucleotide 1488 and be selected from L-Ala, Isoleucine and a leucic amino acid.
Second embodiment according to above-mentioned nucleic acid, described nucleic acid be characterised in that it from 5 ' terminal to 3 ' end comprise (i) comprise be used for polynucleotide of interest the regulatory region of the conditioning signal of described product silk gland cell specifically expressing and (ii) coding be in the zone of the modified signal peptide under the described regulatory region control, described nucleic acid comprises sequence SEQ ID № 1 from 5 ' end to 3 ' end Nucleotide 1 to the polynucleotide of Nucleotide 2026 and wherein the Nucleotide 1486 of sequence SEQ ID № 1 can encode to the trinucleotide of Nucleotide 1488 and be selected from L-Ala, Isoleucine and a leucic amino acid.
According to the 3rd specific embodiment, nucleic acid of the present invention comprises nucleic acid as described above, and wherein said nucleic acid is modified by sequence SEQ ID № 2 polynucleotide that import at least one and be less than 4 copies between sequence SEQ ID № 1 Nucleotide 1 and 1379.For a copy of calling sequence SEQ ID № 2 polynucleotide, people can be inserted into sequence SEQ ID № 10 or SEQ ID № 11 Nucleotide 1 and 1379 and particularly between the Nucleotide 1378 and 1379 of sequence SEQ ID № 1.The invention still further relates to its sequence and defined any nucleic acid complementary nucleic acid above.
As stating, one of purpose of the present invention is to obtain producing the intracellular high speed of silk by the polynucleotide of interest encoded polypeptide behind silkworm to express.
In addition, the objective of the invention is to comprise the nucleic acid of polynucleotide of interest of desired polypeptides of encoding, it merges mutually with the defined regulon nucleic acid that instructs desired polypeptides to produce the silk cell expressing specifically behind silkworm of this specification sheets.For the present invention, this kind comprised the nucleic acid called after " expression cassette " of the polynucleotide of the desired polypeptides of encoding.
In addition, the objective of the invention is to comprise the expression cassette of the polynucleotide of interest under the regulon nucleic acid control that is in as hereinbefore defined.
Expression cassette of the present invention
According to first embodiment, expression cassette of the present invention is characterised in that the polynucleotide of interest coded polypeptide that it comprises.
Alternatively, the polynucleotide of interest fusion polypeptide of can encoding, described fusion polypeptide comprises from N-end to C-end:
At least 172-terminal amino acids (Tanaka K. of-fibroin L peptide sequence, Kajiyama N., Ishikura K., Waga 5., I ikuchi A, Ohtomo K., TakagiT. and Mizuno S, Determination of the site of disulfide linkagebetween heavy and light chains of silk fibroin produced byBombyx mori.Biochimica et Biophysica Anta 1432 (1999) 92-103)) and
-desired polypeptides.
Preferably, desired polypeptides is selected from the spider's thread protein of spider for example (spider belongs to (Nephila)), Galleria fibroin or any other polypeptide that more usually can silky fibre, for example, the ductility intensity or the elastic properties of described silky fibre are modified.
Polynucleotide of interest can also be encoded and be had the soluble polypeptide of biomedical purpose, for example hormone, antigen, enzyme, somatomedin even acceptor.
Particularly, the polynucleotide of interest human interleukin-12 of can encoding.
Expression cassette of the present invention can be at one end or each end surround by insulator, all can express no matter described insulator is intended to make polynucleotide of interest to be incorporated into what position.As suiting of insulator, can mention gypsy insulator (Gerasimova T.I. and the Corces V.G.:Polycomb and trithorax group proteins mediate the function of achromatin Insulator.Cell 1998 of fruit bat (Drosophila), 92:511-521), scs/scs ' (Kellum R. and SchedlP.1991 A position effect assay for boundaries of higher orderchromosomal domains.Cell 64:941-950) or even 5 ' HS4 insulator (Chung J.H. of chicken beta globin genes, Bell A.c. and Felsenfeld G. (1997) Characterization ofthe chicken beta-globin insulator.Proc.Nat.Acad.Sci, USA 94:575-580).
Recombinant vectors of the present invention
The present invention relates to contain the regulon nucleic acid of definition as mentioned or the recombination and integration carrier of expression cassette.Therefore the present invention comprises recombinant vectors, wherein inserted the regulon nucleic acid of the present invention that comprises conditioning signal, polynucleotide of interest produced the silk gland cell expression specifically when this regulon nucleic acid made under these polynucleotide being placed its control behind silkworm becomes possibility.
Part of the present invention also comprises the recombination and integration carrier, and it comprises
A) as the defined regulon nucleic acid of specification sheets of the present invention and
B) place a) polynucleotide of interest under the regulon nucleic acid control of definition.
Polynucleotide of interest comprises coding can detect polypeptide or labeling polypeptide, for example the polynucleotide of the proteinic polypeptide of encoding D s-Red.
With the corresponding recombination and integration carrier of the present invention of top definition be that the preserving number that was deposited in the state-run microbial preservation center (CNCM) of Institute Pasteur (Institut Pasteur) on February 20th, 2003 is the Pig fhx (sp of CNCM I-2975
*) DsRed-(3xP3-GFP).
The invention still further relates to and comprise the recombinant cloning vector of the nucleotide sequence of recombination and integration carrier as mentioned above.
Regulate sequence in order to obtain of the present invention any one, can utilize technology and primer described in the example.
The preserving number that the expert of the art can also utilize on February 20th, 2003 to be deposited in state-run microbial preservation center (CNCM) is the Pig fhx (sp of CNCM I-2975
*) DsRed-(3xP3-GFP)
The general feature of recombinant vectors of the present invention
" carrier " is defined as randomly cyclic DNA or the RNA molecule for strand or double chain form in the meaning of the present invention.Support according to the present invention can be cloning vector or integrative vector.
According to an embodiment preferred of the present invention, recombinant vectors of the present invention is the integrative vector that the copy of the dna sequence dna in this carrier can be inserted in the animal gene group of silkworm species.
Advantageously, recombinant vectors or, in other cases, the expression cassette that comprises in the carrier also can comprise the untranslated 3 ' sequence of known conduct " terminator ".Can be used in the middle of the terminator in the present invention's structure, can mention SV40 polyadenylation sequence.The preferred integrative vector of the present invention is the piggyBac transposon (Cary that exists with native state these species that obtains from cabbage looper (Trichoplusia ni), L, C, Deng, Transposon mutagenesis of baculoviruses:analysis ofTrichoplusia ni transposon IFP2 insertions within the FF-locus of nuclearpolyhedrosis viruses, Virology 172,156-69 (1989)).
The piggyBac transposon comprises the dna fragmentation that can move to another part based on swivel base from a genomic locus.The piggyBac transposon comprises right and a left side " foot ", and the described right side and a left side " foot " comprises the inverted repeats of two weak points, and the gene of itself and its locomotive function of encoding is in same frame.
The integrative vector that the present invention describes is through the piggyBac transposon of modifying, and wherein purpose nucleic acid has replaced the part of the gene of the removed transposon locomotive function of coding.
Integrative vector can be united use with assistant carrier, and described assistant carrier can make the jumping gene that is suppressed in integrative vector express.Integrative vector combines with assistant carrier and can nucleic acid of the present invention or expression cassette be incorporated in the genome of host cell based on swivel base.
Integrative vector of the present invention can also be from Hobo element of fruit bat or the minos transposon of fruit bat.
According to another embodiment, used recombinant cloning vector so that the nucleotide sequence of amplification integrative vector as described above.According to another embodiment, above-mentioned recombination and integration carrier inserts the cloning site of accepting carrier after linearizing or the described multidigit point of accepting carrier is linearized.Employed cloning vector is bacterium or viral source.These carriers are for example PUC, pBR or pSK type plasmid, and they are that the expert of the art is known but need not be described in detail.
Recombinant host cell of the present invention
For the polynucleotide of interest that is under the regulon nucleic acid of the present invention control can be expressed, in this manual Ding Yi regulon nucleic acid, expression cassette or even recombinant vectors must import in the host cell.Use the well-known method of expert of the art, can polynucleotide of the present invention be imported to host cell at external (in vitro)." host cell that comprises nucleic acid " is defined as the cell with the one or more copies that are incorporated into its genome amplifying nucleic acid.
In addition, another object of the present invention is to comprise above defined regulon nucleic acid, expression cassette or even the host cell of recombinant vectors.This host cell preferably belongs to silkworm moth and belongs to (Bombyx), more preferably is the zooblast of eggs of silkworm, and does not consider strain system.Host cell can also be to belong to other lepidopterous insects thing species, for example Philosamia cynthia (Philosamia cynthia) or be more generally that silkworm moth belongs to, tussah belongs to (Antheraea), galleria mellonella waxmoth and belongs to the zooblast that (Galleria), Philosamia cynthia belong to (Philosamia), Spodoptera (Spodoptera) or Drosophila.
More preferably, produce silk gland cell after host cell of the present invention comprises silkworm.
Transgenic animal of the present invention
In addition, nucleic acid, expression cassette, carrier or the recombinant host cell that the objective of the invention is as hereinbefore defined belongs to purposes in the transgenic nonhuman animal the silkworm moth that obtains producing silk.
Preferably, described transgenic animal belong to eggs of silkworm and secrete target protein matter with the silky fibre form.The invention still further relates to and comprise above defined one or more host cells, expression cassette or even the many cells transgenic organism of recombinant vectors.Further purpose of the present invention is a transgenic nonhuman animal, and it comprises the defined nucleic acid of this specification sheets or the expression cassette of integration form in its genome.
Usually, the invention still further relates to above defined regulon nucleic acid, expression cassette, recombinant vectors or even the purposes of host cell in obtaining transgenic animal.
The invention still further relates to the transgenic nonhuman animal of fibroin L hyposecretion, it comprises the expression cassette that is integrated into its genome form, and described expression cassette comprises the polynucleotide of interest of the fusion polypeptide of encoding, and wherein said fusion polypeptide comprises from N-end to C-end
At least 172-terminal amino acids of-fibroin L peptide sequence and
-desired polypeptides.
Fibroin L defective silkworm is known.Can mention Nd-s or Nd-sD mutant (Kazuyuki Mori as an example, Kazunori Tanaka, Yoshimi Kikuchi, Miho Waga, Shou Wage and Shigeki Mizuno:production of a chimeric fibroin light-chainpolypeptide in a fibroin secretion-deficient naked pupa mutant of thesilkworm Bombyx mori.J.Mol.Biol. (1995) 251,217-228.).
Even so, those skilled in the art can provide fibroin L the animal of defective by means of engineered routine techniques.
As an example, wish that those skilled in the art of the animal of realization fibroin L defective can produce mutagenesis in silk, then screen the animal of fibroin L defective.Mutagenesis can be to use the classical mutagenesis of UV or chemomorphosis or even the site-directed mutagenesis of fibroin L gene.
By cocoon being carried out the screening of RNA trace or western blotting realization to the animal of fibroin L defective.
Alternatively, those skilled in the art will recognize that the animal that can also produce fibroin L defective with the RNA perturbation technique.Usually, be three kinds of not homopolypeptides by the fibroin that produces the chamber generation of sericterium (gland) back: the mixture of heavy chain (fibroin H), light chain (fibroin L) and fibrohexamerin.
Is essential by disulfide linkage connecting fiber heart protein H and fibroin L for fibroin being secreted to lumen of gland.Different and this sudden change fibroin L that the C-zone of the fibroin L that produces by mutant homozygote Nd-s or Nd-sD and wild-type produce can not with fibroin H formation disulfide linkage (owing to the intron of the exon that comprises the halfcystine that participates in disulfide linkage recombinate eliminate this fact of disulfide linkage).This hyposecretion does not change the cocoon of being made up of sericin basically (compare fibroin with normal cocoon and be less than 0.3%) that viability still causes the eccrine growth of mutant silkworm to delay and generation is very thin of animal.
Therefore, the present invention relates to the transgenic nonhuman animal of fibroin L hyposecretion, it comprises the expression cassette that is integrated into its genome form, and described expression cassette comprises the polynucleotide of interest of the fusion polypeptide of encoding, and wherein said fusion polypeptide comprises from N-end to C-end
At least 172-terminal amino acids of-fibroin L peptide sequence and
-desired polypeptides.
Particular of the present invention makes can obtain transgenic animal, and in described transgenic animal, the amount of the target protein matter that is produced is to be subjected to the existence of the part of fibroin L in the fusion polypeptide and the control that lacks of the fibroin L that natural animal produces.
Kind and genus that transgenic animal of the present invention have been listed above preferably belonging to about animal host's origin of cell of the present invention.In addition, the objective of the invention is to comprise the cocoon of target protein matter, wherein the expression of target protein matter be instruct by above-mentioned nucleic acid, expression cassette or carrier and wherein target protein matter exist with the concentration that surpasses the 5ng/mg cocoon and be preferably more than the 70ng/mg cocoon.
Produced the 141mg cocoon that comprises 1-10 μ g interleukin II by transgenic animal of the present invention according to embodiment 7 described methods.
Another purpose is the method that obtains the transgenic animal of eggs of silkworm, it is characterized in that comprising following step:
A) before the blastoderm formation ovum is injected nucleic acid mentioned above, expression cassette or carrier in the back of laying eggs after 1-5 hour;
B) integration is described the transgenic animal of defined nucleic acid or expression cassette as this in the selection genome.
Another object of the present invention is the method that obtains the transgenic animal of eggs of silkworm, it is characterized in that comprising following additional step:
Two transgenic animal hybridization that c) will in step b), obtain;
D) select genetically modified homozygote animal.
Another object of the present invention is the method that obtains silky fibre, it is characterized in that comprising following step:
A) raise as the defined transgenic animal of this specification sheets;
B) reclaim the silk that transgenic animal produce.
Another object of the present invention is the method that obtains target protein matter, it is characterized in that comprising following step:
A) raise as the defined transgenic animal of this specification sheets;
B) reclaim the silk that transgenic animal produce;
C) from silky fibre, reclaim target protein matter.
Additional nucleic acid of the present invention
The invention still further relates to instruct and reduce fibroin H synthetic purpose RNA produces specifically expressing in the silk gland cell behind silkworm nucleic acid, wherein said nucleic acid comprises to contain and is useful on the regulatory region of polynucleotide of interest at the conditioning signal of described product silk gland cell specifically expressing, and the Nucleotide 1150 of described nucleic acid and sequence SEQ ID № 1 to the polynucleotide of Nucleotide 1451 have at least 90% identity.
One group of General Definition that the type nucleic acid provides corresponding to the first part at specification sheets and particularly corresponding to two sequences between the definition of identity percentage ratio.
Produce success that silk gland cell produces exogenous protein and mobilize with this protein especially that to be used for its synthetic protein synthesis relevant.
Optimize extraneous protein and express the expression that a kind of mode of speed is a kind of silk protein of reduction.Wherein, only fibroin H just account for produce that sericterium produces proteinic 80% and be the main protein of silk.Because its structure, it is responsible for the physics and chemistry and the mechanical characteristics of silky fibre: intensity, elasticity, insoluble.
This is proteinic removes and uses target protein matter for example spider's thread protein, fibroin H Galleria or already mentioned other target protein matter are replaced and made and particularly can improve and therefore produce new biomaterial to the characteristic of silky fibre.
For the fibroin H gene of stable inhibition the (therefore can pass to the offspring), preferably use RNA to disturb (iRNA) method in product sericterium rear section.As if the general phenomenon that all confirms in prokaryotic organism and eukaryote be the mRNA of the degrading genes by the external source double-stranded RNA is made a response and special and effective this gene of inhibition.
Employed purpose RNA can be can with the mRNA of fibroin H pairing with the RNA of the siRNA type (siRNA) that forms double-stranded RNA or can not form the shRNA (bobby pin RNA) of double-stranded RNA with the messenger RNA(mRNA) pairing of fibroin H.
Preferably, the nucleotide sequence of coding purpose RNA is sequence SEQ ID № 12.
Sequence SEQ ID № 12 is that origin comes from the inversion of fibroin H gene and the multiple nucleotide sequence is formed and will more detailed discussion in embodiment 9.
As embodiment 9 and shown in Figure 13, the contriver finds to transform the generation that silkworm can obviously reduce fibroin H with above-described nucleic acid.The reduction of this kind protein synthesis is based on the principle of RNA interfering, and by the post-treatment of transcribing that the double-stranded RNA transfered cell is triggered, it causes gene inactivation or engineering noise in the sequence specific mode.
Rely on this kind nucleic acid can reduce the generation of fibroin H specifically, and do not influence other proteinic generations that enter the silky fibre composition.
Have no intention to be subjected to the constraint of particular theory, the contriver believes that purpose RNA forms hairpin structure, can stablize RNA polymerase and therefore reduce the synthetic of fibroin H.
Being reduced in of domestic silk core albumen H synthesis rate produced sizable interest in the context of the invention.Rely on the nucleic acid of above just having described, provide such animal with those nucleic acid that instruct desired polypeptides to express, wherein the cell resource is redirected in target protein matter synthetic.This embodiment of the present invention satisfies the needs of the industrial instrument operability that is used for the mass production recombinant protein fully.Transgenic animal comprise the nucleic acid of several types, those nucleic acid for example mentioned above and the nucleic acid of describing in detail more hereinafter.Preferably, nucleic acid of the present invention comprises to contain and is useful on the regulatory region of polynucleotide of interest at the conditioning signal of described product silk gland cell specifically expressing, and described nucleic acid terminally comprises the polynucleotide of the Nucleotide 1150 of sequence SEQ ID № 1 to Nucleotide 1451 to 3 ' end from 5 '.
Preferably, nucleic acid of the present invention comprises to contain and is useful on the regulatory region of polynucleotide of interest at the conditioning signal of described product silk gland cell specifically expressing, and described nucleic acid terminally comprises the polynucleotide of the Nucleotide 1 of sequence SEQ ID № 1 to Nucleotide 1451 to 3 ' end from 5 '.
Another object of the present invention is by the nucleic acid of forming such as nucleic acid defined above, and described nucleic acid is modified by at least one and no more than four copies that import polynucleotide with sequence SEQ ID № 2 between the Nucleotide 1 and 1379 of sequence SEQ ID № 1.
The invention still further relates to the nucleic acid that has with all nucleic acid complementary sequences described above.
Additional expression cassette of the present invention
The invention still further relates to and comprise coding purpose RNA and place expression of nucleic acids box under as described above the nucleic acid control.
Preferably, purpose RNA is by the nucleic acid encoding with sequence SEQ ID № 12.
Expression cassette one end of the present invention or each end surround by insulator, be incorporated into what position polynucleotide of interest is expressed no matter this insulator is intended to this expression cassette.Example as insulator, can mention gypsy insulator (Gerasimova T.I. and the Corces V.G.:Polycomband trithorax group proteins mediate the function of a chromatininsulator of fruit bat, Cell 1998,92:511-521), scs/scs ' (Kellum R. and Schedl be Aposition effect assay for boundaries of higher order chromosomal domains.Cell 64:941-960 P.1991) or even chicken beta globin genes 5 ' H S4 insulator (Chung J.H., BellA.c. with Felsenfeld G. (1997) Characterisation of the chicken beta-globininsulator.Proc.Nat.Acad.Sci, USA 94:575-580).
Additional carrier of the present invention
Another object of the present invention is the recombination and integration carrier that comprises nucleic acid mentioned above or expression cassette.
Therefore the present invention includes recombinant vectors, wherein inserted the regulon nucleic acid that comprises conditioning signal of the present invention, when the nucleic acid of coding purpose RNA is under the described regulon nucleic acid control, can reduces fibroin H synthetic purpose RNA and behind silkworm, produce specifically expressing in the silk gland cell.The present invention also comprises the recombination and integration carrier that comprises with the lower section:
A) regulon nucleic acid as hereinbefore defined and
B) be in a) nucleic acid of the coding purpose RNA under the regulon nucleic acid control of definition.
Recombination and integration carrier of the present invention is corresponding in the General Definition of title for the carrier described in the part of " general feature of recombinant vectors of the present invention ".
The invention still further relates to and comprise the recombinant cloning vector of recombination and integration carrier core nucleotide sequence as mentioned above.
In order to obtain any one adjusting sequence of the present invention, technology and the primer described in can use-case.The expert of the art just can reduce the nucleotide sequence that the DS-red protein synthesis is instructed in the replacement of fibroin H synthetic purpose RNA synthetic sequence with instructing.
Recombinant host cell of the present invention
For the fibroin H synthetic RNA that can reduce that is under the control of regulon nucleic acid that makes above-mentioned definition expresses, must be with regulon nucleic acid defined above, expression cassette or even recombinant vectors importing host cell.
Use the well-known method of those skilled in the art polynucleotide of the present invention to be imported host cell external." host cell that comprises nucleic acid " is defined as the cell of integrating one or more copy of nucleic acid in its genome.
In addition, another purpose of the present invention is to comprise above defined regulon nucleic acid, expression cassette or even the host cell of recombinant vectors.
This host cell preferably silkworm moth belongs to, and more specifically is eggs of silkworm and do not consider the zooblast of its strain system.Host cell can also be to belong to another lepidopterous insects species, for example Philosamia cynthia or be typically the cell of silkworm moth genus, tussah genus, galleria mellonella waxmoth genus, Philosamia cynthia genus, Spodoptera or Drosophila animal more.
More preferably, produce silk gland cell after host cell of the present invention comprises silkworm.
Additional transgenic animal of the present invention
Further, the nucleic acid that the objective of the invention is to define as mentioned, expression cassette, carrier belong to purposes in the transgenic animal obtaining silkworm moth.
Preferably, described transgenic animal belong to eggs of silkworm and secrete target protein matter with silky fibre.
The invention still further relates to of comprising as hereinbefore defined and a plurality of host cells, expression cassette or even the many cells non-human transgenic biology of recombinant vectors.
Further, the objective of the invention is the non-human transgenic animal, it contains to be integrated into the lower part of its genomic form:
(i.) the first part of specification sheets describe can instruct target protein matter express and excretory nucleic acid, expression cassette or carrier and
(ii.) guidance in the definition of the second section of specification sheets can reduce fibroin H synthetic RNA synthetic nucleic acid, expression cassette or carrier.
The availability of transgenic animal has important industrial interest as mentioned above.In fact, in this kind animal, fibroin H synthetic reduces can make therefore intracellular resource is redirected does not influence silky fibre in the synthetic of target protein matter secretion.Embodiment of the present invention satisfy the needs of the industrial instrument availability that is used for the mass production recombinant protein fully.
The invention still further relates to the non-human transgenic animal:
(i.) hyposecretion of fibroin L,
(ii.) comprise expression cassette that the fused protein that can instruct at least 172-terminal amino acids of fibroin L peptide sequence and purpose peptide that is incorporated into its genomic form expresses and
(iii.) guidance in the definition of the second section of specification sheets can reduce fibroin H synthetic RNA synthetic nucleic acid, expression cassette or carrier.
This specific embodiments of the present invention provides the transgenic animal with one group of advantage related to the present invention; The amount of the target protein matter that in other words is exactly wherein to be produced be in check and wherein the cell resource turn to target protein matter synthetic animal.Therefore, the invention provides the direct available industrial instrument that is used for the cost large-scale production of recombinant proteins matter that reduces.
Usually, the invention still further relates to as hereinbefore defined regulon nucleic acid, expression cassette, recombinant vectors or even the purposes of host cell in obtaining the non-human transgenic animal.
About the source of animal host's cell of the present invention, kind that above-described transgenic animal have been listed above preferably belonging to and genus.
According to the identical method of describing in specification sheets first part of those methods, can obtain above-described transgenic animal.
In order easily to distinguish the animal that comprises at least a nucleic acid of describing as the application, the contriver also develops the one group of primer that describes below.
There is adopted primer to comprise 5 ' end be equivalent to encode three Nucleotide of L-Ala, Isoleucine or leucine Nucleotide.
Select antisense primer by any means because it on 3 ' complementary strand with adopted primer hybridization is arranged.
By means of carrying out under the controllable condition of primer in strictness, this can make only amplification comprise cysteine mutation to become L-Ala, Isoleucine or leucic dna sequence dna in polymerase chain reaction (PCR).
Correspondingly, transgenic animal can bring and characterize by appear in the wild-type animal absent variable in southern blotting technique.
Can also use sequence SEQ ID № 13 and SEQ ID № 14 to come the amplification of nucleotide acid sequence, use the cysteine residues that has or lack sudden change in the product that above defined probe in detecting increased then.
In addition, the present invention illustrates the present invention by accompanying drawing and the following examples, and these accompanying drawings and the following examples never limit the present invention, wherein:
Fig. 1 represents to make up the scheme of carrier of the present invention;
Fig. 2 is illustrated in the scheme of replacing the halfcystine codon with the Isoleucine codon in the gene signal peptide of coding firbrohexamerin;
Fig. 3 represents to modify by 3 copies of the polynucleotide that add sequence SEQ ID № 2 scheme of the promotor of firbrohexamerin;
Fig. 4 sequence illustrated SEQ ID № 3;
Fig. 5 represents to be in the proteinic specific tissue of the Ds-Red accumulation under the nucleic acid control of the present invention;
Fig. 6 is illustrated in and detects the Ds-Red protein that is under the nucleic acid control of the present invention in the product sericterium of silkworm;
Fig. 7 represents to be in the special accumulation of Ds-Red protein in silky fibre under the nucleic acid control of the present invention;
Fig. 8 is illustrated in and detects the Ds-Red protein that is under the nucleic acid control of the present invention in the silk cocoon;
Fig. 9 represents to use the sem observation fiber as seen to have Ds-Red protein to exist at fiber surface; Described protein shows as bag by the spherical particle of the variable size of fiber (number that depends on aggregation);
Figure 10 represents to detect the IL-2 protein that is in the cocoon of transgenosis heterozygosis silkworm under the nucleic acid control of the present invention;
Figure 11 represents to detect the heterozygosis transgenic silkworm and produces greater wax moth (Galleria mellonella) the fibroin H protein that is in sericterium (A) and the cocoon (B) under the nucleic acid control of the present invention;
Figure 12 represents to make up and comprises the method that guidance can reduce the nucleic acid carrier of fibroin H synthetic rna expression;
Figure 13 represents to use that fibroin H synthetic reduces in the animal that the represented carrier of Figure 12 transforms.
Embodiment
Normally used in an embodiment general material and method
A)
Material
Employed animal belongs to the eggs of silkworm Nistari strain from Unit é Nationale S é ricole (25 quaiJean-Jacques Rousseau, 69350, La Mulatiere, France).It is the polyvoltinism strain that does not have embryonic diapause in growing.
The bacterium that is used to clone is intestinal bacteria (E.coli) the DH5 α Ti Max.Efficiency from INVITROGEN company (reference number 440097).
B) method
*The raising condition of animal
In " Animalerie A1 " zoosphere, under the controlled condition of (between 22-25 ℃) and 75% humidity under the variable temp of setting according to the larva age, raise silkworm.Be used in the artificial medium for preparing on the mulberry leaves particulate basis and raise silkworm.
*The extraction of nucleic acid
The Qiaprep Spin Miniprep (reference number 27 106) that use derives from Qiagen extracts the DNA of plasmid construction body from bacterium.Use phenol-chloroform-primary isoamyl alcohol that the silkworm extracting genome DNA is come out.
*The generation of transgenic animal
Lay eggs the back after 2-5 hour, and blastoderm is injected integrative vector before forming in the animal ovum.Preparation comprises the solution that integrative vector and equivalent " are assisted " carrier in containing the 0.5mM phosphoric acid buffer (pH7.0) of 5mM KCl.Use Eppendorf microsyringe (Transjector 5246), this kind solution of 1-5nL is injected.The hole that forms by injection places 25 ℃ so that make ovum continue to grow with glue (LoctiteSuperglue 3, Henkel France S.A.) sealing and with ovum then.
*The selection of transgenic animal
Filter out and have Pigfhx (sp by means of 3xP3-GFP gene early expression in the eye of animal
*) transgenic animal of DsRed-(3xP3-GFP) carrier.
*Be used for detecting the detection test that Ds-Red protein exists transgenic animal
Be equipped with exciting light (558nm) launching system and descending original position (producing silk gland cell) and in cocoon, manifest Ds-Red protein at the emission optical filter at 582nm place and the Leica fluorescence amplifier (MZFL2) of digitally captured device.Analysis for cocoon fluorescence is carried out under drying regime.Place a little PBS under amplifier, to observe the product sericterium of drying.In the different steps of growing and particularly the 5th larval stage in synthetic period detects Ds-Red protein in a large number at silk protein.
Use two kinds of antibody by western blotting: the anti-Ds-Red antibody of rabbit (Clonetech, reference number 8370-1) and the goat anti-rabbit antibodies of coupling peroxidase (Biorad, reference number 170-6515) protein that extracts from produce sericterium or silk cocoon is detected.
*Realize proteinic detection test by western blotting with deriving from the sample that produces sericterium, cocoon and silk cocoon clothing to Ds-Red
Proteinic detection test comprises several steps to Ds-Red by western blotting:
The extraction of-protein example
With the 3rd cocoon or or a pair of product sericterium (secernent separates with holder) take out and be transferred in the microminiature tube.Then cocoon is broken into small pieces or in 1mL 10M LiSCN, squeezes body of gland broken; Then the protein extract incubated at room was regularly stirred with vortice at least in 1 hour simultaneously.Then protein example is diluted with 4/5 with Tris damping fluid 10mM-2%SDS-5% beta-mercaptoethanol.
The shell of cocoon or husks is transferred in the microminiature tube, in microminiature tube, adds 150 μ L H
2O is to clean husks.By sample ultrasonic being handled the Ds-Red protein that reclaimed solubility in 10 minutes.
Before last sample, with protein extract sex change preparation in 5 minutes sample in 100 ℃ of following Laemmli 5x damping fluids.
-electrophoresis
Protein extract is splined on the precast gel (INVITROGEN Novex Tris glycine, 10 holes, 1.0mm).Use migration damping fluid (INVITROGEN Novex SDS electrophoretic buffer (10X)) at 120V, the 40mA electrophoresis was realized proteinic migration in 2 hours 30 minutes.
-protein shifts
Protein transduction is moved on the nitrocellulose filter of 9cm * 6cm (Hybond ECLAmersham Pharmacia).The size of employed whatman filter paper is 10cm * 7.5cm.Use transfering buffering liquid (InVitrogen Novex Tris glycine transfering buffering liquid (25X)) at 120V, carry out under the 250mA protein being shifted in 1 hour 15 minutes.
-with ponceau to protein staining
In order to make the protein that shifted as seen, hatched nitrocellulose filter 1 minute with ponceau (Sigma).If necessary, use pen with big or small slug tape label.
The sealing of-film
Under 4 ℃ with film overnight incubation in PLT (PBS-0.2%Tween-2% milk).
-use the one-level antibody incubation
With the PLT dilution anti-Ds-Red antibody of one-level (Living Color Dspeptide Clontech) to 1/500 and at room temperature hatched 2 hours.Hatching the back washed 2 times 5 minutes with PBS-0.02% tween.
-hatch with secondary antibody
(goat anti-rabbit igg (H+L), horseradish peroxidase conjugate is Biorad) to 1/3000 and at room temperature hatched 1 hour to dilute secondary antibody with PLT.Hatching the back washed 4 times 10 minutes with PBS-0.02%tween.
-film development
Solution A (5mL Tris 100mM, pH8.5,22 μ L coumaric acids and the 50 μ l luminol,3-aminophthalic acid cyclic hydrazides that add in the dark) and solution B (5mL Tris 100mM, pH8.5,3 μ L H
2O
2).The mixture that temporarily prepares solution A and solution B is in the dark hatched film then and was developed 1 minute in A and B solution.Film exposed on the radioautograph film about 3 minutes and after exposure with film development.
Embodiment 1: comprise the structure of the integrative vector that places the polynucleotide expression cassette that contains encoding D s-Red polypeptide under the regulon nucleic acid control of the present invention, especially comprise the structure of the expression cassette of sequence SEQ ID № 1 part.
The first step that makes up is expressed as Figure 1A.In this first step, need two kinds of plasmids.
First kind of plasmid is the pDsRed1-N1 (reference number 6921-1) that Clonetech provides, and it comprises from 5 ' end to 3 ' end:
-CMV IE promotor;
-Ds-Red gene;
-SV40 polyadenylation signal;
-kalamycin resistance gene;
-pUC plasmid replication starting point.
Nucleotide sequence by AgeI and NotI Restriction Enzyme is delimited Ds-Red protein at its end.
Employed second kind of plasmid is called pfhx (sp
*) GFP, it comprises from 5 ' end to 3 ' end:
-modified fibrohexamerin promotor;
The gene of-encoding green fluorescent protein (GFP);
-SV40 polyadenylation signal;
-kalamycin resistance gene;
The replication orgin of-pUC plasmid.
Represented as Figure 1A, pfhx (sp
*) the GFP plasmid also comprises the nucleotide sequence that delimit, that place fibrohexamerin gene promoter downstream by two kinds of Restriction Enzyme AgeI and NotI.To obtain from pDSred1-N1 and AgeI-NotI fragment that comprise the proteinic sequence of encoding D s-Red is cloned into pfhx (sp in AgeI-NotI sequence site
*) in the GFP plasmid.
The plasmid that obtains from this clone is called pfhx (sp
*) DsRed, be shown in Figure 1B and its and terminally comprise to 3 ' end from 5 ':
The promotor of-modified fibrohexamerin;
-Ds-Red gene;
-SV40 polyadenylation signal;
-kalamycin resistance gene;
The replication orgin of-pUC plasmid.
Shown in Figure 1B, modified fibrohexamerin promotor and Ds-Red gene are contained in the fragment of delimiting by BglII and NaeI sequence.Use and identical method mentioned above, this fragment cloning is gone into to come from pPigA3cyp6a2-(3xP3-GFP) 2 carriers of piggyBac transposon in the BglII-StuI fragment, the structure of pPigA3cyp6a2-(3xP3-GFP) 2 carriers is shown in Figure 1B.PPigA3cyp6a2 (3xP3-GFP) 2 carriers comprise left side foot (L) and the right foot (R) that is shown in Figure 1B.
Clone the carrier that obtains when finishing for the second time at this and be shown in Fig. 1 C, it is called Pigfhx (sp
*) DsRed-(3xP3-GFP), corresponding to the definition of carrier of the present invention.
Embodiment 2: with the method for halfcystine codon in the gene of Isoleucine codon replacement coding fibrohexamerin signal peptide.Employed method is shown in Fig. 2 and comprises several steps.At first, digestion separates the fibrohexamerin promoter fragment with the PstI Restriction Enzyme by using HindIII.Second step---being called PCR1 among Fig. 2 A---used sequence SEQ ID № 4 shown in Fig. 2 A and the primer of SEQ ID № 5 to carry out the polymerase chain reaction.The 3rd step---being called PCR2 among Fig. 2 A---used the primer of sequence SEQ ID № 6 and SEQ ID № 7 to carry out the polymerase chain reaction.The primer of sequence SEQ ID № 5 and SEQ ID № 6 comprises the Isoleucine codon of the natural signals peptide halfcystine codon that is used for alternative fibrohexameri.Then the nucleic acid of amplification is connected and obtain the nucleic acid that SacII and PstI Restriction Enzyme are delimited that passes through shown in Fig. 2 B.This nucleic acid is included in the Isoleucine codon of the natural halfcystine codon of the replacement of representing with bold face type among Figure 1B.Then this fragment is cloned to replace pfhx (sp
*) corresponding native sequences in the GFP plasmid.
Embodiment 3:
By between the Nucleotide 1-1279 of sequence SEQ ID № 1, adding three schemes that copy modification fibrohexamerin promotors in conjunction with sequence SEQ ID № 2 polynucleotide of SGF-1/ jaw transcription factor; This scheme is shown in Fig. 3.
In the first step, carried out the polymerase chain reaction of fibrohexamerin promoter sequence.A, B, C and D primer that use is shown among Fig. 3 have been realized this amplification, and the B primer is used for sequence SEQID № 3, and the C primer is used for sequence SEQ ID № 9.
In second step, resulting nucleic acid is connected so that obtain nucleic acid, its coding strand is shown in Fig. 3.
Resulting nucleic acid comprises the part of the promoter sequence of the fibrohexamerin that modifies by 3 copies that add sequence SEQ ID № 2 polynucleotide.Then with this nucleic acid clone to pfhx (sp
*) in the position of the fibrohexamerin promotor native sequences that defines by HindIII and PstI restriction site in the GFP plasmid.
Sequence SEQ ID № 3 is illustrated in Fig. 4.
Sequence SEQ ID № 3 comprises from 5 ' end to 3 ' end:
The promotor of-fibrohexamerin gene (1);
-three of SGF1/ jaw element (3) except native sequences adhere to sequence (2);
-fibrohexamerin exons 1 (4), its halfcystine codon is through modifying;
-fibrohexamerin introne 1 (5);
The polynucleotide (6) that-help sequence SEQ ID № 1 makes up;
-encoding D s-Red reports sub proteinic polynucleotide (7).
Embodiment 4: use the pPigFbx (sp that comprises sequence SEQ ID № 1 that comes from the piggyBac transposon
*) the proteinic accumulation of Ds-Red in the product sericterium of the genetically modified silkworm of DsRed-(3xP3-GFP) carrier.Be shown in photo a, b, c, d, e and f representative proteinic tissue specific expression of Ds-Red among Fig. 5 with the genetically modified silkworm of carrier that comprises sequence SEQ ID № 3 that derives from the piggyBac transposon.Photo a, b and c are that three times under sight are amplified.Use the scheme of above describing to detect the test that Ds-Red protein exists.Photo d, e and f still obtain under the proteinic fluorescence spectrum of Ds-Red corresponding to photo a, b and c.
These photos can confirm that the proteinic fluorescence of Ds-Red and its expression are confined to produce silk gland cell (PSG, 2) behind the silkworm.
Produce silk gland cell (SSG, 1) at the middle part and do not observe proteinic fluorescence corresponding to Ds-Red.Can infer that nucleic acid driving purposes polypeptide of the present invention produces specifically expressing in the silk cell behind lepidopterous insects.
This result confirms by western blotting shown in Figure 6.The western blotting that is carried out under above-described condition helps to detect the proteinic existence of Ds-Red at the product silk gland cell of silkworm.With SDS-PAGE gel (14%; The Tris-glycine; 0.1%SDS) be transferred on the nitrocellulose filter and and dye with Coomassie blue.Nitrocellulose filter is shown in Fig. 6 A.With the same filter membrane anti-Ds-Red antibody incubation of one-level polyclone, hatch with the secondary goat anti-rabbit antibodies of puting together horseradish peroxidase then then.The results are shown in Fig. 6 B.Band by chemiluminescence detection is corresponding to the proteinic existence of Ds-Red.
Visible to deriving from proteinic 4 kinds of settlings that silkworm produces sericterium on filter membrane shown in Fig. 6 A and the 6B.Use above-described method to extract protein from producing sericterium.Four kinds of settlings are as follows:
Swimming lane 1: derive from the middle protein that produces sericterium of non-transgenic silkworm;
Swimming lane 2: the source is for the protein that produces sericterium in the middle of the silkworm that is included in sequence SEQ ID № 1 transfer genetic heterozygosis;
Swimming lane 3: derive from the protein that produces sericterium behind the non-transgenic silkworm;
Swimming lane 4: the source is for the protein of the back product sericterium of the silkworm that is included in sequence SEQ ID № 1 transfer genetic heterozygosis.
The intensity of the band that the filter membrane shown in Fig. 6 A shows is suitable between swimming lane.Can reach a conclusion: sedimentary protein is equivalent on the SDS-PAGE gel.Also can comprise visible size mark in the left-hand component of filter membrane.Same filter membrane is shown in Fig. 6 B after with antibody treatment mentioned above.Can observe at back product sericterium and have quite a large amount of Ds-Red protein.Ds-Red protein also is present in middle product sericterium but degree is less.The contriver thinks that this existence is because the secreted Ds-Red protein of silk cell is produced in the back produces due to the sericterium in the middle of migrating to and produce in the middle of not being due to the synthetic Ds-Red protein of sericterium.
Embodiment 5: with the Pigfhx (sp that comprises sequence SEQ ID № 1 that derives from the PiggyBac transposon
*) the proteinic accumulation of Ds-Red in the genetically modified silkworm of carrier of DsRed-(3xP3 GFP)
Detect the test that Ds-Red protein exists according to described scheme above.As seen in Figure 7, silky fibre has the fluorescence that exists corresponding to Ds-Red protein.Can derive the secretion of nucleic acid driving purposes polypeptide of the present invention the lepidopterous insects silk from this.
This result confirms by western blotting shown in Figure 8.With SDS-PAGE gel (14%; The Tris-glycine; 0.1%SDS) be transferred on the nitrocellulose filter and and dye with ponceau.
Nitrocellulose filter is shown in Fig. 8 A.With this same film and the anti-Ds-Red antibody incubation of one-level polyclone, hatch with the secondary goat anti-rabbit antibodies of puting together horseradish peroxidase then then.The results are shown in Fig. 6 B.Can see the band by chemiluminescence detection, it is corresponding to the proteinic existence of Ds-Red.Proteinic 9 settlings that derive from silk cocoon can be observed on the filter membrane shown in Fig. 8 A and the 8B.Use method mentioned above to extract protein.They are:
Swimming lane C1 and C2: the protein that derives from the non-transgenic silk cocoon;
Swimming lane T1 and T2: derive from protein to the silk cocoon of transgenosis heterozygosis described in the sequence SEQ ID № 1.
Preceding 6 the settling correspondences that begin from the big or small mark that is shown in the filter membrane left side derive from except that the decapsidate protein of silkworm cocoon of (being called husks).Three protein that the settling correspondence obtains from the silk cocoon husks with water rinse in back.From the filter membrane shown in Fig. 8 A can be observed originally on the SDS-PAGE gel sedimentary protein be equivalent.To be shown in Fig. 8 B after same the filter membrane processing as indicated above.As seen there is quite a large amount of Ds-Red protein in the part that rinsing obtains to transgenosis silk cocoon husks.Can the secretion of inference nucleic acid driving purposes of the present invention polypeptide the lepidopterous insects silk from this.
Embodiment 6: with the proteinic accumulation of Ds-Red on the genetically modified silk fiber of the structure surface of describing among the embodiment 1
Fig. 9 represents to find that with sem observation Ds-Red protein is present in the surface of fiber; Protein is occurred by the form of the spherical particle of the variable size of fiber (depending on the number of aggregation) with bag.
After with transgenosis cocoon water solution wetted, show that from the proteinic measurement of concetration (using conventional Bradford method) of corresponding supernatant liquor there are about 100 microgram Ds-Red in each cocoon.
In Fig. 9 visible in secretion process the Ds-red protein of non-fiber be sent to the outside of silky fibre.This phenomenon helps extracting the purpose nonfibrous protein.For for example embodiment 7 Human Inter Leukin-2's of description generation can be led to the same conclusion.
Embodiment 7: human IL-2's generation
The generation of the transgenic silkworm of secretion human interleukin-12 (IL-2)
The gene of coding IL-2 is replaced the proteinic gene of encoding D s-Red in the integrative vector described in the embodiment 1.
The selection of transgenic animal
By means of early expression 3xP3-GFP gene Selection transgenic animal in the eye of animal.Figure 10 is illustrated in the proteinic detection of IL-2 that places in the transgenosis heterozygosis silk cocoon under the nucleic acid control of the present invention.
Produce the test that detects IL-2 in sericterium or the silk cocoon transgenic animal
Use double antibody by western blotting: polyclone people anti-IL 2 antibodies detects from producing the protein extract that sericterium or silk cocoon obtain with the goat anti-rabbit antibodies (Biorad, reference number 170-6515) of puting together peroxidase.The test that detects exogenous protein by western blotting comprises and identical electrophoresis, transfer and protein staining, the film development step of step with respect to description in proteinic material of Ds-Red and the method part.
Unique step and and therefore below more clearly description different about the description of Ds-Red corresponding to the protein example extraction.
The extraction of protein example
Begin with the 141mg cocoon:
-the 46mg cocoon is carried out total extraction (sample number 1)
-19mg forms husks (shell of cocoon) (sample number 2)
-76mg the cocoon is come unstuck (removing bag by the protein thread glue protein of fiber) obtains two kinds of components:
-a kind of be insoluble (sample number 3)
-another kind is soluble (sample number 4).
The processing of sample:
-sample number 1: cocoon is being broken into small pieces and is being dissolved in 10M LiSCN (250 μ L) after 1 hour in room temperature, with 4/5 dilution, spending the night and hatched 30 minutes at 37 ℃ in incubated at room with Tris 10mM damping fluid-2% SDS-5% beta-mercaptoethanol.
-sample number 2: with husks room temperature be dissolved in 10M LiSCN (200 μ L) 1 hour and incubated at room spend the night and under 37 ℃, hatch 30 minutes then with sample with Tris 10mM damping fluid-2%SDS-5% beta-mercaptoethanol with 4/5 dilution.
-sample 3 and 4: cocoon is hatched 1 hour (removing sericin) under 100 ℃ in 2ml suds (7g/L).With soluble constituent (supernatant liquor that contains sericin) spend the night in incubated at room (sample number 4).Insoluble component (comprising fibroin) is dissolved among the LiSCN 10M (500 μ L) in ambient temperature overnight then sample diluted with 4/5 with Tris 10mM damping fluid-2%SDS-5% beta-mercaptoethanol.Soluble part (supernatant liquor that comprises sericin) incubated at room spend the night (sample number 4).Insoluble part (comprising fibroin) is dissolved among the LiSCN10 (500 μ L) in ambient temperature overnight dilutes (sample number 3) with Tris 10mM damping fluid-2%SDS-5% beta-mercaptoethanol with 4/1 with sample then.
Before deposition, the sample that will comprise protein extract in Laemmli 5x damping fluid in 100 ℃ of following sex change 5 minutes.
Following step is with identical to the described step of Ds-Red.
Western blotting shown in Figure 10 shows no matter sample is which kind of type all exists IL-2 protein.
-swimming lane 1: the protein (sample number 1) that obtains from the general extractive of cocoon;
-swimming lane 3: the protein that from the extract of husks, obtains (sample number 2);
-swimming lane 4: the protein (fibroin) (sample number 3) that obtains from the indissolvable component of cocoon;
-swimming lane 5: the protein (sericin) (sample number 4) that obtains from the soluble constituent of cocoon;
-swimming lane 2: the protein that obtains from the general extractive of the contrast cocoon (negative control) handled with sample number 1 the same terms;
The deposition of-swimming lane S:10ng human IL-2 standardized solution.
About swimming lane, the amount of the IL-2 that can produce each cocoon is carried out sxemiquantitative and is estimated.According to the transgenic lines of being analyzed, the amount of the IL-2 that each cocoon produces is 1-10 μ g.This embodiment proves that clearly in the generation for the molecule Ro 24-7472/000 with medical interests, the present invention is effective.In addition, the nonfibrous protein with biomedical interests is purifying easily, because it is sent to the outside of silky fibre when nonfibrous protein is synthetic, this is verified about Ds-Red protein in embodiment 6.
Embodiment 8: the generation of the fibroin H of greater wax moth
The generation of the transgenic silkworm of the fibroin H of secretion greater wax moth
The proteinic gene of encoding D s-Red in the integrative vector of describing among the gene replacement embodiment 1 with coding greater wax moth fibroin H.Figure 11 represents placing the detection of the greater wax moth fibroin H under the nucleic acid control of the present invention in the product sericterium (A) of heterozygote transgenic silkworm and the cocoon (B).
In the product sericteriums of transgenic animal or cocoon, detect the test of greater wax moth fibroin H
Use double antibody by the western blotting method: the anti-FibHGm antibody of polyclone people detects the protein extract that obtains from produce sericterium or cocoon with the goat anti-rabbit antibodies (Biorad, reference number 170-6515) of puting together peroxidase.The test that detects exogenous protein by western blotting is made up of electrophoresis, transfer and protein staining, the film development step identical with those steps of partly describing with respect to the proteinic material of Ds-Red and method.Unique step of extracting corresponding to protein example and different and therefore description more clearly below about the description of Ds-Red.
The extraction of protein example and processing
The a pair of product sericterium (secernent separates with holder) that will take out from the transgenic animal of the 5th larval stage is transferred in the microminiature tube, and crushing was also hatched under 60 ℃ 1 hour in hatching 20 minutes under the room temperature in 500 μ L LiSCN then.Sample is diluted with 4/5 with Tris 10mM damping fluid-2%SDS-5% beta-mercaptoethanol.
The cocoon (20mg) that is broken into small pieces is dissolved in LiSCN 10M (250 μ L) under 65 ℃ diluted with 4/5 with Tris 10mM damping fluid-2%SDS-5% beta-mercaptoethanol in 30 minutes then.
Before deposition, the sample that will contain protein extract in Laemmli 5x damping fluid in 100 ℃ of following sex change 5 minutes.Following step is with identical to the described step of Ds-Red.
-western blotting shown in Figure 11 show exist in product sericterium (holder and secernent) at the transgenosis individuality and the cocoon FibHGm (size: 115KDa), and handle under the condition identical with condition mentioned above from the control sample that the non-transgenic animal obtains, lack this protein.
Swimming lane 1: produce the protein that the secernent extracting section of sericterium obtains from transgenic animal;
Swimming lane 2: the holder that produces sericterium from transgenic animal partly extracts the protein that obtains;
Swimming lane 3: from the resulting protein of transgenic animal cocoon general extractive;
Swimming lane 1
*: produce the protein that the secernent extracting section of sericterium obtains from control animal;
Swimming lane 2
*: the holder that produces sericterium from control animal partly extracts the protein that obtains;
Swimming lane 3
*: the protein that obtains from control animal cocoon general extractive.
This embodiment proves that clearly the present invention is effective for the generation that the true tumor material for example has the silk mixture of intensity and elastic new mechanical characteristics.
Embodiment 9: make up the scheme that comprises the nucleic acid carrier that instructs reduction fibroin H synthetic rna expression
In order to suppress fibroin H, produced as the transgenosis piggyBac carrier described in the embodiment 1, it comprises two fragments that are in reverse multiple goal gene.To derive from the justice that has of fibroin H gene---antisense sequences separates by the indifferent equilibrium sequence and is under the control of fibrohexamerin promotor (Figure 12).This carrier drives and produces the double-stranded sRNA that is made up of a chain, and a described chain folding forms the secondary structure that is called " hairpin structure ", and described double-stranded sRNA target is positioned the part (Figure 12) of the 465bp tumor-necrosis factor glycoproteins of fibroin H gene extron 2.These sequences can be alternatively from repeating and the formation of amorphous structure territory.As shown in figure 12, the size of fibroin H gene is 17kbp.Encoding sequence is made up of two exons: one is 15 for 67bp and another, 750bp.The feature of this gene is an exon 2 camber tumor-necrosis factor glycoproteins.Each repeat region comprises the subdomain of 208bp, and this subdomain is with multiple Ua and Ub motif tissue.Being used for target decides the segmental position of 465bp of mRNA and represents by arrow.That transcribes mRNA that the back produces repeats to be expressed as gray area among the figure.(Zhou etc., 2000 Fine organization of Bombyxmori fibroin heavy chain gene.Nucleic Res 28,2413-2419).
Described common injection with assistant carrier (helper) " general material and method part ", this structure allows to set up transgenic silkworm system, is the amount of having measured the fibroin H that produces in the cocoon for this.
Secretion has produced measurable influence to fibroin H for (histogram) as shown in figure 13, RNA mechanism, and this influence is variable between transgenic lines.
With respect to the contrast cocoon, comprise less fibroin H to 20% by the weave silk cocoon that forms of transgenic silkworm.
Sequence table
＜110〉Centre National de La Recherche Scientifique (CNRS)
＜120〉instruct desired polypeptides behind lepidopterous insects, to produce nucleic acid and the application of expressing in the sericterium thereof
<130>P443FR
<160>13
＜170〉PatentIn version 3 .1
<210>1
<211>2721
<212>DNA
＜213〉artificial sequence
<400>1
aagcttagat?aattcggcat?tgtgcgccac?tgagtcgcat?tatgctctgt?aattggaaac 60
taccaaacat?tgtgtaccct?ttaatgatat?tctaatctat?atatataaaa?atgaattgct 120
gttcgttagt?ctcgctaaaa?ctcgagaacg?gccggaccga?tttggctaat?tttggtcttg 180
aattatttgt?ggaagtccag?agaagattta?gaaggtttaa?ataaatatga?aaatgctcgg 240
aattaaataa?aaataacaat?tttgtttttt?ctttgatgtg?ttcccgtcgg?acggattcct 300
ttagtctttt?atttatcgac?tagcgacccg?ccgcttcgct?tcggaaacat?taaaatacac 360
atgataccaa?aaaaattaaa?taattttttt?ttaaaaaaag?tagcctatgt?tcatcaggta 420
caatgtcggc?ttctaatgga?aaaagaattt?ttcaaatcgg?tccagtagtt?tcggagccta 480
ttcgaaacaa?acaaacaaac?aaatctttcc?tctttataat?attagtatag?atagtataga 540
ttgaggcact?acgaagtctg?ccgggtcagc?tagtatactc?ataaataagg?tcaactttaa 600
tacagtatta?catttaatta?ggcatatttg?gtctttaggt?tcacgcttta?atcttttttg 660
gttgatttta?atttctggct?gatcgctatg?gcggttattg?agcaatgcct?tcgtccaaca 720
gttgacatct?gttgatgatg?gtgatatctt?caaaattacc?ttagcgcaat?gtagacttat 780
acagtatttc?tgttttccta?agttaattac?cgctgtagcc?aataccgtct?ttaccataag 840
cgcacacggg?gcccggtcca?gggccgagtg?tcgtcgaggg?ggcccgaaag?accggcaagt 900
tctctcacac?gtttattccc?aaaacatttt?tgtcgggcac?attacacttt?ttccacaaat 960
ccgtaatcag?aaggtattta?gcaaggcata?tactatgcct?ataatagaag?attttgctca 1020
acagaaatcc?cgagagaaac?cgttatcgaa?atcgtaacca?aaaaaccagc?agcattctaa 1080
tatcattaat?gacatattat?atcatactgt?atttgattac?ctataataaa?gggtcatact 1140
cagtaaaaaa?atgttaatat?aattcgcttt?ttttactttc?caaaagggcc?tcaaattctt 1200
gtgtgtccaa?gggccccatc?ttagtttaag?acgtccctgg?ctgtagccca?gttactgcca 1260
cacaaacatg?cttaactccc?cgcctacgtc?gaggagaaca?ttttgcgcct?tagaaaataa 1320
aatggcgtcg?ccgcggcgca?acaataagaa?cttaattcgt?gcaattgttt?ccacgacgct 1380
atttatttaa?cgttattcgt?tgtgaggaac?aatactttgt?ataattaatg?ttgatcagtg 1440
cctaacgacg?cagttgttta?ttattcgcgc?aacatgctgg?cgcggatcct?agctgtagcc 1500
gctgtggcag?ttttggcttc?tgcaggtaag?tccgatgttt?cttgattagg?tcaattataa 1560
ctacacgtaa?cgctttgtga?ttaatcggga?agactattga?ttaattatat?tccgcaacag 1620
ggggtatatt?agtcctggtg?ttaaacagtt?gtcgaagtcc?attgactttc?cattgagttt 1680
cgccgacatc?ctcatcaggc?aacactgttc?gaaggaattc?ttcccaaatc?cggatggtgc 1740
gtggcaaaat?taacatctag?aaaagtattg?aacgcagctg?ttcagatagt?aggtttgaag 1800
ttaaatctag?tgctggacgg?tacggtggta?aaaaggagat?ggttgaatca?tttccactaa 1860
tgtcttacag?aagtcgttct?ggtacaaaac?ccagaggacc?tgtctgaatt?acaagtgaag 1920
gacggctgtt?ttctctgcag?cagaagtggt?aggatgtgga?ccccactgca?tgcgcactaa 1980
caagaagccc?caccactcat?tgctcatata?taatcttgta?ccacaggacc?ggtcgccacc 2040
atggtgcgct?cctccaagaa?cgtcatcaag?gagttcatgc?gcttcaaggt?gcgcatggag 2100
ggcaccgtga?acggccacga?gttcgagatc?gagggcgagg?gcgagggccg?cccctacgag 2160
ggccacaaca?ccgtgaagct?gaaggtgacc?aagggcggcc?ccctgccctt?cgcctgggac 2220
atcctgtccc?cccagttcca?gtacggctcc?aaggtgtacg?tgaagcaccc?cgccgacatc 2280
cccgactaca?agaagctgtc?cttccccgag?ggcttcaagt?gggagcgcgt?gatgaacttc 2340
gaggacggcg?gcgtggtgac?cgtgacccag?gactcctccc?tgcaggacgg?ctgcttcatc 2400
tacaaggtga?agttcatcgg?cgtgaacttc?ccctccgacg?gccccgtaat?gcagaagaag 2460
accatgggct?gggaggcctc?caccgagcgc?ctgtaccccc?gcgacggcgt?gctgaagggc 2520
gagatccaca?aggccctgaa?gctgaaggac?ggcggccact?acctggtgga?gttcaagtcc 2580
atctacatgg?ccaagaagcc?cgtgcagctg?cccggctact?actacgtgga?ctccaagctg 2640
gacatcacct?cccacaacga?ggactacacc?atcgtggagc?agtacgagcg?caccgagggc 2700
cgccaccacc?tgttcctgta?g 2721
<210>2
<211>12
<212>DNA
＜213〉silkworm (Bombyx mori)
<400>2
ctatttattt?aa 12
<210>3
<211>2772
<212>DNA
＜213〉artificial sequence
<400>3
aagcttagat?aattcggcat?tgtgcgccac?tgagtcgcat?tatgctctgt?aattggaaac 60
taccaaacat?tgtgtaccct?ttaatgatat?tctaatctat?atatataaaa?atgaattgct 120
gttcgttagt?ctcgctaaaa?ctcgagaacg?gccggaccga?tttggctaat?tttggtcttg 180
aattatttgt?ggaagtccag?agaagattta?gaaggtttaa?ataaatatga?aaatgctcgg 240
aattaaataa?aaataacaat?tttgtttttt?ctttgatgtg?ttcccgtcgg?acggattcct 300
ttagtctttt?atttatcgac?tagcgacccg?ccgcttcgct?tcggaaacat?taaaatacac 360
atgataccaa?aaaaattaaa?taattttttt?ttaaaaaaag?tagcctatgt?tcatcaggta 420
caatgtcggc?ttctaatgga?aaaagaattt?ttcaaatcgg?tccagtagtt?tcggagccta 480
ttcgaaacaa?acaaacaaac?aaatctttcc?tctttataat?attagtatag?atagtataga 540
ttgaggcact?acgaagtctg?ccgggtcagc?tagtatactc?ataaataagg?tcaactttaa 600
tacagtatta?catttaatta?ggcatatttg?gtctttaggt?tcacgcttta?atcttttttg 660
gttgatttta?atttctggct?gatcgctatg?gcggttattg?agcaatgcct?tcgtccaaca 720
gttgacatct?gttgatgatg?gtgatatctt?caaaattacc?ttagcgcaat?gtagacttat 780
acagtatttc?tgttttccta?agttaattac?cgctgtagcc?aataccgtct?ttaccataag 840
cgcacacggg?gcccggtcca?gggccgagtg?tcgtcgaggg?ggcccgaaag?accggcaagt 900
tctctcacac?gtttattccc?aaaacatttt?tgtcgggcac?attacacttt?ttccacaaat 960
ccgtaatcag?aaggtattta?gcaaggcata?tactatgcct?ataatagaag?attttgctca 1020
acagaaatcc?cgagagaaac?cgttatcgaa?atcgtaacca?aaaaaccagc?agcattctaa 1080
tatcattaat?gacatattat?atcatactgt?atttgattac?ctataataaa?gggtcatact 1140
cagtaaaaaa?atgttaatat?aattcgcttt?ttttactttc?caaaagggcc?tcaaattctt 1200
gtgtgtccaa?gggccccatc?ttagtttaag?acgtccctgg?ctgtagccca?gttactgcca 1260
cacaaacatg?cttaactccc?cgcctacgtc?gaggagaaca?ttttgcgcct?tagaaaataa 1320
aatggcgtcg?ccgcggcgca?acaataagaa?cttaattcgt?gcaattgttt?ccacgacgct 1380
atttatttaa?cttatctatt?tatttctatt?tatttccacg?acgctattta?tttaacgtta 1440
tcgttattcg?ttgtgaggaa?caatactttg?tataattaat?gttgatcagt?gcctaacgac 1500
gcagttgttt?attattcgcg?caacatgctg?gcgcggatcc?tagctgtagc?cgctgtggca 1560
gttttggctt?ctgcaggtaa?gtccgatgtt?tcttgattag?gtcaattata?actacacgta 1620
acgctttgtg?attaatcggg?aagactattg?attaattata?ttccgcaaca?gggggtatat 1680
tagtcctggt?gttaaacagt?tgtcgaagtc?cattgacttt?ccattgagtt?tcgccgacat 1740
cctcatcagg?caacactgtt?cgaaggaatt?cttcccaaat?ccggatggtg?cgtggcaaaa 1800
ttaacatcta?gaaaagtatt?gaacgcagct?gttcagatag?taggtttgaa?gttaaatcta 1860
gtgctggacg?gtacggtggt?aaaaaggaga?tggttgaatc?atttccacta?atgtcttaca 1920
gaagtcgttc?tggtacaaaa?cccagaggac?ctgtctgaat?tacaagtgaa?ggacggctgt 1980
tttctctgca?gcagaagtgg?taggatgtgg?accccactgc?atgcgcacta?acaagaagcc 2040
ccaccactca?ttgctcatat?ataatcttgt?accacaggac?cggtcgccac?catggtgcgc 2100
tcctccaaga?acgtcatcaa?ggagttcatg?cgcttcaagg?tgcgcatgga?gggcaccgtg 2160
aacggccacg?agttcgagat?cgagggcgag?ggcgagggcc?gcccctacga?gggccacaac 2220
accgtgaagc?tgaaggtgac?caagggcggc?cccctgccct?tcgcctggga?catcctgtcc 2280
ccccagttcc?agtacggctc?caaggtgtac?gtgaagcacc?ccgccgacat?ccccgactac 2340
aagaagctgt?ccttccccga?gggcttcaag?tgggagcgcg?tgatgaactt?cgaggacggc 2400
ggcgtggtga?ccgtgaccca?ggactcctcc?ctgcaggacg?gctgcttcat?ctacaaggtg 2460
aagttcatcg?gcgtgaactt?cccctccgac?ggccccgtaa?tgcagaagaa?gaccatgggc 2520
tgggaggcct?ccaccgagcg?cctgtacccc?cgcgacggcg?tgctgaaggg?cgagatccac 2580
aaggccctga?agctgaagga?cggcggccac?tacctggtgg?agttcaagtc?catctacatg 2640
gccaagaagc?ccgtgcagct?gcccggctac?tactacgtgg?actccaagct?ggacatcacc 2700
tcccacaacg?aggactacac?catcgtggag?cagtacgagc?gcaccgaggg?ccgccaccac 2760
ctgttcctgt?ag 2772
<210>4
<211>25
<212>DNA
＜213〉silkworm
<400>4
ggcgtcgccg?cggcgcaaca?ataag 25
<210>5
<211>25
<212>DNA
＜213〉silkworm
<400>5
tacgaccgcg?cctaggatcg?acatc 25
<210>6
<211>25
<212>DNA
＜213〉artificial sequence
<400>6
atgctggcgc?ggatcctagc?tgtag 25
<210>7
<211>20
<212>DNA
＜213〉silkworm
<400>7
cgtcaaaacc?gaagacgtcc 20
<210>8
<211>36
<212>DNA
＜213〉silkworm
<400>8
ggtgctgcga?taaataaatt?ggaatagata?aataaa 36
<210>9
<211>36
<212>DNA
＜213〉silkworm
<400>9
ctatttattt?ccacgacgct?atttatttaa?cgttat 36
<210>10
<211>16
<212>DNA
＜213〉artificial sequence
<400>10
ctatttattt?actagt 16
<210>11
<211>37
<212>DNA
＜213〉artificial sequence
<400>11
ctatttattt?aacgttattc?gttgtgagga?acactag 37
<210>12
<211>18
<212>DNA
＜213〉silkworm
<400>12
gtttccacga?cgctattt 18
<210>13
<211>18
<212>DNA
＜213〉silkworm
<400>13
ctgcagaagc?caaaactg 18
Claims (33)
1. nucleic acid, it instructs target protein matter to produce specifically expressing in the silk gland cell behind silkworm, wherein said nucleic acid from 5 ' terminal to 3 ' end comprise (i) comprise be used for polynucleotide of interest the regulatory region of the conditioning signal of described product silk gland cell specifically expressing and (ii) coding be in the zone of the modified signal peptide under the described regulatory region control, the Nucleotide 1150 of wherein said nucleic acid and sequence SEQ ID № 1 to the polynucleotide of Nucleotide 2026 on Nucleotide, have at least 90% identity and wherein the Nucleotide 1486 of sequence SEQ ID № 1 encode to the trinucleotide of Nucleotide 1488 and be selected from L-Ala, Isoleucine and a leucic amino acid.
2. according to the nucleic acid of claim 1, wherein said nucleic acid from 5 ' terminal to 3 ' end comprise (i) comprise be used for polynucleotide of interest the regulatory region of the conditioning signal of described product silk gland cell specifically expressing and (ii) coding be in the zone of the modified signal peptide under the described regulatory region control, wherein said nucleic acid from 5 ' the terminal Nucleotide 1150 that comprises sequence SEQ ID № 1 to 3 ' end to the polynucleotide of the Nucleotide 2026 and wherein Nucleotide 1486 of sequence SEQ ID № 1 is encoded to the trinucleotide of Nucleotide 1488 and is selected from L-Ala, Isoleucine and a leucic amino acid.
3. according to the nucleic acid of claim 1, wherein said nucleic acid from 5 ' terminal to 3 ' end comprise (i) comprise be used for polynucleotide of interest the regulatory region of the conditioning signal of described product silk gland cell specifically expressing and (ii) coding be in the zone of the modified signal peptide under the described regulatory region control, wherein said nucleic acid from 5 ' the terminal Nucleotide 1 that comprises sequence SEQ ID № 1 to 3 ' end to the polynucleotide of the Nucleotide 2026 and wherein Nucleotide 1486 of sequence SEQ ID № 1 is encoded to the trinucleotide of Nucleotide 1488 and is selected from L-Ala, Isoleucine and a leucic amino acid.
4. at least one of any one nucleic acid of claim 1-3, the wherein said nucleic acid polynucleotide by importing sequence SEQ ID № 2 between the Nucleotide 1 and 1379 of sequence SEQ ID № 1 and no more than four copy are modified.
5. nucleic acid, it has any described nucleic acid complementary sequence with claim 1-4.
6. expression cassette, it comprises the polynucleotide of interest that places under any one the nucleic acid control of claim 1-5.
7. according to the expression cassette of claim 6, polynucleotide of interest coded polypeptide wherein.
8. according to the expression cassette of claim 6, polynucleotide of interest coding fusion polypeptide wherein, it comprises from N-end to C-end:
At least 172-terminal amino acids of-fibroin L peptide sequence and
-desired polypeptides.
9. according to the expression cassette of claim 7 or 8, wherein desired polypeptides is selected from following protein:
-spider spider's thread protein;
-galleria mellonella waxmoth belong to animal fibroin and
-Human Inter Leukin-2.
10. recombination and integration carrier, it comprises according to any one nucleic acid of claim 1-5 or the expression cassette any according to claim 6-9.
11. recombinant cloning vector, it comprises the nucleotide sequence of the recombination and integration carrier of claim 10.
12. according to the recombination and integration carrier of claim 10, wherein said recombination and integration carrier is the state-run microbial preservation center (CNCM) that was kept at Institute Pasteur on February 20th, 2003, the Pigfhx (sp that preserving number is CNCM I-2975
*) DsRed-(3xP3-GFP).
13. recombinant host cell, it comprises any one nucleic acid of claim 1-5 or any described expression cassette of claim 6-9 or any one carrier of claim 10-12.
14., it is characterized in that described recombinant host cell is the product silk zooblast that is selected from silkworm moth genus, tussah genus, galleria mellonella waxmoth genus, Philosamia cynthia genus, Spodoptera or Drosophila according to the recombinant host cell of claim 13.
15. according to the recombinant host cell of claim 13, wherein said recombinant host cell is a bombyx mori cell.
16. according to the recombinant host cell of claim 13, wherein said recombinant host cell is the back product silk cell of silkworm.
17. the purposes of the recombinant host cell that carrier that any one expression cassette, the claim 10-12 of any one nucleic acid, the claim 6-9 of claim 1-5 is any or claim 13-16 are any is used to obtain silkworm moth and belongs to transgenic animal.
18. according to the purposes of claim 17, wherein the eggs of silkworm transgenic animal are secreted into target protein matter in the silky fibre.
19. transgenic nonhuman animal, it comprises any one nucleic acid of the claim 1-5 that is incorporated into its genome form, any one expression cassette or any one carrier of claim 10-12 of claim 6-9.
20. transgenic nonhuman animal, it lacks the secretion of fibroin L, and described transgenic nonhuman animal comprises the claim 8 that is incorporated into its genome form or 9 expression cassette.
21. cocoon, it comprises target protein matter, described protein expression is subjected to the guidance of any one carrier of any one expression cassette of any one nucleic acid of claim 1-5, claim 6-9 or claim 10-12 and wherein said target protein matter with greater than the 5ng/mg cocoon, and the concentration that is preferably more than the 70ng/mg cocoon exists.
22. obtain the method for eggs of silkworm transgenic animal, wherein this method comprises following step:
A) any one any one expression cassette or any one carrier of claim 10-12 of nucleic acid, claim 6-9 of silkworm egg injection claim 1-5 before the back 1 to 5 hour of laying eggs, blastoderm form;
B) select to integrate in the genome transgenic animal of any one expression cassette of any one nucleic acid of claim 1-5 or claim 6-9.
23. the method that obtains animal of claim 21, wherein this method comprises extra the following step:
C) two transgenic animal hybridization that step b) obtained;
D) select genetically modified homozygote animal.
24. obtain the method for silky fibre, it is characterized in that this method comprises following step:
A) raising is according to the transgenic animal of claim 19;
B) reclaim the silk that described transgenic animal produce.
25. obtain the method for target protein matter, it is characterized in that this method comprises following step:
A) raising is according to the transgenic animal of claim 19;
B) reclaim the silk that described transgenic animal produce;
C) reclaim target protein matter from silky fibre.
26. nucleic acid, its guidance reduction fibroin H synthetic purpose RNA produces the specifically expressing in the silk gland cell behind silkworm, wherein said nucleic acid contains to comprise and is used for the regulatory region of polynucleotide of interest at the conditioning signal of described product silk gland cell specifically expressing, and the Nucleotide 1150 of described nucleic acid and sequence SEQ ID № 1 to the polynucleotide of Nucleotide 1451 have at least 90% Nucleotide identity.
27. nucleic acid, its nucleic acid by claim 26 is formed, and at least one of the polynucleotide of wherein said nucleic acid by importing sequence SEQ ID № 2 between sequence SEQID № 1 Nucleotide 1 and 1379 and no more than 4 copies are modified.
28. expression cassette, it comprises the nucleic acid of coding purpose RNA, and wherein said expression cassette places under the control of nucleic acid of claim 26 or 27.
29. the recombination and integration carrier, it comprises the nucleic acid of claim 26 or 27 or the expression cassette of claim 28.
30. recombinant host cell, it comprises nucleic acid or the expression cassette of claim 28 or the carrier of claim 29 of claim 26 or 27.
31. the purposes of the carrier of the nucleic acid of claim 26 or 27 or the expression cassette of claim 28 or claim 29 is used to obtain silkworm moth and belongs to transgenic animal.
32. transgenic nonhuman animal, wherein said animal comprise its genome form that is incorporated into
I) any one carrier of any one expression cassette of any one nucleic acid, the claim 6-8 of claim 1-5 or claim 9-11 and
The nucleic acid or the expression cassette of claim 28 or the carrier of claim 29 that ii) comprise claim 26 or 27.
33., it is characterized in that described animal comprises the claim 26 that is incorporated into its genome form or 27 nucleic acid or the expression cassette of claim 28 or the carrier of claim 29 according to the transgenic nonhuman animal of claim 20.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR03/03137 | 2003-03-13 | ||
| FR0303137A FR2852325B1 (en) | 2003-03-13 | 2003-03-13 | NUCLEIC ACID DIRECTING THE EXPRESSION OF A POLYPEPTIDE OF INTEREST IN POST-LEPIDOPTERA SERICIGENIC GLANDS, AND USES THEREOF |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1774507A true CN1774507A (en) | 2006-05-17 |
Family
ID=32893287
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200480010261 Pending CN1774507A (en) | 2003-03-13 | 2004-03-12 | Nucleic acid controlling the expression of a useful polypeptide in the posterior silk glands of a lepidoptera and application thereof |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1606410A2 (en) |
| JP (1) | JP2006521802A (en) |
| CN (1) | CN1774507A (en) |
| FR (1) | FR2852325B1 (en) |
| WO (1) | WO2004083445A2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102187845A (en) * | 2010-03-05 | 2011-09-21 | 中国科学院上海生命科学研究院 | Transgenic method for improving silk yield |
| CN102242147A (en) * | 2011-05-13 | 2011-11-16 | 浙江同点生物科技有限公司 | Method for synthesizing and secreting rabies virus nucleoprotein in middle silkworm silk-gland cells |
| CN102286529A (en) * | 2011-05-13 | 2011-12-21 | 浙江大学 | Method for synthesizing silkworm silk gland cells capable of secreting rabies virus glucoprotein |
| CN107027718A (en) * | 2017-04-12 | 2017-08-11 | 广西壮族自治区蚕业技术推广总站 | The method that Diapause transgenic bombyx mori is made by instant corona |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006016323A (en) * | 2004-06-30 | 2006-01-19 | Hiroshima Industrial Promotion Organization | Physiologically active biomaterial |
| GB0710614D0 (en) * | 2007-06-04 | 2007-07-11 | Lonza Biologics Plc | Mammalian expression vector with a highly efficient secretory signal sequence |
| WO2013056664A1 (en) * | 2011-10-20 | 2013-04-25 | 西南大学 | Method and uses for bombyx mori silk fibroin heavy chain gene mutation sequence and mutant |
| CN105683373A (en) * | 2013-10-25 | 2016-06-15 | 国立研究开发法人农业生物资源研究所 | Exogenous gene expression enhancer |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09316095A (en) | 1996-05-27 | 1997-12-09 | Mitsubishi Chem Corp | New signal peptide |
| AU772650B2 (en) | 1999-08-06 | 2004-05-06 | Bioneer A/S | Method of isolating secretion signals in lactic acid bacteria and novel secretion signals isolated from lactococcus lactis |
| JP4701336B2 (en) | 2001-04-18 | 2011-06-15 | 独立行政法人農業生物資源研究所 | Transformed silkworm producing human collagen |
| JP2003026599A (en) | 2001-06-11 | 2003-01-29 | Univ Nagoya | Vaccine adjuvant to which il-15 gene is applied |
-
2003
- 2003-03-13 FR FR0303137A patent/FR2852325B1/en not_active Expired - Fee Related
-
2004
- 2004-03-12 EP EP04720052A patent/EP1606410A2/en not_active Withdrawn
- 2004-03-12 CN CN 200480010261 patent/CN1774507A/en active Pending
- 2004-03-12 JP JP2006505851A patent/JP2006521802A/en active Pending
- 2004-03-12 WO PCT/FR2004/050107 patent/WO2004083445A2/en active Application Filing
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102187845A (en) * | 2010-03-05 | 2011-09-21 | 中国科学院上海生命科学研究院 | Transgenic method for improving silk yield |
| CN102187845B (en) * | 2010-03-05 | 2013-06-05 | 中国科学院上海生命科学研究院 | Transgenic method for improving silk yield |
| CN102242147A (en) * | 2011-05-13 | 2011-11-16 | 浙江同点生物科技有限公司 | Method for synthesizing and secreting rabies virus nucleoprotein in middle silkworm silk-gland cells |
| CN102286529A (en) * | 2011-05-13 | 2011-12-21 | 浙江大学 | Method for synthesizing silkworm silk gland cells capable of secreting rabies virus glucoprotein |
| CN107027718A (en) * | 2017-04-12 | 2017-08-11 | 广西壮族自治区蚕业技术推广总站 | The method that Diapause transgenic bombyx mori is made by instant corona |
Also Published As
| Publication number | Publication date |
|---|---|
| EP1606410A2 (en) | 2005-12-21 |
| FR2852325B1 (en) | 2005-06-03 |
| JP2006521802A (en) | 2006-09-28 |
| WO2004083445A9 (en) | 2010-12-09 |
| WO2004083445A3 (en) | 2005-06-16 |
| FR2852325A1 (en) | 2004-09-17 |
| WO2004083445A2 (en) | 2004-09-30 |
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