CN1740325A - A fusion gene, it expressed protein and producing process thereof - Google Patents
A fusion gene, it expressed protein and producing process thereof Download PDFInfo
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- CN1740325A CN1740325A CN 200410056850 CN200410056850A CN1740325A CN 1740325 A CN1740325 A CN 1740325A CN 200410056850 CN200410056850 CN 200410056850 CN 200410056850 A CN200410056850 A CN 200410056850A CN 1740325 A CN1740325 A CN 1740325A
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Abstract
This invention belongs to polypeptide drug producing skill by genetic engineering of biological technique pharmaceutical engineering. It constitutes fusion gene of CTB and human insulin B chain. It also has a high-efficient CTB and human insulin B chain fusion gene expression in silkworm larvae, pupae and strawberry. By animal orally test, it has obvious inhibition effect on diabetes for mice and becomes a kind of new method of producing anti-I type diabetes drugs.
Description
Technical field
The present invention relates to a kind of fusion gene, its expressed proteins and preparation method.Specifically, the present invention relates to CTB and insulin human B chain fusion gene, its expressed proteins with and preparation method thereof.
Background technology
(insulin-dependent diabetes IDDM) is produced by defect of insulin secretion type i diabetes, and it is a kind of autoimmunity eqpidemic disease at beta Cell of islet.Though cause the autoantigen of IDDM to comprise multiple antigens such as Regular Insulin L-Glutamic decarboxylase, Regular Insulin, heat shock protein(HSP), carboxypeptidase H; but only give the oral a kind of autoantigen-Regular Insulin of animal in the experiment; promptly induced immunological tolerance; produced provide protection (Science; 1994,265:1237).This is because oral antigen can be induced the regulatory T cells secretion SC factor, as TGF-β, to by each other irrelevant various antigen inductions but suppress in same special organogenetic all pathology immune responses, promptly antigen drives the onlooker and suppresses (Antigen-drivenBystander Suppression).Inducing that the onlooker suppresses is antigen-specific, and the effect right and wrong are special, this for autoimmune diseases such as prevention type i diabetes provide a new approach (Immunol Today, 1998,19:173).Simultaneously, studies show that b subunit of cholera toxin (CTB) can be used as the powerful mucous membrane delivery system of inducing oral tolerance, and the CTB coupled antigen may relate to the heavy dose of oral autoantigen of tradition and produce the visibly different immune regulation mechanism of immunological tolerance effect, may relate to the variation (NatBiotechnol of the mobile behavior of inflammatory cells, 1998,16,292), hint CTB coupling autoantigen not only makes autoimmunity eqpidemic diseases such as using oral tolerance control type i diabetes have more actual application value, and also have potential to treat prospect (Proc Natl Acad Sci USA, 1997,94:4610).
Summary of the invention
One of purpose of the present invention provides a kind of CTB and insulin human B chain fusion gene.
Two of purpose of the present invention provides CTB and insulin human B chain fusion gene expressed proteins.
Three of purpose of the present invention provides a kind of proteic method of expressing CTB and insulin human B chain fusion gene for preparing.
The present invention realizes by following technical solution.
(1) makes up transferring plasmid pBac-CTBINB and the pBI-CTBINB that contains CTB and insulin human B chain fusion gene.Designing 2 primer P1 and P2, is that template, P1 and P2 are that primer increases with pBac-CTBINS plasmid (seeing 200310121132.1 patent application specifications), obtains fusion gene CTB-INB.Fusion gene sequence and amino acid sequence coded thereof are respectively shown in SEQ ID NO.1 and SEQ ID NO.2.The purpose fragment behind BamHI and EcoRI double digestion, is connected equally by on the transfer vector pBacPAK8 of BamHI and EcoRI double digestion, makes up the baculovirus transferring plasmid pBac-CTBINB that contains CTB and insulin human B chain fusion gene.Further with pBac-CTBINB behind BamHI and SacI double digestion, be connected equally by on the transfer vector pBI121 of BamHI and Sac I double digestion, make up the plant transferring plasmid pBI-CTBINB that contains CTB and insulin human B chain fusion gene.
P1<SEQ?ID?NO.3>：5′CGGGATCCATGATTAAATTAAAATTTGG3′
P2<SEQ?ID?NO.4>：5′GGGAATTCTTAGGTCTTGGGTGTGTAGA3′
(2) contain the acquisition of the recombinant silkworm baculovirus BmBacCTBINB of CTB and insulin human B chain fusion gene: transferring plasmid pBac-CTBINB and Bombyx mori nuclear polyhydrosis virus DNA are carried out cotransfection silkworm (Bombyx mori) cells BmN, carry out the screening of plaque and Southern dot blot, filter out the recombinant baculovirus BmBacCTBINB that contains CTB and insulin human B chain fusion gene.This virus has submitted to China Committee for Culture Collection of Microorganisms common micro-organisms center to preserve, and preservation date is on April 22nd, 2004, and deposit number is CGMCC No.1138.
(3) CTB and the expression of insulin human B chain fusion protein in silkworm: recombinant baculovirus BmBacCTBINB is infected the BmN bombyx mori cell carry out virus amplification, the recombinant baculovirus BmBacCTBINB after will increasing then is injected in silkworm larva and the pupal cell.
(4) CTB and the expression of insulin human B chain fusion protein in strawberry: the plant transferring plasmid pBI-CTBINB electricity consumption method that swashs is imported agrobacterium tumefaciens, cultivate altogether, at kantlex substratum screening transfer-gen plant by agrobacterium tumefaciens and stem apex.
The invention has the beneficial effects as follows: efficiently express CTB and insulin human B chain fusion protein with silkworm and strawberry, active higher than CTB that directly from prokaryotic organism, expresses and insulin human B chain fusion protein, silkworm is can aseptic extensive raising economic insects, can raise on a large scale at low cost, and can not cause public hazards, multiple natural protein protective material is arranged in the silkworm body, expression product is had provide protection, make gene expression product very stable; Strawberry is cultivation on a large scale then, can eat (and potato and tobacco etc. can not be eaten raw) raw.Can silkworm and the fusion rotein produced of strawberry can be used as oral or eat vaccine raw, removed the misery and the infected threat of injection for the patient.
Description of drawings
Fig. 1 is the insect baculovirus transferring plasmid pBac-CTB-INB (descriptive name: AcMNPV/AcNPV: autographa california polyhedrosis virus polyhedron promotor 5 ' end/3 ' end flanking sequence that contains CTB and insulin human B chain fusion gene; P: autographa california polyhedrosis virus polyhedron promotor; A (Polyhedin Poly A+signal) polyhedron gene polyadenylation signal; M13 ori:M13 phage replication starting point; Amp: penbritin gene; The ori:pUC replication origin)
Fig. 2 is that the SDS-PAGE of silkworm expression product analyzes collection of illustrative plates, and wherein: M represents the standard protein molecular weight marker; The silkworm hemolymph that 1 expression BmCTBINB infects; The silkworm hemolymph of 2 expression wild virus infections.
Fig. 3 is that the Western of silkworm expression product analyzes, and wherein: M represents the standard protein molecular weight marker; The silkworm hemolymph that 1 expression BmCTBINB infects; The silkworm hemolymph of 2 expression wild virus infections.
Embodiment
Further set forth the present invention below by embodiment, but do not limit protection scope of the present invention.
(embodiment 1) contains the transferring plasmid pBac-CTBINB of fusion gene and the structure of pBI-CTBINB
With pBac-CTBINS plasmid (seeing 200310121132.1 patent application specifications) is that template, P1 and P2 are that primer increases, reaction conditions is 94 ℃ of pre-sex change 5 minutes, during amplification 94 ℃ 1 minute, 57 ℃ 1 minute, 72 ℃ 1 minute, react 30 circulations, last 72 ℃ the insulation 10 minutes, obtain the big or small purpose fusion gene CTB-INB that is about about 500bp, and this gene 5 ' and 3 ' hold to have BamHI and EcoR I site respectively.The above-mentioned purpose fusion gene is carried out being connected in behind BamHI and the EcoRI double digestion equally on the pBacPAK8 (CLONTECH company) that is cut by BamHI and EcoRI enzyme, structure contains the baculovirus transferring plasmid pBac-CTBINB of CTB and insulin human B chain gene, after restriction analysis and the correct insertion of PCR identified gene, show that through automatic sequence mensuration the fusion gene sequence of structure is entirely true.PBac-CTBINB behind BamHI and SacI double digestion, is connected equally by on the transfer vector pBI121 of BamHI and SacI double digestion (CLONTECH company), makes up the plant transferring plasmid pBI-CTBINB that contains CTB and insulin human B chain fusion gene.
The acquisition of the recombinant baculovirus of (embodiment 2) CTB and insulin human B chain fusion gene
Get insect baculovirus transferring plasmid pBac-CTBINB and the wild Bombyx mori nuclear polyhydrosis virus DNA of 6ul that 5ul contains CTB and insulin human B chain fusion gene and carry out cotransfection.The TC-100 substratum of getting 6ul Lipofectin (GIBCOBRL company) adding 100ul serum-free is mixed.With BmN cell TC-100 (GIBCOBRL company) the substratum washed twice of serum-free of cultivating in advance in 35mm Dish, and dropwise add transferring plasmid and Lipofectin mixture, cultivated 4-5 days, and collected supernatant and carry out first round plaque select for 27 ℃.Get the BmN cell among the 5ul supernatant infection 35mm Dish, supernatant discarded adds the TC-100 substratum and the low melting-point agarose of balanced mix after 1 hour.Picking plaque after 4-5 days infected Bm N cell 3-4 days, preserved supernatant, and cell is used for Southern hybridization with the NaOH cracking, and the supernatant of getting positive colony carries out 10 and takes turns plaque select.The supernatant of getting positive colony infects Bm N cell amplification.Can obtain a large amount of recombinant baculovirus that contains CTB and insulin human B chain fusion gene.
The recombinant baculovirus that contains CTB and insulin human B chain fusion gene that amplification is got in (embodiment 3) CTB and the expression of insulin human B chain fusion gene in silkworm larva and pupa be expelled to five age silkworm (Bombyx mori) larva and pupa in, (titre is 1 * 10 about every injection 2ul
7/ ml), get the silkworm lymph and the pupa blood of expressing in 24,48,72,96,120 and 144 hours respectively, get supernatant after 5000rpm 5min is centrifugal, after 10 times of PBS pH7.4 dilutions, add isopyknic 2 * protein sample-loading buffer (100Mm Tris.HCl, 4%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), get 20ul and carry out the SDS-PAGE analysis, see accompanying drawing 2.The result shows that CTB and insulin human B chain fusion protein obtain to efficiently express in silkworm and pupa, show that through Western hybridization (accompanying drawing 3) the expression product molecular weight is 18kD.Silkworm larva and pupal cell are contained the CTB of expression and the hemolymph of insulin human B chain fusion protein, and the high speed frozen centrifugation is got supernatant.Coated elisa plate after diluting 1000 times; be standard substance with CTB simultaneously; ELISA step operation according to routine; wherein the anti-CT one of rabbit anti-(available from SIGMA company) dilution is 5000 times; 1000 times of goat antirabbit two anti-(available from Huamei Bio-Engrg Co.) dilutions with OPD (available from Shanghai chemical reagents corporation) colour developing, are measured the OD value in the 492nm place; the result shows that the target protein that larva and pupal cell are expressed has very strong immunocompetence, and expression amount reaches 380 μ g/ml and 400 μ g/ml respectively.
(embodiment 4) CTB and the expression of insulin human B chain fusion protein in strawberry
The plant transferring plasmid pBI-CTBINB electricity consumption method that swashs is imported agrobacterium tumefaciens lba4404 (CLONTECH company), cultivate altogether in its importing strawberry by agrobacterium tumefaciens and strawberry stem tip, on screening culture medium, screen, screening culture medium is to contain 0.6mg/LIAA, 1.5mg/L BA, the MS nutrient agar of 500mg/L carboxylic benzyl mycin and 100mg/L kantlex etc., at 28 ℃, after the light dark period of 16h/8h is cultivated 2-4 month down, the transgenosis seedling is transferred on the MS substratum that contains the 100mg/L kantlex, it is taken root, at last, the seedling of will regenerating moves on to the greenhouse and has obtained transfer-gen plant, confirms that by Southern hybridization and round pcr CTB and insulin human B chain fusion gene have been incorporated in the strawberry genome, and the primer that round pcr is identified is P1 and P2, according to " (molecular cloning " (Sambrook etc., translations such as Jin Dongyan, Science Press, 1992) ordinary method operate.By the transfer-gen plant fruit is extracted protein, add isopyknic 2 * protein sample-loading buffer (100Mm Tris.HCl, 4%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), get 20ul and carry out SDS-PAGE analysis and Wersten engram analysis.As can be seen from the results, CTB and insulin human B chain fusion gene expression product in strawberry fruit are about 18kD, and be consistent with the prediction size.Expression product to CTB and the fusion of insulin human B chain carries out immunocompetence evaluation (working method is with embodiment 3), the result shows that expression product has very strong immunocompetence, can calculate expression amount in strawberry fruit by the CTB standard substance and reach and account for 0.5% of soluble protein content.
The animal experiment of CTB that expresses in (embodiment 5) silkworm larva silkworm chrysalis and the strawberry fruit and insulin human B chain fusion protein effect
Express CTB and insulin human B chain fusion protein at silkworm larva and pupal cell, through filtering (100KD ultrafiltration), 16000rmp and 35000rmp are centrifugal after after the lyophilize, with silkworm larva and silkworm pupa is that stopping composition is mixed with oral pharmaceutical, is directly used in oral animal experiment after strawberry fruit then grinds.With 4 ages in week female NOD mouse be divided into 4 groups at random, 5 every group, since feeding in the 5th week, dosage is 100 μ g/ mouse, administration is 2 times weekly, feeds for 5 weeks continuously.To normal silkworm hemolymph and the negative contrast of strawberry fruit, the 10th week was put to death mouse, got mouse islets and made tissue pathological slice with oral, and a situation arises to observe insulitis, and do corresponding scoring.The result shows that the scorching index of the mouse islets of administration group is 1.5 ± 1.3, and mice in control group insulitis index is up to being 4.2 ± 1.7, and administration group insulitis significantly is lower than control mice.
Untitledl.ST25.txt
SEQUENCE?LISTING
＜110〉Zhejiang University
＜120〉a kind of fusion gene, its expressed proteins and preparation method thereof
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ggaacacctc?aaaatattac?tgatttgtgt?gcagaatacc?acaacacaca?aatacatacg 120
ctaaatgata?agatattttc?gtatacagaa?tctctagctg?gaaaaagaga?gatggctatt 180
attactttta?agaatggtgc?aacttttcaa?gtagaagtac?caggtagtca?acatatagat 240
tcacaaaaaa?aagcgattga?aaggatgaag?gataccctga?ggattgcata?tcttactgaa 300
gctaaagtcg?aaaagttatg?tgtatggaat?aataaaacgc?ctcatgcgat?tgccgcaatt 360
agtatggcaa?atggccccgg?cccctttgtg?aaccaacacc?tgtgcggctc?acacctggtg 420
gaagctctct?acctagtgtg?cggggaacga?ggcttcttct?acacacccaa?gacctaa 477
Untitledl.ST25.txt
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Met?Ile?Lys?Leu?Lys?Phe?Gly?Val?Phe?Phe?Thr?Val?Leu?Leu?Ser?Ser
1 5 10 15
Ala?Tyr?Ala?His?Gly?Thr?Pro?Gln?Asn?Ile?Thr?Asp?Leu?Cys?Ala?Glu
20 25 30
Tyr?His?Asn?Thr?Gln?Ile?His?Thr?Leu?Asn?Asp?Lys?Ile?Phe?Ser?Tyr
35 40 45
Thr?Glu?Ser?Leu?Ala?Gly?Lys?Arg?Glu?Met?Ala?Ile?Ile?Thr?Phe?Lys
50 55 60
Asn?Gly?Ala?Thr?Phe?Gln?Val?Glu?Val?Pro?Gly?Ser?Gln?His?Ile?Asp
65 70 75 80
Ser?Gln?Lys?Lys?Ala?Ile?Glu?Arg?Met?Lys?Asp?Thr?Leu?Arg?Ile?Ala
85 90 95
Tyr?Leu?Thr?Glu?Ala?Lys?Val?Glu?Lys?Leu?Cys?Val?Trp?Asn?Asn?Lys
100 105 110
Thr?Pro?His?Ala?Ile?Ala?Ala?Ile?Ser?Met?Ala?Asn?Gly?Pro?Gly?Pro
115 120 125
Phe?Val?Asn?Gln?His?Leu?Cys?Gly?Ser?His?Leu?Val?Glu?Ala?Leu?Tyr
130 135 140
Leu?Val?Cys?Gly?Glu?Arg?Gly?Phe?Phe?Tyr?Thr?Pro?Lys?Thr
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Untitledl.ST25.txt
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Claims (3)
1, a kind of CTB and insulin human B chain fusion gene is characterized in that it has the base sequence shown in the SEQ IDNO.1.
2, CTB and insulin human B chain fusion gene expressed proteins is characterized in that it has the protein sequence shown in the SEQ ID NO.2.
3, a kind of expression CTB and the proteic method of insulin human B chain fusion gene comprise the steps:
(1) 2 primer P1 of design and P2, with the pBac-CTBINS plasmid is template, P1 and P2 are that primer increases, obtain fusion gene CTB-INB, with the purpose fragment that obtains behind BamHI and EcoRI double digestion, be connected equally by on the transfer vector pBacPAK8 of BamHI and EcoRI double digestion, structure contains the baculovirus transferring plasmid pBac-CTBINB of CTB and insulin human B chain fusion gene, further with pBac-CTBINB behind BamHI and SacI double digestion, be connected equally by on the transfer vector pBI121 of BamHI and SacI double digestion, make up the plant transferring plasmid pBI-CTBINB that contains CTB and insulin human B chain fusion gene;
Wherein, primer sequence is respectively:
P1＜SEQ?ID?NO.3＞5′CGGGATCCATGATTAAATTAAAATTTGG3′
P2＜SEQ?ID?NO.4＞5′GGGAATTCTTAGGTCTTGGGTGTGTAGA3′
(2) transferring plasmid pBac-CTBINB and Bombyx mori nuclear polyhydrosis virus DNA are carried out cotransfection bombyx mori cell BmN, filter out the recombinant baculovirus BmBacCTBINB that contains CTB and insulin human B chain fusion gene by plaque and Southern dot blot;
(3) recombinant baculovirus BmBacCTBINB is infected the BmN bombyx mori cell and carry out virus amplification, the recombinant baculovirus BmBacCTBINB after will increasing then is injected in silkworm larva and the pupal cell, extracts silkworm larva and pupa lymph blood respectively;
Perhaps, the plant transferring plasmid pBI-CTBINB electricity consumption method that swashs is imported agrobacterium tumefaciens, cultivate altogether, at kantlex substratum screening transfer-gen plant by agrobacterium tumefaciens and stem apex.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101353663B (en) * | 2008-03-12 | 2012-04-25 | 浙江大学 | Anti-I type diabetes fuse protein and preparation thereof |
| CN103820477A (en) * | 2014-01-15 | 2014-05-28 | 浙江大学 | Fusion protein of CTB (Cellulose Tribenzoate), human insulin and glutamic acid decarboxylase 3p531 fragments and application thereof |
| WO2016161983A1 (en) * | 2015-04-10 | 2016-10-13 | 中国医学科学院药物研究所 | Fusion carrier protein and application thereof in promoting target protein or polypeptide expression |
| WO2018188179A1 (en) * | 2017-04-14 | 2018-10-18 | 北京百华百汇生物科技有限公司 | Glucagon-like peptide-1 receptor agonist fusion protein and use thereof |
| CN110256578A (en) * | 2019-06-24 | 2019-09-20 | 王跃驹 | The application of plant production people b subunit of cholera toxin (CTB) and the fusion protein quick results Jiangtang capsule of proinsulin |
-
2004
- 2004-08-25 CN CNB200410056850XA patent/CN100471955C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101353663B (en) * | 2008-03-12 | 2012-04-25 | 浙江大学 | Anti-I type diabetes fuse protein and preparation thereof |
| CN103820477A (en) * | 2014-01-15 | 2014-05-28 | 浙江大学 | Fusion protein of CTB (Cellulose Tribenzoate), human insulin and glutamic acid decarboxylase 3p531 fragments and application thereof |
| WO2016161983A1 (en) * | 2015-04-10 | 2016-10-13 | 中国医学科学院药物研究所 | Fusion carrier protein and application thereof in promoting target protein or polypeptide expression |
| WO2018188179A1 (en) * | 2017-04-14 | 2018-10-18 | 北京百华百汇生物科技有限公司 | Glucagon-like peptide-1 receptor agonist fusion protein and use thereof |
| CN110256578A (en) * | 2019-06-24 | 2019-09-20 | 王跃驹 | The application of plant production people b subunit of cholera toxin (CTB) and the fusion protein quick results Jiangtang capsule of proinsulin |
| CN110256578B (en) * | 2019-06-24 | 2021-04-23 | 王跃驹 | Application of plant produced human cholera toxin B subunit (CTB) and proinsulin fusion protein quick-acting oral hypoglycemic capsule |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100471955C (en) | 2009-03-25 |
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