CN1629288A - Recombinant Baculovirus for expressing CTB and human insulin fusion protein and use thereof - Google Patents
Recombinant Baculovirus for expressing CTB and human insulin fusion protein and use thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to the preparation of polypeptides medicament through genetic engineering, which comprises restructuring fusion gene with CTB and human insuline with Bombyx mori Nuclear polyhedrosis Virus, obtaining the recombinant viruses, inoculating the recombinant virus to domestic silkworm larva and aurelia for expression, freeze drying to obtain the oral medication. Animal test has shown that, the medicament has appreciable actions for lowering rat blood sugar and repressing diabetes, thus a novel method for preparing anti-I type diabetes oral medication can be achieved.
Description
Technical field
The present invention relates to a kind of recombinant baculovirus, prepare fusion rotein and the application in preparation anti-I type diabetes oral pharmaceutical thereof by this recombinant virus.
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 (ImmunolToday, 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 variation (the Nat Biotechnol 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).
Expressed fusion protein mainly is to be undertaken by transgenic plant at present, and its defective is that expression amount is too low, only accounts for the 0.01-0.4% of soluble protein content, and the tolerance effect of generation is also low.
Summary of the invention
The baculovirus expression vector system that one of purpose of the present invention provides a kind of CTB and human insuline recombinant gene and contains this recombination.
Two of purpose of the present invention provides a kind of CTB and insulin human's fusion rotein and preparation method thereof.
Three of purpose of the present invention provides a kind of with the silkworm larva of recombinant silkworm baculovirus infection and anti-I type diabetes oral pharmaceutical of pupa preparation and preparation method thereof.
The present invention realizes by following technical solution.
Baculovirus expression vector system (Bombyx mori Nuclear polyhedrosis Virus) is an eukaryotic expression system, this technology is since initiatives such as nineteen eighty-three Smith, existing hundred kinds of foreign genes are in this system expression (" insect viruses molecular biology ", 1998,547).Because a large amount of proteic provide protection of silkworm lymph principal body and the effect of some unknown mechanisms, the albumen of silkworm expression can be produced oral pharmaceutical.The present invention uses molecule clone technology to obtain CTB and insulin human's fusion gene, adopt baculovirus expression vector system (Bombyxmori Nuclear polyhedrosis Virus), and in silkworm larva and pupa, express this fusion rotein, through separation and purification, oral pharmaceutical are made in lyophilize.
Concrete technical scheme is:
(1) construction recombination plasmid pBac-INS: (Science 1980 according to the human insulin gene sequence; 208:57) 6 primers of design, concrete sequence sees Table 1, and wherein F1, F2, F3 are forward primer, R1, R2, R3 reverse primer, the 5 ' end of F1 is added with the BamHI site, and the 5 ' end of R1 is added with EcoRI site (black matrix is represented).Wherein the C peptide between B chain and the A chain is replaced by 6 amino acid RRGSKR, so that can (Gene 1981 by protease hydrolyzed; 16:63).Method by chain extension reaction and twice PCR obtains to connect on transfer vector pBacPAK8 behind the aim sequence, obtains recombinant plasmid pBac-INS.
Table 1
| Primer | Nucleotide sequence | ??SIN |
| ??F1 | ??5’-GGGGATCCATGTTTGTGAACCAACACCTGTGCGGCTCACA-3’ | ????3 |
| ??F2 | ??5’-CCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGG-3’ | ????4 |
| ??F3 | ??5’-CTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCC-3’ | ????5 |
| ??R1 | ??5’-GGGAATTCTTAGTTGCAGTAGTTCTCCAGCTGGTAGAGG-3’ | ????6 |
| ??R2 | ??5’-TCCAGCTGGTAGAGGGAGCAGATGCTGGTACAGCATTGTTCCACAATGCC-3’ | ????7 |
| ??R3 | ??5’-TTGTTCCACAATGCCACGCTTGGAGCCCCGGCGGGTCTTGGGTGTGTAGA-3’ | ????8 |
*SIN：SEQ?ID?NO.
(2) gene fusion construct CTB-INS: design 3 primers, concrete sequence sees Table 2.Primer Pct and P1 use terminator that removes the CTB gene and the sequence that adds upper binding head at its 3 ' end; Primer P2 and R1 use initiator codon that removes human insulin gene and the sequence (black matrix is represented) that adds upper binding head at its 5 ' end.(this plasmid is open by Chinese 01144502.5 patent application " method for making of b subunit of cholera toxin " with the pBac-CTB plasmid, and preserve at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.0594) be that template, Pctb and P1 are that primer increases, make between CTB gene and the human insulin gene to be connected, obtain fragment CTB-GPGP with flexible tetrapeptide (GPGP).With the pBac-INS plasmid is that template, P2 and R1 are primer, obtains INS-GPGP by amplification.With CTB-GPGP and INS-GPGP each other primer and template carry out chain extension reaction, obtain product and be designated as S1, be that template, Pctb and R1 are that primer increases again with S1, obtain size and be about fusion gene CTB-INS about 560bp.The fusion gene sequence is shown in SEQ ID NO.1, and its amino acid sequence coded is shown in SEQ ID NO.2.
Table 2
| Primer | Nucleotide sequence | ????SEQ?ID?NO. |
| ??Pctb | ????5’-CGGGATCCATGATTAAATTAAAATTTGG-3’ | ????9 |
| ??P1 | ????5’-GGGGCCGGGGCCATTTGCCATACTAATTG-3’ | ????10 |
| ??P2 | ????5’-GGCCCCGGCCCCTTTGTGAACCAACACCT-3’ | ????11 |
(3) make up the insect baculovirus transferring plasmid pBac-CTB-INS contain fusion gene: the purpose fragment (CTB-INS) that PCR is obtained is 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-CTB-INS of CTB and insulin human's fusion gene, and its structure is seen Fig. 1;
(4) contain the acquisition of the recombinant silkworm baculovirus BmBacCTBINS of CTB and insulin human's fusion gene: transferring plasmid pBac-CTB-INS and Bombyx mori nuclear polyhydrosis virus DNA are carried out cotransfection silkworm (Bombyxmori) cells BmN, carry out 10 and take turns plaque and the screening of Southern dot blot, filter out the recombinant baculovirus BmBacCTBINS that contains CTB and insulin human's 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 November 7th, 2003, and deposit number is CGMCC No.1033;
(5) express CTB and insulin human's fusion rotein: recombinant baculovirus BmBacCTBINS is infected the BmN bombyx mori cell carry out virus amplification, recombinant baculovirus BmBacCTBINS after will increasing then is injected in silkworm larva and the pupal cell, get silkworm larva and pupa lymph blood respectively, after 5000rpm 5 minutes is centrifugal, get supernatant, add isopyknic 2 * albumen sample-loading buffer after diluting 10 times, carry out 12% SDS-PAGE analysis.With coomassie brilliant blue R250 dyeing, the results are shown in Figure 2.From SDS-PAGE and Wersten trace (Fig. 3) result as can be seen, CTB and the expression product of insulin human's fusion gene in silkworm larva and pupa are about 21kD, and be consistent with the prediction size.Expression product to CTB and insulin human's fusion carries out the immunocompetence evaluation, the result shows that expression product has very strong immunocompetence, the expression amount that can calculate in larva and pupal cell by the CTB standard substance reaches 500 μ g/ml and 600 μ g/ml respectively, be much higher than the expression amount in transgenic plant, realized efficiently expressing.
CTB and the insulin human's fusion rotein expressed at silkworm larva and pupal cell are through filtering, after centrifugal, the lyophilize, being that stopping composition is mixed with oral pharmaceutical with silkworm larva and silkworm pupa.
The invention has the beneficial effects as follows: efficiently express CTB and insulin human's fusion rotein with silkworm, active higher than the fusion rotein of directly from prokaryotic organism, expressing, than the productive rate height of expressing by transgenic plant; 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: the fusion rotein that silkworm produces can be used as oral pharmaceutical, has 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-INS (descriptive name: AcMNPV/AcNPV: autographa california polyhedrosis virus polyhedron promotor 5 ' end/3 ' end flanking sequence that contains CTB and insulin human's 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, wherein: M, standard protein molecular weight marker; 1, the silkworm hemolymph of BmCTBINS infection; 2, the silkworm hemolymph of wild virus infection.
Fig. 3 is that the Western of silkworm expression product analyzes, wherein: M, standard protein molecular weight marker; 1, the silkworm hemolymph of BmCTBINS infection; 2, the silkworm hemolymph of wild virus infection.
Embodiment
Further set forth the present invention below by embodiment, but do not limit protection scope of the present invention.
The structure of (embodiment 1) CTB and insulin human's fusion gene
At first, (Science 1980 according to 6 primers of the human insulin gene sequences Design of having delivered; 208:57), wherein the C peptide between B chain and the A chain is replaced by 6 amino acid whose " mini-C " peptides (RRGSKR) that (Gene 1981; 16:63) wherein F1, F2, F3 are forward primer, R1, R2, R3 reverse primer (table 1), and the 5 ' end of F1 is added with the BamHI site, and the 5 ' end of R1 is added with EcoRI site (black matrix is represented).
Method by chain extension reaction and twice PCR obtains the insulin human: (1) chain extension: reaction system contains primers F 3 and R3, the Taq enzyme of 5 units and other conventional PCR reagent (worker company is given birth in Shanghai); Reaction conditions is 94 ℃ of sex change 10 minutes, and 55 ℃ of 5 minutes relief F3 of annealing and R3 primer and template each other extended 5 minutes at 72 ℃, obtained product and were designated as SegI; (2) PCR for the first time: with SegI is template, F2 and R2 are primer, reaction conditions is 94 ℃ of pre-sex change 5 minutes, during amplification 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, react 30 circulations, last 72 ℃ of insulations 5 minutes, this moment, product was designated as SegII, and with DNA cleaning agents box (the clean biotech company of Hangzhou Wei Te) this product of purifying; (3) the 2nd PCR: the SegII with purifying is a template, and F1 and R1 are primer, and reaction conditions is 94 ℃ of pre-sex change 5 minutes, during amplification 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, react 30 circulations, last 72 ℃ of insulations 10 minutes obtain the PCR product at last and are designated as SegIII.Once more after this purpose fragment of purifying, reclaim gene fragment with BamHI and EcoRI double digestion low melting-point agarose gel, be connected with same pBacPAK8 carrier segments through BamHI and EcoRI double digestion, cut and PCR evaluation acquisition recombinant plasmid pBac-INS through enzyme, entirely true by the nucleotide sequence of order-checking reference insulin gene.
Be gene fusion construct, design 3 primer Pctb, P1 and P2 again, make between CTB gene and the human insulin gene to be connected with flexible tetrapeptide (GPGP).With the pBac-CTB plasmid is template, and Pctb and P1 are primer, and reaction conditions is 94 ℃ of pre-sex change 5 minutes, during amplification 94 ℃ 1 minute, 59 ℃ 1 minute, 72 ℃ 1 minute, react 30 circulations, last 72 ℃ of insulations 10 minutes obtain fragment and are designated as CTB-GPGP; Be template with the pBac-INS plasmid equally, P2 and R1 are primer, and 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 ℃ of insulations 10 minutes obtain fragment and are designated as GPGP-INS.At last with fragment CTB-GPGP and GPGP-INS gene fusion construct, at first carry out chain extension reaction: CTB-GPGP that purifying is crossed in the reaction system and GPGP-INS be primer and template each other, reaction conditions is 94 ℃ of sex change 10 minutes, annealed 5 minutes for 59 ℃, 72 ℃ were extended 5 minutes, and obtained product and be designated as S1.Be template with S1 again, Pctb and R1 are primer, and reaction conditions is 94 ℃ of pre-sex change 5 minutes during amplification, during amplification 94 ℃ 1 minute, 59 ℃ 1 minute, 72 ℃ 1 minute, react 30 circulations, last 72 ℃ of insulations 10 minutes obtain the big or small purpose fusion gene CTB-INS that is about about 560bp.
The structure of the insect baculovirus transferring plasmid of (embodiment 2) CTB and insulin human's fusion gene
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-CTB-INS of CTB and human insulin 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, fusion gene sequence and amino acid sequence coded thereof are shown in SEQ ID NO.1 and SEQ ID NO.2.
The acquisition of the recombinant baculovirus of (embodiment 3) CTB and insulin human's fusion gene
Get insect baculovirus transferring plasmid pBac-CTB-INS and the wild Bombyx mori nuclear polyhydrosis virus DNA of 6ul that 5ul contains CTB and insulin human's 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 the 10th 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's fusion gene, this virus has submitted to China Committee for Culture Collection of Microorganisms common micro-organisms center to preserve, the address is in the BeiJing ZhongGuanCun, and preservation date is on November 7th, 2003, and deposit number is CGMCC No.1033.
(embodiment 4) CTB and the expression of insulin human's fusion gene in silkworm larva and pupa
The recombinant baculovirus that contains CTB and insulin human's fusion gene of getting amplification 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 (100MmTris.HCl, 4%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), get 20ul and carry out the SDS-PAGE analysis.The result shows that CTB and insulin human's fusion rotein obtain to efficiently express in silkworm and pupa, show that through Western hybridization the expression product molecular weight is 21kD.
The CTB that (embodiment 5) silkworm is expressed and the immunocompetence of insulin human's fusion rotein are measured
Silkworm larva and pupal cell are contained the CTB of expression and the hemolymph of insulin human's fusion rotein, 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 500 μ g/ml and 600 μ g/ml respectively.
The CTB of (embodiment 6) expression in silkworm larva and pupa and the preparation of insulin human's fusion rotein oral pharmaceutical
With recombinant virus BmBacCTBINS percutaneous puncture-inoculation silkworm larva in five ages and pupal cell (applicant raises voluntarily), collect the silkworm lymph blood and the pupal cell of expressing the 5th day, the 12000rmp high speed centrifugation is got supernatant and is used for determination of activity.Supernatant is through filtering (100KD ultrafiltration), the 12000rmp high speed centrifugation, after 35000rmp is centrifugal, get the supernatant lyophilize again after, be that stopping composition is mixed with oral pharmaceutical with the silkworm pupa.
CTB and insulin human's fusion rotein that (embodiment 7) silkworm larva silkworm chrysalis is expressed carry out animal experiment
With 4 the week ages female NOD mouse be divided into 4 groups at random, preceding two groups every group 5, do pathological analysis, the back two groups every group 10, do diabetes monitoring.Since the 5th all oral administrations, dosage is 100 μ g/ mouse, and administration is 2 times weekly.Do pathological analysis, fed for 5 weeks continuously, the feeding 25 weeks continuously of diabetes monitoring.Before and after one group of negative contrast is all arranged in two groups, orally give normal silkworm hemolymph.Pathological analysis: the 10th week was put to death mouse, got its tail vein and made antibody titers mensuration, measured the insulin antibody level that oral back mouse produces; And get mouse islets and make tissue slice, a situation arises to observe insulitis, and do corresponding scoring.Diabetes detect: from the 10th week, detect the glucose in urine situation of NOD mouse urine, positive person continues to survey blood sugar, suffers from diabetes if continuous two all blood-sugar contents, then are judged to be this mouse more than or equal to 16.7mmol/L.Respectively mouse morbidity in two groups is compared statistical study.Mouse islets is made the tissue slice result and is shown that administration group insulitis significantly is lower than contrast NOD mouse; The negative 90% contrast NOD mouse in 25 week backs has suffered from diabetes, and the administration group then has only the 20%NOD mouse to suffer from diabetes.
Sequence table
＜110〉Zhejiang University
＜120〉the recombinant silkworm baculovirus of expression CTB and insulin human's fusion rotein and the application in preparation anti-I type diabetes oral pharmaceutical thereof
<160>11
<170>PatentIn?version?3.1
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<211>558
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<213>Artificial
<400>1
atgattaaat?taaaatttgg?tgtttttttt?acagttttac?tatcttcagc?atatgcacat?????60
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?gacccgccgg????480
ggctccaagc?gtggcattgt?ggaacaatgc?tgtaccagca?tctgctccct?ctaccagctg????540
gagaactact?gcaactaa??????????????????????????????????????????????????558
<211>185
<212>PRT
<213>Artificial
<400>2
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?Ash?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?Arg?Arg
145?????????????????150?????????????????155?????????????????160
Gly?Ser?Lys?Arg?Gly?Ile?Val?Glu?Gln?Cys?Cys?Thr?Ser?Ile?Cys?Ser
165?????????????????170?????????????????175
Leu?Tyr?Gln?Leu?Glu?Asn?Tyr?Cys?Asn
180?????????????????185
<210>3
<211>40
<212>DNA
<213>Artificial
<400>3
ggggatccat?gtttgtgaac?caacacctgt?gcggctcaca????????????????????????????40
<210>4
<211>45
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<400>4
cctgtgcggc?tcacacctgg?tggaagctct?ctacctagtg?tgcgg??????????????????????45
<210>5
<211>50
<212>DNA
<213>Artificial
<400>5
ctacctagtg?tgcggggaac?gaggcttctt?ctacacaccc?aagacccgcc?????????????????50
<210>6
<211>39
<212>DNA
<213>Artificial
<400>6
gggaattctt?agttgcagta?gttctccagc?tggtagagg?????????????????????????????39
<210>7
<211>50
<212>DNA
<213>Artificial
<400>7
tccagctggt?agagggagca?gatgctggta?cagcattgtt?ccacaatgcc?????????????????50
<210>8
<211>50
<212>DNA
<213>Artificial
<400>8
ttgttccaca?atgccacgct?tggagccccg?gcgggtcttg?ggtgtgtaga?????????????????50
<210>9
<211>28
<212>DNA
<213>Artificial
<400>9
cgggatccat?gattaaatta?aaatttgg?????????????????????????????????????????28
<210>10
<211>29
<212>DNA
<213>Artificial
<400>10
ggggccgggg?ccatttgcca?tactaattg????????????????????????????????????????29
<210>11
<211>29
<212>DNA
<213>Artificial
<400>11
ggccccggcc?cctttgtgaa?ccaacacct????????????????????????????????????????29
Claims (7)
1. a fusion gene is characterized in that being formed by human insulin gene and CTB gene recombination, has the base sequence shown in the SEQ ID NO.1.
2. recombinant silkworm baculovirus is characterized in that including the fusion gene of base sequence shown in the SEQ ID NO.1, and deposit number is CGMCC No.1033.
3. the preparation method of the described recombinant silkworm baculovirus of claim 2 comprises the steps:
(1) construction recombination plasmid pBac-INS:(a) with F3 and R3 primer and template each other, it is template with SegI that extension obtains product S egI (b), F2 and R2 are primer, it is template with the SegII of purifying that amplification obtains product S egII (c), F1 and R1 are primer, amplification obtains PCR product S egIII, once more after this purpose fragment of purifying, reclaim gene fragment with BamHI and EcoRI double digestion low melting-point agarose gel, be connected with same pBacPAK8 carrier segments, obtain recombinant plasmid pBac-INS through BamHI and EcoRI double digestion;
(2) be that template, Pctb and P1 are that primer increases gene fusion construct CTB-INS:(a) with the pBac-CTB plasmid, obtaining fragment CTB-GPGP (b) is that template, P2 and R1 are primer with the pBac-INS plasmid, primer and template are carried out chain extension reaction each other with CTB-GPGP and INS-GPGP to obtain fragment INS-GPGP (c) by amplification, obtain product S 1, be that template, Pctb and R1 are that primer increases again with S1, obtain fusion gene CTB-INS;
(3) above-mentioned fusion gene is carried out be connected in behind BamHI and the EcoRI double digestion equally on the pBacPAK8 that is cut by BamHI and EcoRI enzyme, make up the baculovirus transferring plasmid pBac-CTB-INS that contains CTB and human insulin gene;
(4) get the insect baculovirus transferring plasmid pBac-CTB-INS and the wild Bombyx mori nuclear polyhydrosis virus DNA that contain CTB and insulin human's fusion gene and carry out the recombinant silkworm baculovirus BmBacCTBINS that cotransfection obtains to contain CTB and insulin human's fusion gene;
Wherein, primers F 1 sequence is＜SEQ ID NO.3 〉:
5’-GGGGATCCATGTTTGTGAACCAACACCTGTGCGGCTCACA-3’
Primers F 2 sequences are＜SEQ ID NO.4 〉:
5’-CCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGG-3’
Primers F 3 sequences are＜SEQ ID NO.5 〉:
5’-CTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCC-3’
Primer R1 sequence is＜SEQ ID NO.6 〉:
5’-GGGAATTCTTAGTTGCAGTAGTTCTCCAGCTGGTAGAGG-3’
Primer R2 sequence is＜SEQ ID NO.7 〉:
5’-TCCAGCTGGTAGAGGGAGCAGATGCTGGTACAGCATTGTTCCACAATGCC-3’
Primer R3 sequence is＜SEQ ID NO.8 〉:
5’-TTGTTCCACAATGCCACGCTTGGAGCCCCGGCGGGTCTTGGGTGTGTAGA-3’
Primer Pctb sequence is＜SEQ ID NO.9 〉: 5 '-CGGGATCCATGATTAAATTAAAATTTGG-3 '
Primer P1 sequence is＜SEQ ID NO.10 〉: 5 '-GGGGCCGGGGCCATTTGCCATACTAATTG-3 '
Primer P2 sequence is＜SEQ ID NO.11 〉: 5 '-GGCCCCGGCCCCTTTGTGAACCAACACCT-3 '.
4. a fusion rotein is characterized in that being formed by insulin human and CTB reorganization, has the aminoacid sequence shown in the SEQ ID NO.2.
5. the preparation method of the described fusion rotein of claim 4 comprises the steps:
(1) construction recombination plasmid pBac-INS;
(2) gene fusion construct CTB-INS;
(3) make up the insect baculovirus transferring plasmid pBac-CTB-INS that contains fusion gene;
(4) acquisition contains the recombinant silkworm baculovirus BmBacCTBINS of CTB and insulin human's fusion gene;
(5) express CTB and insulin human's fusion rotein: recombinant baculovirus BmBacCTBINS is infected the BmN bombyx mori cell carry out virus amplification, the recombinant baculovirus BmBacCTBINS after will increasing then is injected in silkworm larva and the pupal cell, gives expression to fusion rotein.
6. a medicine for the treatment of type i diabetes is characterized in that comprising fusion rotein as claimed in claim 4 and silkworm weighting agent.
7. the preparation method of the described treatment type i diabetes of claim 6 medicine is characterized in that and will express the silkworm larva of CTB and insulin human's fusion rotein and pupa after filtration, make after the centrifugal and lyophilize.
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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 |
| CN101392027B (en) * | 2008-07-03 | 2012-04-25 | 浙江大学 | Fusion protein for treating Alzheimer disease and preparation method 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 |
| 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 |
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2003
- 2003-12-15 CN CNB2003101211321A patent/CN1277925C/en not_active Expired - Fee Related
Cited By (7)
| 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 |
| CN101392027B (en) * | 2008-07-03 | 2012-04-25 | 浙江大学 | Fusion protein for treating Alzheimer disease and preparation method 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 |
| 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 |
| WO2020259110A1 (en) * | 2019-06-24 | 2020-12-30 | 王跃驹 | Application of plant-produced fast-acting oral hypoglycemic capsules of fusion protein of human cholera toxin b subunit (ctb) and 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 |
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| Publication number | Publication date |
|---|---|
| CN1277925C (en) | 2006-10-04 |
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