CN1170927C - Method for producing medicine using silkworm expressed numan epidermal growth factor - Google Patents
Method for producing medicine using silkworm expressed numan epidermal growth factor Download PDFInfo
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- CN1170927C CN1170927C CNB011241233A CN01124123A CN1170927C CN 1170927 C CN1170927 C CN 1170927C CN B011241233 A CNB011241233 A CN B011241233A CN 01124123 A CN01124123 A CN 01124123A CN 1170927 C CN1170927 C CN 1170927C
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Abstract
The present invention belongs to the technical field of producing polypeptide mdicine by gene engineering in biotechnological pharmaceutical engineering. The present invention realizes that a human EGF gene is cloned to a transfer vector pBacPAK8 of a bombyx mori nuclearpolyhedrosis virus and homologously recombined with the linearized bombyx mori nuclearpolyhedrosis virus in a bombyx mori cell, so a recombination bombyx mori nuclearpolyhedrosis virus BmBacEGF with a gene of a human epidermal growth factor is obtained. The virus is submitted to the common micro-organism center of the microscobial preservation and management committee of China to be preserved, the address is zhongguancun of beijing, the preserving date is 02 july, 2001, and the preserving number is 0593. Stab inoculation is carried out to a larva and a pupal cell of a bombyx mori by the recombination bombyx mori nuclearpolyhedrosis virus to realize that the human epidermal growth factor is effectively expressed in the larva and the pupal cell of the bombyx mori, and the larva and the pupal cell which effectively express the human epidermal growth factor are developed into oral medicine. Animal experiments indicate that the medicine has obvious action on treating peptic ulcers and provides a new method for treating peptic ulcer diseases. Compared with the prior art, the present invention has the advantages of easy raw material acquisition, low production cost and good therapeutic effect.
Description
Technical field
The present invention relates to silkworm expressed numan epidermal growth factor (Epidermal growth Factor, EGF) method of production medicine.
Background technology
Urogastron (EGF) be by Cohen nineteen fifty-nine study nerve growth factor (nervegrowth factor, serendipitous in the time of NGF), from mouse submandibular gland, separated to obtain in 1962.Vivo and vitro experimental results show that it has the function that promotes propagation to multiple tissue-derived epithelial cell, and people's Urogastron is equaled to be purified to from people's urine in 1975 by Gregory the earliest, has identical biologic activity by discovering with mouse EGF.The most potential purposes of EGF is in clinical application, and EGF has the function that promotes epithelium regeneration, can be used to promote the healing of surgical incision and other surface of a wound, as the scratch of dermatoplasty, skin, burn, erosion etc.; Promote the propagation of corneal epithelial cell, be used for treating the survival of corneal injury, ulcer, soda acid burn and promotion corneal transplant.EGF plays very vital role in human gi-tract, duodenal mucosa ulcer healing process, stomach, duodenal mucosa damage that multiple stimulation is caused all have certain protection and therapeutic action.EGF mainly is in expression in escherichia coli (Yadwad etc., 1989 at present; Shimizum etc., 1991) be essentially injecting drug use, the production cost height does not have oral dosage form basically on the market.Baculovirus expression vector system (Bombyx moriNuclear 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 at this system expression (" insect viruses molecular biology "; 1998; 547-578); because a large amount of proteic provide protection of silkworm lymph principal body and the effect of some unknown mechanisms are arranged; the albumen of silkworm expression can be produced oral pharmaceutical; and because EGF directly stimulates the hyperplasia of epithelium; promote tissue repair, so oral EGF makes, and high density is another exploration approach of clinical peptide ulceration in its maintenance stomach.
Summary of the invention
For this reason, the present invention's chemical synthesis process synthetic people EGF gene, be cloned into pBluscriptSK, after order-checking is justified this gene clone is arrived among silkworm baculovirus (Bombyx mori Nuclearpolyhedrosis Virus) the transfer vector pBacPAK8, with cut through the AocI enzyme after the linearizing silkworm baculovirus, in bombyx mori cell, carry out homologous recombination, obtain the recombinant silkworm baculovirus BmBacEGF of band human epidermal growth factor 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, preservation date is July 2 calendar year 2001, deposit number is CGMCC NO:0593, with recombinant silkworm baculovirus stab inoculation silkworm larva and pupal cell, make the human epidermal growth factor in silkworm larva and pupa, obtain to efficiently express.The larva and the silkworm chrysalis that efficiently express the human epidermal growth factor are developed into oral pharmaceutical, show the remarkable effect that the treatment peptide ulceration is had through animal experiment.
The invention provides structure with chemosynthesis people's EGF gene and plasmid pSK-EGF.According to known person EGF gene order (Proc.Natl.Acad.Sci.USA, 1983, VOL80,7462), oligonucleotide chain with dna synthesizer and synthetic 4 the coding EGF of tris phosphite method, with the urea-denatured gel electrophoresis separation and purification of polyacrylamide-7mol/L, the extension of 5 ' terminal phosphateization of oligonucleotide chain, renaturation, chain, connection, EcoRI endonuclease digestion, and be cloned into the EcoRI site of pBluscript SK, construction recombination plasmid pSK-EGF, entirely true through its nucleotide sequence of order-checking proof.
The present invention also provides the baculovirus transferring plasmid that contains people EGF gene pBacEGF.The plasmid pSK-EGF that will contain people EGF gene fragment is after BamHI and EcoRV enzyme are cut, low melting point glue reclaims gene fragment and is connected through the transfer vector pBacPAK-8 of BamHI and SmaI double digestion (CLONTECH company), obtain recombinant transfer vector plasmid pBacEGF (Fig. 1), correct through the restriction analysis identified gene.
The present invention also provides the recombinant baculovirus BmBacEGF that contains people EGF gene.With recombinant transfer vector pBacEGF DNA and linearizing viral BmBacPAK6 (applicant makes up voluntarily) DNA through the AocI linearization for enzyme restriction by liposome-mediated cotransfection silkworm cultured cell, homologous recombination can take place in the two in cell, after treating that cotransfection tangible virus infection symptom occurred after 5～7 days, get supernatant and carry out plaque screening.Filter out the recombinant virus BmBacEGF (Fig. 2) that contains the hEGF gene by 2 plaque screenings of taking turns and DNA dot blot again, 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, preservation date is July 2 calendar year 2001, deposit number CGMCC NO:0593.
The present invention also provides the expression product of people EGF gene in silkworm five-age larva and pupa.Recombinant baculovirus BmBacEGF infected silkworm cell is carried out virus amplification, recombinant baculovirus BmBacEGF after will increasing then be injected to five age silkworm larva and pupal cell in, get 24,48,72,96 and 120 hours silkworm and pupa hemolymph respectively, after 5000rpm 5 minutes is centrifugal, get supernatant, add isopyknic 2 * albumen sample-loading buffer after diluting 10 times, get 20ul and carry out 20% SDS-PAGE and analyze, Western hybridization show can with the EGF antibodies, molecular weight is about 6KD.ELASA detects and shows that infecting the 120th hour every the silkworm average expression amount in back is 0.42mg, and infecting the 120th hour each the pupa average expression amount in back is 0.5mg (Fig. 3).
The present invention has also carried out bioactive evaluation to people EGF expression of gene product.The biological activity of EGF is used the Balb/c3T3 cell, adopt MTT desmoenzyme connection instrument to detect, with BmBacEGF percutaneous puncture-inoculation silkworm larva in five ages and pupal cell, collect silkworm blood and pupal cell hemolymph in infecting the back different time, survey the activity of the EGF in the hemolymph behind the high speed centrifugation, the EGF ED50 (Effective dose 50: make cell yield reach the concentration of maximum value one half EGF) after the 120th hour that infects in silkworm blood and the pupal cell hemolymph is respectively 0.6ng/ml and 0.88ng/ml.
The present invention also carries out experimentation on animals to making oral pharmaceutical with silkworm larva that efficiently expresses EGF and pupa.The silkworm chrysalis that efficiently expresses people EGF is carried out the roughing out purifying be developed into oral pharmaceutical; feed the SD rat with various dose; after three days (once a day); with finding behind the dehydrated alcohol filling stomach; compare with control group; gastric mucosa of rat is obviously reduced by the degree of ethanol damage, and its protection stomach mucous membrane effect is at 50-200ug/kg.
-1Be dose-effect relationship (table 1) in the scope, show with silkworm larva of expressing hEGF and pupa and make the gastric mucosal protective effect that oral pharmaceutical have tangible anti-ethanol damage, become a kind of oral pharmaceutical of novel treatment human peptic ulcer.
Comprehensively described, advantage of the present invention is as follows: because the existence of disulfide linkage and other multiple functional groups in the hEGF molecule, thereby the product purity of chemosynthesis and productive rate can't satisfy suitability for industrialized production.The production of hEGF at present mainly obtains in the intestinal bacteria yeast by genetic engineering technique, but expression product often forms inclusion body, relates to protein denaturation and annealing issues during separation and purification, and the multistep operation of bringing procreation for aftertreatment and purifying process.In order to overcome above-mentioned difficulties, the present invention as bio-reactor, efficiently expresses the human epidermal growth factor with silkworm, expression amount reaches the 0.5mg/ pupa, make oral pharmaceutical with the silkworm larva and the pupa of expressing hEGF,, determine its treatment function gastroenteritic ulcer through animal experiment.
Description of drawings
Fig. 1, contain the silkworm baculovirus transfer vector pBacEGF (descriptive name: 3 ' FS/5 ' FS: autographa california polyhedrosis virus polyhedron promotor 3 ' end/5 ' end flanking sequence: P: autographa california polyhedrosis virus polyhedron promotor of people EGF gene; A (Polyhedin Poly A
+Signal) polyhedron gene polyadenylation signal; M13ori:M13 phage replication starting point; Amp: penbritin gene; The ori:pUC replication origin.)
Fig. 2 second take turns the recombinant virus of plaque screening DNA dot blot result (1, positive control 2, negative control 3, wild-type virus 4, normal bombyx mori cell DNA 5-8, recombinant virus
EGF content in Fig. 3 BmBacEGF infected silkworm larva hemolymph
Embodiment
The present invention is further elaborated by following examples, but does not limit the scope of the invention.
The artificial chemosynthesis of embodiment 1, people EGF gene and the structure of escherichia coli vector pSK-EGF
According to the EGF gene order (Proc.Natl.Acad.Sci.USA that has reported, 1983, VOL80,7462), oligonucleotide chain with dna synthesizer and synthetic 4 the coding EGF of tris phosphite method, with the urea-denatured gel electrophoresis separation and purification of polyacrylamide-7mol/L, its sequence is respectively: oligonucleotide chain 1:5 '-GGA ATT CGT TAA CTC CGA CTC CGA ATG TCC ATT GTCCCA CGA CGG TTA CTG TTT GCA CGA-3 ', oligonucleotide chain 2:5 '-AAGCTT TGG ACA AGT ACG CCT GTA ACT GTG TTG TTG GTT ACATCG GTG AAA GAT GTC AAT A-3, oligonucleotide chain 3:5 '-GGA ATT CTCATT ATT CCC ACC ACT TCA AGT CTC TGT ATT GA ATC TTT CACCGA TGT-3, oligonucleotide chain 4:5 '-GGC GTA CTT GTC CAA AGC TTCGAT GTA CAT ACA AAC ACC GTC GTG CAA ACA GTA ACCGTC-3 ', 5 ' terminal phosphateization of oligonucleotide chain, renaturation, the extension of chain, connect, the EcoRI endonuclease digestion, and be cloned into EcoRI (Bao Ling Man) site of pBluscript SK (CLONTECH company) plasmid, construction recombination plasmid pSK-EGF, entirely true through its nucleotide sequence of order-checking proof.
The structure of the silkworm baculovirus transferring plasmid of embodiment 2, people EGF gene
The plasmid pSK-EGF that will contain people EGF gene fragment is after BamHI (Bao Ling Man) and EcoRV enzyme are cut, reclaim gene fragment with low melting point glue, and be connected through the transfer vector pBacPAK8 of BamHI and SmaI double digestion (CLONTECH company), obtain recombinant transfer vector plasmid pBacEGF (Fig. 1), correct through the restriction analysis identified gene.
The acquisition of the recombinant baculovirus of embodiment 3, people EGF gene
Get 5ul and contain the silkworm baculovirus transferring plasmid pBacEGF of people EGF gene and the 6ul modification virus BmBacPAK6 (applicant makes up voluntarily) through AocI (Bao Ling Man) linearization for enzyme restriction, TC-100 (GIBCOBRL company) substratum that adds the 100ul serum-free is mixed.The TC-100 substratum of getting 6ulDosper (Bao Ling Man) adding 100ul serum-free is mixed.The TC-100 substratum washed twice of BmN cell (Zhejiang University's strain that biochemical research is preserved) the usefulness serum-free in the 35mm plate will be cultivated in advance, and dropwise add transferring plasmid and Dosper mixture, cultivated 4-5 days, and collected supernatant and carry out first round plaque screening for 27 ℃.Get the bombyx mori cell in the 5ul supernatant infection 35mm plate, 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 silkworm cell 3-4 days is preserved supernatant.The supernatant of getting positive colony carries out second and takes turns plaque screening, cell is used for DNA dot blot (see figure 2) with the NaOH cracking, make template random primer probe mark test kit (Bao Ling Man) label probe with the EGF gene, hybridizing method is according to " molecular cloning " (Science Press, 1995).The supernatant of getting positive colony carries out second and takes turns the plaque screening (see figure 2).Get the supernatant infected silkworm cell amplification of positive colony, can obtain a large amount of recombinant baculovirus BmBacEGF that contains people EGF gene.This virus energy infected silkworm cell is the morbidity shape at the microscopically cell, and available EGF gene is made probe by DNA hybridization calibrating.This virus has submitted to China Committee for Culture Collection of Microorganisms common micro-organisms center to preserve, and the address is in the BeiJing ZhongGuanCun, and preservation date is July 2 calendar year 2001, and deposit number is CGMCC NO:0593.
Embodiment 4, the expression of people EGF gene in silkworm larva and pupa
With recombinant baculovirus BmBacEGF be expelled to five age silkworm larva and pupa (Bombyx mori) (applicant raises voluntarily) in, (titre is 1 * 10 about 2ul in every injection
7/ ml), get 24 respectively, 48,72, the silkworm lymph blood of expressing in 96 and 120 hours, get supernatant after 5000rpm 5min is centrifugal, after 10 times of PBSpH7.4 dilutions, add isopyknic 2 * protein sample-loading buffer (100Mm Tris.HCl, 4%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), boiling sex change gets 20ul and carries out the SDS-PAGE electrophoresis, electrifying with pvdf membrane (Bao Ling Man) turns over night, by with one anti-(mouse-anti people EGF, Shenzhen brilliant U.S. company), two anti-(sheep anti-mouse iggs, Shanghai bio-engineering corporation) hybridization, the result shows that Western hybridization band molecular weight is 6KI) about.ELASA detects and shows that infecting the 120th hour every the silkworm average expression amount in back is 0.42mg, and infecting the 120th hour each the pupa average expression amount in back is 0.5mg (Fig. 3).
The biological activity determination of the people EGF that embodiment 5, silkworm are expressed
The biological activity of EGF is used Balb/c3T3 cell (Chinese biological goods calibrating institute), adopts MTT desmoenzyme connection instrument to detect.Getting one of aseptic 96 orifice plate, is blank with No. 1 hole, and 2 good holes are contrast, add substratum 50ul, and 4 parallel group in each sample respectively adds testing sample 50ul in No. 1 hole and No. 3 holes, since No. 3 half-and-half dilutions to the right; Select one bottle in logarithmic phase cell, trysinization is washed once with 0.5% substratum behind the cell harvesting, adjusts cell with 0.5% substratum and counts to desired concn, and every hole adds cell suspension 50ul (each hole of control group adds the 50ul substratum), 37 ℃, 5%CO
2Incubator was cultivated 40-50 hour, added MTT (Sigma company) 20ul then, and 37 ℃, 5%CO
2Continue to cultivate 4 hours, add 10%SDS-0.01NH
4Cl100ul spends the night, and microplate reader is surveyed OD value, λ=490nm.The result shows, with BmBacEGF percutaneous puncture-inoculation silkworm larva in five ages and pupal cell, collects the silkworm hemolymph in infecting the back different time, surveys the activity of the EGF in the hemolymph behind the high speed centrifugation, the EGF ED after the 120th hour that infects in silkworm blood and the pupal cell hemolymph
50(Effective dose 50: make cell yield reach the concentration of maximum value one half EGF) is respectively 0.6ng/ml and 0.88ng/ml.
Embodiment 6, usefulness efficiently express the silkworm chrysalis of people EGF and make the experimentation on animals that oral pharmaceutical carry out
With efficiently express people EGF five age silkworm larva, pupal cell, 4 ℃ of grindings are got supernatant behind the 12000rpm high speed centrifugation, the 40000rpm ultracentrifugation is got the supernatant lyophilize and is made oral pharmaceutical again.Laboratory animal is the SD rat, and available from the Academy of Medical Sciences, Zhejiang animal center, totally 50, every body weight 220-250g is divided into 5 groups at random, 10 every group, is respectively hEGFI group (50ug/kg.d
-1Oral), hEGFII organizes (100ug/kg.d
-1Oral), hEGF III organizes (200ug/kg.d
-1Oral), Cimitidine Type A/AB group (30mg/kg.d
-1Oral), control group (0.9% physiological saline 1ml, oral).Test and played rat in preceding 3 days and give perfusion or subcutaneous injection medicine in the gastral cavity respectively, all medicines all with the dilution of 0.9% physiological saline, divide 3 times on the one, at every turn isometric(al) filling mouse.Test and played fasting in preceding 2 days, but can't help water, each mouse is only irritated stomach with dehydrated alcohol 1ml/ after 48 hours on an empty stomach, irritates ethanol and gets blood after 1 hour, puts to death rat, dissects and takes out stomach, fixes with 1% formaldehyde solution.Cut off and flatten along the greater gastric curvature side, the visual inspection degree of impairment is measured damage length, and histopathologic slide's row light microscopy checking is expressed and done to degree of injury with ulcer index.Damage index score standard: the wire damage, every mm length-gauge 1 minute, damage width surpass 2mm person's score to be doubled, and all score sum that adds up is as the ulcer index (table 1) of every rat pipe film injury.The result shows with silkworm larva of expressing hEGF and pupa and makes the gastric mucosal protective effect that oral pharmaceutical have tangible anti-ethanol damage, becomes a kind of novel method for the treatment of the oral pharmaceutical of human peptic ulcer.
Table 1, express the experimentation on animals result of EGF oral pharmaceutical with silkworm
| Group | Number of elements | Scope | Ulcer index | The P value |
| Control group | 10 | ?33～70 | ?58 | |
| (dosage is 50 ug/kg.d to the administration group -1hEGF) | 10 | ?10～67 | ?50 | ＞0.05 |
| (dosage is 100 ug/kg.d to the administration group -1hEGF) | 10 | ?11～53 | ?43 | ＜0.01 |
| (dosage is 200 ug/kg.d to the administration group -1hEGF) | 10 | ?0～26 | ?13 | ＜0.01 |
| Cimitidine Type A/AB | 10 | ?30～55 | ?35 | ＜0.05 |
Annotate: the P value is that each medication group and control group ulcer index compare
Claims (2)
1. method for preparing medicine with silkworm expressed numan epidermal growth factor, it is characterized in that: the silkworm with recombinant baculovirus that obtains the band human epidermal growth factor gene of reorganization by gene engineering method, this virus has submitted to Chinese bacterial classification center to preserve, deposit number is 0593, and carry out expressing human EGF with this virus inoculation silkworm larva and pupa, the expression product that is obtained is that stopping composition is mixed with oral pharmaceutical through lyophilize with the silkworm pupa.
2. method according to claim 1, it is characterized in that the expression product of described people EGF gene in silkworm five-age larva and pupa be by recombinant baculovirus BmBacEGF be injected to five age silkworm larva and pupal cell in express to produce.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100362103C (en) * | 2004-12-21 | 2008-01-16 | 苏州大学 | Human epidermis factor nucleic sequence and use thereof |
| CN101481702B (en) * | 2009-01-22 | 2013-05-01 | 天津耀宇生物技术有限公司 | Recombinant vector containing polyhedrosis gene, and method for expressing and purifying protein |
| CN103695468A (en) * | 2013-11-06 | 2014-04-02 | 江苏恒顺醋业股份有限公司 | Method for expressing EGF-TCSKDEL fusion protein by bombyx mori baculovirus Bac-to-Bac system |
| CN104073518A (en) * | 2014-04-21 | 2014-10-01 | 广西医科大学 | Method for preparing recombinant human epidermal growth factor by adopting castor silkworm chrysalis as bioreactor |
| CN110713532B (en) * | 2019-11-06 | 2021-04-13 | 吉林省蚕业科学研究院 | Truncated protein AP-EGF of tussah epidermal growth factor and preparation method and application thereof |
| CN112852876B (en) * | 2021-03-04 | 2022-11-29 | 西南大学 | Silkworm silk gland recombinant expression vector for expressing human epidermal growth factor and preparation method and application thereof |
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