CN100362103C - Human epidermis factor nucleic sequence and use thereof - Google Patents
Human epidermis factor nucleic sequence and use thereof Download PDFInfo
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- CN100362103C CN100362103C CNB200410065878XA CN200410065878A CN100362103C CN 100362103 C CN100362103 C CN 100362103C CN B200410065878X A CNB200410065878X A CN B200410065878XA CN 200410065878 A CN200410065878 A CN 200410065878A CN 100362103 C CN100362103 C CN 100362103C
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Abstract
The present invention discloses a nucleic acid sequence of a human epidermal growth factor and the expression thereof in colibacillus and domestic silkworms. A new HCEGF nucleotide sequence is designed and artificially synthesized according to the amino acid sequence of the human epidermal growth factor and the codon preference of domestic silkworms on the reference of optimized codon of colibacillus, and the sequence can effectively express human epidermal growth factors in the colibacillus and the domestic silkworms. The expression quantity of the sequence in the colibacillus can reach more than 30% of total bacterial protein and can reach 28.4 mu g per millimeter pupa haemolymph in domestic silkworms. The composition sequence is different from the gene sequence of the human epidermal growth factor, and the product expressed by the composition sequence is completely identical with the expression product of a natural human epidermal growth factor. Thus, the composition sequence has all characteristics of the natural human epidermal growth factor.
Description
Technical field
The present invention relates to genetically engineered and polypeptide drug production field, be specifically related to a kind of human epidermis factor nucleic sequence that intestinal bacteria and silkworm efficiently express that is used for of synthetic, with and expression vector and the application in the preparation human epidermal growth factor.
Background technology
(Human epidermal growth factor hEGF) with the separating of urogastrone (Urogastrone), finds that these two kinds of materials have similar amino acid and form and biologic activity along with the human epidermal growth factor.Research afterwards confirms that further hEGF and urogastrone are with a kind of material.HEGF is that molecular weight is the small molecular protein about 6000Da, and multiple pharmacological effect is arranged.Having 53 amino acid, is one of important somatomedin, also is to find the earliest and try out in one of clinical somatomedin.Clinic trial result shows, hEGF can impel ectoderm cell and capillary vessel growth, promote the healing of burn, wound and surgical wound, quicken to transplant the growth of epidermis and corneal transplant, therefore, there is important practical to be worth to treatments such as the treatment of wound burn, skin and corneal transplantation, skin ulcerous, stomach ulcer, also can be used for promoting the healing of surgical incision.In addition, epidermic cell is active to be reduced the people along with the age increases, and cause the aging of skin gradually, and hEGF can stimulate epidermal growth, plays anti-aging effects, thereby also be widely used in the makeup now.
The sequence number that human epidermal growth factor (hEGF) gene is published among the GenBank is X04571, and the nucleic acid position of human epidermal growth factor's bioactive peptide correspondence is 3347-3505, and the relative position of polypeptide chain is at AA949-1001.
Existing both at home and abroad several units in intestinal bacteria (E.coli) successful expression the human epidermal growth factor, nucleotide sequence human epidermal growth factor (hEGF) gene that it adopts, but it is lower in the expression in escherichia coli amount, and what obtain simultaneously is injecting drug use basically.
Silkworm-baculovirus expression system is an eukaryotic expression system, because a large amount of proteic provide protection of silkworm lymph principal body, the albumen of silkworm expression can be produced oral pharmaceutical.Chinese invention patent application CN1405310A discloses a kind of method of producing medicine with silkworm expressed numan epidermal growth factor, it is according to the people's of report EGF gene order, EGF gene with dna synthesizer chemosynthesis people, be cloned in the transfer vector, and acquisition recombinant silkworm baculovirus, virus inoculation to silkworm larva or pupa, is expressed producing the human epidermal growth factor.
Yet silkworm or intestinal bacteria are different with human body to the preference of gene-code, and it is recombinant expressed that aforesaid method all adopts people's gene to carry out, though can obtain the human epidermal growth factor, expression level is low, cause the actual fabrication yield low, the cost height.
Advantages such as silkworm-baculovirus expression system has the expression level height, and the expression product natural sex is good.In order to improve human epidermal growth factor's expression level, we are according to human epidermal growth factor (hEGF) gene order, preferences with reference to the silkworm codon, and suitably with reference to the preferred codon of intestinal bacteria, designed and synthesized human epidermal growth factor gene (HCEGF), by the expression in intestinal bacteria and silkworm, the result confirms that our institute's synthetic sequence has all obtained expressing efficiently in intestinal bacteria and silkworm, broken through the low bottleneck of human epidermal growth factor's expression amount.
Summary of the invention
First purpose of the present invention provides a kind of new human epidermis factor nucleic sequence, by the change to gene order, makes and greatly improves expression level in the preparation, obtains identical human epidermal growth factor's protein simultaneously again.
Second purpose of the present invention provides the carrier that contains described nucleotide sequence.
The 3rd purpose of the present invention provides the process of utilizing above-mentioned preparing carriers human epidermal growth factor.
For achieving the above object, the technical solution used in the present invention is: the horizontal acid sequence of a kind of human epidermal growth factor, it has nucleotide sequence shown in the SEQ ID NO.1.
This nucleotide sequence has 162 Nucleotide, is obtained by synthetic, and wherein, last three TAA are terminator codon.Compare with the people's who announces epidermis factor nucleic sequence, 22% base difference (except that adding the TAA terminator codon) is arranged, and its contrast table is as follows, wherein, HCEGF is a nucleotide sequence of the present invention, and HEGF is the people's of announcement epidermis factor nucleic sequence.
HCEGF?(1) AACAGCGACAGCGAATGCCCGTTGAGCCACGACGGCTACTGCTTGCACGACGGCGTGTGCATGTACATTG
HEGF (1) AATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCATGTATATTG
HCEGF?(71)?AAGCGTTGGACAAAATACGCGTGCAACTGCGTGGTGGGCTACATTGGCGAACGCTGCCATACCGCGACTT
HEGF (71)?AAGCATTGGACAAGTATGCATGCAACTGTGTTGTTGGCTACATCGGGGAGCGATGTCAGTACCGAGACCT
HCEGF?(141)GAAATGGTGGGAATTGCGCTAA
HEGF (141)GAAGTGGTGGGAACTGCGC
And at final amino acid levels, both have obtained on all four result:
HCEGF NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR
HEGF NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR
The present invention provides the carrier that comprises nucleotide sequence shown in the SEQ ID NO.1 simultaneously.
Further, described carrier is the expression vector pET-28a that comprises nucleotide sequence shown in the SEQ ID NO.1.
And described carrier is the transfer vector pBacPAK-His that comprises nucleotide sequence shown in the SEQ ID NO.1.
The present invention discloses a kind of human epidermal growth factor's of preparation method, adopt the transfer vector pBacPAK-His that comprises nucleotide sequence shown in the SEQ IDNO.1 to make up recombinant baculovirus, be seeded in silkworm larva or the pupa, obtain the human epidermal growth factor.
The human epidermal growth factor's of preparation of the present invention method also can be to adopt the expression vector pET-28a that comprises nucleotide sequence shown in the SEQ ID NO.1, transformed into escherichia coli, acquisition human epidermal growth factor.
Because the technique scheme utilization, the present invention compared with prior art has following advantage:
Advantages such as silkworm-baculovirus expression system has the expression level height, and the expression product natural sex is good.In order further to improve human epidermal growth factor's expression level, we are according to human epidermal growth factor gene (hEGF) sequence, with reference to the silkworm codon-bias, and suitably with reference to the preferred codon of intestinal bacteria, designed and synthesized human epidermal growth factor gene (HCEGF), by the expression in intestinal bacteria and silkworm, the result confirms that our institute's synthetic sequence has all obtained expressing efficiently in intestinal bacteria and silkworm, broken through the low bottleneck of human epidermal growth factor's expression amount.
This sequence can efficiently express the human epidermal growth factor in intestinal bacteria and silkworm body, can reach more than 30% of bacterial protein in the expression in escherichia coli amount, can reach 28.4 μ g/ml pupa hemolymphs in silkworm.This composition sequence is different from the human epidermal growth factor gene sequence, but expressed product is identical with natural human epidermal growth factor's expression product, thereby has all characteristics of natural human Urogastron.
Embodiment
Below in conjunction with embodiment the present invention is further described:
Embodiment one: the synthetic of human epidermis factor nucleic sequence:
According to the gene (HCEGF) that nucleotide sequence shown in the SEQ ID NO.1 constitutes, adopt solid-phase synthesis on 3900 full-automatic dna synthesizers, to press synthetic 4 the long-chain primers of operational manual, its sequence is respectively:
EGF-F1：GGATCCAACAGCGACAGCGAATGCCCGTTGAGCCACGACGGCTACTGCTTGCACG
EGF-R1：GCACGCGTATTTGTCCAACGCTTCAATGTACATGCACACGCCGTCGTGCAAGCAGTAGCCG
EGF-F2：TGGACAAATACGCGTGCAACTGCGTGGTGGGCTACATTGGCGAACGCTGCCAATAC
EGF-R2：GAATTCTTAGCGCAATTCCCACCATTTCAAGTCGCGGTATTGGCAGCGTTCGCC
Respectively EGF-F1, EGF-R1 and EGF-F2, EGF-R2 are carried out first round polymerase chain reaction (pcr amplification reaction).
Reaction 1: adopt following raw materials according to carry out amplified reaction
0.5 the thermophilic archaeal dna polymerase of microlitre (Taq polymerase)
Four kinds of mononucleotide mixtures (dNTPs) of 1 microlitre, 10 mmoles
5.0 microlitre 10 * PCR damping fluid
The MgCl of 3 microlitres, 25 mmoles
2
The mixture of 2 microlitres, 25 micromolar EGF-F1 and EGF-Rl
37.5 the water of microlitre
Earlier carry out pre-reaction of degeneration 1 minute under 94 ℃, carry out denaturation renaturation-extension circulating reaction again 30 times, each circulation comprises that 94 ℃ were reacted 1 minute, and 55 ℃ were reacted 1 minute, and 72 ℃ were reacted 1 minute.
Reaction 2: identical with reaction 1, just react the mixture that object becomes 2 microlitres, 25 micromolar EGF-F2, EGF-R2.
Product with two reactions mixes again, carries out the pcr amplification reaction second time, and reaction conditions and above-mentioned reacting phase are together.
After the amplification PCR product is carried out gel and reclaim, carry out the TA clone again.Picking ten clones check order, and the right-on result that will check order picks out, and just obtains the HCEGF of synthetic.
Embodiment two: the expression in intestinal bacteria of construction of prokaryotic expression vector and gene
The design of synthetic HCEGF sequence two ends has BamH I, EcoR I site among the embodiment one, and after enzyme was cut, the clone advanced among the pET-28a, obtains prokaryotic expression carrier pET-28a-HCEGF.
The abduction delivering of thalline: the pET-28a-HCEGF DNA of reorganization is converted among E.coli (intestinal bacteria) BL21, transformed bacteria was inoculated in the LB substratum that contains kantlex with 1: 100, cultivated 3 hours for 37 ℃, add IPTG to final concentration be 1mmol/mL, carried out abduction delivering 3 hours.
The expression of results of HCEGF in E.coli detects: will identify correct expression plasmid pET-28a-HCEGF Transformed E .coli recipient bacterium BL21, and use the IPTG abduction delivering.SDS-PAGE and Western blot detect and show, the molecular weight 10kDa of recombinant protein, and the recombinant protein of expression accounts for more than 30% of total bacterial protein.
The single step purification of prokaryotic expression product: will express the laggard row metal a flat iron plate for making cakes of bacterium liquid supersound process and close affinity chromatography.Carry out SDS-PAGE after purified product concentrates and analyze that can to detect 1 molecular weight be the 10kDa protein band.
Embodiment three: the expression in silkworm larva and pupa of the structure of eucaryon transfer vector and gene
The structure of recombinant baculovirus transfer vector: synthetic HCEGF is cloned among the pBacPAK-His through BamH I, EcoR I site, obtains pBacPAK-His-HCEGF.
Virus reorganization operation: viral Bm-BacPAK6 DNA adopts liposome (Lipofectin) method, with pBacPAK-His-HCEGF cotransfection BmN cell after Bsu36 I cutting.The cotransfection nutrient solution carries out the plaque purifying, filters out recombinant virus Bm-HCEGF, and after identifying with PCR, the amplification back is stand-by in cell.
The collection of recombinant protein sample in silkworm larva or the pupa: with the tender pupa that recombinant virus 2 μ L injection inoculations play silkworm or just pupated five ages, the check plot is injection water and Bm-BacPAK6 respectively, collects the hemolymph of silkworm or pupa after 120 hours respectively, and-20 ℃ of preservations are to be measured.
The screening of recombinant virus Bm-HCEGF and expression product detect: pBacPAK-His-HCEGF with through the Bm-BacPAK6 DNA of Bsu36 I linearization for enzyme restriction cotransfection BmN cell, obtain recombinant virus Bm-HCEGF by plaque select and infect the BmN cell, the total DNA of extracting cell, PCR can amplify 170bp left and right sides DNA and insert fragment.
SDS-PAGE analyzes and infects the silkworm pupa hemolymph of recombinant virus after 120 hours, can about 10kDa, detect the protein component suitable with the EGF theoretical molecular, then do not have in the hemolymph of infection wild-type virus, show that foreign gene has obtained expressing in the silkworm body.ELISA detects, virus inoculation after 120 hours expression amount be 28.4 μ g/ml pupa hemolymphs.
＜110〉University Of Suzhou
＜120〉human epidermis factor nucleic sequence and application thereof
<160>1
<210>1
<211>162
<212>DNA
＜213〉artificial sequence
<220>
<221>cDS
<222>(1)…(162)
<400>1
aacagcgaca?gcgaatgccc?gttgagccac?gacggctact?gcttgcacga?cggcgtgtgc?60
atgtacattg?aagcgttgga?caaatacgcg?tgcaactgcg?tggtgggcta?cattggcgaa?120
cgctgccaat?accgcgactt?gaaatggtgg?gaattgcgctaa 162
Claims (6)
1. human epidermis factor nucleic sequence, it has nucleotide sequence shown in the SEQ ID NO.1.
2. the carrier that comprises the nucleotide sequence of claim 1.
3. the expression vector pET-28a that comprises the nucleotide sequence of claim 1.
4. the transfer vector pBacPAK-His that comprises the nucleotide sequence of claim 1.
5. a method for preparing the human epidermal growth factor comprises with the described transfer vector of claim 4 making up recombinant baculovirus, is seeded in silkworm larva or the pupa, obtains the human epidermal growth factor.
6. a method for preparing the human epidermal growth factor comprises with the described expression vector transformed into escherichia coli of claim 3, expresses obtaining the human epidermal growth factor.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101759806B (en) * | 2009-07-31 | 2012-12-19 | 山东省科学院中日友好生物技术研究中心 | Cytoplasm leader peptide (CTD)-containing human epidermal growth factor fusion protein (CTD-hEGF) prepared by using hydroxylamine cutting method and application thereof |
| 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 |
| US10588972B2 (en) * | 2014-06-02 | 2020-03-17 | Li-Cor, Inc. | Phthalocyanine probes and uses thereof |
| CN111273030A (en) * | 2020-02-25 | 2020-06-12 | 芜湖天明生物技术有限公司 | Kit for quantitatively detecting recombinant human epidermal growth factor by direct competition ELISA method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1279288A (en) * | 2000-07-19 | 2001-01-10 | 深圳市华生元基因工程发展有限公司 | Recombination human epidermal growth factor gene, its expression product and its application |
| CN1296976A (en) * | 1999-11-23 | 2001-05-30 | 上海博容基因开发有限公司 | Polypeptide-human epidermal growth factor receptor protein 25 and polynucleotide for coding said polypeptide |
| CN1358749A (en) * | 2000-12-13 | 2002-07-17 | 上海博德基因开发有限公司 | Novel polypeptide-human epidernal growth factor 10.12 the polynucleotide for encoding said polypeptide |
| CN1360022A (en) * | 2000-12-19 | 2002-07-24 | 中国科学院上海生物化学研究所 | Recombinant human epidermal growth factor and its preparing process and medicinal composition |
| CN1405310A (en) * | 2001-08-15 | 2003-03-26 | 浙江中奇生物药业股份有限公司 | Method for producing medicine using silkworm expressed numan epidermal growth factor |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1296976A (en) * | 1999-11-23 | 2001-05-30 | 上海博容基因开发有限公司 | Polypeptide-human epidermal growth factor receptor protein 25 and polynucleotide for coding said polypeptide |
| CN1279288A (en) * | 2000-07-19 | 2001-01-10 | 深圳市华生元基因工程发展有限公司 | Recombination human epidermal growth factor gene, its expression product and its application |
| CN1358749A (en) * | 2000-12-13 | 2002-07-17 | 上海博德基因开发有限公司 | Novel polypeptide-human epidernal growth factor 10.12 the polynucleotide for encoding said polypeptide |
| CN1360022A (en) * | 2000-12-19 | 2002-07-24 | 中国科学院上海生物化学研究所 | Recombinant human epidermal growth factor and its preparing process and medicinal composition |
| CN1405310A (en) * | 2001-08-15 | 2003-03-26 | 浙江中奇生物药业股份有限公司 | Method for producing medicine using silkworm expressed numan epidermal growth factor |
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