CN103740730A - Optimized king cobra venom protease inhibitor gene OH-TCI as well as expression method and application thereof - Google Patents
Optimized king cobra venom protease inhibitor gene OH-TCI as well as expression method and application thereof Download PDFInfo
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- CN103740730A CN103740730A CN201410001201.3A CN201410001201A CN103740730A CN 103740730 A CN103740730 A CN 103740730A CN 201410001201 A CN201410001201 A CN 201410001201A CN 103740730 A CN103740730 A CN 103740730A
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Abstract
The invention provides an optimized king cobra venom protease inhibitor gene (i)OH-TCI (/i) as well as expression carrier and an application thereof. The optimized king cobra venom protease inhibitor gene (i)OH-TCI (/i) is synthesized, recombinant plasmids pUC18-T-OH-TCI and pET43.1-OH-TCi are constructed and are transferred into escherichia coli, positive converters are screened, and the experiment results show that the recombinant escherichia coli secretion expresses fusion protein NusA-OH-TCI, and after enzyme digestion of thrombin, recombinant rOH-TCI which can remarkably inhibit activity of trypsin and chymotrypsin. A king cobra venom protease inhibitor OH-TCI with high expression amount and high activity can be obtained through an escherichia coli expression system, and the defects that the cost is high and the expression amount is small when OH-TCI is obtained in other modes are overcome. The inhibitor has wide application prospects in industry such as medicine and chemical engineering.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of king cobra toxin protease inhibitor gene OH-TCI and expression method and application of optimization.
Background technology
From domestic Elapidae Ophiophagus hannan (Cantor), separating and obtained snake venom Kunitz-type proteinase inhibitor OH-TCI first at present, is that unique that current nature is found has trypsinase and chymotrypsin inhibitor activity simultaneously and the two is suppressed to active essentially identical Kunitz-family member.OH-TCI and Trypsin inhibitor,Trasylol Aprotinin belong to Knitz-type proteinase inhibitor together, and the mature peptide of the two forms by 58 amino-acid residues.But the inhibition activity of the two trypsinase and Chymotrypsin has obvious difference.Trypsin inhibitor,Trasylol (Aprotinin) is a kind of wide spectrum serpin, has plasmin, kallikrein and the hematoblastic effect of protection of inhibition.Clinically, be mainly used in treating acute pancreatitis and reduce the amount of bleeding in art.
OH-TCI extracts acquisition on a small quantity from eyes boa snake venom.Due to its source difficulty, can not carry out suitability for industrialized production, taking DNA recombinant technology to produce proteinase inhibitor OH-TCI is the effective way that solves its imbalance between supply and demand.
Summary of the invention
The present invention is directed to the deficiency that the expression that utilizes escherichia expression system to produce the existing foreign protein of king cobra toxin protease inhibitor OH-TCI in prior art is undesirable at cell periplasmic space, translation post-treatment, yield poorly, a kind of king cobra toxin protease inhibitor gene OH-TCI and expression method and application of optimization are provided.The present invention adopts escherichia expression system can obtain high expression level amount, highly active rOH-TCI, is not subject to the restriction in starting material sources, has overcome otherwise to obtain that the cost of OH-TCI is high, expression amount is low and active low shortcoming.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
The king cobra toxin protease inhibitor gene OH-TCI that the invention provides a kind of optimization, it has the nucleotide sequence of sequence table SEQ ID NO:1.
The Auele Specific Primer of cloning described gene is:
Primers F 1:5 '-ACGAATTCAAGGTTTTTGAAAGATGTGA-3 ';
Primer R1:5 '-ACGCGGCCGCTTAACCACCACCACCACCAA-3 '.
5 ' the end at described OH-TCI gene is added with SmaI restriction enzyme site, and 3 ' end is added with HandIII restriction enzyme site.
The proteinase inhibitor OH-TCI that the present invention also provides described king cobra toxin protease inhibitor gene OH-TCI coding to produce, it has the aminoacid sequence shown in sequence table SEQ ID NO:2.
The recombinant vectors that contains described king cobra toxin protease inhibitor gene OH-TCI, described recombinant vectors is pUC18-OH-TCI and pET43.1-OH-TCI.
The present invention also provides the application of described king cobra toxin protease inhibitor OH-TCI in medicine or the healthcare products of the amount of bleeding in preparation treatment acute pancreatitis and minimizing art.Described king cobra toxin protease inhibitor OH-TCI has the inhibition activity to trypsinase and Chymotrypsin.
The present invention selects king cobra toxin protease inhibitor gene OH-TCI, according to the Preference of e. coli codon, gene order is optimized, and the OH-TCI gene after synthetic optimization, after it is connected with pUC18-T carrier, proceed to and in intestinal bacteria, obtain recombinant plasmid pUC18-OH-TCI, take recombinant plasmid pUC18-OH-TCI as template, with F1, R1 primer, carry out pcr amplification and obtain goal gene fragment OH-TCI.King cobra toxin protease inhibitor gene OH-TCI after synthetic optimization is connected with carrier pET43.1a, has built recombinant plasmid pET43.1-OH-TCI.Recombinant plasmid pET43.1-OH-TCI is forwarded in e. coli bl21 through chemical transformation, containing microbiotic Ampicillin screening, obtaining positive transformant, positive transformant is inoculated in LB liquid nutrient medium, by bacterial strain by 2% inoculum size access LB substratum, in 37 ℃, when 210r/min shaking table is cultured to OD600 value and reaches 0.5-0.6, add IPTG, abduction delivering at 28 ℃ of temperature carries out the exploration of best abduction delivering condition simultaneously, to determine the top condition of expression.After induction finishes, centrifugal collection somatic cells.Ultrasonication, centrifugal.Cellular lysate thing supernatant carries out SDS-PAGE, obtains and expects that consistent size is about the object band of 73kD.Ni-NTA affinitive layer purification fusion rotein NusA-OH-TCI; Fusion rotein NusA-OH-TCI junction is designed with the restriction enzyme site of thrombin, with thrombin enzyme, cuts fusion rotein, by HPLC method, prepares restructuring rOH-TCI.Adopt substance assistant laser desorpted ionized time-of-flight mass spectrometry (TOFMS) to measure the molecular weight of restructuring rOH-TCI.Because thrombin enzyme is cut, at the N of restructuring rOH-TCI, hold residual two amino acid Gly-Ser, result shows, recombinant protein rOH-TCI molecular weight of albumen and theoretical basically identical (6490.40Da).It is active that restructuring rOH-TCI has obvious inhibition to trypsinase, Quimotrase.
Compared with prior art, advantage of the present invention and positively effect are: the present invention utilizes escherichia expression system to be used as producing the expression system of king cobra toxin protease inhibitor OH-TCI, it has, and production cost is low, simple to operate, the advantages such as the leavening property rapid, expression efficiency is high and good of growing, and the proteinase inhibitor OH-TCI that the gene of the king cobra toxin protease inhibitor OH-TCI after optimization of the present invention produces through escherichia expression system has advantages of high reactivity, high expression level amount.
Read by reference to the accompanying drawings after the specific embodiment of the present invention, it is clearer that the other features and advantages of the invention will become.
Accompanying drawing explanation
Fig. 1 is that original OH-TCI sequence and the present invention optimize rear OH-TCI gene order contrast collection of illustrative plates.
Fig. 2 is recombinant expression plasmid pET43.1-OH-TCI signal collection of illustrative plates in the present invention.
Fig. 3 is the order-checking collection of illustrative plates of recombinant expression plasmid pET43.1-OH-TCI in the present invention.
Fig. 4 is the SDS-PAGE collection of illustrative plates of pET43.1-OH-TCI protein expression in the present invention, 1, before pET43.1-OH-TCI/BL21 induction; 2, after pET43.1-OH-TCI/BL21 induction; 3, the ultrasonic centrifugal supernatant of thalline after pET43.1-OH-TCI/BL21 induction; 4, the ultrasonic centrifugation of thalline after pET43.1-OH-TCI/BL21 induction; 5, Ni-NTA resin purification NusA-OH-TCI fusion rotein; M, MAKER molecular weight of albumen.
Fig. 5 is the HPLC purifying collection of illustrative plates of recombinant protein rOH-TCI (rOT) in the present invention.
Fig. 6 is rOH-TCI mass spectroscopy figure in the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in further detail.
One, the selection of OH-TCI gene, optimization and synthetic
1, the selection of OH-TCI gene
In Ophiophagus hannah (Cantor)., separate novel protein enzyme inhibitors OH-TCI, belong to Kunitz-family, ripe molecule is comprised of 58 amino acid, and it is to have reported at present unique trypsinase and Chymotrypsin to be had to the active protein molecular of good inhibition simultaneously.
2, the optimization of OH-TCI gene order
The intrinsiccharacteristic of foreign gene as codon preference be the important factor that affects foreign protein effective expression.The present invention is according to e. coli codon Preference, by the codon optimized one-tenth high frequency of the intestinal bacteria low frequency codon of OH-TCI gene, to improve translation speed and the expression amount of target protein.In the nucleotide sequence of OH-TCI gene and Genbank, OH-TCI gene order contrasts as shown in Figure 1.
In Fig. 1, above collection of illustrative plates, a sequence is OH-TCI gene order in Genbank (NCBI sequence number: EU246692.1), one is the OH-TCI gene order after optimizing below, and the horizontal line part after both contrasts is identical base, and marking part is divided into difference base.
Fig. 1 shows, compared with OH-TCI sequence in Genbank, the OH-TCI after optimization has 38 bases that displacement has occurred.
By professional software, analyze, 5 ' end of described OH-TCI gene is added with SmaI restriction enzyme site (CCCGGG), and 3 ' end is added with HandIII restriction enzyme site (AAGCTT).Shown in underscore in the Nucleotide of restriction enzyme site as shown in SEQ ID No:1.
CCCGGGGCAGCGGTCGTCCGAAATTCTGTGAACTGCCAGCTGTGAGCGGTTTTTGT
AAGGCGTACATTCCGTCCTTCTATTACAACCCGGACGCATCTGCGTGCCAGAAATTC
ATCTACGGCGGCTGCGGTGGTAACGCCAATAAATTCAAAACCATCGAAGAGTGCCA
CCGCACTTGCGTTGGCTAA
AAGCTT
3, synthesizing of the OH-TCI gene order of optimizing
The Auele Specific Primer of cloning described gene is:
Primers F 1:5 '-ACGAATTCAAGGTTTTTGAAAGATGTGA-3 ' (SEQ ID NO:3);
Primer R1:5 '-ACGCGGCCGCTTAACCACCACCACCACCAA-3 ' (SEQ ID NO:4).
OH-TCI gene order after described optimization is synthetic by Shanghai Sheng Gong company.Synthetic OH-TCI gene complete sequence is as shown in SEQ ID No:1.
By the nucleotide sequence coded OH-TCI protein sequence of SEQ ID No:1 as SEQ ID No:2:
GRPKFCELPAVSGFCKAYIPSFYYNPDASACQKFIYGGCGGNANKFKTIEECHRTCVG。
Its molecular weight is 6490.40Da.
Two, the structure of recombinant plasmid pUC18-OH-TCI
The carrier adopting is the pUC18-T carrier that business is bought, and the host cell of carrier construction is DH5 α.
1, new synthetic OH-TCI gene is connected with pUC18-T carrier
Described synthetic OH-TCI gene is connected with pUC18-T carrier, and linked system is as follows:
After centrifugal mixing, 16 ℃ of low temperature water-baths connect spends the night.Connect product and be directly used in conversion.
2, the preparation of DH5 α competent cell
DH5 α competent cell is purchased from TIANGEN Biotech (Beijing) Co., Ltd..
3, the connection product of synthetic OH-TCI gene after being connected with pUC18-T carrier transforms
The above-mentioned connection product making is transformed in DH5 α competent cell.Step of converting is as follows:
(1) the connection product of acquisition 10 μ L are added in 200 μ L competent cells, establish negative control simultaneously, and do not add the competent cell contrast of any plasmid DNA;
(2) after mixing gently, ice bath 30min immediately;
(3) centrifuge tube that mixture is housed is moved in the circulator bath of pre-heating to 42 ℃ to lucky standing 90s;
(4) by centrifuge tube fast transfer in ice bath, place 2min;
(5), in super clean bench, the mixture in centrifuge tube is added in the LB liquid nutrient medium that 800 μ L are preheated to 37 ℃;
(6) put upside down and mix gently, by mixture in 37 ℃ of shaking culture 45min;
(7) by the bacterium liquid of cultivating with the centrifugal 5min of 3000rpm, in super clean bench, discard part supernatant, stay approximately 100 μ L supernatants, with sample injector resuspended bacterial sediment;
(8) get bacterium liquid and coat on the LB flat board containing Amp, be inverted overnight incubation for 37 ℃.
4, the sequencing of recombinant plasmid
In order further to determine, the recombinant plasmid that above-mentioned steps obtains positive bacteria liquid is carried out to sequencing by order-checking company.Correct recombinant plasmid called after pUC18-OH-TCI will finally be identified.
Three, the structure of recombinant plasmid pET43.1-OH-TCI
The coli expression carrier adopting is pET43.1, and the host cell of carrier construction is bacillus coli DH 5 alpha.
1, the clone of OH-TCl gene
First with recombinant clone bacterial strain BL21/pUC18-OH-TCI, be inoculated in LB substratum, 37 ℃ of shaking culture are spent the night; utilize bacterial plasmid to reclaim test kit plasmid purification pUC18-OH-TCI; take pUC18-OH-TCI as template, carry out endonuclease reaction with F1, R1 primer, endonuclease reaction system is as follows:
The enzyme of gained is cut product and is run 0.8% agarose gel electrophoresis, cuts object band, then with sepharose DNA, reclaims test kit and reclaims.
3, the purifying of PCR product and recovery
To after preliminary evaluation, all run 0.8% agarose gel electrophoresis by remaining PCR product, cut object band, and adopt UNIQ-10 pillar DNA glue to reclaim test kit and carry out to specifications purifying recovery, acquire object fragment OH-TCI gene.
4, the enzyme of coli expression carrier pET43.1 cuts back to close
The structure collection of illustrative plates of recombinant expression vector pET43.1-OH-TCI as shown in Figure 2.With SmaI, two kinds of restriction enzymes double zyme cutting pET43.1 carriers of HandIII, it is as follows that enzyme is cut system:
Reclaim enzyme and cut product.
5, the preparation of DH5 α competent cell
DH5 α competent cell is purchased from TIANGEN Biotech (Beijing) Co., Ltd..
6, the conversion that is connected of object fragment OH-TCI and pET43.1 carrier endonuclease bamhi
OH-TCI and pET43.1 endonuclease bamhi are mixed, then with the connection of spending the night of 16 ℃ of T4DNA ligase enzymes.Linked system is as follows:
Then connection product is transformed in DH5 α competent cell, step of converting is described above, obtains recombinant expression plasmid.
7, the extraction of recombinant expression plasmid
Adopt alkaline lysis to extract in a small amount recombinant expression plasmid DNA.
9, the sequencing of recombinant plasmid
In order further to determine the recombinant plasmid obtaining, positive bacteria liquid is delivered to Shanghai Sheng Gong company and carry out sequencing.Correct recombinant plasmid called after pET43.1-OH-TCI will finally be identified.Sequencer map as shown in Figure 3.
Four, the structure of coli expression carrier
1, chemical method recombinant plasmid pET43.1-OH-TCI Transformed E .coli BL21
2, sequencing is identified recombinant bacterial strain
In culture medium flat plate, grow single bacterium colony with the toothpick picking of sterilizing, by plasmid extraction method, extract plasmid, give the order-checking of Shanghai biotechnology company limited and determine.
3, abduction delivering (seeing Fig. 4)
(1) the mono-clonal recombination bacillus coli BL21/pET43.1-OH-TCI transforming is inoculated in respectively the LB(Amp containing 2mL) in liquid nutrient medium, 37 ℃, incubated overnight in the shaking table of 210rpm.
(2) in the ratio of 2:100, get bacterium liquid 2mL and join the LB(Amp containing 200mL) in liquid nutrient medium, 37 ℃, in the shaking table of 210rpm, cultivate.
(3) work as OD
600value reaches at 0.5 o'clock, IPTG1mL, 28 ℃, 250rpm, 4h abduction delivering.
(4) ultrasonication, centrifugal 12000rpm, 15min.
(5) on cellular lysate thing and lysate cleer and peaceful precipitation through SDS-PAGE electrophoresis, the solvability of comparison object albumen in various expression system ,-20 ℃ save backup.
Five, the HPLC of rOH-TCI separates with molecular weight and determines
Purifying and the enzyme of 2.3NusA-OH-TCI fusion rotein are cut
The further affinitive layer purification of Ni-NTA resin, detects through 12%SDS-PAGE, is shown as the band of the about 70kDa of molecular weight, and its purity more than 85% (is shown in Fig. 4).NusA-OH-TCI fusion rotein discharges NusA chaperone and rOH-TCI target protein through zymoplasm cracking, recombinant protein rOH-TCI than original OH-TCI sequence many 2 amino acid (Gly-Ser).NusA-OH-TCI enzyme is cut to mixture and carry out ultrafiltration, first use 30kDa ultrafiltration membrance filter, then by 3kDa ultra-filtration membrane desalination for filtered solution, subsequently concentrated solution is separated with anti-phase high-pressure liquid phase HPLC, freeze-drying, obtains object product rOH-TCI (seeing Fig. 5).The average yield of rOH-TCI is that every milliliter of bacterium liquid is 1.5mg/L.By MALDI-TOF-MS, determine that rOH-TCI molecular weight is 6485.20Da, with its theoretical molecular (6490.40Da) be basically identical (seeing Fig. 6).
Six, the determination of activity of rOH-TCI
Measure the inhibition activity of rOH-TCI to trypsinase and Chymotrypsin, result shows that purifying gained rOH-TCI has stronger inhibition activity to trypsinase and Quimotrase.It is 7.603 × 10 that rOH-TCI suppresses constant (Ki) to Chymotrypsin
-8m, and be 2.92 × 10 to tryptic inhibition constant (KI)
-7m.
Above embodiment is only in order to technical scheme of the present invention to be described, but not is limited; Although the present invention is had been described in detail with reference to previous embodiment, for the person of ordinary skill of the art, the technical scheme that still can record previous embodiment is modified, or part technical characterictic is wherein equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
SEQUENCE LISTING
<110> Weifang Medical College
The king cobra toxin protease inhibitor gene OH-TCI of a <120> optimization and expression method and application
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 194
<212> DNA
<213> artificial sequence
<400> 1
cccggggcag cggtcgtccg aaattctgtg aactgccagc tgtgagcggt ttttgtaagg 60
cgtacattcc gtccttctat tacaacccgg acgcatctgc gtgccagaaa ttcatctacg 120
gcggctgcgg tggtaacgcc aataaattca aaaccatcga agagtgccac cgcacttgcg 180
ttggctaaaa gctt 194
<210> 2
<211> 58
<212> PRT
<213> artificial sequence
<400> 2
Gly Arg Pro Lys Phe Cys Glu Leu Pro Ala Val Ser Gly Phe Cys Lys
1 5 10 15
Ala Tyr Ile Pro Ser Phe Tyr Tyr Asn Pro Asp Ala Ser Ala Cys Gln
20 25 30
Lys Phe Ile Tyr Gly Gly Cys Gly Gly Asn Ala Asn Lys Phe Lys Thr
35 40 45
Ile Glu Glu Cys His Arg Thr Cys Val Gly
50 55
<210> 3
<211> 28
<212> DNA
<213> artificial sequence
<400> 3
acgaattcaa ggtttttgaa agatgtga 28
<210> 4
<211> 30
<212> DNA
<213> artificial sequence
<400> 4
acgcggccgc ttaaccacca ccaccaccaa 30
Claims (9)
1. the king cobra toxin protease inhibitor gene of an optimization
oH-TCI, it has the nucleotide sequence of sequence table SEQ ID NO:1.
2. king cobra toxin protease inhibitor gene according to claim 1
oH-TCI, it is characterized in that the Auele Specific Primer of cloning described gene is:
Primers F 1:5 '-ACGAATTCAAGGTTTTTGAAAGATGTGA-3 ';
Primer R1:5 '-ACGCGGCCGCTTAACCACCACCACCACCAA-3 '.
3. king cobra toxin protease inhibitor gene according to claim 1
oH-TCI, it is characterized in that described
oH-TCI5 ' end of gene is added with
smai restriction enzyme site, 3 ' end is added with
handiII restriction enzyme site.
4. king cobra toxin protease inhibitor gene claimed in claim 1
oH-TCIthe proteinase inhibitor OH-TCI that coding produces, it has the aminoacid sequence shown in sequence table SEQ ID NO:2.
5. contain king cobra toxin protease inhibitor gene claimed in claim 1
oH-TCIrecombinant vectors.
6. recombinant vectors according to claim 4, is characterized in that described recombinant vectors is pUC18-OH-TCI and pET43.1-OH-TCI.
7. the expression method of king cobra toxin protease inhibitor OH-TCI claimed in claim 4, is characterized in that it comprises the following steps: by described
oH-TCIgene proceeds to after being connected with pUC18-T carrier and in intestinal bacteria, obtains recombinant plasmid pUC18-OH-TCI, take pUC18-OH-TCI as template, with F1, R1 primer, carry out pcr amplification and obtain goal gene fragment OH-TCI, construction recombination plasmid pET43.1-OH-TCI is also forwarded in e. coli bl21, screening obtains positive transformant, and positive transformant is inoculated in liquid nutrient medium, and bacterial strain is accessed in LB substratum by 2% inoculum size, in 37 ℃, 210 r/min shaking tables are cultured to OD
600when value reaches 0.5-0.6, add IPTG, abduction delivering at 28 ℃ of temperature, after induction finishes, centrifugal collection somatic cells, purified fusion protein NusA-OH-TCl, cuts fusion rotein with thrombin enzyme and obtains recombinant protein enzyme inhibitors rOH-TCl.
8. the application in medicine or the healthcare products of the amount of bleeding of king cobra toxin protease inhibitor OH-TCI according to claim 4 in preparation treatment acute pancreatitis and minimizing art.
9. the application in medicine or the healthcare products of the amount of bleeding of king cobra toxin protease inhibitor OH-TCI according to claim 4 in preparation treatment acute pancreatitis and minimizing art, is characterized in that described king cobra toxin protease inhibitor OH-TCI has the inhibition activity to trypsinase and Chymotrypsin.
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Cited By (1)
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| CN107540745A (en) * | 2017-08-23 | 2018-01-05 | 湖南甲骨文生物医药有限公司 | A kind of polypeptide and its expression for suppressing function with trypsase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186646A (en) * | 2007-10-26 | 2008-05-28 | 中国科学院昆明动物研究所 | Application of king cobra toxin protease inhibitor and its derivatives |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186646A (en) * | 2007-10-26 | 2008-05-28 | 中国科学院昆明动物研究所 | Application of king cobra toxin protease inhibitor and its derivatives |
Non-Patent Citations (2)
| Title |
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| GENBANK: "IVBIT_OPHHA", 《GENBANK》 * |
| 钱友存 等: "蛇神经毒素的表达和鉴定", 《生物工程学报》 * |
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| CN107540745A (en) * | 2017-08-23 | 2018-01-05 | 湖南甲骨文生物医药有限公司 | A kind of polypeptide and its expression for suppressing function with trypsase |
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