CN102242127B - Preparation method of serial antibacterial peptide Alloferon-1 and application of rAlloferon-1-K - Google Patents
Preparation method of serial antibacterial peptide Alloferon-1 and application of rAlloferon-1-K Download PDFInfo
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- CN102242127B CN102242127B CN 201110131135 CN201110131135A CN102242127B CN 102242127 B CN102242127 B CN 102242127B CN 201110131135 CN201110131135 CN 201110131135 CN 201110131135 A CN201110131135 A CN 201110131135A CN 102242127 B CN102242127 B CN 102242127B
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Abstract
The invention relates to a preparation method and application of an antibacterial peptide. The invention aims to provide a preparation method of an antibacterial peptide Alloferon-1, which has simple steps and high expression efficiency and keeps the in-vitro anti-tumor activity of the antibacterial peptide, and provide application of rAlloferon-1-K. The technical scheme is as follows: the preparation method of the serial antibacterial peptide Alloferon-1 comprises the following steps of: (1) designing a pair of terminal single-stranded DNAs (deoxyribonucleic acids); (2) generating a concatemer in a PCR (polymerase chain reaction) system; (3) carrying out electrophoretic screening on a 14 Alloferon-1 concatemer; (4) constructing a prokaryotic recombinant expression system E.coliBL21DE3pET42a-14Alloferon-1-K; (5) expressing and purifying recombinant protein 14rAlloferon-1-K; and (6) performing trypsin digestion on the 14rAlloferon-1-K, and purifying to obtain the rAlloferon-1-K.
Description
Technical field
The enzyme that the present invention relates to a kind of foundation, reorganization tandem polypeptide of series connection prokaryotic expression method of peptide is cut, purifying and application; It specifically is the series connection prokaryotic expression method of antibacterial peptide Alloferon-1; Obtain recombinant peptide monomer rAlloferon-1-K with tryptic digestion, resist swollen tumor promotion to detect rAlloferon-1-K.
Background technology
Antibacterial peptide is extensively to be present in the intravital no antigen small peptide of multiple biology, and various mikrobes and protozoon are all had the inhibition of broad-spectrum high efficacy or kill activity, and some antibacterial peptides also have antitumor action and injuring normal cell not.Therefore, antibacterial peptide is the research focus of anti-infective at present and anti-cancer agent.
Alloferon-1 at first finds in calliphora erythrocephala blood, by 13 amino acid form, molecular weight is 1481.53 novel antibacterial peptide.But its lower natural expression output is difficult to satisfy industrialized requirement.Bibliographical information is arranged, adopt efficiently that prokaryotic expression can obtain antibacterial peptides such as a large amount of Magainin-II expediently, and confirm that its BA is similar with natural product.Therefore, feasible on the research Alloferon-1 reorganization series connection high efficiency expression technology.Experimentation on animals confirms that this antibacterial peptide Alloferon-1 has stronger antitumor, antiviral activity, and therefore, the antitumor applied research of research Alloferon-1 reorganization series connection high efficiency expression product has realistic meaning.
Summary of the invention
The purpose of this invention is to provide that a kind of step is simple and direct, expression efficiency is high and keep the antibacterial peptide Alloferon-1 preparation method of its anti tumor activity in vitro.
Another object of the present invention is the application of above-mentioned expression product rAlloferon-1-K.
Technical scheme provided by the invention is: the preparation method of series antimicrobial peptide Alloferon-1 specifically comprises following steps:
1) a pair of non-mirror image symmetry of design contains complementary sticky end single stranded DNA; Positive chain-ordering: 5 '-phosphorylation-CATGGCGTGAGCGGCCATGGCCAGCATGGCGTGCATGGCAAA-3 ', minus strand sequence: 5 '-phosphorylation-GCCATGGCCGCTCACGCCATGTTTGCCATGCACGCCATGCTG-3 ';
2) in the PCR reaction system, through annealing, obtain single copy double-stranded DNA, utilize the series connection at random in the PCR system of complementary sticky end, generate the concatermer of different copies;
3) T-A clone product agarose electrophoresis screening 14 copy Alloferon-1 concatermers, order-checking is identified;
4) identify that with order-checking correct T-A clone 14 copy Alloferon-1 concatermers make up the protokaryon recombinant expression system
E.coliBL21DE3
PET42a-14 * Alloferon-1-K
5) IPTG induces purpose recombinant protein 14rAlloferon-1-K to express, and the Ni-NTA affinity chromatography is purified and collected the purpose recombinant protein;
6) tryptic digestion 14rAlloferon-1-K, Sephadex G-50 chromatography is purified and is obtained rAlloferon-1-K, concentrates the back and detects rAlloferon-1-K concentration.
Recombinant protein rAlloferon-1-K behind the series connection prokaryotic expression 14 copy Alloferon-1 of described antibacterial peptide Alloferon-1 has nucleotide sequence shown in the sequence 3 and aminoacid sequence.
The recombinant protein rAlloferon-1-K that the preparation method of series antimicrobial peptide Alloferon-1 obtains is applied to antitumor.
The invention has the beneficial effects as follows: show the recombinant protein rAlloferon-1-K that preparation method provided by the invention obtains, the effect that has obvious inhibition KB, SGC and the growth of HL-60 cell and breed through external anti-swollen test of tumour.Above-mentioned result of study shows that we have successfully set up antibacterial peptide Alloferon-1 series connection protokaryon preparation method, and its preparation product rAlloferon-1-K is expected to become polypeptide class anti-cancer agent.In addition, the present invention adopts enzyme to cut the higher trypsinase of efficient to replace the enteropeptidase enzyme to cut can reducing preparation cost, help applying.
Embodiment
The sequence table explanation
Sequence 1 be find in the calliphora erythrocephala blood the aminoacid sequence of a kind of novel antibacterial peptide Alloferon-1.
Sequence 2 is the aminoacid sequence of antibacterial peptide Alloferon-1 and the corresponding nucleotide sequence of synthetic (band Trypsin restriction enzyme site Methionin), and lastrow is a nucleotide sequence, next behavior aminoacid sequence.
Sequence 3 is rAlloferon-1-K tandem sequences; The rAlloferon-1-K tandem sequence of 14 copies; Every row's lastrow is a nucleotide sequence; Next behavior aminoacid sequence, it is the Alloferon-1 sequence of 14 copies that the square frame district is arranged, amino acid and nucleotides sequence are classified tryptic digestion site Methionin and password thereof as in the.）
According to bibliographical information; The Alloferon-1 aminoacid sequence is that HGVSGHGQHGVHG has antiviral, anti-tumor activity; The aminoacid sequence of Alloferon-2 then is the GVSGHGQHGVHG non-activity; Both sequence differences only are embodied in the N end, and active key position belongs to so the N end is Alloferon-1 most probably.We add difficult its BA that influences of tryptic digestion site Methionin codon tandem expression through the C end of Alloferon-1; The tandem expression product can obtain a plurality of single rAlloferon-1-K (HGVSGHGQHGVHGDDDDK) through tryptic digestion K site, can keep the activity of Alloferon-1 when improving output.In order to obtain the recombinant expressed output of higher purpose, we make up and screening obtains to contain 14 protokaryon recombinant expression systems that repeat tandem expression 14rAlloferon-1-K
E.coliBL21DE3
PET42a-14 * Alloferon-1-K, adopt Ni-NTA and Sephadex G-50 chromatography, the purpose of purifying fast tandem expression product.T-A clone back sequencing result shows that we clone's Alloferon-1-K sequence is entirely true.Adopt to connect primer PCR and T-A clone technology, we have obtained artificial fusion gene fragment and the clone thereof identical with expected sequence.
The protokaryon recombinant expression system pET42a-14Alloferon-1-K-that makes up
E.coliBLDE3 induces down at IPTG, can express tandem purpose recombinant protein 14rAlloferon-1-K efficiently, and its output is about 30% of total bacterial protein, after the Ni-NTA post is purified, in polyacrylamide gel, is rendered as single protein band.14rAlloferon-1-K is behind tryptic digestion, and with Sephadex G-50 post purification rAlloferon-1-K, this provides good basis for further studying antitumor, the antiviral isoreactivity in rAlloferon-1-K inside and outside.
With direct chemical synthetic Alloferon-1 (cAlloferon-1) and the Alloferon-1 (cAlloferon-1-K) that contains the tryptic digestion site as control test the effect of rAlloferon-1-K vitro inhibition KB, SGC and HL-60 growth of tumour cell and propagation.The result shows that rAlloferon-1-K has the effect of obvious inhibition KB, SGC and growth of HL-60 cell and propagation, and with cAlloferon-1 and cAlloferon-1-K restraining effect no significant difference.
Embodiment
The preparation method of series antimicrobial peptide Alloferon-1 is:
1) obtains the aminoacid sequence of calliphora erythrocephala natural A lloferon-1 from GenBank: N-His Gly Val Ser Gly His Gly Gln His Gly Val His Gly-C (the Alloferon-1 sequence does not contain l-arginine and Methionin);
2) a pair of non-mirror image symmetry of design contains complementary sticky end single stranded DNA; Alloferon-1 aminoacid sequence and intestinal bacteria preference codon according to report; The Alloferon-1 that considers tandem expression simultaneously separates; Positive chain-ordering: 5 '-phosphorylation-CATGGCGTGAGCGGCCATGGCCAGCATGGCGTGCATGGCAAA-3 '; Establish tryptic digestion site Methionin codon, minus strand sequence: 5 '-phosphorylation-GCCATGGCCGCTCACGCCATGTTTGCCATGCACGCCATGCTG-3 ' at positive chain DNA Alloferon-1 gene 3 ' end.Minus-strand dna and normal chain are staggered complementary; Entrust Invitrogen company synthetic.
3) the double-stranded formation of purpose peptide nucleotide sequence; In 1 * T4 DNA ligase enzyme damping fluid (TaKaRa), 225 pmol normal chains are mixed with the equivalent minus-strand dna; 95 ℃ of 3 min in the PCR appearance; Powered-down makes reactant naturally cooling 30 min; Add T4 dna ligase (TaKaRa) behind 55 ℃ of 10 min, 16 ℃ of connections are spent the night, with breach in the DNA plerosis two strands.Employing Ex-Taq PCR test kit (TaKaRa) is with placed in-line Alloferon-1-K double-stranded DNA two ends strand polishing and connect 3 '-A, reaction parameter: 94 ℃ of 5min, 55 ℃ of 10 min, 72 ℃ of 30 min.
4) T-A clone: adopt T-A clone's test kit (TaKaRa) that dna fragmentation is connected with pMD19-T (outsourcing), be transformed into
E.coliIncrease among the DH101B, extract recombinant plasmid, after endonuclease BamH I and Hind III (TaKaRa) the double digestion preliminary evaluation, entrust the order-checking of Shanghai Invitrogen company with plasmid extraction kit (Simgen).
5) structure and the evaluation of purpose peptide series connection recombinant expression vector: choosing the correct 14 * Alloferon-1-K DNA of sequence according to sequencing result is template; Adopt circulation cooling pcr amplification purpose fragment; Design according to synthetic Alloferon-1 nucleotide sequence contains restriction endonuclease Nde I and Xho I site, opens the upstream and downstream primer in beginning password ATG and termination codon TAA and tryptic digestion site; Pcr amplification, reaction parameter: 94 ℃ of 5 min; 94 ℃ of 30 s, 68 ℃ of 30 s, 72 ℃ of 1 min, 5 circulations; 94 ℃ of 30 s, 66 ℃ of 30 s, 72 ℃ of 1 min, 5 circulations; 94 ℃ of 30 s, 64 ℃ of 30 s, 72 ℃ of 1 min, 5 circulations; 94 ℃ of 30 s, 62 ℃ of 30 s, 72 ℃ of 1 min, 15 circulations; 72 ℃ of 10 min.The PCR product detects with 1.5% agarose gel electrophoresis, by last method the purpose fragment and the pMD19-T that reclaim is connected to form recombinant plasmid pMD19-T
14 * Alloferon-1-KProkaryotic expression carrier pET42a (Novagen) and pMD19-T
14 * Alloferon-1-KSpend the night with Nde I and 37 ℃ of double digestions of Xho I respectively, the 14 * Alloferon-1-K that reclaims behind the agarose gel electrophoresis is connected 16 h with linearizing pET42a for 16 ℃ with T4 DNA ligase enzyme, connects product and is transformed into
E.coliForm expression system among the BL21DE3 (Novagen)
E.coliBL21DE3
PET42a-14 * Alloferon-1-K, extract pET42a
14 * Alloferon-1-KEntrust the order-checking of Shanghai Invitrogen company.
6) the purpose recombinant products is expressed, enzyme is cut and purify:
E.coliBL21DE3
PET42a-14 * Alloferon-1-KInduce purpose recombinant protein 14 * rAlloferon-1-K to express containing 30 ℃ of 0.5 mmol/L IPTG (Sigma) LB nutrient solutions, SDS-PAGE and Bio-Rad gel images systems inspection expression and output.Adopt Ni-NTA affinity column purification 14 * rAlloferon-1-K; Be dissolved in pH8.0,0.01 mol/L phosphoric acid (PB) damping fluid; Per 10 mg, 14 * rAlloferon-1-K adds 0.1 mg trypsinase, and 37 ℃ of enzymes are crossed Sephadex G-50 post after cutting 2 h, collects to contain monomeric the 3rd elution peak of rAlloferon-1-K; Elutriant is above-mentioned PB, the determined by ultraviolet spectrophotometry protein concentration.RAlloferon-1 purification and concentration detection method are the same.
7) the purpose peptide is synthetic: trust Invitrogen company directly synthesizes Alloferon-1 small peptide HGVSGHGQHGVHG (cAlloferon-1) and contains the small peptide HGVSGHGQHGVHGK (cAlloferon-1-K) in tryptic digestion site.
The application of rAlloferon-1-K: be target cell with human oral squamous epithelium cancer cell KB, people's adenocarcinoma of stomach cell SGC and marrow series leukemia cell HL-60 respectively, detect the external growth-inhibiting effect of different al loferon-1 to tumour cell.The every hole 1 * 10 of 96 well culture plates
4KB, 2 * 10
4SGC, 1 * 10
5Add 1 * 10 respectively in the K562 cell
5, 1 * 10
5, 1 * 10
6The BALB/c mouse splenocyte adds different concns rAlloferon-1-K, then at 5%CO
2Under 37 ℃ of conditions, KB and K562 cell cultures 24 h, SGC cell cultures 72 h measure OD behind adding CCK-8 reagent colour development 1 h
450Value is also calculated tumor cell destruction.CAlloferon-1 and cAlloferon-1-K positive control are set in the experiment simultaneously reach the normal control that does not add any medicine.
Be the series connection prokaryotic expression of identifying antibacterial peptide Alloferon-1 technological feasibility and reduction rAlloferon-1 preparation cost; We replace enteropeptidase with the active trypsinase that more high price is cheaper when restriction enzyme site is set, promptly 3 ' end immediately following the Alloferon-1 gene contains trypsin K when the positive chain DNA of design) codon.Anti-tumor activity test is the result show, genetic engineering technique obtains crAlloferon 1, the cAlloferon 1-K anti-tumor activity there was no significant difference of rAlloferon 1-K, rAlloferon 1-EK and artificial chemosynthesis.Feasible and the available trypsinase that the series connection prokaryotic expression technology of the antibacterial peptide Alloferon-1 that sets up is described replaces enteropeptidase to reduce preparation cost.
Following each table is seen in concrete test:
1.cAlloferon-1 with the direct antitumor action of cAlloferon-1-K
In 0.1-1000 ng/mL scope, the activity of the equal unrestraint KB of cAlloferon-1 and cAlloferon-1-K, SGC and K562 growth of tumour cell and propagation (
P>0.05) (table 1).
The direct antitumous effect of table 1 cAlloferon-1 and cAlloferon-1-K
2.cAlloferon-1 with the indirect antitumor action of cAlloferon-1-K
In 0.1-10 ng/mL scope and in the presence of the mouse boosting cell; CAlloferon-1 and cAlloferon-1-K act on KB and K562 tumour cell 24 h respectively, have all shown the activity of obvious suppression growth of tumour cell and propagation when acting on SGC tumour cell 72 h (P < 0.01), and above-mentioned cAlloferon-1 and cAlloferon-1-K extracorporeal anti-tumor cytoactive no significant difference (P>0.05) (table 2).
The indirect antitumous effect of table 2 cAlloferon-1 and cAlloferon-1-K
* with
#: compare with splenocyte+tumour cell
P<0.01
3.rAlloferon-1-K indirect antitumor action
In 0.1-10 ng/mL scope and in the presence of the mouse boosting cell, rAlloferon-1-K act on KB and K562 tumour cell 24 h respectively, all shown the activity of obvious suppression growth of tumour cell and propagation when acting on SGC tumour cell 72 h (
P<0.01), this anti-tumor activity and cAlloferon-1 and cAlloferon-1-K no significant difference (
P>0.05) (table 3).
The indirect antitumous effect of table 3 rAlloferon-1-K
* with
#: compare with splenocyte+tumour cell
P<0.01
Conclusion:
1. CCK-8 method detected result: in 0.1 ng/mL～10 ng/mL concentration ranges, cAlloferon-1, cAlloferon-1-K, rAlloferon-1-K have obvious raising mouse boosting cell kill and wound K562, KB and SGC growth of tumour cell and propagation BA (
P<0.01), action effect and drug level are irrelevant, rAlloferon-1-K anti-tumor activity and cAlloferon-1 and cAlloferon-1-K comparison there was no significant difference (
P>0.05).Point out each Alloferon-1 to show antitumor action through activating mouse boosting cell, rAlloferon-1-K molecule C end adds that (Lys K) does not influence its anti-tumor activity to Methionin.
2. the CCK-8 method detects principle: contained WST-8 is 2-(2-methoxyl group-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2 in the CCK-8 reagent; 4-disulfonic acid benzene)-2H-tetrazolium list sodium salt; Can be under the effect of electron carrier 1-methoxyl group-5-toluphenazine methyl-sulfate (1-Methoxy PMS) by cell mitochondrial in desaturase be reduced to yellow first Za product (Formazan) with high water soluble, the first Za thing amount and the viable cell quantity of generation have favorable linearity positive correlation.
3. tumour cell is selected: KB, SGC and K562 cell are respectively squamous cell carcinoma, people's adenocarcinoma of stomach and leukemia cell, can represent three kinds of different types of tumors cells respectively.
4. CCK-8 method detected object: the rAlloferon-1-K that purifies in the direct synthetic cAlloferon-1 of chemical method, cAlloferon-1-K and recombinant expressed back is to the restraining effect of above-mentioned three kinds of dissimilar growth of tumour cell and breeding.
Sequence table
< 110>Yan Jie
< 120>application of the preparation method of series antimicrobial peptide Alloferon-1 and rAlloferon-1-K
<160>3
<170> WinBlast v.0.2.0
<210>1
< 211>amino acid/11 3
<212>PRT
< 213>calliphora erythrocephala (Calliphora vicina)
<220>
< 221>aminoacid sequence of antibacterial peptide Alloferon-1
< 222>aminoacid sequence (1) ... (13)
<400>1
His Gly Val Ser Gly His Gly Gln His Gly Val His Gly 16
1 5 10
< 110>Yan Jie
< 120>application of the preparation method of series antimicrobial peptide Alloferon-1 and rAlloferon-1-K
<160>3
<170> WinBlast v.0.2.0
<210>2
< 211>Nucleotide 42, amino acid/11 4
<212>DNA/PRT
< 213>synthetic
<220>
< 221>the corresponding nucleotide sequence of the aminoacid sequence of antibacterial peptide Alloferon-1 and synthetic (band tryptic digestion site)
< 222>nucleotide sequence (1) ... (42), aminoacid sequence (1) ... (14)
<400>1
CAT GGC GTG AGC GGC CAT GGC CAG CAT GGC GTG CAT GGC AAA 48
His Gly Val Ser Gly His Gly Gln His Gly Val His Gly Lys 16
1 5 10 15
< 110>Yan Jie
< 120>application of the preparation method of series antimicrobial peptide Alloferon-1 and rAlloferon-1-K
<160>3
<170> WinBlast v.0.2.0
<210>3
< 211>Nucleotide 588, amino acid/11 96
<212>DNA/PRT
< 213>synthetic
<220>
The Alloferon-1-K tandem sequence of < 221>14 copies
< 222>nucleotide sequence (1) ... (588), aminoacid sequence (1) ... (196)
<400>1
CAT GGC GTG AGC GGC CAT GGC CAG CAT GGC GTG CAT GGC AAA CAT GGC
His Gly Val Ser Gly His Gly Gln His Gly Val His Gly Lys His Gly
1 5 10 15
GTG AGC GGC CAT GGC CAG CAT GGC GTG CAT GGC AAA CAT GGC GTG AGC
Val Ser Gly His Gly Gln His Gly Val His Gly Lys His Gly Val Ser
20 25 30
GGC CAT GGC CAG CAT GGC GTG CAT GGC AAA CAT GGC GTG AGC GGC CAT
Gly His Gly Gln His Gly Val His Gly Lys His Gly Val Ser Gly His
35 40 45
GGC CAG CAT GGC GTG CAT GGC AAA CAT GGC GTG AGC GGC CAT GGC CAG
Gly Gln His Gly Val His Gly Lys His Gly Val Ser Gly His Gly Gln
50 55 60
CAT GGC GTG CAT GGC AAA CAT GGC GTG AGC GGC CAT GGC CAG CAT GGC
His Gly Val His Gly Lys His Gly Val Ser Gly His Gly Gln His Gly
65 70 75 80
GTG CAT GGC AAA CAT GGC GTG AGC GGC CAT GGC CAG CAT GGC GTG CAT
Val His Gly Lys His Gly Val Ser Gly His Gly Gln His Gly Val His
85 90 95
GGC AAA CAT GGC GTG AGC GGC CAT GGC CAG CAT GGC GTG CAT GGC AAA
Gly Lys His Gly Val Ser Gly His Gly Gln His Gly Val His Gly Lys
100 105 110
CAT GGC GTG AGC GGC CAT GGC CAG CAT GGC GTG CAT GGC AAA CAT GGC
His Gly Val Ser Gly His Gly Gln His Gly Val His Gly Lys His Gly
115 120 125
GTG AGC GGC CAT GGC CAG CAT GGC GTG CAT GGC AAA CAT GGC GTG AGC
Val Ser Gly His Gly Gln His Gly Val His Gly Lys His Gly Val Ser
130 135 140
GGC CAT GGC CAG CAT GGC GTG CAT GGC AAA CAT GGC GTG AGC GGC CAT
Gly His Gly Gln His Gly Val His Gly Lys His Gly Val Ser Gly His
145 150 155 160
GGC CAG CAT GGC GTG CAT GGC AAA CAT GGC GTG AGC GGC CAT GGC CAG
Gly Gln His Gly Val His Gly Lys His Gly Val Ser Gly His Gly Gln
165 170 175
CAT GGC GTG CAT GGC AAA CAT GGC GTG AGC GGC CAT GGC CAG CAT GGC
His Gly Val His Gly Lys His Gly Val Ser Gly His Gly Gln His Gly
180 185 190
GTG CAT GGC AAA
Val His Gly Lys
195
Annotate: is that amino acid and nucleotides sequence are classified tryptic digestion site Methionin and password thereof as.
Claims (2)
1. the preparation method of series antimicrobial peptide Alloferon-1 specifically comprises following steps:
1) a pair of non-mirror image symmetry of design contains complementary sticky end single stranded DNA; Positive chain-ordering: 5 '-phosphorylation-CATGGCGTGAGCGGCCATGGCCAGCATGGCGTGCATGGCAAA-3 ', minus strand sequence: 5 '-phosphorylation-GCCATGGCCGCTCACGCCATGTTTGCCATGCACGCCATGCTG-3 ';
2) in the PCR reaction system, through annealing, obtain single copy double-stranded DNA, utilize the series connection at random in the PCR system of complementary sticky end, generate the concatermer of different copies;
3) T-A clone product agarose electrophoresis screening 14 copy Alloferon-1 concatermers, order-checking is identified;
4) identify that with order-checking correct T-A clone 14 copy Alloferon-1 concatermers make up the protokaryon recombinant expression system
E.coliBL21DE3
PET42a-14 * Alloferon-1-K
5) IPTG induces purpose recombinant protein 14rAlloferon-1-K to express, and the Ni-NTA affinity chromatography is purified and collected the purpose recombinant protein;
6) tryptic digestion 14rAlloferon-1-K, Sephadex G-50 chromatography is purified and is obtained rAlloferon-1-K, concentrates the back and detects rAlloferon-1-K concentration.
2. the preparation method of series antimicrobial peptide Alloferon-1 according to claim 1; It is characterized in that the recombinant protein rAlloferon-1-K after said antibacterial peptide Alloferon-1 series connection prokaryotic expression 14 copies Alloferon-1, be the aminoacid sequence shown in the sequence 3.
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| CN103948910A (en) * | 2014-02-24 | 2014-07-30 | 张家港市第一人民医院 | Use of immunomodulatory polypeptide ZL-1 in preparation of anti-tumor drugs |
| CN106560475A (en) * | 2016-04-01 | 2017-04-12 | 天演益合（厦门）生物技术有限公司 | Active short peptide gene engineering biosynthesis process |
| RU2667128C2 (en) * | 2016-12-29 | 2018-09-14 | Герман Петрович Беккер | Composition for preparation of anti-tumor medication and method for preparation of anti-tumor medication based on it |
| WO2022002060A1 (en) * | 2020-06-29 | 2022-01-06 | Vamos Biotech (Shanghai) Co., Ltd | Method for antiviral and cytokine storm treatment |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1273248A (en) * | 1999-11-24 | 2000-11-15 | 刘建宁 | Efficient gene engineering process for preparing polypeptide medicines |
| CN1531551A (en) * | 2001-07-19 | 2004-09-22 | \ | Production of insulinotropic secretion peptide GLP-1(7-36) and GLP-1 analog |
| CN1911957A (en) * | 2006-08-16 | 2007-02-14 | 深圳职业技术学院 | Series polypeptide of blood pressure lowering peptide, expression carrier, polynucleotide sequence and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1273248A (en) * | 1999-11-24 | 2000-11-15 | 刘建宁 | Efficient gene engineering process for preparing polypeptide medicines |
| CN1531551A (en) * | 2001-07-19 | 2004-09-22 | \ | Production of insulinotropic secretion peptide GLP-1(7-36) and GLP-1 analog |
| CN1911957A (en) * | 2006-08-16 | 2007-02-14 | 深圳职业技术学院 | Series polypeptide of blood pressure lowering peptide, expression carrier, polynucleotide sequence and application |
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