CN107236030A - A kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process - Google Patents
A kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process Download PDFInfo
- Publication number
- CN107236030A CN107236030A CN201710614785.5A CN201710614785A CN107236030A CN 107236030 A CN107236030 A CN 107236030A CN 201710614785 A CN201710614785 A CN 201710614785A CN 107236030 A CN107236030 A CN 107236030A
- Authority
- CN
- China
- Prior art keywords
- protein
- magnetic bead
- peptidoglycan
- 500mmnacl
- silkworm
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Microbiology (AREA)
- Insects & Arthropods (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process, belong to Insect immunity field.Comprise the following steps:MRNA is extracted from silkworm fat-body, reverse transcription is into cDNA, cultivated silkworm peptidoglycan is expanded from cDNA by designing primer and recognizes the gene of Protein S 1, cultivated silkworm peptidoglycan is recognized into the gene cloning of Protein S 1 into PET 30a using the method for molecular cloning and BL21 (DE3) is converted.By BL21 (DE3) incubated overnight containing target gene, by 1:It is 0.5 or so that 100 dilution bacterium solutions, which shake bacterium to OD values, adds final concentration 0.8mMIPTG and 20% glucose induction 5h, and supernatant is abandoned in centrifugation, collects thalline.Prokaryotic expression system genetic map used in the present invention is clear and definite, it is easy to operate, and saves the time, being capable of high-caliber expression foreign protein.
Description
Technical field
The invention belongs to Insect immunity field, more particularly to a kind of cultivated silkworm peptidoglycan identification albumen(PGRP-S1)Protokaryon
Expression.
Background technology
Silkworm is a kind of important economic insects, and silk is used for making silk, sells at home and abroad, driven relevant enterprise
Development, greatly facilitated the economic development of China.Contain abundant protein, edible in silkworm chrysalis.What silkworm excrement and silkworm sloughed off
Micromicro is used as medicine, with important medical value.So in order to improve silkworm survival rate, enhancing silkworm is resisted to pathogenic microorganism
Ability, immune research silkworm is essential.And had increasingly on the document that microbial pathogens is immunized silkworm
It is reported more.
Silkworm belongs to lepidopterous insects, is both important economic insects, is important model organism again.Silkworm lacks the day after tomorrow
It is immune, a set of fairly perfect immunologic mechanism i.e. congenital immunity is formd during the long-term evolution with microorganism, for
The invasion of imperial pathogenic microorganism.Silkworm congenital immunity rely primarily on pattern recognition receptors identification pathogen associated molecular pattern and then
Congenital immunity path is activated, so as to kill microorganism.These pathogen associated molecular patterns only exist pathogenic microorganism without depositing
In host, such as lipopolysaccharides, peptide glycan, these pathogen associated molecular patterns are recognized by corresponding pattern recognition receptors, from
And activate the signal path in downstream.Peptidoglycan recognition protein(PGRP)Family is the most important pattern recognition receptors of silkworm, and it can
To recognize the peptide glycan of pathogenic microorganism, and then the immune pathway of silkworm is triggered, so as to activate or suppress the expression of antibacterial peptide.Have
Research shows that silkworm infection Gram-negative bacteria activation Imd paths obtain immune response, gram-positive bacteria activation Imd paths
Immune response is obtained, but also report shows to connect each other between both approach on evidence.
12 peptidoglycan identification protein genes are identified in the genome of silkworm, these peptidoglycan recognition proteins have one
Individual PGRP domains, for combining the peptide glycan of pathogenic microorganism, and then start or suppress the synthesis of antibacterial peptide.Table on evidence
Some bright peptidoglycan recognition proteins can also trigger the activation of phenol oxidase path, promote melanism effect.Some peptide glycans are recognized
Albumen can also kill bacterium directly as bactericide.
The content of the invention
The invention provides a kind of structure of the prokaryotic expression carrier of cultivated silkworm peptidoglycan identification Protein S 1.
The present invention is realized by following technical proposal:
The method that cultivated silkworm peptidoglycan recognizes the prokaryotic expression of Protein S 1, extracts total serum IgE from silkworm fat-body tissue, passes through reverse transcription
CDNA is synthesized, as template, expand from silkworm fat-body tissue by designing primer and obtains cultivated silkworm peptidoglycan identification albumen
S1 genes.
The method that cultivated silkworm peptidoglycan recognizes the prokaryotic expression of Protein S 1,
The sense primer for designing primer is 5'- CGCAGGATCCGACATGGCCCGCCTCC -3'(BamH1), (SEQ ID
NO.1);
Anti-sense primer is 5'- ATTAGCGGCCGCTCTTGATGGAGTCCACGTTCT -3'(Not1)(SEQ ID NO.2).
Restriction enzyme site is inserted in the primer of design, in order to which cultivated silkworm peptidoglycan is recognized into the gene cloning of Protein S 1 with the method for molecular cloning
Into prokaryotic expression carrier PET-30a, the prokaryotic expression carrier built is converted into DH5 α, by bacterium solution PCR and digestion verification
Positive colony sequencing, will be sequenced correct positive colony and extracts plasmid conversion BL21(DE3).
The method that cultivated silkworm peptidoglycan recognizes the prokaryotic expression of Protein S 1, will be sequenced correct BL21(DE3)Incubated overnight, then
With 1:100 are diluted with fresh culture, continue to cultivate to OD values about 0.5 or so, add final concentration of 0.8mM IPTG with
Mass concentration is 20% glucose induction, and induction time is 5h.Because the albumen of expression carries His labels, nickel ion can be used
Chelating magnetic bead is purified.
The method that nickel ion chelating magnetic bead carries out purifying inclusion body protein, is carried out as steps described below:
(1)The bacterium solution of induction is centrifuged, precipitation is collected, precipitation is washed twice with 1 × PBS, supernatant is abandoned in centrifugation, precipitation adds suitable
1 × PBS of amount simultaneously suspends, ultrasonic on ice(Power:25%, operation 3 seconds, interval 8 seconds, 25 times)Cracking, centrifugation, takes supernatant respectively
12%SDS-PAGE is carried out with precipitation, to determine the solubility of destination protein.
(2)Sample treatment:Precipitate with appropriate inclusion body cleaning solution(1M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl, PH
=7.8)It is resuspended, supernatant is abandoned in centrifugation.Precipitate with appropriate inclusion body lysate(6M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl,
PH=7.8)It is resuspended, centrifugation obtains supernatant.
(3)Magnetic bead is pre-processed:Use Buffer A(8M urea, 20mM sodium phosphates, 500mMNaCl, PH=7.8)Balance nickel from
Son chelating magnetic bead magnetic bead is combined with destination protein:Added in magnetic bead in sample handling procedure and centrifuge obtained supernatant, make purpose
Albumen is combined with magnetic bead.
(4)Magnetic bead is washed:Use Buffer B(8M urea, 20mM sodium phosphates, 500mMNaCl, PH=6.0)With Buffer C
(8M urea, 20mM sodium phosphates, 500mMNaCl, PH=5.3)Magnetic bead is washed, with except foreigh protein removing.
(5)Destination protein is eluted:Use BufferD(8M urea, 20mM sodium phosphates, 500mMNaCl, PH=4.0)Elute purpose
Albumen.
(6)Destination protein renaturation:Renaturation is carried out to inclusion body using dialysis.
In place of the advantage of the present invention:The tedious steps that albumen is extracted from Tissues of Silkworm Bombyx Moril are eliminated, experiment is greatly saved
Cost and time.
Brief description of the drawings
Fig. 1 is that PCR is expanded in the result that cultivated silkworm peptidoglycan recognizes the gene of Protein S 1, figure:1-6:Using fat-body as template, ladder
Spend PCR amplification S1 genes;
Fig. 2 is bacterium solution PCR results, in figure:1:DL5000DNA Marker;2-3:The bacterium solution PCR primer of one bacterium colony;3-4:Separately
The bacterium solution PCR primer of one bacterium colony;
Fig. 3 is the double digestion result of recombinant plasmid, in figure:1:DL5000DNA Marker;2-3:The double digestion production of recombinant plasmid
Thing;
Fig. 4 is induced for IPTG in the result of destination protein expression, figure:1:Albumen Marker;2:Bacterium solution is not induced;3:IPTG is induced
Bacterium solution;
Fig. 5 is determines that destination protein whether there is in precipitation, in figure:1:Bacterium solution is not induced;2:The bacterium solution of IPTG inductions;3:It is super
Total bacterium solution is taken after sonication;4:Supernatant is taken after ultrasonically treated;5:Precipitation is taken after ultrasonically treated;
Fig. 6 for purifying destination protein SDS-PAGE results, in figure:1:Albumen Marker;2:Bacterium solution is not induced;3:Before purification
Albumen;4:The albumen of purifying;
Fig. 7 is the destination protein Western blot result figures of purifying, in:1:Bacterium solution is not induced;2:Albumen before purification;3:
The albumen of purifying.
Embodiment
In order to be best understood from the purpose of the present invention and advantage, the present invention will be further explained in conjunction with the embodiments.Institute
Only it is used for explaining the present invention for embodiment, but does not limit the present invention.
Embodiment 1:Obtain the cultivated silkworm peptidoglycan identification gene of Protein S 1
TaKaRa companies are purchased from for the high-fidelity enzyme used in PCR amplification genes, primer is limited by Shanghai JaRa bioengineering
Company synthesizes.
First, the nucleotide sequence announced according to GenBank(NM_001043371)Primer is designed, sense primer is 5'-
CGCAGGATCCGACATGGCCCGCCTCC -3'(BamH1), anti-sense primer is 5'- ATTAGCGGCCGCTC
TTGATGGAGTCCACGTTCT -3'(Not1).Secondly, fractionation of fatty body extracts mRNA from silkworm, and reverse transcription is
cDNA.Using cDNA as template, performing PCR is entered by designed primer, the cultivated silkworm peptidoglycan identification gene of Protein S 1 is amplified.With 1%
Ago-Gel is detected(Referring to Fig. 1).
Embodiment 2:Cultivated silkworm peptidoglycan recognizes the structure of the gene expression bacterium of Protein S 1
Host Strains DH5 α and BL21 for structure(DE3), PET-30a plasmids preserve by this laboratory, DH5 α and BL21(DE3)
Competence bacterium is made.BamH1 and Not1, ligase is purchased from TaKaRa companies.Plasmid extraction kit is public purchased from OMAGA
Department.
Double digestion is carried out to the peptidoglycan recognition protein S1 genes and PET-30a of amplification, using quick ligase by double enzymes
Cut peptidoglycan recognition protein S1 genes to be connected on the PET-30a of same double digestion, in 16 DEG C of water-bath 2h, and convert DH5 α impressions
State bacterium.The DH5 α of conversion are coated on containing kanamycins(30μg/mL)LB solid mediums on, 37 DEG C of inversions were cultivated
Night.Bacterium colony on flat board is subjected to bacterium solution PCR checkings, verified using two pairs of primers(Referring to Fig. 2).Bacterium solution PCR is verified
Correct bacterium colony incubated overnight, extracts plasmid with plasmid extraction kit and is verified for double digestion(Referring to Fig. 3), checking is correct
Purpose cloning and sequencing.Successful purpose cloned plasmids conversion BL21 is sequenced(DE3), filter out the bacterium colony containing target gene.
Embodiment 3:Induce the expression of destination protein
By the BL21 containing target gene(DE3)Bacterium colony is chosen in containing kanamycins(30μg/mL)LB fluid nutrient mediums in, 37 DEG C
Overnight incubation, then by 1:100 dilutions, which are inoculated in, new contains kanamycins(30μg/mL)LB fluid nutrient mediums in 37 DEG C culture
To OD values about 0.5 or so, final concentration of 0.8mM IPTG and 20% glucose induction is added, induction time is 5h, 4 DEG C
4500rpm centrifuges 25min, collects thalline, carries out 12%SDS-PAGE and verifies whether expression(Referring to Fig. 4).
Embodiment 4:Purify destination protein
Nickel ion chelating magnetic bead is purchased from castor nanosecond science and technology(Suzhou)Co., Ltd.
By the bacterium of collection with 20mL1 × PBS twice, 4 DEG C of 4500rpm centrifuge 25min, collect thalline.With
Thalline is resuspended in 10mL1 × PBS, ultrasonic on ice(Power:25%, operation 3 seconds, interval 8 seconds, 25 times)Cracking, 4 DEG C of 12000rpm
10min is centrifuged, takes supernatant precipitation to carry out 12%SDS-PAGE respectively, to determine the solubility of destination protein(Referring to Fig. 5).
Sample treatment:Precipitation 10mL inclusion body cleaning solutions(1M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl, PH=7.8)
It is resuspended, supernatant is abandoned in centrifugation, is repeated once.Precipitation 10mL inclusion body lysates(6M guanidine hydrochlorides, 20mM sodium phosphates,
500mMNaCl,PH=7.8)It is resuspended, centrifugation obtains supernatant.
Magnetic bead is pre-processed:Magnetic bead is fully mixed, 1mL suspension containing magnetic beads is drawn in centrifuge tube, centrifuge tube is placed in magnetic
On separator, preservation liquid is removed after solution is clarified.Centrifuge tube is removed from magnetic separator.Add 10mLBufferA(8M urinates
Element, 20mM sodium phosphates, 500mMNaCl, PH=7.8)Magnetic bead is suspended again, centrifuge tube is placed on magnetic separator, magnetic point
From removing supernatant, repeated washing 2 times is with balance nickel ion chelating magnetic bead.
Magnetic bead is combined with destination protein:Added in magnetic bead in sample handling procedure and centrifuge obtained supernatant, by centrifuge tube
It is placed on rotary mixer and rotates 20min, destination protein is combined with magnetic bead.Centrifuge tube is placed on magnetic separator afterwards,
Magnetic Isolation, removes supernatant.
Magnetic bead is washed:Sequentially add 10mLBufferB(8M urea, 20mM sodium phosphates, 500mMNaCl, PH=6.0)With
BufferC(8M urea, 20mM sodium phosphates, 500mMNaCl, PH=5.3)Magnetic bead is washed, with except foreigh protein removing.Finally plus
Entering 10mLBufferC makes magnetic bead suspend again, suspension containing magnetic beads is transferred in new centrifuge tube, to prevent the pollution of foreign protein.
Destination protein is eluted:Use 5mLBufferD(8M urea, 20mM sodium phosphates, 500mMNaCl, PH=4.0)Elute purpose
Albumen.
Destination protein renaturation:Using dialysis to inclusion body carry out renaturation, extracellular fluid dialysis be respectively A (20mM sodium phosphates,
500mMNaCl, 4M urea, pH 4.0), B (20mM sodium phosphates, 500mMNaCl, 2M urea, pH 4.0), C (20mM phosphoric acid
Sodium, 500mMNaCl, 1M urea, pH 4.0), D (20mM sodium phosphates, 500mMNaCl, 0M urea, pH 4.0), every time dialysis
Time is 4 hours.
Embodiment 5:12%SDS-PAGE is analyzed and Western blot checkings
Primary antibody(anti-His)Secondary antibody(Sheep anti-mouse igg)Purchased from Nanjing Vazyme Biotechnology Co., Ltd.
12%SDS-PAGE is analyzed:The destination protein for taking 100 μ L to purify adds 20 μ L 5 × SDS Loading Buffer and boiled
5min, 12000rpm centrifuge 5min, take 20 μ L loadings.12%SDS-PAGE is carried out, 2h is dyed with coomassie brilliant blue staining liquid, is used
Coomassie brilliant blue destainer decolourizes to stay overnight(Referring to Fig. 6).
Western blot are verified:By on the albumen of purifying to PDVF films, 1h is closed with 5% skimmed milk power, is washed with TBST
Wash 4 times, add primary antibody(anti-His 1:1000 dilutions), 4 DEG C overnight, and PDVF films are washed 4 times with TBST, adds secondary antibody(Goat-anti
Mouse IgG 1:5000 dilutions)1h, TBST are washed 4 times, add nitrite ion, ECL colour developings(Referring to Fig. 7).
The condition that above-described embodiment more optimizes for the present invention, provable by Fig. 6 and Fig. 7, the albumen being purified into is preferable.This
The experimental method of invention is simultaneously not restricted to the described embodiments, on the premise of experimental principle of the present invention, the modification done and is changed
Enter to belong within protection scope of the present invention.
SEQUENCE LISTING
<110>Jiangsu University
<120>A kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process
<160>2
<170> Patent In version 3.3
<210>SEQ ID NO.1
<211>26
<212>DNA
<213>Artificial sequence
<220>
<221>
<222>(1)..(26)
<400>1
CGCAGGATCC GACATGGCCC GCCTCC 26
<110>Jiangsu University
<210>SEQ ID NO.2
<211>33
<212>DNA
<213>Artificial sequence
<220>
<221>
<222>(1)..(33)
<400>2
ATTAGCGGCC GCTCTTGATG GAGTCCACGT TCT 33
Claims (3)
1. the method that cultivated silkworm peptidoglycan recognizes the prokaryotic expression of Protein S 1, it is characterised in that:
The sense primer for designing primer is 5'- CGCAGGATCCGACATGGCCCGCCTCC -3',
Anti-sense primer is 5'- ATTAGCGGCCGCTCTTGATGGAGTCCACGTTCT -3;
Restriction enzyme site is inserted in the primer of design, in order to which cultivated silkworm peptidoglycan is recognized into the gene of Protein S 1 with the method for molecular cloning
It is cloned into prokaryotic expression carrier PET-30a, the prokaryotic expression carrier built is converted into DH5 α, bacterium solution PCR and digestion are tested
The positive colony sequencing of card, will be sequenced correct positive colony and extracts plasmid conversion BL21(DE3).
2. the method that cultivated silkworm peptidoglycan recognizes the prokaryotic expression of Protein S 1, it is characterised in that correct BL21 will be sequenced(DE3)Overnight
Culture, then takes 40 μ L bacterium solutions to add the new nutrient solutions of 4mL, continues to cultivate to OD values about 0.5 or so, adds final concentration of
0.8mM isopropylthiogalactoside(Abbreviation IPTG)With the glucose induction that mass concentration is 20%, induction time is 5h;
Because the albumen of expression carries His labels, it can be purified with nickel ion chelating magnetic bead.
3. the method that nickel ion chelating magnetic bead carries out purifying inclusion body protein, it is characterised in that carry out as steps described below:
(1)The bacterium solution of induction is centrifuged, precipitation is collected, precipitation is washed twice with 1 × PBS, supernatant is abandoned in centrifugation, precipitation adds suitable
1 × PBS of amount simultaneously suspends, ultrasonic on ice(Power:25%, operation 3 seconds, interval 8 seconds, 25 times)Cracking, centrifugation, takes supernatant respectively
12%SDS-PAGE is carried out with precipitation, to determine the solubility of destination protein;
(2)Sample treatment:Precipitate with appropriate inclusion body cleaning solution(1M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl, PH=
7.8)It is resuspended, supernatant is abandoned in centrifugation;
Precipitate with appropriate inclusion body lysate(6M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl, PH=7.8)It is resuspended, centrifuges
To supernatant;
(3)Magnetic bead is pre-processed:Use Buffer A(8M urea, 20mM sodium phosphates, 500mMNaCl, PH=7.8)Balance nickel ion chela
Magnetic bead magnetic bead is closed to be combined with destination protein:Added in magnetic bead in sample handling procedure and centrifuge obtained supernatant, make destination protein
Combined with magnetic bead;
(4)Magnetic bead is washed:Use Buffer B(8M urea, 20mM sodium phosphates, 500mMNaCl, PH=6.0)With Buffer C(8M urinates
Element, 20mM sodium phosphates, 500mMNaCl, PH=5.3)Magnetic bead is washed, with except foreigh protein removing;
(5)Destination protein is eluted:Use BufferD(8M urea, 20mM sodium phosphates, 500mMNaCl, PH=4.0)Elute purpose egg
In vain;
(6)Destination protein renaturation:Renaturation is carried out to inclusion body using dialysis.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710614785.5A CN107236030A (en) | 2017-07-26 | 2017-07-26 | A kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710614785.5A CN107236030A (en) | 2017-07-26 | 2017-07-26 | A kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process |
Publications (1)
Publication Number | Publication Date |
---|---|
CN107236030A true CN107236030A (en) | 2017-10-10 |
Family
ID=59989768
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710614785.5A Pending CN107236030A (en) | 2017-07-26 | 2017-07-26 | A kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107236030A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108586589A (en) * | 2018-01-16 | 2018-09-28 | 南京农业大学 | A kind of preparation method and applications of the Swollenin albumen from Guizhou trichoderma |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101619102A (en) * | 2008-12-16 | 2010-01-06 | 中国科学院海洋研究所 | Preparation and application of chlamys farreri peptidoglycan recognition protein (Cf-PGRP) with sterilizing activity |
CN104531712A (en) * | 2014-12-07 | 2015-04-22 | 浙江大学 | Preparation and application of Bemisia tabaci peptidoglycan recognition protein with bactericidal activity |
-
2017
- 2017-07-26 CN CN201710614785.5A patent/CN107236030A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101619102A (en) * | 2008-12-16 | 2010-01-06 | 中国科学院海洋研究所 | Preparation and application of chlamys farreri peptidoglycan recognition protein (Cf-PGRP) with sterilizing activity |
CN104531712A (en) * | 2014-12-07 | 2015-04-22 | 浙江大学 | Preparation and application of Bemisia tabaci peptidoglycan recognition protein with bactericidal activity |
Non-Patent Citations (1)
Title |
---|
OCHIAI,M.等: "AB016249.1", 《GENBANK》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108586589A (en) * | 2018-01-16 | 2018-09-28 | 南京农业大学 | A kind of preparation method and applications of the Swollenin albumen from Guizhou trichoderma |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101687024A (en) | trypanosoma antigens, vaccine compositions, and related methods | |
CN104693310B (en) | A kind of chimeric protein, virus-like particle and its application | |
CN106854247A (en) | A kind of preparation method of the bacterial virus catenase that can crack Escherichia coli and salmonella | |
CN107533066A (en) | Mk system, particularly for baculovirus expression subunit antigen Mk system | |
CN104292339A (en) | Recombinant protein containing SARS virus RBD antigen and baculovirus displaying RBD protein | |
CN101016541A (en) | Method of producing brucella vaccine antigen protein | |
CN108794583A (en) | The vaccine that Raccoon dog parvovirus virus-like particle, preparation method are prepared with application and the virus-like particle | |
CN101441217B (en) | Hog cholera antibody ELISA diagnosis reagent kit | |
CN107236030A (en) | A kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process | |
CN111777683A (en) | Fusion protein of nervous necrosis virus MCP and Edwardsiella ictaluri ompN1 and preparation method thereof | |
CN106754981B (en) | Method for preparing gosling plague virus-like particles by using escherichia coli system | |
CN104630184B (en) | A kind of alkalescent xylanase and its encoding gene and application | |
CN102492714A (en) | Method for preparing recombinant Chinese rural dog interferon-alpha | |
CN103614387B (en) | The carrying Cap gene of porcine circovirus type 2 gene optimized and recombinant plasmid and application | |
CN109180818A (en) | A method of inclusion body is purified using GEM | |
CN108003233A (en) | Long oyster domain protein containing DM9 CgDM9CP-2, preparation method and application | |
CN103387992A (en) | Gene for coding recombinant porcine beta-defensin-1 and preparation method of porcine beta-defensin-1 | |
CN104531712B (en) | The preparation and application of Bemisia tabaci peptidoglycan recognition protein with bactericidal activity | |
CN104292338A (en) | Recombinant protein containing SARS virus N antigen and baculovirus displaying N protein | |
CN106366201A (en) | Gene sequence, carrier, recombination strain and recombination protein of fusion protein DAMP4-IGF-1 and preparing method thereof | |
CN102604993A (en) | Immunologic adjuvant-Helicobacter pylori antigen fused protein oral vaccine and preparation method thereof | |
CN110128545A (en) | A kind of fusion, recombinant expression carrier, antigen and its preparation method and application | |
CN103626878A (en) | Newcastle disease virus F protein and enterotoxin LTB fusion protein and application thereof | |
CN107936107A (en) | Long 1 gene recombinant proteins of oyster interferon regulatory factor CgIRF, preparation method and application | |
CN107936106A (en) | Long oyster domain protein containing DM9 CgDM9CP 4, preparation method and application |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171010 |