CN101139599A - Cultivated silkworm peptidoglycan identification protein gene - Google Patents
Cultivated silkworm peptidoglycan identification protein gene Download PDFInfo
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- CN101139599A CN101139599A CNA2007100925426A CN200710092542A CN101139599A CN 101139599 A CN101139599 A CN 101139599A CN A2007100925426 A CNA2007100925426 A CN A2007100925426A CN 200710092542 A CN200710092542 A CN 200710092542A CN 101139599 A CN101139599 A CN 101139599A
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- gene
- silkworm
- peptidoglycan
- pgrps
- protein gene
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Abstract
The invention provides an identifying protein gene for a silkworm peptidoglycan. First of all, predicating PGRPs gene by using a silkworm gene database and EST database source, then extracting PGA-induced 5-instar silkworm epiploon, synthesizing CDNA, and cloning gene. PGRPs gene can obviously kill bacteria, and PGRPs pertain solution of low concentration can also suppress bacteria growth. The congenital immunity identifying receptor of silkworm activates the congenital immunity and adjusts the antibiotic peptide expression, and can be used for culturing antibiotic silkworms.
Description
Technical field
The present invention relates to biological technical field, particularly relate to and a kind ofly come from silkworm congenital immunity identification receptor, have identification bacterial cell wall peptidoglycan and activate the silkworm immunity system and antibacterial protein gene.
Background technology
China just began silkworm rearing before 5000, be the source region of sericulture.Along with the continuous development of biotechnology means, with the level of comprehensive utilization that improves silkworm.The moiety of microorganism wall is autarcetic efficient activated molecule, and the peptidoglycan of these molecules such as gram positive bacterium (peptidoglycan, PGN).The polymkeric substance that PGN is made up of aminosugar skeleton and peptide chain, its structural unit is by N-acetyl-glucosamine and-acetylmuramic acid disaccharide unit and the tetrapeptide subunit and the peptide bridge composition of β-(1,4)-connection.Inferior peptide unit is different with a peptide bridge upper amino acid composition and arrangement, has determined the diversity of peptidoglycan kind.Natural immune system is discerned microorganism surface standpatter's pattern by at the conservative a series of pattern recognition acceptors of evolution camber, and wherein PGRPs is a kind of important pattern recognition acceptor, is bringing into play important effect in the process of opposing pathogenic agent invasion.The PGRPs family member has a C end PGRP structural domain, and 165 amino acid are arranged approximately, demonstrates the conservative property of height from the insect to the Mammals.But outside the PGRP structural domain, aminoacid sequence differs greatly.According to their structure, peptidoglycan recognition protein can be divided into two hypotypes, i.e. elongated peptidoglycan recognition protein (PGRP-L) and short type peptidoglycan recognition protein (PGRP-S).The PGRP-L of insect mainly is expressed in hemocyte, and PGRP-S is present in hemolymph, epidermis and internal organ, and a spot of expression is also arranged in hemocyte.Wherein some PGRPs is the constructive expression, and it is inducible expressions that some PGRPs (mainly be PGRP-L, but except PGRP-LB) are also arranged, and can raise its expression at infectation of bacteria or injection PGN.
PGRP-L expresses the most extensively among people's the PGRPs, except that liver and tire liver are expressed, at transverse colon, lymphoglandula, heart, thymus gland, pancreas, descending colon, stomach and testis low the expression is arranged.PGRP-I α also expresses at tonsilla and thymus gland, and at stomach, descending colon, rectum and brain utmost point low expression level is arranged also except that the oesophagus high expression level.PGRP-I β only expresses in oesophagus, tonsilla and thymus gland.PGRP-S is at the marrow high expression level, and in tire liver and white corpuscle low expression level, only neutrophil leucocyte is expressed in peripheral blood.Because " pollution " of the neutrophil leucocyte in peripheral blood effect, make PGRP-S might be in spleen and jejunum low expression level, this may be exactly to cause PGRP-S in the rat spleen that the reason of low expression level is arranged.What is interesting is that find that the mouse sleep insuffience can raise the expression of its PGRP, this explanation PGRP may play certain effect aspect the self-control of sleep.The pro-phenoloxidase cascade reaction is the part of insect antibacterial defence, the pattern recognition acceptor is the startup person of pro-phenoloxidase activation system, when the pattern recognition acceptor with form a mixture after the microorganism wall composition combines, the activation proenzyme of this mixture activating pro-phenoloxidase, make it become the pro-phenoloxidase activating enzyme, the pro-phenoloxidase activating enzyme activates pro-phenoloxidase, becomes activated phenol oxidase.Phenol oxidase is a kind of copper bearing oxygenant, can be oxidized to quinone to the substrate that contains phenol, and non-enzymatic reaction is converted into melanochrome through several steps again, and melanochrome is virose to microorganism.Can also can induce the expression of its antibacterial peptide simultaneously by the control Rel transcription factor Relish of family.All peptidoglycan recognition protein energy binding peptide glycan and bacterium, this shows that PGRP has the significance of identification and kill bacteria in the natural immunity.Although but PGRPs is named as peptidoglycan recognition protein, it is identification polypeptide glycan or gram-positive microorganism not only, can also discern some other molecule.From a few days ago on, still there is not the research report of relevant cultivated silkworm peptidoglycan identification protein gene.
Summary of the invention
The object of the present invention is to provide a kind of cultivated silkworm peptidoglycan identification protein gene, utilize the obvious killing action of PGRPs albumen, further cultivate the silkworm resistant variety bacterium.
The present patent application people at first utilizes domestic silkworm gene group database and est database resource, has finished the prediction of PGRPs gene, extract through PGN inductive silkworm fatty body RNA in three days five ages and with become CDNA, finish gene clone and correlative study.Concrete grammar is as follows:
(1) relevant CDNA obtains: three days five ages, silkworm was made greatly, got fatty body after PGN induces 12 hours, preserved in frozen and the liquid nitrogen; RNAiso reagent (TaKaRa) extracts RNA, and synthetic CDNA;
(2) BmPGRPs gene ORF obtains and the clone: according to domestic silkworm gene group database and est database, dope PGRPs gene ORF, and the design primer, the BmPGRPs gene fragment obtained by pcr amplification, amplified fragments is cloned into PMD18-T Simple Vector, carries out the sequence checking then;
(3) sequence homology analysis: the similarity comparison is finished with BLAST on the NCBI website, the BLAST analysis revealed, and the BmPGRPs genetic comparison is conservative, in the silkworm correlative study, do not report, be a new gene;
(4) BmPGRPs expression of gene protein and purifying: PGRPs full length gene ORF fragment cloning to prokaryotic expression carrier Pet50, and is finished abduction delivering; This albumen induces 6h can obtain soluble protein IPTG final concentration 0.5mM, 30 ℃; To express gained albumen and pass through method for purifying proteins such as post, obtain the PGRPs gene protein.
The BmPGRPs full length gene ORF that aforesaid method obtains and the aminoacid sequence of deduction thereof are as follows:
CCT?AGT?CCA?ATA?AAA?TTA?GTT?AGT?GAC?TAT?TCC?TTC?CCC?TTC?GTA
P S P I K L V S D Y S F P F V
AGC?CGA?GAG?GAA?TGG?GGT?GCA?AGG?CCA?CCA?ACA?ATG?ACA?TCA?CCA
S R E E W G A R P P T M T S P
CTC?AAA?GTC?AGT?CCA?GTG?CCC?ATA?GTA?GTG?ATT?CAC?CAC?AGC?TAC
L K V S P V P I V V I H H S Y
ATT?CCC?AAA?ATA?TGC?TTA?GTT?CGA?GCA?GAC?TGT?GAG?AGA?GAT?TTG
I P K I C L V R A D C E R D L
CGT?AAT?ATG?CAA?AGA?GTG?CAC?CAA?GTG?ACT?AAT?GGC?TGG?GAA?GAT
R N M Q R V H Q V T N G W E D
ATT?GGA?TAT?AGT?TTC?GCA?GTC?GGC?GGT?GAA?GGA?ACT?GTG?TTC?GAG
I G Y S F A V G G E G T V F E
GGC?CGT?GGC?TGG?AGT?TCA?ATT?GGT?GCT?CAC?GCG?TTC?GGC?GTC?AAC
G R G W S S I G A H A F G V N
ACC?CGA?AGC?ATC?GGA?ATC?TTG?TTG?ATT?GGA?GAT?TTT?ATT?ACT?AAT
T R S I G I L L I G D F I T N
CAA?CCG?CCC?CAG?GCG?CAG?CTA?CAA?TCT?GTC?AAG?GAC?CTG?ATC?GAG
Q P P Q A Q L Q S V K D L I E
GCT?GGT?GTG?AGG?CTC?GGT?CAC?ATC?CGA?TCC?GAT?TAC?AAA?CTC?ATC
A G V R L G H I R S D Y K L I
GGA?CAC?AGA?CAA?GTC?ACT?CCC?ACT?GAA?TGC?CCT?GGA?CAG?AGA?CTG
G H R Q V T P T E C P G Q R L
TTT?GAT?GAA?ATC?TCA?AAA?TGG?GAT?CAC?TTT?TCA?CTG?AAT?TGG?GAT
F D E I S K W D H F S L N W D
GAT
D
The advantage of invention is:
PGRPs albumen has tangible killing action to bacterium, and the PGRPs protein solution of low concentration has the effect of obvious bacteria growing inhibiting equally; Silkworm congenital immunity identification receptor activates congenital immunity and regulates and control peptide expression, can cultivate the silkworm resistant variety.
Embodiment:
Embodiment:
Domestic silkworm gene group database and est database are predicted, obtain the total length ORF of PGRPs gene, according to inferior design primer, upstream primer sequence: 5 ' CGCG GAT CCT AGT CCA ATA AAA TTA GTT AG, 3 ' downstream primer sequence: 5 ' CCCAAG CTT ATC ATC CCA ATT CAG TGA AA 3 '; Three days five ages, silkworm was made greatly, got fatty body after PGN induces 12 hours, preserved in frozen and the liquid nitrogen; RNAiso reagent (TaKaRa) extracts RNA, and synthetic CDNA; Obtain the BmPGRPs gene fragment by pcr amplification, amplified fragments is cloned into PMD18-T Simple Vector, carry out the sequence checking then; The similarity comparison is finished with BLAST on the NCBI website, the BLAST analysis revealed, and the PGRPs genetic comparison is conservative, in the silkworm correlative study, do not report, be a new gene; PGRPs full length gene ORF fragment cloning to prokaryotic expression carrier Pet50, and is finished abduction delivering; This albumen induces 6h can obtain soluble protein IPTG final concentration 0.5mM, 30 ℃; To express gained albumen and pass through method for purifying proteins such as post, obtain the BmPGRPs gene protein.
Claims (2)
1. cultivated silkworm peptidoglycan identification protein gene is characterized in that its sequence is as follows:
CCTAGTCCAA?TAAAATTAGT?TAGTGACTAT?TCCTTCCCCT?TCGTAAGCCG?AGAGGAATGG 60
GGTGCAAGGC?CACCAACAAT?GACATCACCA?CTCAAAGTCA?GTCCAGTGCC?CATAGTAGTG?120
ATTCACCACA?GCTACATTCC?CAAAATATGC?TTAGTTCGAG?CAGACTGTGA?GAGAGATTTG?180
CGTAATATGC?AAAGAGTGCA?CCAAGTGACT?AATGGCTGGG?AAGATATTGG?ATATAGTTTC?240
GCAGTCGGCG?GTGAAGGAAC?TGTGTTCGAG?GGCCGTGGCT?GGAGTTCAAT?TGGTGCTCAC?300
GCGTTCGGCG?TCAACACCCG?AAGCATCGGA?ATCTTGTTGA?TTGGAGATTT?TATTACTAAT?360
CAACCGCCCC?AGGCGCAGCT?ACAATCTGTC?AAGGACCTGA?TCGAGGCTGG?TGTGAGGCTC?420
GGTCACATCC?GATCCGATTA?CAAACTCATC?GGACACAGAC?AAGTCACTCC?CACTGAATGC?480
CCTGGACAGA?GACTGTTTGA?TGAAATCTCA?AAATGGGATC?ACTTTTCACT?GAATTGGGAT?540
GAT 543
2. cultivated silkworm peptidoglycan identification protein gene encoded protein matter is characterized in that its sequence is as follows:
PSPIKLVSDY?SFPFVSREEW?GARPPTMTSP?LKVSPVPIVV?IHHSYIPKIC?LVRADCERDL 60
RNMQRVHQVT?NGWEDIGYSF?AVGGEGTVFE?GRGWSSIGAH?AFGVNTRSIG?ILLIGDFITN 120
QPPQAQLQSV?KDLIEAGVRL?GHIRSDYKLI?GHRQVTPTEC?PGQRLFDEIS?KWDHFSLNWD 180
D 181
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100925426A CN101139599A (en) | 2007-08-07 | 2007-08-07 | Cultivated silkworm peptidoglycan identification protein gene |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100925426A CN101139599A (en) | 2007-08-07 | 2007-08-07 | Cultivated silkworm peptidoglycan identification protein gene |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484468A (en) * | 2013-09-11 | 2014-01-01 | 华南农业大学 | Diamondback moth peptidoglycan recognition protein, preparation method and application thereof |
| CN104203977A (en) * | 2012-02-15 | 2014-12-10 | 诺和诺德A/S（股份有限公司） | Antibodies that bind peptidoglycan recognition protein 1 |
| CN106749589A (en) * | 2016-12-22 | 2017-05-31 | 沈阳药科大学 | Peptidoglycan recognition protein SA and preparation method thereof, pattern recognition function and application |
-
2007
- 2007-08-07 CN CNA2007100925426A patent/CN101139599A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104203977A (en) * | 2012-02-15 | 2014-12-10 | 诺和诺德A/S（股份有限公司） | Antibodies that bind peptidoglycan recognition protein 1 |
| CN108530535A (en) * | 2012-02-15 | 2018-09-14 | 诺和诺德股份有限公司 | In conjunction with the antibody of peptidoglycan recognition protein 1 |
| US10150809B2 (en) | 2012-02-15 | 2018-12-11 | Bristol-Myers Squibb Company | Antibodies that bind peptidoglycan recognition protein 1 |
| US10906965B2 (en) | 2012-02-15 | 2021-02-02 | Novo Nordisk A/S | Methods of treating autoimmune disease or chronic inflammation wtih antibodies that bind peptidoglycan recognition protein 1 |
| CN108530535B (en) * | 2012-02-15 | 2021-02-26 | 诺和诺德股份有限公司 | Antibody binding to peptidoglycan-recognizing protein 1 |
| CN103484468A (en) * | 2013-09-11 | 2014-01-01 | 华南农业大学 | Diamondback moth peptidoglycan recognition protein, preparation method and application thereof |
| CN106749589A (en) * | 2016-12-22 | 2017-05-31 | 沈阳药科大学 | Peptidoglycan recognition protein SA and preparation method thereof, pattern recognition function and application |
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