CN101139607A - Cultivated silkworm microsporidian polar tube protein PTP1 gene - Google Patents
Cultivated silkworm microsporidian polar tube protein PTP1 gene Download PDFInfo
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Abstract
The invention provides an important protein gene PTP1 relative with growth and infection of microsporidia of silkworms. The gene sequence CDS for coding the pole-tube protein PTP1 for microsporidia of silkworms is got based on the 9-magification gene data of microsporidia of silkworm and by collinear analysis. The pole-tube protein PTP1 got by the invention is of high importance for studying the breeding and infection mechanism of microsporidia, hence can play role in preventing and treating microsporidia in production practice. According RNAi interference, after the pole-tube protein PTP1 being interfered, the microsporidia loses the capability for infecting the host. The pole-tube protein PTP1 and its potential controlling gene of microsporidia will become a new factor for treating microsporidia infection. In addition, by means of transgenosis, the expression of the water-channel protein gene can be enriched, so that the water-channel protein gene can be used to control such destructive insects as locusts, etc.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of breeding that comes from nosema bombycis and infect relevant cultivated silkworm microsporidian polar tube protein PTP 1 gene.
Background technology
Microsporidium is widespread distribution obligatory parasitism protozoon in natural cell, kind surplus nearly 150 genus 1200, host very widely being arranged, comprise vertebrates and invertebrates, is the fatal cause of disease of economic insects, fish, Lagomorpha, product hair animal, rodents and primates.And nosema bombycis (Nosema bombycis), classification position belongs to Protozoa, monofilament Sporozoa, Microsporidea.Parasitize the economic insects silkworm and cause that serious prion is sick by microsporidium, and, classified as silkworm by the sericulture countries and regions always and already produce legal Quarantine Objects because little spore protozoon can be propagated by the embryo in downward generation.List promptly reaches more than the up to ten million units because of the direct economic loss of being with seed culture of viruses and pebrine to bring with regard to China every year.Capturing the chronic disease of this silk industry of pebrine, is the emphasis and the difficult point research field of silkworm industry.
Pebrine is the disease that is caused by the microsporidium parasitic host.Microsporidium is with its unique mode of infection, extensively parasitic and infection has insect, fish, rodents, Lagomorpha, fleece animal, primates and household pet such as the dog and the bird of economic worth, cause serious disease even death, seriously restricting world silkworm and already producing and the animal child care.Even can also opportunistic ground aids infection (HIV) patient, organ transplantation patient, take immune suppressant drug patient and innate immunity incomcopetence person, cause malignant diseases such as respiratory system, urinary system and skin ulceration, aggravate one's illness, quicken patient's death.Over more than 100 year, although used the number of chemical medicine to treat, as: imidazoles, fumidil, song miaow pyridine alkali, benzene Lai Te, thiophanate methyl or the like, result of treatment is unsatisfactory, finds to have the specific medicament of treatment microsporidiosis so far not yet.
Utmost point pipe is nosema bombycis breeding and the vitals that infect, plays key effect nosema bombycis in the infecting of silkworm.Utmost point length of tube is generally at 50～150 μ m, and diameter is generally at 0.1～0.2 mu m range.Utmost point pipe has snappiness, and its pipe diameter can be increased to 0.4 μ m in transporting sporoplasmic process, and sporoplasm transports its length of back that finishes can shorten 5～10%.In the nosema bombycis phagocytic process, utmost point pipe injects sporoplasm its tenuigenin simultaneously suitably stimulating rapid ejection the in back also " to thrust " in the bombyx mori cell, finishes the invasion to silkworm.And its moiety mainly is an albumen, and the polar tube protein (ptp) PTP1 main integral part that is it.Polar tube protein (ptp) PTP1 is a kind of concanavalin A, is the major protein of utmost point pipe, and the proteic content of PTP1 accounts for 70% of whole polar tube protein (ptp) in E.hellom.Dye analysis PTP1 by SDA-PAGE and silver and find that the PTP1 molecular size of different types of microsporidium does not wait.To proteic studies show that of different microsporidium PTP1, PTP1 albumen has hydrophobicity, high proline content and immunizing antigen etc. such as feature (Delbac et al., 1998).And PTP2 and PTP3 also are the important component that constitutes utmost point pipe.Have research to think, in utmost point pipe ejection process, PTP1 and PTP2 were class crystalloid protein monomers before spore activates, and form polymer in the germination process on utmost point pipe top, and PTP3 influenced the ejection of utmost point pipe by the polymeric conformational state of regulation and control PTP1-PTP2.In addition, the glycosylation of polar tube protein (ptp) PTP1 plays an important role to the structure of utmost point pipe, thereby the remote effect microsporidium is to the invasion of host cell.But the research about the microsporidium polar tube protein (ptp) only is confined to a small amount of microsporidium that part microsporidiums such as Encephalitozoon, Glugea, Ameson and Nosema belong at present.
Summary of the invention
The present invention intends based on the genome analysis data, and its purpose is to provide a kind of nosema bombycis polar tube protein (ptp) PTP1 gene.By analyzing function, composition and the characteristic of PTP1, understand fully the effect of polar tube protein (ptp) PTP1 in nosema bombycis utmost point pipe ejection process, verify nosema bombycis and infect the morbific mechanism of host silkworm, production practice and diseases prevention and treatment are extremely important.
The present patent application people has obtained the encoding sequence of this protein gene by the clone to cultivated silkworm microsporidian polar tube protein PTP 1 gene, and concrete grammar is as follows:
(1) Collinearity Diagnosis Analysis:, find that the arrangement of homologous gene on genome has very strong homogeny, i.e. collinearity by to the arranging situation on nosema bombycis and the two genome of rabbit encephalitis microsporidium; By the 6th karyomit(e) of rabbit encephalitis microsporidium is had the Superscaffold000055 sequence Collinearity Diagnosis Analysis of the nosema bombycis of homologous genes with it, obtained coding nosema bombycis polar tube protein (ptp) PTP1 gene order CDS;
(2) design of primers: utilize primer-design software Primer 5.0 to carry out design of primers, add the BamHI restriction enzyme site at forward primer, add Sal I restriction enzyme site at reverse primer, primer sequence is respectively:
PTP1-primer-F：5′-CGGGATCCATGGCATGTTCACCCGGAAAT-3′
PTP1-primer-R：5′-CGGTCGACGTTTAGCACACATGGATTATTGGC-3′
(3) polar tube protein PTP1 pcr amplification and clone: utilize the little spore genome that extracts directly as template, adopt genome pcr amplification purpose fragment, amplified fragments is cloned on pMD18-T (TaKaRa) carrier, carry out sequencing (Shanghai is handsome) then, thus the polar tube protein PTP1 complete encoding sequence that obtains;
(4) sequence homology analysis: the homology comparison is finished with BLAST on the NCBI website, protein does not have significant homology in BLAST analysis revealed nosema bombycis PTP1 and the NCBI protein library, and PTP1 is the important albumen of nosema bombycis utmost point pipe, so the PTP1 gene is a kind of newfound protein gene.
The PTP1 full-length cDNA gene that aforesaid method obtains and the aminoacid sequence of deduction thereof are as follows:
atgagaattagatccttcaaacttctaagtctagtagcttacatt
M R I R S F K L L S L V A Y I
aaactgaactcagctaacatggcatgtgcacccggaaatggaact
K L N S A N M A C A P G N G T
cctgttgtagcaattactcaacctggtggcgtagaaaattgtgca
P V V A I T Q P G G V E N C A
acaacatcacccgctcctcttgtttatgaaagttcaaatggacaa
T T S P A P L V Y E S S N G Q
aatcctctcaataactttactccagattgtttagtaaactcacca
N P L N N F T P D C L V N S P
ggtgtaccagttcaaagctacccagtaataggtgtcccagtctca
G V P V Q S Y P V I G V P V S
ggaacacccggaactccaggagctcccggaacagcaggaaataat
G T P G T P G A P G T A G N N
tatggaccaacagaatgtgtacccacctcaggaggtggatcttat
Y G P T E C V P T S G G G S Y
cctccaggatactttaatcctccaatggctactactattattaat
P P G Y F N P P M A T T I I N
cctccaacatatgctcctccaggcacacccggatcaccaagcgca
P P T Y A P P G T P G S P S A
aacaatccaccagctggtttccatgcaccaccaccagccggacca
N N P P A G F H A P P P A G P
ccagcaaagaatgaagaacaatgtactgtaaccactaagcttgat
P A K N E E Q C T V T T K L D
tgtgaacctgtatcaccaggagtccctgctgttactgctccacca
C E P V S P G V P A V T A P P
gccggaccaccaattgaatctgtaggtgttcctcctcaaaaagtc
A G P P I E S V G V P P Q K V
gctttagtgactcctgtcgcaagtgtagcaagtgtagtagctgta
A L V T P V A S V A S V V A V
ccaccatcccatgctactggatcaggtgttccttgtattccagta
P P S H A T G S G V P C I P V
tcttcaaccggtggccatccaagtaatctcgttggcggtggtttc
S S T G G H P S N L V G G G F
ccaagtaatgcacaagtcactcaagccccatgtgtcccatcacaa
P S N A Q V T Q A P C V P S Q
gctcatcatccagtagcaagtgttccagtaacgagtgtcccagta
A H H P V A S V P V T S V P V
aatgctgttccaatgaccagtgcccctgttacaccacttggccct
N A V P M T S A P V T P L G P
tcttattacggatcttcaagccttccaccatcaggctctcatcct
S Y Y G S S S L P P S G S H P
actgctccttgtggtgctcctcagaacgcacttagttctccttgt
T A P C G A P Q N A L S S P C
gctactaataatgcttcggctggaaacacttatacttcatcgaat
A T N N A S A G N T Y T S S N
gtcggagctgcttgttcacttccaaatgctgaaaattgtcttatg
V G A A C S L P N A E N C L M
aaagcccttgagaatgtaggcagaccctccccacaagaagccgca
K A L E N V G R P S P Q E A A
tcatgtgttactgcacaaaaacaacctatggtcgagattggtatg
S C V T A Q K Q P M V E I G M
atgttgcctgttaccgacgcttattctaatgcagccaataatcca
M L P V T D A Y S N A A N N P
tgtgtgctaaac
C V L N
Advantage of the present invention is:
(1) because microsporidium polar tube protein (ptp) PTP1 is a major protein of forming microsporidium utmost point pipe, and utmost point pipe is the vitals that microsporidium is bred and infected, insect, fish, rodents, Lagomorpha, fleece animal, primates and the household pet such as dog and the bird that have economic worth by its extensively parasitic also infection, cause serious disease even death, seriously restricting world silkworm and already producing and the animal child care.Even can also opportunistic ground aids infection (HIV) patient, organ transplantation patient, take immune suppressant drug patient and innate immunity incomcopetence person, cause malignant diseases such as respiratory system, urinary system and skin ulceration, aggravate one's illness, quicken patient's death.Therefore the polar tube protein (ptp) PTP1 that is obtained has its vital role to breeding of research microsporidium and infection mechanism.By analyzing function, composition and the characteristic of PTP1, understand fully the effect of polar tube protein (ptp) PTP1 in nosema bombycis utmost point pipe ejection process, verify nosema bombycis and infect the morbific mechanism of host silkworm, production practice and diseases prevention and treatment are extremely important.
(2) analysis of experiments such as RNAi interference shows that after polar tube protein PTP1 was interfered with, microsporidium lost the ability of infection host.So will becoming the treatment microsporidium, the polar tube protein PTP1 of microsporidium and its potential regulatory gene infect new factor.Can also make it its abundant expression by genetically modified means, thereby murder part insects such as locust.
Embodiment:
Embodiment:
Using Glimmer3.0 software relatively predicts the nosema bombycis gene in conjunction with homology, analyze the arranging situation of gene on nosema bombycis and the two genome of rabbit encephalitis microsporidium, find that the arrangement of homologous gene on genome has very strong collinearity feature, nosema bombycis polar tube protein (ptp) PTP1 obtains encoding; Use Primer5.0 design primer software design and go out a pair of Auele Specific Primer, be respectively PTP1-F5, CGGGATCCATGGCATGTTCACCCGGAAAT-3 ' and PTP1-R5 ' CGGTCGACGTTTAGCACACATGGATTATTGGC-3, the amplification segment is cloned on the pMD18-T carrier, sequencing is finished by Shanghai English fine horse, and the homology comparison is finished with BLAST on the NCBI website, and sequential analysis shows that it is a new protein gene.
Claims (2)
1. cultivated silkworm microsporidian polar tube protein PTP 1 gene is characterized in that its sequence is as follows:
atgagaatta?gatccttcaa?acttctaagt?ctagtagctt?acattaaact?gaactcagct 60
aacatggcat?gtgcacccgg?aaatggaact?cctgttgtag?caattactca?acctggtggc 120
gtagaaaatt?gtgcaacaac?atcacccgct?cctcttgttt?atgaaagttc?aaatggacaa 180
aatcctctca?ataactttac?tccagattgt?ttagtaaact?caccaggtgt?accagttcaa 240
agctacccag?taataggtgt?cccagtctca?ggaacacccg?gaactccagg?agctcccgga 300
acagcaggaa?ataattatgg?accaacagaa?tgtgtaccca?cctcaggagg?tggatcttat 360
cctccaggat?actttaatcc?tccaatggct?actactatta?ttaatcctcc?aacatatgct 420
cctccaggca?cacccggatc?accaagcgca?aacaatccac?cagctggttt?ccatgcacca 480
ccaccagccg?gaccaccagc?aaagaatgaa?gaacaatgta?ctgtaaccac?taagcttgat 540
tgtgaacctg?tatcaccagg?agtccctgct?gttactgctc?caccagccgg?accaccaatt 600
gaatctgtag?gtgttcctcc?tcaaaaagtc?gctttagtga?ctcctgtcgc?aagtgtagca 660
agtgtagtag?ctgtaccacc?atcccatgct?actggatcag?gtgttccttg?tattccagta 720
tcttcaaccg?gtggccatcc?aagtaatctc?gttggcggtg?gtttcccaag?taatgcacaa 780
gtcactcaag?ccccatgtgt?cccatcacaa?gctcatcatc?cagtagcaag?tgttccagta 840
acgagtgtcc?cagtaaatgc?tgttccaatg?accagtgccc?ctgttacacc?acttggccct 900
tcttattacg?gatcttcaag?ccttccacca?tcaggctctc?atcctactgc?tccttgtggt 960
gctcctcaga?acgcacttag?ttctccttgt?gctactaata?atgcttcggc?tggaaacact 1020
tatacttcat?cgaatgtcgg?agctgcttgt?tcacttccaa?atgctgaaaa?ttgtcttatg 1080
aaagcccttg?agaatgtagg?cagaccctcc?ccacaagaag?ccgcatcatg?tgttactgca 1140
caaaaacaac?ctatggtcga?gattggtatg?atgttgcctg?ttaccgacgc?ttattctaat 1200
gcagccaata?atccatgtgt?gctaaac 1227
2. cultivated silkworm microsporidian polar tube protein PTP 1 gene encoded protein matter is characterized in that its sequence is as follows:
Met?Arg?Ile?Arg?Ser?Phe?Lys?Leu?Leu?Ser?Leu?Val?Ala?Tyr?Ile 15
Lys?Leu?Asn?Ser?Ala?Asn?Met?Ala?Cys?Ala?Pro?Gly?Asn?Gly?Thr 30
Pro?Val?Val?Ala?Ile?Thr?Gln?Pro?Gly?Gly?Val?Glu?Asn?Cys?Ala 45
Thr?Thr?Ser?Pro?Ala?Pro?Leu?Val?Tyr?Glu?Ser?Ser?Asn?Gly?Gln 60
Asn?Pro?Leu?Asn?Asn?Phe?Thr?Pro?Asp?Cys?Leu?Val?Asn?Ser?Pro 75
Gly?Val?Pro?Val?Gln?Ser?Tyr?Pro?Val?Ile?Gly?Val?Pro?Val?Ser 90
Gly?Thr?Pro?Gly?Thr?Pro?Gly?Ala?Pro?Gly?Thr?Ala?Gly?Asn?Asn 105
Tyr?Gly?Pro?Thr?Glu?Cys?Val?Pro?Thr?Ser?Gly?Gly?Gly?Ser?Tyr 120
Pro?Pro?Gly?Tyr?Phe?Asn?Pro?Pro?Met?Ala?Thr?Thr?Ile?Ile?Asn 135
Pro?Pro?Thr?Tyr?Ala?Pro?Pro?Gly?Thr?Pro?Gly?Ser?Pro?Ser?Ala 150
Asn?Asn?Pro?Pro?Ala?Gly?Phe?His?Ala?Pro?Pro?Pro?Ala?Gly?Pro 165
Pro?Ala?Lys?Asn?Glu?Glu?Gln?Cys?Thr?Val?Thr?Thr?Lys?Leu?Asp 180
Cys?Glu?Pro?Val?Ser?Pro?Gly?Val?Pro?Ala?Val?Thr?Ala?Pro?Pro 195
Ala?Gly?Pro?Pro?Ile?Glu?Ser?Val?Gly?Val?Pro?Pro?Gln?Lys?Val 210
Ala?Leu?Val?Thr?Pro?Val?Ala?Ser?Val?Ala?Ser?Val?Val?Ala?Val 225
Pro?Pro?Ser?His?Ala?Thr?Gly?Ser?Gly?Val?Pro?Cys?Ile?Pro?Val 240
Ser?Ser?Thr?Gly?Gly?His?Pro?Ser?Asn?Leu?Val?Gly?Gly?Gly?Phe 255
Pro?Ser?Asn?Ala?Gln?Val?Thr?Gln?Ala?Pro?Cys?Val?Pro?Ser?Gln 270
Ala?His?His?Pro?Val?Ala?Ser?Val?Pro?Val?Thr?Ser?Val?Pro?Val 285
Asn?Ala?Val?Pro?Met?Thr?Ser?Ala?Pro?Val?Thr?Pro?Leu?Gly?Pro 300
Ser?Tyr?Tyr?Gly?Ser?Ser?Ser?Leu?Pro?Pro?Ser?Gly?Ser?His?Pro 315
Thr?Ala?Pro?Cys?Gly?Ala?Pro?Gln?Asn?Ala?Leu?Ser?Ser?Pro?Cys 330
Ala?Thr?Asn?Asn?Ala?Ser?Ala?Gly?Asn?Thr?Tyr?Thr?Ser?Ser?Asn 345
Val?Gly?Ala?Ala?Cys?Ser?Leu?Pro?Asn?Ala?Glu?Asn?Cys?Leu?Met 360
Lys?Ala?Leu?Glu?Asn?Val?Gly?Arg?Pro?Ser?Pro?Gln?Glu?Ala?Ala 375
Ser?Cys?Val?Thr?Ala?Gln?Lys?Gln?Pro?Met?Val?Glu?Ile?Gly?Met 390
Met?Leu?Pro?Val?Thr?Asp?Ala?Tyr?Ser?Asn?Ala?Ala?Asn?Asn?Pro 405
Cys?Val?Leu?Asn
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| WO2010128465A1 (en) * | 2009-05-05 | 2010-11-11 | Beeologics, Llc | Prevention and treatment of nosema disease in bees |
| US8097712B2 (en) | 2007-11-07 | 2012-01-17 | Beelogics Inc. | Compositions for conferring tolerance to viral disease in social insects, and the use thereof |
| US8962584B2 (en) | 2009-10-14 | 2015-02-24 | Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. | Compositions for controlling Varroa mites in bees |
| CN108913720A (en) * | 2018-08-20 | 2018-11-30 | 西南大学 | A kind of carrier and method of eukaryon soluble-expression Bm cell line pole pipe glycoprotein |
| US10378012B2 (en) | 2014-07-29 | 2019-08-13 | Monsanto Technology Llc | Compositions and methods for controlling insect pests |
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- 2007-08-07 CN CNA2007100925430A patent/CN101139607A/en active Pending
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| US8097712B2 (en) | 2007-11-07 | 2012-01-17 | Beelogics Inc. | Compositions for conferring tolerance to viral disease in social insects, and the use thereof |
| US8507457B2 (en) | 2007-11-07 | 2013-08-13 | Beeologics Inc. | Compositions for conferring tolerance to viral disease in social insects, and the use thereof |
| US10888579B2 (en) | 2007-11-07 | 2021-01-12 | Beeologics Inc. | Compositions for conferring tolerance to viral disease in social insects, and the use thereof |
| US9932579B2 (en) | 2009-05-05 | 2018-04-03 | Beeologics Inc | Prevention and treatment of Nosema disease in bees |
| US8822426B2 (en) | 2009-05-05 | 2014-09-02 | Beeologics Inc. | Prevention and treatment of nosema disease in bees |
| WO2010128465A1 (en) * | 2009-05-05 | 2010-11-11 | Beeologics, Llc | Prevention and treatment of nosema disease in bees |
| AU2010244122B2 (en) * | 2009-05-05 | 2015-05-14 | Beeologics Inc. | Prevention and treatment of Nosema disease in bees |
| US8962584B2 (en) | 2009-10-14 | 2015-02-24 | Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. | Compositions for controlling Varroa mites in bees |
| US10801028B2 (en) | 2009-10-14 | 2020-10-13 | Beeologics Inc. | Compositions for controlling Varroa mites in bees |
| US9662348B2 (en) | 2009-10-14 | 2017-05-30 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Compositions for controlling Varroa mites in bees |
| US10597676B2 (en) | 2013-07-19 | 2020-03-24 | Monsanto Technology Llc | Compositions and methods for controlling Leptinotarsa |
| US11377667B2 (en) | 2013-07-19 | 2022-07-05 | Monsanto Technology Llc | Compositions and methods for controlling Leptinotarsa |
| US11091770B2 (en) | 2014-04-01 | 2021-08-17 | Monsanto Technology Llc | Compositions and methods for controlling insect pests |
| US10378012B2 (en) | 2014-07-29 | 2019-08-13 | Monsanto Technology Llc | Compositions and methods for controlling insect pests |
| US11124792B2 (en) | 2014-07-29 | 2021-09-21 | Monsanto Technology Llc | Compositions and methods for controlling insect pests |
| US10968449B2 (en) | 2015-01-22 | 2021-04-06 | Monsanto Technology Llc | Compositions and methods for controlling Leptinotarsa |
| CN108913720A (en) * | 2018-08-20 | 2018-11-30 | 西南大学 | A kind of carrier and method of eukaryon soluble-expression Bm cell line pole pipe glycoprotein |
| CN116925198A (en) * | 2023-08-15 | 2023-10-24 | 西南大学 | Recombinant protein of microsporidian polar tube protein EcPTP1, and preparation method and application thereof |
| CN116925198B (en) * | 2023-08-15 | 2024-05-14 | 西南大学 | Recombinant protein of microsporidian polar tube protein EcPTP and preparation method and application thereof |
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