CN1782073A - High efficiency expression of smell molecule combined protein of migratory locust in pronucleus system - Google Patents

High efficiency expression of smell molecule combined protein of migratory locust in pronucleus system Download PDF

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CN1782073A
CN1782073A CN 200410009964 CN200410009964A CN1782073A CN 1782073 A CN1782073 A CN 1782073A CN 200410009964 CN200410009964 CN 200410009964 CN 200410009964 A CN200410009964 A CN 200410009964A CN 1782073 A CN1782073 A CN 1782073A
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obp
protein
scent molecule
expression
conjugated protein
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CN100424173C (en
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张龙
班丽萍
于艳雪
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China Agricultural University
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China Agricultural University
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Abstract

The present invention discloses a method of expressing recombinant odorant binding protein (OBP) of insect in prokaryotic cell, and vector containing target nucleic acid sequence is transformed to competent prokaryotic cell. The present invention also discloses new OBP-coding nucleic acid sequence obtained from Locusta migratoria antenna.

Description

Conjugated protein the efficiently expressing in prokaryotic system of scent molecule in the migratory locusts
Invention field
The invention belongs to the genetically engineered field, is a kind of method by the procaryotic cell expression recombinant protein.Be specifically related to express the protein-bonded method of insect scent molecule in the intestinal bacteria (E.coli) by carrier construction is transformed into.In addition, the present invention also relates to the nucleotide sequence of a kind of new coding OBP.
Background technology
Behaviors such as migratory locusts are the important pests in China and even the worldwide, take place that area is big, harm is serious, and it gets food, mating, lay eggs, migrate are all closely related with the sense of smell of migratory locusts prosperity.The albumen that in migratory locusts smell receptor official's lymph liquid, exists a class in the signal transmission, to have vital role, this proteinoid is called conjugated protein (the Odorant binding proteins of scent molecule, OBPs), be present in (Pelosi, 1998 in vertebrates nasal mucosa liquid and the insect susceptor lymph liquid with high density; Steinbrecht, 1998).Iso-electric point is low, molecular weight less (15~20kDa), reversibly in conjunction with scent molecule.About the research of scent molecule in the insect conjugated protein (OBP), all concentrate in the lepidopteran moth class at first.This proteic separation purification method generally adopts methods such as gel permeation chromatography, ultrafiltration, dialysis, ion exchange chromatography.Although these methods can access the target protein of comparison purifying, proteic loss amount is all bigger, is difficult to reach the required protein content of further investigation.In addition there are many kinds of insect feelers less, therefrom the protein-bonded amount of scent molecule that goes out of separation and purification can not be satisfied with further research work especially.Therefore set up the proteic system gesture of a kind of efficient great expression necessary.At present about the prokaryotic expression of reorganization OBP, also mainly concentrate in the insects such as lepidopteran, Diptera in the insect, relevant report research is also arranged in the incomplete metamorphosis insect.Since the nineties in 20th century, develop rapidly and widespread use along with biological chemistry and Protocols in Molecular Biology, can obtain at present the expression of heterologous protein by Heterogeneous systems, and then the research of aspects such as the protein-bonded function of insect scent molecule, structure and biochemical characteristic become possibility, experience mechanism for exploring the insect smell, and then announcement vertebrates olfactory transduction machinery provides material.
Summary of the invention
All to the expression of albumen in prokaryotic system, especially in the insect olfaction conduction, play collaborative protein-bonded prokaryotic expression method of scent molecule and condition thereof of transporting the effect of extraneous chemical information thing and study both at home and abroad.But its expression amount is greatly between 10~20mg/L, and exists with the form of inclusion body, need utilize ultrasonic disruption or stronger denaturing agent soluble protein, still can not be quickly and easily provides high-abundance proteins for the research of insect olfaction mechanism.One of the object of the invention is to realize the conjugated protein efficient great expression (expression amount reaches 40mg/L) in prokaryotic system of scent molecule, for the research of OBP physiological function and insect olfaction mechanism is given security.Two of the object of the invention provides the resulting new sequence of cloning and sequencing (SEQ ID NO.1) from migratory locusts Locusta migratoria feeler:
gacgtgaaca tgaaacttac tgggcgcatt atggatgctg caaaagaagt ggaccacaca 60
tgccgctcat ctactggggt tccaagagac atgctccata gatatgctga aggtcaaact 120
gtagatgatg atgatttcaa gtgttacctg aagtgtatta tggttgagtt taattcactc 180
tcagatgatg gagtttttgt tttagaagaa gaattagaaa atgttcctcc agaaataaag 240
gaagaaggcc atagggttgt acacagctgc aaacacataa atcatgatga agcttgcgaa 300
acagcttacc agatccatca gtgctataaa cagagtgatc ctgagttgta cagcctggta 360
gttcgtgcat ttgatgcaac cattggtgac taataggaag ttgttactgc aggcagctta 420
cagtgaaagg actgtcaagc tgtctatntg aggtgcgaag tattacaaag gtgctttcct. 480
aaatgatttt agtgaagtgt gttgcaccat atgctgaaat taatatctgt acnccncaat 540
aatatgacca aataaacaat ggcatcncaa agtacaaaaa aaaaaaaaaa aaaaaaaaa 599
Wherein: n=a or c or g or t
The present invention is on the basis of original prokaryotic system express recombinant protein, start with from the preparation condition of competent cell, find out the condition that original expression system is suitable for that is different from, increased substantially the Recombinant Protein Expression amount, for the further investigation of protein purification and physiological function provides competent protein content.Adopt this method marking protein program comparatively simple, save time.The technical scheme of the efficient great expression reorganization OBP of the present invention in prokaryotic system may further comprise the steps:
1. the preparation of competent cell:
Preparation competent cell E.coli BL21: it is cultivated OD at 37 ℃ 600Be at 0.4~0.6 o'clock, centrifugal, abandon supernatant, will precipitate and use 0.1M CaCl 2Dissolving.Recentrifuge is abandoned supernatant, uses 0.1M CaCl 2Dissolution precipitation once more, packing, the 0.1ml/ pipe ,-70 ℃ of storages are standby.
2. vector construction:
Expression vector pET-5b is carried out double digestion, mix mutually with the purpose fragment, the connection of at room temperature spending the night obtains making up the back carrier.
3. be transformed into competent cell:
To make up the back carrier and be transformed into competent cell: will make up the back carrier and mix mutually with competent cell, 42 ℃ of heat shock 45~90s behind the ice bath 30min are hatched 1hr for 37 ℃, coated plate, 37 ℃ of overnight incubation.
4. choose single bacterium colony and cultivate in a large number, and it is carried out abduction delivering:
Choose single bacterium colony and cultivate in a large number, and it is carried out abduction delivering: choose single bacterium colony and in the LB nutrient solution, cultivate in a large number, at OD 600Reach at 0.4~0.6 o'clock and add IPTG, its final concentration is 0.2uM, cultivates 1~3hr for 25~28 ℃.
5. electrophoresis detection expression of results:
Get the abduction delivering product and the bacterium liquid of abduction delivering not, obtain precipitation after centrifugal, with SDS-PAGE sample buffer dissolution precipitation, 100 ℃ of boiling water bath 5min carry out the SDS-PAGE electrophoresis then to detect expression of results.
6., the cracking purifying of recombinant protein:
The enrichment post precipitation, in intestinal bacteria, add lysis buffer 37 ℃ with resuspended precipitation, get supernatant, will precipitate and further carry out ultrasonic disruption.With Superose-12 and Mono-Q separation and purification supernatant.
Lysis buffer: 10mM Tris, 8M Urea, 1mM PMSF
Description of drawings
Fig. 1. the reorganization scent molecule is conjugated protein expresses in prokaryotic system E.coli and the synoptic diagram of purge process
Fig. 2. the collection of illustrative plates of expression plasmid
Below in conjunction with accompanying drawing and specific embodiment the present invention is further detailed, it has no intention to limit by any way scope of the presently claimed invention.
The preparation of embodiment 1 competent cell
1. single bacterium colony of a coli strain E.coli of picking BL21 (available from Promega company) from the fresh flat board of 37 ℃ of cultivation 16~20hr is inoculated in the 5mL LB nutrient solution, and 37 ℃ of concussions are spent the night.
2. 1mL bacterium liquid is transferred in the 100mL LB nutrient solution, and concussion is cultured to OD 600Be 0.4~0.6.
3. under aseptic condition, bacterium liquid is transferred in the sterilization centrifuge tube, put 10min on ice, make culture be cooled to 0 ℃.
4. the centrifugal 10min of 4000rpm removes supernatant, adds the 0.1mol/L CaCl of 10mL sterilization precooling 2Suspend gently and precipitate, put 10min on ice.
5. the centrifugal 10min of 4000rpm removes supernatant, adds the 0.1mol/LCaCl of 2mL precooling 2Suspend gently and precipitate, 100 μ L packing are put standby on ice or-70 ℃ of preservations.
Embodiment 2 vector constructions
Expression vector pET-5b is carried out double digestion with NdeI, EcoRI, and at room temperature spending the night with the purpose fragment that end is respectively NdeI, EcoRI sequence is connected, and obtains making up the back carrier.
Embodiment 3 will contain the segmental carrier of purpose and be transformed into competent cell
1. add 1 μ L and connect product in 100 μ L competent escherichia coli cells, ice bath 30min.
2. 42 ℃ of water-bath heat shock 90s put 5min on ice immediately.
3. add 400 μ L LB substratum, cultivate 60min for 37 ℃.
4. be coated with LB/agar flat board (containing 100 μ g/mL Amp, 20 μ g/mL X-gal and IPTG), place 37 ℃ of incubators to cultivate 16~18hr.
Embodiment 4 abduction deliverings
Choose single bacterium colony and cultivate in a large number, and it is carried out abduction delivering: choose single bacterium colony and in the LB nutrient solution, cultivate in a large number, at OD 600Reach at 0.4~0.6 o'clock and add IPTG, its final concentration is 0.2uM, cultivates 3hr for 25~28 ℃.
Embodiment 5 electrophoresis detection expressions of results
Get the bacterium liquid and the bacterium liquid of abduction delivering not behind the 200ul abduction delivering respectively, obtain precipitation after centrifugal, with 30ul SDS-PAGE sample buffer dissolution precipitation, 100 ℃ of boiling water bath 5min carry out the SDS-PAGE electrophoresis.By Marker albumen control test expression of results.
The cracking purifying of embodiment 6 recombinant proteins
Enrichment post precipitation, every gram intestinal bacteria (weight in wet base) add the 3ml lysis buffer 37 ℃ of resuspended precipitations, get supernatant, will precipitate and further carry out ultrasonic disruption.With Superose-12 and Mono-Q separation and purification supernatant.Lysis buffer: 10mM Tris, 8MUrea, 1mMPMSF.
Comparing embodiment:
Compare by the traditional method express recombinant protein and with embodiment of the present invention, except that following condition, other condition is all identical with operation.
Comparison point New expression system Original expression system
The preparation of competent cell 42 ℃ of heat shock 45~90s 42 ℃ of heat shock 120s
The consumption of IPTG 0.2uM 0.4uM
The abduction delivering temperature 25~28℃ 30~37℃
The abduction delivering time 1~3hr 2.5~24hr
The OBP expression amount 40mg/L 10mg/L
From last table as seen, compare with the prokaryotic system of original express recombinant protein, the new expression system of the present invention has increased considerably the Recombinant Protein Expression amount, makes the expression amount of OBP be increased to four times of original expression system.
Sequence table
<110>Organization
ming,xing
<120〉conjugated protein the efficiently expressing in prokaryotic system of scent molecule in the migratory locusts
<160>1
<170>PatentIn version 3.1
<210>1
<211>599
<212>DNA
<213>Locusta migratoria
<223〉n=a or c or g or t
<400>1
gacgtgaaca tgaaacttac tgggcgcatt atggatgctg caaaagaagt ggaccacaca 60
tgccgctcat ctactggggt tccaagagac atgctccata gatatgctga aggtcaaact 120
gtagatgatg atgatttcaa gtgttacctg aagtgtatta tggttgagtt taattcactc 180
tcagatgatg gagtttttgt tttagaagaa gaattagaaa atgttcctcc agaaataaag 240
gaagaaggcc atagggttgt acacagctgc aaacacataa atcatgatga agcttgcgaa 300
acagcttacc agatccatca gtgctataaa cagagtgatc ctgagttgta cagcctggta 360
gttcgtgcat ttgatgcaac cattggtgac taataggaag ttgttactgc aggcagctta 420
cagtgaaagg actgtcaagc tgtctatntg aggtgcgaag tattacaaag gtgctttcct. 480
aaatgatttt agtgaagtgt gttgcaccat atgctgaaat taatatctgt acnccncaat 540
aatatgacca aataaacaat ggcatcncaa agtacaaaaa aaaaaaaaaa aaaaaaaaa 599

Claims (5)

  1. One kind by prokaryotic expression systems produce scent molecule conjugated protein (Odorantbinding proteins, method OBPs) comprises step:
    A). prokaryotic organism are prepared competent cell;
    B). the nucleotide sequence of the OBP that will encode links to each other with expression vector;
    C). use b) in the recombinant expression vector that the obtains competent cell in being transformed into a), 42 ℃ of heat shock 45~90s behind the ice bath 0.5hr are hatched 1hr for 37 ℃, coated plate, 37 ℃ of overnight incubation;
    D). in nutrient solution, use inductor abduction delivering and purification of recombinant proteins.
  2. 2. the method for preparing scent molecule conjugated protein (OBP) according to claim 1 is characterized in that: inductor is the IPTG of 0.2uM, and inducing temperature is 25~28 ℃, and induction time is 1~3hr.
  3. 3. the method for preparing scent molecule conjugated protein (OBP) according to claim 1 and 2 is characterized in that: prokaryotic organism are E.coli BL21.
  4. 4. the method for preparing scent molecule conjugated protein (OBP) according to claim 1 and 2, the nucleotide sequence of the OBP that it is characterized in that encoding is SEQ ID NO.1.
  5. 5. coding scent molecule protein-bonded nucleic acid molecule, it contains sequence SEQ IDNO.1.
CNB2004100099649A 2004-12-03 2004-12-03 High efficiency expression of smell molecule combined protein of migratory locust in pronucleus system Expired - Fee Related CN100424173C (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102399773A (en) * 2010-09-16 2012-04-04 中国农业科学院植物保护研究所 Method for separation and identification of odorant binding protein (OBP) genes in insect's antenna
CN103472102A (en) * 2013-09-29 2013-12-25 浙江大学 Preparing method and application of odorant binding protein sensor based on impedance analysis
CN104845976A (en) * 2015-02-17 2015-08-19 中国农业科学院植物保护研究所 Vegetable leafminer odorant-binding protein and application thereof
CN106841629A (en) * 2015-12-03 2017-06-13 中国科学院上海微系统与信息技术研究所 A kind of odor identification biology sensor based on silicon nanowires
CN107646808A (en) * 2017-10-13 2018-02-02 东北师范大学 Green plants source ambrostoma quadriimapressum field trapper design based on AquaOBP4
CN116769786A (en) * 2022-09-07 2023-09-19 贵州大学 Migratory locust smell binding protein LmOBP11 gene and encoding protein and application thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5128246A (en) * 1988-03-30 1992-07-07 The Johns Hopkins University Methods for isolating and expressing gene for odorant binding protein

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102399773A (en) * 2010-09-16 2012-04-04 中国农业科学院植物保护研究所 Method for separation and identification of odorant binding protein (OBP) genes in insect's antenna
CN103472102A (en) * 2013-09-29 2013-12-25 浙江大学 Preparing method and application of odorant binding protein sensor based on impedance analysis
CN103472102B (en) * 2013-09-29 2015-08-12 浙江大学 Based on preparation method and the application of the OBP sensor of impedance analysis
CN104845976A (en) * 2015-02-17 2015-08-19 中国农业科学院植物保护研究所 Vegetable leafminer odorant-binding protein and application thereof
CN104845976B (en) * 2015-02-17 2020-03-13 中国农业科学院植物保护研究所 Liriomyza sativae odor binding protein and application thereof
CN106841629A (en) * 2015-12-03 2017-06-13 中国科学院上海微系统与信息技术研究所 A kind of odor identification biology sensor based on silicon nanowires
CN106841629B (en) * 2015-12-03 2019-10-22 中国科学院上海微系统与信息技术研究所 A kind of odor identification biosensor based on silicon nanowires
CN107646808A (en) * 2017-10-13 2018-02-02 东北师范大学 Green plants source ambrostoma quadriimapressum field trapper design based on AquaOBP4
CN116769786A (en) * 2022-09-07 2023-09-19 贵州大学 Migratory locust smell binding protein LmOBP11 gene and encoding protein and application thereof

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