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

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.

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