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

A technology for binding proteins and molecules, which is applied in the field of encoding OBP nucleic acid sequences and expressing insect odor molecule binding proteins. It can solve the problems of small insect antennae, large protein loss, and difficulty in reaching protein levels, so as to achieve simple procedures and improve expression. volume effect

Inactive Publication Date: 2006-06-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can obtain relatively purified target proteins, the loss of protein is relatively large, and it is difficult to achieve the amount of protein required for in-depth research.
What's more, there are many kinds of insects with small antennae, and the amount of odorant molecule-binding proteins isolated and purified from them is not enough for further research work.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The preparation of embodiment 1 competent cell

[0033] ①Pick a single colony of Escherichia coli strain E.coli BL21 (purchased from Promega) from a fresh plate cultured at 37°C for 16-20hr, inoculate it in 5mL LB culture medium, and shake overnight at 37°C.

[0034] ②Transfer 1mL of bacteria liquid to 100mL of LB culture medium, shake and culture to OD 600 0.4 to 0.6.

[0035] ③Under sterile conditions, transfer the bacterial solution to a sterilized centrifuge tube, place it on ice for 10 minutes, and cool the culture to 0°C.

[0036] ④Centrifuge at 4000rpm for 10min, pour off the supernatant, add 10mL sterilized and pre-cooled 0.1mol / L CaCl 2 Gently suspend the precipitate and place on ice for 10 min.

[0037] ⑤Centrifuge at 4000rpm for 10min, discard the supernatant, add 2mL of pre-cooled 0.1mol / LCaCl 2 Gently suspend the precipitate, aliquot 100 μL, store on ice or store at -70°C.

Embodiment 2

[0038] Embodiment 2 vector construction

[0039] The expression vector pET-5b was double-digested with NdeI and EcoRI, and ligated with the target fragments whose ends were respectively NdeI and EcoRI sequences at room temperature overnight to obtain the constructed vector.

Embodiment 3

[0040] Example 3 The vector containing the fragment of interest is transformed into competent cells

[0041] ① Add 1 μL of the ligation product to 100 μL of E. coli competent cells, and bathe in ice for 30 minutes.

[0042] ②Heat-shock in a water bath at 42°C for 90 seconds, and immediately put it on ice for 5 minutes.

[0043] ③ Add 400 μL LB medium and incubate at 37°C for 60 minutes.

[0044] ④ Spread LB / agar plates (containing 100 μg / mL Amp, 20 μg / mL X-gal and IPTG), and culture them in a 37°C incubator for 16-18 hours.

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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

field of invention [0001] The invention belongs to the field of genetic engineering and relates to a method for expressing recombinant protein through prokaryotic cells. It specifically relates to a method for expressing the insect odorant molecule binding protein by transforming the constructed vector into Escherichia coli (E.coli). In addition, the present invention also relates to a novel nucleic acid sequence encoding OBP. Background technique [0002] Migratory locusts are important pests in my country and even in the world. They occur in a large area and cause serious damage. Their feeding, mating, oviposition, and migration behaviors are closely related to the developed sense of smell of migratory locusts. In the lymph fluid of the olfactory receptors of migratory locusts, there is a class of proteins that play an important role in signal transmission. These proteins are called odorant binding proteins (Odorant binding proteins, OBPs), which exis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/63C12P21/02
Inventor 张龙班丽萍于艳雪
Owner CHINA AGRI UNIV
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