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Preservation method of cotton bollworm sterol carrier protein-2

A carrier protein and preservation method technology, applied in the field of preservation of cotton bollworm sterol carrier protein 2, can solve problems such as time-consuming and laborious, HaSCP-2 protein content and activity do not meet the requirements, and HaSCP-2 protein expression instability, etc. Long-term use and storage, save the effect of cumbersome procedures

Active Publication Date: 2017-09-19
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, general experimenters will induce and cultivate engineered bacteria according to the amount of required HaSCP-2 protein. The disadvantage of this approach is that each time HaSCP-2 protein is needed, the engineered bacteria must be induced and cultivated again. The cycle is generally 2 to 3 days, which is time-consuming and labor-intensive; on the other hand, the expression of HaSCP-2 protein is unstable. Due to the degradation of the strain and the error of each operation, the HaSCP-2 protein induced and expressed each time has a large The difference often occurs in the laboratory. The concentration and activity of the HaSCP-2 protein obtained by the first expression meet the requirements, while the second time the content and activity of the HaSCP-2 protein obtained after the expression of the genetically engineered bacteria does not meet the requirements

Method used

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  • Preservation method of cotton bollworm sterol carrier protein-2
  • Preservation method of cotton bollworm sterol carrier protein-2
  • Preservation method of cotton bollworm sterol carrier protein-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Preparation of Genetic Engineering Bacteria PET28a-HaSCP2-BL21

[0016] A. Total RNA was extracted from the midgut of the fifth instar larvae of cotton bollworm:

[0017] The fifth instar cotton bollworm larvae were dissected to take out the midgut, and placed in ice-cold TRIzol reagent at a ratio of 25-50 mg / 500 μl to extract total RNA from the larval midgut.

[0018] B, utilize RT-PCR technology to amplify the sterol carrier protein 2 gene of cotton bollworm:

[0019] Firstly, the total RNA of the larval midgut in step A was reverse-transcribed using the promega reverse transcription kit to obtain cDNA.

[0020] Then use cDNA as a template to carry out PCR amplification, and the PCR amplification system is shown in Table 1:

[0021] The reaction program of PCR amplification is shown in Table 2:

[0022] After PCR amplification, the target HaSCP-2 gene is obtained, and the sequence of the HaSCP-2 gene is shown in SEQID No.1 in the sequence listing.

[0023] Table 1...

Embodiment 2

[0033] High expression of target protein

[0034] (1) Activation: The positive clones of PET28a-HaSCP2-BL21 genetically engineered bacteria were first inoculated in 5mL LB medium and cultured at 37°C with constant temperature and shaking. When the medium OD 600 When it is 0.5, add final concentration of 1mM IPTG and incubate at 37°C for 2 hours with constant temperature shaking, then shake at room temperature overnight, and use SDS-PAGE gel electrophoresis to detect the induced expression of the target protein.

[0035] (2) Large-scale induced expression: when the expression of HaSCP-2 protein accounts for more than 40% of the total protein of the cells, the activated bacteria are inoculated in 12L LB medium, and cultured with constant temperature shaking at 37°C. When the medium OD 600 When the concentration is 0.5, add final concentration of 1mM IPTG and incubate at 37°C for 2 hours, then incubate overnight at room temperature, and use SDS-PAGE gel electrophoresis to detect ...

Embodiment 3

[0037]Preservation and use of target protein

[0038] Use a stainless steel medicine spoon to dig out 15g of bacteria each time and put them into liquid nitrogen. Under the rapid cooling of liquid nitrogen, the bacteria are frozen into solid spherical bacteria balls, and the obtained bacteria are frozen into several bacteria balls by liquid nitrogen. , take out the balls and store them in a refrigerator at -80°C; according to the amount of protein needed, take out an appropriate amount of balls from the refrigerator at -80°C each time, dissolve the balls in PBS buffer, and then dissolve them in order The target protein was obtained after cell disruption and nickel column purification.

[0039] Research and measure the decrease of the content and activity of the HaSCP-2 protein in the bacterium balls stored in the refrigerator at -80°C: the specific method is as follows:

[0040] On March 1, 2015, a batch of HaSCP-2 bacterium was obtained according to the storage method of the...

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Abstract

The invention relates to the field of biotechnology and in particular to a preservation method of cotton bollworm sterol carrier protein-2. The preservation method of the cotton bollworm sterol carrier protein-2 designed by the invention comprises the following steps: (1) constructing genetic engineering bacteria; (2) abundantly expressing target protein; (3) digging 10-20g thallus with a medicine spoon each time, placing the thallus in liquid nitrogen, freezing the thallus into solid spherical bacterium balls under the action of rapidly cooling the liquid nitrogen, and after the obtained thallus is frozen into a plurality of bacterium balls by the liquid nitrogen, placing all the bacterium balls in a refrigerator at the temperature of 80 DEG C below zero for preservation; and (4) according to the required protein quantity, taking a proper amount of the bacterium balls out of refrigerator with the temperature of 80 DEG C below zero per time, dissolving the bacterium balls into a buffer solution, enabling the bacterium balls to be sequentially subjected to cell disruption and protein purification after complete dissolution, and obtaining the target protein. The preservation method can ensure the content and the activity of the protein used in each test study to keep relatively consistent and also saves tedious procedure required for each induction culture.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preserving sterol carrier protein 2 of cotton bollworm. Background technique [0002] Cotton bollworm is a common agricultural pest, harming more than 100 agricultural crops, and has developed strong resistance to various chemical insecticides and even Bt. Therefore, it is urgent to develop environmentally friendly and efficient new insecticides. When researchers develop biological insecticides for cotton bollworm, they choose to use the bollworm sterol carrier protein 2 (HaSCP-2) as the target site, for example, "Cloning, expression and functional research of the bollworm sterol carrier protein , Du Xin, Central China Normal University, 2012 "The research on HaSCP-2 mentioned in "HaSCP-2 protein will be used many times in the whole research process (generally 5-10 years), because HaSCP-2 protein is removed from the bacterial body After extraction, the storage time in t...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/12C12N1/21C12N1/04C12R1/19
CPCC07K14/43563C12N1/04C12N15/70
Inventor 杜馨蔡俊
Owner HUBEI UNIV OF TECH
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