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Preparation method for bumblebee peptidoglycan recognition protein

A technology for identifying proteins and peptidoglycan, applied in the field of molecular biology, can solve problems such as hindering the research and application of new antibacterial and immune drugs of bumblebees, and achieve the effects of small protein damage, mild operation process, and simple and fast method.

Active Publication Date: 2017-08-29
BEE RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above studies have not formed an effective method for large-scale preparation of insect PGRP-SA protein and purification of high-purity protein
[0005] Bumblebees are effective pollinators of many wild plants and crops, and have important economic and ecological values. At the same time, the health of bumblebees is threatened by various pathogenic microorganisms such as bacteria. The important immune molecule peptidoglycan recognition protein PGRP in their innate immune system - The in vitro preparation of SA has not been reported, which seriously hinders the research and application of new antibacterial and immune drugs in bumble bees

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  • Preparation method for bumblebee peptidoglycan recognition protein
  • Preparation method for bumblebee peptidoglycan recognition protein
  • Preparation method for bumblebee peptidoglycan recognition protein

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preparation example Construction

[0023] The present invention relates to the establishment of a peptidoglycan recognition protein PGRP-SA in vitro expression, folding renaturation and multi-step purification method; in particular, it relates to the important recognition molecule PGRP-SA in the innate antibacterial immune response of wild plants and agricultural crops. In vitro preparation method of SA protein.

[0024] A preparation method for bumblebee peptidoglycan recognition protein, comprising the steps of:

[0025] 1), connecting the gene fragment of bumble bee PGRP-SA to an expression vector and transforming it into Escherichia coli for expression, collecting the expressed inclusion body protein;

[0026] 2), after washing the inclusion body protein, adding a protein denaturant to dissolve it, and then performing refolding treatment with a refolding solution;

[0027] 3) After the protein is concentrated, it is sequentially eluted and purified through a molecular sieve column and an anion exchange col...

Embodiment 1

[0086] The sequence of the protein expressed in this embodiment is obtained by the following methods:

[0087] The total RNA of Bombus flamingo was extracted by conventional methods, and then reverse-transcribed to obtain cDNA, and the cDNA was used as a template to amplify the bumblebee PGRP-SA gene. The gene fragment size is about 500bp, and the sequence is shown in SEQ ID NO:1.

[0088] The upstream primer used in the amplification is shown in SEQ ID NO:2, and the downstream primer is shown in SEQ ID NO:3.

[0089] Amplification was carried out with LATaq enzyme from Takara Company, and the amplification conditions were as follows:

[0090] ① Denaturation at 95°C for 5 minutes;

[0091] ② Denaturation at 95°C for 30s;

[0092] ③Annealing at 58°C for 1min;

[0093] ④ Extend at 72°C for 30s;

[0094] Repeat ②~④30 times;

[0095] ⑤ Extend at 72°C for 10 minutes;

[0096] ⑥ Keep warm at 4°C.

[0097] The above gene fragments were connected to PET21a+ vector (endonuclease...

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Abstract

The invention relates to the field of molecular biology, and concretely relates to a preparation method for a bumblebee peptidoglycan recognition protein (PGRP). The method comprises the following steps: (1) a gene fragment of bumblebee PGRP-SA is connected with an expression vector and transformed into colibacillus for expression, and an inclusion body protein obtained by the expression is collected; (2) the inclusion body protein is washed, and then a protein denaturant is added so as to dissolve the inclusion body protein, and subsequently a renaturation solution is used to conduct renaturation treatment; and (3) protein concentration is performed, and then a molecular sieve column and an anion-exchange column are successively employed for elution purification. According to the invention, expression conditions of the bumblebee PGRP-SA protein are optimized, the formulas of important reagents in every process are preferably selected, and renaturation by using chromatography is combined. The method is simple and rapid, the operation process is gentle, and harm to the protein is small, so that the high-purity active protein is obtained.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for preparing a bumble bee peptidoglycan recognition protein. Background technique [0002] Insects, as the most diverse animals on earth, currently have about 1 million known species. Most of them live in harsh environments full of pathogenic microorganisms. Under the pressure of long-term evolutionary selection, insects have formed a unique immune defense system to fight against Invasion of these pathogenic microorganisms. [0003] Insects lack acquired immunity and rely solely on innate immunity as their defense mechanism. For foreign invading pathogenic microorganisms, insects resist through innate immunity. The first step is to recognize pathogen-associated molecular patterns through pattern recognition receptors, thereby activating downstream responses. As pattern recognition receptors in insects are: peptidoglycan recognition proteins (Peptidoglycan Recognition ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/70C07K1/18
CPCC07K14/705
Inventor 刘彦杰安建东黄家兴孙成丁桂玲
Owner BEE RES INST CHINESE ACAD OF AGRI SCI