Construction of insect source antibiotic protein/peptide gene and screening method thereof

Abstract

The invention relates to a method for establishing and screening an insect source antibacterial protein / peptide gene library, comprising the following steps: firstly, a cDNA expression library of an insect source antibacterial protein / peptide gene is established; and secondly, the cDNA expression library is screened, which comprises the following steps: a. an SOLR bacterium liquid is prepared; b. a prophage library is transferred into the prepared SOLR bacterium liquid for titer measurement; c. a LB / Amp<+> filtration membrane flat plate is prepared; and d. the transferred SOLR bacterium liquid is taken for being applied to the plate, and cultivation is performed until colonies are grown up to between 0.9 and 1.1 millimeters, the colonies are transferred into a dyeing agar culture medium for dyeing; simultaneously the colonies are compared with colonies which do not undergo IPTG induction culture; blue colonies are selected and stored in 100 milliliters of LB / Amp<+> culture media which contain 20 percent of glycerol at a temperature of 70 DEG C below zero, and then the insect source antibacterial protein / peptide gene library is obtained. By adoption of the method to establish and screen the insect source antibacterial protein / peptide gene library, the pertinence and the efficiency of the screening can be greatly improved.

Description

technical field

[0001] The invention relates to fields including utilization of insect resources, biotechnology and development of medicine and veterinary medicine, and is suitable for screening of various insects. Background technique

[0002] As the most diverse organisms on the earth, insects have the characteristics of fast reproduction and wide adaptability. Except for the ocean, insects are distributed in all ecological environments. Since the 19th century, insects have been studied as immune defense systems, and studies have shown that antimicrobial proteins play a key role in insect humoral immune responses (Boman, 2003; Kimbrell, 2001; Hoffmann et al., 1992; Hultmark, 1993). The existing insects that still exist on the earth have survived the fierce competition for survival. During the long-term evolution process, they have enhanced their ability to resist the infection of various pathogenic bacteria, which screens insects with antibacterial effects for us. Antimic...

Examples

Embodiment 1

[0048] Embodiment 1, a method for constructing and screening an insect-derived antimicrobial protein / peptide gene library, the following steps are performed in sequence:

[0049] 1. Construction of cDNA expression library

[0050] On the basis of extracting insect mRNA, proceed according to the steps specified in the operation guide of the corresponding kit.

[0051] Isolate total RNA from whole or specific organs (tissues) of insects (species can be selected), and obtain poly(A) by purifying mRNA kit + RNA. Using cDNA synthesis kit, Synthesis kits and Gigapack III cloning kit, follow the instructions of the kit to perform each step to construct a cDNA expression library. (kits can be purchased from various biological companies)

[0052] 2. Screening of cDNA library

[0053] (1) Preparation of SOLR bacteria solution

[0054] Burn the inoculation ring on the alcohol lamp until red, dip a small amount of Escherichia coli SOLR glycerol bacteria in the Kan + Streak on th...

technical field

[0001] The invention relates to fields including utilization of insect resources, biotechnology and development of medicine and veterinary medicine, and is suitable for screening of various insects. Background technique

[0002] As the most diverse organisms on the earth, insects have the characteristics of fast reproduction and wide adaptability. Except for the ocean, insects are distributed in all ecological environments. Since the 19th century, insects have been studied as immune defense systems, and studies have shown that antimicrobial proteins play a key role in insect humoral immune responses (Boman, 2003; Kimbrell, 2001; Hoffmann et al., 1992; Hultmark, 1993). The existing insects that still exist on the earth have survived the fierce competition for survival. During the long-term evolution process, they have enhanced their ability to resist the infection of various pathogenic bacteria, which screens insects with antibacterial effects for us. Antimic...

Examples

Embodiment 1

[0048] Embodiment 1, a method for constructing and screening an insect-derived antimicrobial protein / peptide gene library, the following steps are performed in sequence:

[0049] 1. Construction of cDNA expression library

[0050] On the basis of extracting insect mRNA, proceed according to the steps specified in the operation guide of the corresponding kit.

[0051] Isolate total RNA from whole or specific organs (tissues) of insects (species can be selected), and obtain poly(A) by purifying mRNA kit + RNA. Using cDNA synthesis kit, Synthesis kits and Gigapack III cloning kit, follow the instructions of the kit to perform each step to construct a cDNA expression library. (kits can be purchased from various biological companies)

[0052] 2. Screening of cDNA library

[0053] (1) Preparation of SOLR bacteria solution

[0054] Burn the inoculation ring on the alcohol lamp until red, dip a small amount of Escherichia coli SOLR glycerol bacteria in the Kan + Streak on th...

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