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...

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Problems solved by technology

[0007] The main limitations of this research approach: First, because the primers are designed according to the existing antibacterial protein / peptide genes, as a result, the obtained genes have a greater similarity with the existing ones, and the difference is only due to the different insects tested. However, it is difficult to discover new antimicrobial proteins / peptides and their genes; the second is to measure and evaluate the activity of the obtained target gene expressions one by one, which is not efficient

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