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Housefly phylaxin gene, and its cloning method and recombinant application

A cloning method and defensin technology, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve the problems of low molecular weight and low antigenicity

Inactive Publication Date: 2003-03-26
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these antimicrobial peptides show significant structural diversity, they also share many common features: low molecular weight, and therefore low antigenicity; positive charge at physiological pH, and most have amphipathic α-helices or β-helices. Lamellar or both structures

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Adults of housefly (Musca domestica) were reared in the laboratory, and Escherichia coli (Escherichia coli) and Staphylococcus aureus (Staphylococcus auceus) were used as test bacteria.

[0057] 1. Total RNA extraction: Total RNA was extracted by one-step method using existing technology.

[0058] 2. cDNA first-strand synthesis: 5-10 micrograms of total RNA, plus 20 microliters of reaction solution (50 millimoles potassium chloride, 3 millimoles magnesium chloride, 10 millimoles Tris-hydrochloric acid buffer (Tris- HCl) pH 8.3, 1 millimolar dithiothreitol (DTT), 5 micromolar oligodeoxythymonucleotide (Oligod(T) 17 ), 500 micromole deoxyribonucleotide mixture (dNTP), 25 units of RNase inhibitor, 8 units of AMV reverse transcriptase) were reacted at 42°C for 90 minutes. The reaction was terminated at 70°C for 10 minutes.

[0059] 3. PCR reaction: chain polymerase reaction (PCR) reagents and conditions: first mix the following reagents together

[0060] 5 microliters (μ...

Embodiment 2

[0077] As described in Example 1, the difference is that houseflies were collected in the field, and total RNA was extracted using RNA kit. 8 µg of total RNA was then used for cDNA first-strand synthesis.

Embodiment 3

[0079] As described in Example 1, the difference is that the mRNA was isolated and extracted using the mRNA kit. 10 μg of mRNA was then used for cDNA first-strand synthesis. During plasmid purification, 1 ml of the overnight culture solution was collected for plasmid purification. Example 4: Recombinant application of housefly defensin cDNA

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PUM

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Abstract

A housefly alexin cDNA and its clone method is disclosed in the invention. The housefly alexin has sequences of SEQ ID No.1 and SEQ ID NO.2. The clone method of the invention is: distilling general RNA or mRNA from housefly, reverse transcripting into Cdna; designing introduction on the basis of alexin conservative sequence, extending to a DNA segment via the chain-type polymerase reaction, purificating extension outcome, cloning in the purificated outcome to the pGEM-TEasy carrier, converting HD5 alpha cell, flat cultivating after a night. The housefly alexin may use of producing medicine product by gene recombining technology, expressing in the escherichia coli and microzyme and getting the housefly alexin having the antibacterium function.

Description

(1) Technical field [0001] The invention relates to the cloning and expression technology in the technical field of biological gene engineering, in particular to the housefly defensin gene and the cloning and expression technology. (2) Background technology [0002] The application of traditional antibiotics ushered in a new era of anti-infection treatment, but only a few years after antibiotics were used, drug-resistant bacteria appeared and spread widely. The application of each new antibacterial drug is always accompanied by the emergence of drug-resistant bacteria. In order to overcome this situation, people continue to synthesize new drugs. However, more stubborn drug-resistant bacteria and even multi-drug-resistant bacteria (MDR) may also appear. , such as Staphylococcus (MRSA), Mycobacterium tuberculosis, Pneumococcus, etc. are resistant to multiple antibiotics, resulting in many clinically used drugs being ineffective. Especially in recent years, some new pathogenic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/00A01K63/00C07H21/04C07K14/435C12N15/12C12N15/63C12P19/34
Inventor 王金星赵小凡
Owner SHANDONG UNIV
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