Anopheles sinensis transgene system

A transgenic, Anopheles mosquito technology, applied in the field of biomedicine, can solve the problems of the environment, mosquito resistance, pollution, etc., and achieve the effect of no environmental pollution

Inactive Publication Date: 2006-12-20
陈晓光 +1
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Current control strategies for mosquitoes and mosquito-borne diseases are mainly the use of antibiotics, insecticides and repellents (for example, some controlled trials include indoor spraying of insecticides and effective application of pyrethrin-soaked bed nets) and symptomatic treatment of patients. However, with the widespread use of "traditional antibiotics", many new problems continue to arise: allergic reactions to antibiotics and long-term use lead to the emergence of drug-resistant strains; the emergence of mosquito resistance and environmental pollution; In addition, in In countries where mosquito-borne diseases are endemic, the poorest areas are the hardest hit, the above-mentioned control measures are rarely applied effectively and the rate of insecticide resistance has largely outpaced that of The speed with which new insecticides can be produced and brought to market, therefore, the development of new and effective means of controlling mosquito-borne diseases is of paramount importance

Method used

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  • Anopheles sinensis transgene system
  • Anopheles sinensis transgene system
  • Anopheles sinensis transgene system

Examples

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example 1

[0048] Example 1 Construction of Anopheles sinensis genome library

[0049] 1. Extraction of Anopheles sinensis genomic DNA:

[0050]Take 50 adult mosquitoes of Anopheles sinensis, remove the legs and wings, add the adult mosquitoes into a mortar containing a little liquid nitrogen, and quickly grind the adult mosquitoes into powder. After the powder is completely thawed; add an equal volume of extraction Buffer I (50mM Tris-HCl, pH8.0; 50mM EDTA, pH8.0; 100mM NaCl), divide the above homogenate into sterilized 1.5ml EP tubes, add Equal volume of extraction Buffer II (50mM Tris-HCl, pH8.0; 50mM EDTA, pH8.0; 100mM NaCl; 10% SDS; 50-200μg / mL proteinase K), vortexed and mixed. Water bath at 37°C for 3-4 hours until completely dissolved. Centrifuge at 3000 rpm for 3 minutes at room temperature, and transfer the supernatant to a new EP tube. Add an equal volume of phenol to the above-mentioned EP tubes, shake gently for 1 min, centrifuge at 8000 rpm for 5 min at room temperature,...

example 2

[0052] Cloning of Example Two Anopheles sinensis Vg Promoter

[0053] According to the published Drosophila and Aedes aegypti Vg promoter sequences, design a pair of primers:

[0054] P Vg-6838: 5'-ACC AAC CAA ATG GGA AAT GT-3'

[0055] P Vg-7144: 5'-GAC GAT GAA GAG GAA GAT GA-3'

[0056] Using the Anopheles sinensis genome library as a template, PCR amplification was performed with the designed primers. PCR cycle conditions: 95°C, 5min; 95°C denaturation for 1min, 55°C annealing for 1min, 72°C extension for 90s, 30 cycles; 72°C for 8min . After the PCR product was identified by 1% agarose gel electrophoresis, a fragment with a size of about 400bp was obtained, and the gel was cut and purified with a kit to recover the target fragment (the gel recovery kit was from Shanghai Shenergy Gaming Company); refer to the molecular cloning experiment guide ( The second edition, Sambrook J, et al, translated by Jin Dongyan, 55), with CaCl 2 The intermediate host bacteria DH5α compete...

example 3

[0077] Cloning of Example Three Anopheles sinensis Defensin Coding Sequence

[0078] 1. According to the published Aedes aegypti defensin gene sequence, design a pair of primers:

[0079] Def-1: 5′-GAG AAT TCA TGC AGC CCC TCA CTG TC-3′

[0080] Def-2: 5′-AAG GAT CCT TAA TTC CGG CAG ACG CA-3′

[0081] Use Trizol to extract total RNA of Anopheles sinensis, specific steps:

[0082] Take the total RNA (Note: The EP tubes and tips used in the following steps must be treated with DEPC, and the whole process is carried out in an ultra-clean bench with gloves on, and the reagents at the bottom of the reaction have different degrees of toxicity)

[0083] 1. Take 50 adult Anopheles sinensis mosquitoes, remove the legs and wings, add the adult mosquitoes into a mortar containing a little liquid nitrogen, and quickly grind the adult mosquitoes into powder. Add 1ml of Trizol, transfer to a 1.5ml EP tube, shake to dissolve the precipitate, and let stand at room temperature for 5min.

[...

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Abstract

The invention discloses a promoter gene of cloned Chinese Anopheles vitellogenin (Vg) and Chinese Anopheles defending agent coding gene through molecular biological checking method, which is characterized by the following: constructing carrier plasmid; infecting Chinese Anopheles egg through microscopic injection; hatching the egg; observing GFP expression condition and RT-PCR checking result under fluorescent microscope; certifying excellent expression of Vg promoter to drive Defensin gene in gene-transmission Chinese Anopheles body; constructing gene-transmission system of Chinese Anopheles.

Description

technical field [0001] The invention relates to a transgenic system of Anopheles sinensis in the field of biomedicine, and a method for cloning a mosquito vitellogenin (Vg) promoter gene and an Anopheles sinensis defensin (Defensin) gene coding sequence involved in the transgenic system. The construction method of the system and the qualitative analysis method of the transgenic system. Background technique [0002] Mosquitoes belong to the class Insecta, Diptera, and Mosquitoidae. They are an important class of medical insects. So far, there have been 3 subfamilies, 38 genera, and more than 3,350 species and subspecies of mosquitoes in the world. Among them, those related to infectious diseases Mosquito species are Anopheles, Culex, and Aedes. Infectious diseases that use mosquitoes as vectors, such as malaria, filariasis, Japanese encephalitis, and dengue fever, are important diseases that seriously threaten people's lives and health in today's society. Among them, malaria...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/11C12N15/10C12N15/85C12N15/65C12N15/89C12Q1/68C12N15/113
Inventor 陈晓光张亚晶郑学礼
Owner 陈晓光
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