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ALV-J P-miRNA-env recombinant plasmid nano-composite based on chitosan encapsulation and preparing method and application thereof

A technology of cts-p-mir-env and cts-p-mirna-env, which is applied in the fields of biomedicine and virology, can solve the problems of accelerated virus evolution and mutation, unreasonable and unscientific purification measures, and reduce the direct and indirect economic loss

Active Publication Date: 2016-07-06
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, unreasonable and unscientific purification measures will also accelerate the evolution and mutation of the virus, making the disease more complicated. Based on the current breeding situation in our country, it is of great practical significance to seek new technologies and methods for the prevention and control of ALV-J

Method used

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  • ALV-J P-miRNA-env recombinant plasmid nano-composite based on chitosan encapsulation and preparing method and application thereof
  • ALV-J P-miRNA-env recombinant plasmid nano-composite based on chitosan encapsulation and preparing method and application thereof
  • ALV-J P-miRNA-env recombinant plasmid nano-composite based on chitosan encapsulation and preparing method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0035] Transformation of embodiment 1 plasmid and extraction of endotoxin

[0036] Use Tiangen Biotechnology Co., Ltd.'s endotoxin-free plasmid large-scale extraction kit: (the above-mentioned solution P1 and so on are all included in the kit)

[0037] 1.1 Conversion

[0038] (1) Take out DH5α, put it in ice water, add (10 μL) recombinant plasmid DNA to each tube in an ultra-clean bench, mix well, and put it in an ice bath for 30-40 minutes;

[0039] (2) Put the transformation tube in a 42° water bath, and strictly time it for 90 seconds;

[0040] (3) After that, quickly transfer the tube to ice and place it for 2-3 minutes;

[0041] (4) Take it to an ultra-clean bench, add 900 μL LB liquid medium, shake at 37° for 45-60 min,

[0042] (5) Centrifuge at 3000r for 2min, discard 500μL, resuspend the rest, spread on a spectinomycin-resistant plate, incubate at 37° for 40min, and incubate upside down for 16h;

[0043] (6) The next day, pick a single colony in the ultra-clean be...

Embodiment 2

[0055] The preparation method of embodiment 2 chitosan blank nanoparticles

[0056] Prepare chitosan blank nanoparticles by complex coacervation method, drop the solution in 1mL TPP (1mg / ml) solution into 3mL chitosan (2mg / ml) acetic acid solution with a 2ml needle, and the dropping rate is 40d-50d / min , vortexed for 30 seconds after dropping, and incubated at room temperature for 30 minutes. Collect the nanoparticle suspension in the RNAse-free and DNase-free EP tube to obtain chitosan blank nanoparticles.

Embodiment 3

[0057] The preparation of embodiment 3CTS-P-miRNA-env nanocomposite

[0058] The specific method is as follows:

[0059] Preparation of CTS-P-miR-env nanocomposites by complex coacervation method: Prepare CTS-P-miR-env nanoparticles by complex coacervation method, add 60 μg RNase-free P-miR-env (0.225ug / μL) to 1 mL TPP solution Add P-miR-env-TPP solution to 3mL chitosan acetic acid solution with a 2ml needle dropwise at a rate of 40d-50d / min, vortex and mix for 30s after dripping, and incubate at room temperature for 30min. Collect the nanoparticle suspension in RNAse-free and DNase-free EP tubes.

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Abstract

The invention relates to the fields of biological pharmacy and virology, particularly provides a preparative technique and application of a small interfering gene medicine, and more particularly relates to a nano-composite based on natural positive ion carrier chitosan and a J-avian leucosis virus subgroup ALV-JP-miRNA-env gene and a preparing method and application thereof.Results show that by the adoption of the prepared CTS-P-miRNA-env nano-composite, the ALV-J virus replication inhibition effect is more remarkable compared with original naked plasmids, occurrence and spreading of J-avian leucosis virus subgroup can be controlled more effectively, a new antiviral method is provided for avian leukosis control, and technological support is also provided for clinical application of anti-avian leukosis biologicals.

Description

(1) Technical field [0001] The invention relates to the fields of biomedicine and virology, in particular to a chitosan-based ALV-JP-miRNA-env recombinant plasmid nanocomposite and a preparation method and application thereof. (2) Background technology [0002] Subgroup J avian leukemia is a neoplastic disease caused by subgroup J avian leukemia virus (ALV-J). The main features are tumors in multiple tissues and organs. The occurrence of ALV-J in my country is on the rise. Clinical cases have developed from commercial broiler chickens to commercial layer chickens, local breeder chickens and other poultry. The main characteristics of its occurrence include early onset, High infection rate and morbidity rate, wide range of hosts, strong pathogenicity, and diverse tumors. In addition, ALV-J-based poultry tumor diseases can also be mixed with tumor diseases such as Marek's disease and reticuloendothelial hyperplasia, which greatly increases the difficulty of disease prevention an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/36A61P35/02
CPCA61K47/36A61K48/0083
Inventor 成子强贾雪莲张利崔熙尧
Owner SHANDONG AGRICULTURAL UNIVERSITY
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