Method for producing transgene protein medicine of mammary gland expression using gland virus as carrier mammary

A protein drug and adenovirus technology, applied in biochemical equipment and methods, botany equipment and methods, genetic engineering, etc., can solve the problems of low expression level of target genes, complexity, high production costs, etc., to shorten the development time Cycle time, the effect of reducing R&D costs

Inactive Publication Date: 2013-08-07
李青旺 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still inevitable defects in this technology: (1) It requires complex technical procedures to operate, resulting in unstable transgenic results
(2) This method has a very low success rate of genetically modified large animals, resulting in high production costs
(3) Due to the position effect of the transferred exogenous target gene, the expression level of the target gene is very low
(4) The heterologous expression of exogenous genes in animals often causes harm to animal health and affects the production of genetically engineered protein drugs
However, so far, there have been no relevant application reports of adenoviral vectors in the production of recombinant drug proteins.

Method used

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  • Method for producing transgene protein medicine of mammary gland expression using gland virus as carrier mammary
  • Method for producing transgene protein medicine of mammary gland expression using gland virus as carrier mammary
  • Method for producing transgene protein medicine of mammary gland expression using gland virus as carrier mammary

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Using adenovirus as vector to introduce goat mammary gland to express and produce human lactoferrin.

[0052] (1) Isolation and cloning of target drug protein gene sequence

[0053] 1. Extraction of total RNA from human breast tissue

[0054] (1) Cut a small piece of frozen breast tissue, quickly weigh it, and then transfer it into freshly prepared lysis buffer, add 1ml of lysis buffer for every 50mg of tissue.

[0055] (2) Homogenize mammary gland tissue to break cells, pre-filter and centrifuge once, and collect filtrate.

[0056] (3) Add an equal volume of chloroform, vortex and mix well, and absorb the supernatant after centrifugation.

[0057] (4) Add an equal volume of 70% ethanol, mix gently, and centrifuge to obtain total RNA.

[0058] (5) 0.01% DEPC-treated RNase-free water was used to dissolve the precipitate, and the content was determined by conventional methods.

[0059] 2. Synthesis of total cDNA by reverse transcription

[0060] (1) Take 1-2ug mammar...

Embodiment 2

[0144] Using adenovirus as vector to introduce rabbit mammary gland to express and produce human growth hormone. The rabbits used in the experiment came from Changli County, Hebei Province, and the amount of carrier introduced into the rabbit mammary gland was calculated according to the breast volume of the animal itself. Wherein the content of the target protein human growth hormone obtained in step five is higher than the expression level of the recombinant protein in goat milk, which is 2-8g / L. Other operating methods are the same as steps (1), (2), (3) and (4) of Example 1.

Embodiment 3

[0146] Using adenovirus as vector to introduce porcine mammary gland to express and produce human growth hormone. The pigs used in the experiment come from Changli, Hebei. The amount of pig mammary glands introduced into the carrier requires a small amount of multi-nipple injection. Other operating methods are the same as steps (1), (2), (3) and (4) of Example 1. Wherein the expression level of the target protein hGH obtained in step five is 0.3-0.4g / L.

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Abstract

The invention discloses a method to produce transgene albumen medicine that takes galactophore expression using adenovirus as carrier. The feature is that: using copying defect type adenovirus framework plamid vector to carry different target albumen gene sequence, and gaining reconstructed albumen carrier from packaging cell in E1A sequence containing Ad5 adenovirus gene group, and gaining instantaneous high expression external target gene medicine albumen. The operation process includes the following steps: separating and cloning target medicine albumen gene sequence; constructing confluence eukaryon expression carrier to testing target albumen to gain external expression right medicine albumen gene; rebuilding the target albumen gene with the adenovirus gene group frame molecule to gain the adenovirus carrier of rebuilt target medicine albumen gene; inducing into animal galactophore; excreting the needed target medicine albumen from latex. The rebuilt albumen could be directly usedto develop kinds of health products and medicine.

Description

technical field [0001] The invention belongs to the technical fields of cell biology, molecular biology and genetic engineering, in particular to a method for expressing and producing transgenic protein medicine using adenovirus as a carrier mammary gland. Background technique [0002] The transgenic animal technology can express and produce the required target protein through the animal body according to people's wishes, so that the transgenic animal becomes a living "drug factory". Among them, the animal mammary gland bioreactor is one of the most popular researches in the production of transgenic protein drugs. Its principle is to use the promoter element specifically expressed in mammalian mammary glands to construct transgenic animals, guide the expression of foreign genes in animal mammary glands, and generate The required recombinant medicinal or health care proteins are obtained from the milk secreted by the mammary glands of transgenic animals. [0003] The convent...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/861C12P21/02A61K38/17
Inventor 李青旺韩增胜
Owner 李青旺
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