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Method for producing recombinant human BChE (butyrylcholinesterase) from transgenic animals by using gene knock-in and nuclear transfer technologies

A butyrylcholinesterase and genome sequence technology, applied in the field of biopharmaceuticals, can solve the problems of inhomogeneity of recombinant human butyrylcholinesterase products, etc.

Active Publication Date: 2014-08-06
SHANGHAI JENOMED BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In summary, various expression or separation and purification systems in the prior art either cannot produce or separate and purify a sufficient amount of active recombinant human butyrylcholinesterase, or the produced active recombinant human butyrylcholine Esterase products are inhomogeneous, so there is an urgent need in this area to develop a new method for preparing butyrylcholinesterase that is stable, efficient, simple, and suitable for large-scale production and application

Method used

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  • Method for producing recombinant human BChE (butyrylcholinesterase) from transgenic animals by using gene knock-in and nuclear transfer technologies
  • Method for producing recombinant human BChE (butyrylcholinesterase) from transgenic animals by using gene knock-in and nuclear transfer technologies
  • Method for producing recombinant human BChE (butyrylcholinesterase) from transgenic animals by using gene knock-in and nuclear transfer technologies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0181] Example 1. Goat β-casein gene knock-in donor construct No. 1 construction

[0182] 1.1 Construction of the β-casein gene knock-in donor construct integrated in the goat β-casein gene intron 1:

[0183] (i) Use a cis primer containing SacI, NotI, SalI sites and part of the 5'end homology arm sequence of the goat β-casein gene as shown in SEQ ID NO.: 7, and located in the goat β-casein promoter The trans primer containing the BamHI site at the midstream end of intron 1 is shown in SEQ ID NO.: 8. Using genomic DNA isolated from goat blood as a template, PCR amplification was performed to obtain a 2kb amplified product (5' homology arm sequence), the amplified product was then cloned into the pCDNA1-Neo plasmid (Invitrogen) by SacI-BamHI digestion to form pbCN5-Neo.

[0184] (ii) Use a cis primer containing BamHI site, goat β-casein signal sequence and part of human butyrylcholinesterase cDNA sequence as shown in SEQ ID NO.: 9, and containing XhoI, AscI, AgeI, PmeI The trans pri...

Embodiment 2

[0194] Example 2. Goat β-casein gene knock-in donor construct No. 2 construction

[0195] 2.1 Construction of a β-casein gene knock-in donor construct integrated into exon 8 of goat β-casein gene:

[0196] (i) Use a cis primer containing SacI, NotI, SalI sites and part of goat β-casein gene sequence as shown in SEQ ID NO.: 15 and containing AgeI site, part of 2A-polypeptide coding sequence and part of goat β -The trans primer of the casein gene sequence is shown in SEQ ID NO.: 16, using the genomic DNA isolated from goat blood as a template to perform PCR amplification to obtain a 2kb amplified product, which is then passed through SacI -AgeI digestion was cloned into pbCN-BChEKI1-Neo described in Example 1 to form pbCN52-Neo.

[0197] (ii) Use a cis primer containing AgeI site, part of 2A-polypeptide coding sequence, goat β-casein signal sequence and part of human butyrylcholinesterase cDNA sequence as shown in SEQ ID NO.: 17, and containing The trans primers of XhoI, AscI, BamHI ...

Embodiment 3

[0206] Example 3 TALEN vector construction:

[0207] Construct TALEN vector according to the method of gene synthesis ( image 3 ).

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Abstract

The invention discloses a method for producing recombinant human BChE (butyrylcholinesterase) from transgenic non-human mammals by using gene knock-in and nuclear transfer technologies, and particularly provides a donor construct used for specific expression of a mammary gland. The construct integrates DNA (deoxyribose nucleic acid) coding sequences of human BChE to a specific expression protein gene locus of the mammary gland in targeted manner and performs successful transfection on non-human mammal embryo fibroblasts, juvenile and adult somatic cells or embryonic stem cells, clone is performed by using a somatic cell nuclear transfer technology so as to obtain a new individual, and the carried human BChE gene enables the mammary gland of a non-human mammal to express stably and secrete holoenzyme or a monomer of the human BChE. Recombinant protein produced with the method can be used for preventing and treating nerve poison and organic phosphorus compound poisoning as well as apnea caused by cocaine poisoning and succinylcholine, and for detecting and removing residues of organic phosphorus compounds on vegetables, other crops, various article layers and the soil.

Description

Technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to a method for stably producing recombinant human butyrylcholinesterase on a transgenic animal mammary gland bioreactor platform using gene knock-in and somatic cell nuclear transfer technology. Background technique [0002] The so-called genetically modified animal medicine is the introduction of drug protein genes into animals, such as cows or dairy goats, and then the drugs are produced from mammary glands or other organs, and genetically engineered drugs are made through purification and other processes. This method is characterized by high yield, low cost, stable biological activity of the drug, high yield, high quality, low energy consumption, and no pollution; but the technology is more complicated and the initial investment is relatively high. In recent years, due to the continuous improvement of genetic engineering technology and the invention of animal cl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N5/10C12N15/877C12N9/18A01K67/027A61K38/46A61P39/02A61P25/00
Inventor 黄跃进
Owner SHANGHAI JENOMED BIOTECH CO LTD
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