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Method for large-scale production of recombinant human butyrylcholinesterase using transgenic animal mammary gland biological platform

A butyrylcholinesterase and transgenic technology, applied in the field of producing recombinant human butyrylcholinesterase, can solve the problems of low transgenic efficiency, unfavorable quality control and subsequent processing, no in vivo enzymatic activity and the like

Active Publication Date: 2017-08-08
SHANGHAI JENOMED BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many genetically engineered butyrylcholinesterases have little or no in vivo enzymatic activity, and these expression systems are insufficient to produce butyrylcholinesterase in sufficient quantities for practical use
[0019] In addition, although there are studies on the production of butyrylcholinesterase using transgenic goat milk, because the transgenic goats are obtained by microinjection of gametes, the cycle is long and the transgenic efficiency is very low, only about 5%.
In addition, because the recombinant human butyrylcholinesterase produced by transgenic animals is a mixture of tetramer, dimer and monomer (PNAS104: 13603-13608, 2007), it is not conducive to quality control and subsequent processing, so it is difficult to industrialize very big
[0020] 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 new efficient, simple and convenient methods for preparing butyrylcholinesterase suitable for large-scale production and application

Method used

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  • Method for large-scale production of recombinant human butyrylcholinesterase using transgenic animal mammary gland biological platform
  • Method for large-scale production of recombinant human butyrylcholinesterase using transgenic animal mammary gland biological platform
  • Method for large-scale production of recombinant human butyrylcholinesterase using transgenic animal mammary gland biological platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0198] Example 1. Construction and preparation of transgenic expression cassettes

[0199] Experimental steps:

[0200] (i) Plasmid pUC19 (Promega) was cut with SacI and BamHI endonucleases, and ligated to an insulator fragment from the upstream region of the chicken β-globin gene (Proc Natl Acad Sci USA94:575-80, 1997). The insulator fragment is made using cis primers containing SacI, SacII, NotI, SalI sites and part of the chicken β-globin gene upstream region DNA sequence such as SEQ ID NO.: 7 (5'GAC ACT GAG CTC CAC CGC GGA CTG CGG CCG CTC GTC GAC GGG ACAGCC CCC CCC CAA AGC CCC CAG3'), and the reverse primer containing BamHI, XhoI site and part of the chicken β-globin gene upstream region DNA sequence such as SEQ ID NO.: 8 (5'AGC GTC GGA TCC ACT TGC GTC CTC GAG GCC CCATCC TCA CTG ACT CCG TCC TGG AGT TG3'), amplified from chicken genomic DNA by PCR. The PCR product was divided into two parts, cut with SacI-XhoI or SalI-BamHI respectively, and then joined together through S...

Embodiment 2

[0205] Example 2. Production of transgenic sheep

[0206] Embryonic cells were isolated from sheep embryos on day 27-30, mainly containing embryonic fibroblasts, and cultured in Dulbecco's modified Eagle medium at 38°C, 5% carbon dioxide, supplemented with 20% fetal bovine serum (FBS) and 20 μg / ml gentamicin. When the cells reached 70% confluence, they were treated with 0.05% trypsin-EDTA, collected, counted, and frozen in aliquots of 10% DMSO and 90% FBS. A small number of cells were placed on slides for cytogenetic analysis. Chromosomally normal frozen cells were thawed for transfection of transgene expression construct DNA to generate stable cell lines. Stable cell lines were generated by lipid-mediated gene transfer.

[0207] The above-mentioned transgene expression cassette (i.e. pIn / BCN / BCHE / Neo plasmid) DNA fragment of embodiment 1, comprises insulator, goat β-casein promoter, human butyrylcholinesterase cDNA sequence, β-casein gene 3' end and Neo fragments, cleaved...

Embodiment 3

[0211] Example 3. Detection and breeding of transgenic breeding sheep and their offspring

[0212] About 4 days after the birth of the cloned new sheep, blood was taken for PCR analysis to confirm the transgene. There are three sets of PCR reaction primers.

[0213] The first set of primers such as SEQ ID NO.:15 and 16 (5'CTT CCG TGG CCA GAA TGG AT3' / 5'CAT CAGAAG TTA AAC AGC ACA GTT AGT3') from the 3' end of human butyrylcholinesterase cDNA to phase A 510bp DNA fragment was amplified from the β-casein gene exon 7 fragment adjacent to the BCN-BCHE fragment.

[0214] The second set of primers such as SEQ ID NO.: 17 and 18 (5'AGG AGC ACA GTG CTC ATC CAG ATC3' / 5'GAC GCC CCA TCC TCA CTG ACT3') amplifies a 910bp DNA fragment from the insulator fragment.

[0215] The third set of primers such as SEQ ID NO.: 19 and 20 (5'GAG GAA CAA CAG CAA ACA GAG3' / 5'ACCCTA CTG TCT TTC ATC AGC3') amplifies the goat endogenous β-casein gene 360bp fragment to Make sure that the extracted DNA is fre...

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Abstract

The invention provides a method for producing recombinant human butyrylcholine esterase on large scale by utilizing a biological platform of the mammary gland of a transgenic animal. The method comprises the following steps: utilizing the biological platform of the mammary gland of the transgenic animal to efficiently produce holonomic and monomeric recombinant human butyrylcholine esterase on large scale for the first time, namely cut-off type enzyme without containing the last 40 amino acid residues of the holonomic butyrylcholine esterase, and a protein fused with monomeric butyrylcholine esterase and albumin. The produced recombinant protein can be used for preventing and treating apnea caused by poisoning of nerve gas, poisoning of organophosphorus pesticide, poisoning of cocaine and succinylcholine, and is used for detecting and removing residues of organophosphorus pesticide on vegetables, melons, fruits, other crops, surfaces of various articles and soil.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a method for large-scale production of recombinant human butyrylcholinesterase using a transgenic animal mammary gland biological platform. Background technique [0002] Over the past forty years, genetic engineering technology has advanced by leaps and bounds. A large number of genetic medicines have come out one after another, with an annual output value of billions of dollars. [0003] The production methods of genetically engineered drugs can be divided into two categories. [0004] The first type of method is to introduce specific drug genes into engineered bacteria or engineered cells, and obtain them through fermentation and cultivation of bacteria or cells, followed by separation and purification. This method is characterized by simple technology, but high production cost and low biological activity of the drug. [0005] The second type of approa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/63C12N5/10C12N9/18A61K38/46A61P39/02A61P11/00A01K67/027
Inventor 黄跃进
Owner SHANGHAI JENOMED BIOTECH CO LTD
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