Method for utilizing human butyrylcholinesterase minigene to express recombinant human butyrylcholinesterase in mammary gland

A human butyrylcholine and gene technology, applied in the field of genetic engineering, can solve the problems of difficult gene manipulation, low efficiency and the like

Inactive Publication Date: 2015-05-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the human butyrylcholinesterase gene is about 80kb in size, and the efficiency of recombining the BCHE gene (about 59kb in length from the start codon to the stop codon) into the capture vector PBR322 is very low, and it is difficult to operate the gene

Method used

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  • Method for utilizing human butyrylcholinesterase minigene to express recombinant human butyrylcholinesterase in mammary gland
  • Method for utilizing human butyrylcholinesterase minigene to express recombinant human butyrylcholinesterase in mammary gland
  • Method for utilizing human butyrylcholinesterase minigene to express recombinant human butyrylcholinesterase in mammary gland

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Experimental program
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Effect test

Embodiment 1

[0041]Example 1 Construction of mammary gland-specific expression of human butyrylcholinesterase vector pBAC-hLF-hBCHE-neo

[0042] 1. Construction strategy of human butyrylcholinesterase mammary gland-specific expression vector pBAC-hLF-hBCHE-neo

[0043] 1.1 Construction of capture vector pBR322-hLF-hBCHE

[0044] Using hLF BAC and hBCHE BAC as templates to amplify the homologous arms used in the homologous recombination operation, the PCR products of the 4 homologous arms were connected into the pMD19-T vector and sequenced to verify correctness. The four homology arms were connected into a fragment in the order of HA-hLF-F, HA-hBCHE-F, HA-hBCHE-R and HA-hLF-R by fusion PCR method, and connected to pBR322 by NdeI and HindIII double enzyme digestion Among the backbone vectors, the capture vector pBR322-hLF-hBCHE was obtained.

[0045] 1.2. Modification of human butyrylcholinesterase BAC carrier

[0046] The genomic DNA of human butyrylcholinesterase is about 80kb in lengt...

Embodiment 2

[0087] Example 2 Establishment and detection of human butyrylcholinesterase transgenic mouse model

[0088] 1. Production of Transgenic Mice by Microinjection

[0089] For the preparation process of transgenic mice, please refer to "Experimental Guidelines for Mouse Embryo Operation" (A. Nagy et al., 2004, Science Press). Transgenic efficiency. The production process of transgenic mice is as follows: Image 6 shown.

[0090] Microinjection is used to produce transgenic mice. For ordinary small fragment vectors, the linear integration efficiency is higher than that of circular ones. Therefore, the constructed expression vectors must be enzymatically cut into linear shapes for microinjection. However, due to its large structure, the large-fragment BAC vector is prone to breakage during the recovery process after linearization. If it is not recovered, the same high transgenic efficiency can be obtained, which will greatly increase the success rate and save working time. In ou...

Embodiment 3

[0099] Determination of recombinant human butyrylcholinesterase in the milk sample of embodiment 3 transgenic mice

[0100] 1. Western blotting detection of transgenic mouse milk samples

[0101] The normal milk of F0 generation 4, 5 and F1 generation 7, 9, 11 mice was collected, and the milk of negative mice was used as a negative control. The milk was collected one week after the birth of the mice, and the mother mice were separated from the pups for more than 3 hours before collection, and a certain dose of oxytocin was injected into the abdominal cavity. Whole milk and milk samples treated with defatted and decaseinized milk were electrophoresed on 10% SDS-PAGE gel. After the electrophoresis was completed, transfer to the membrane for 80 minutes using a Bio-Rad wet-transfer instrument at 350 mA. After the transfer was completed, block overnight with 5% skimmed milk powder, then incubate with rabbit anti-human primary antibody (diluted 1:500) for 1 h, wash the membrane wi...

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Abstract

The present invention provides a method for utilizing human butyrylcholinesterase minigene to express recombinant human butyrylcholinesterase in mammary gland. According to the method, the human butyrylcholinesterase minigene is adopted to completely replace the target gene on the artificial chromosome of the mammal mammary gland specific expression protein by using the recombinant technology, such that the exogenous gene acquires the complete regulation sequence highly expressing the target gene in the mammary gland; under the premise of no influence on transcription and translation of the butyrylcholinesterase gene, the size of the human butyrylcholinesterase gene is reduced through the modification on the artificial chromosome so as to obtain the human butyrylcholinesterase minigene; and the obtained recombinant artificial chromosome is transformed into the mammal so as to express the recombinant human butyrylcholinesterase in the animal mammary gland. According to the present invention, the disadvantages that the position effect of the exogenous gene expression and easy silencing of the cDNA expression exogenous gene are overcome, the in vitro recombination operation is easy to perform, the vector construction is convenient, and the new strategy is provided for the expression of the large structure gene protein.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for expressing recombinant human butyrylcholinesterase in mammary glands using a human butyrylcholinesterase mini-gene. Background technique [0002] Butyrylcholinesterase, also known as pseudocholinesterase (BCHE), belongs to the family of serine esterases. It is mainly distributed in serum and liver in the body, and a small amount is also present in muscle and brain tissue. This protein is a natural antidote in the human body. It can hydrolyze many esters, peptides and amides, participate in the metabolism of certain drugs, and it can also promote cell growth. [0003] BCHE has good specificity to butyrylcholine and benzoylcholine, and can also specifically combine with organophosphate poisons or pesticides, and participate in the metabolism and biotransformation of some ester compounds, such as cocaine and succinylcholine wait. BCHE has a detoxification effect on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/85
CPCC12N9/18C12Y301/01008
Inventor 李宁鲁丹李秋艳刘燊商圣哲吴芳芳
Owner CHINA AGRI UNIV
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