CHO (Chinese hamster ovary) cell strain with high-efficiency expression of CD2V protein of African swine fever (ASF)

An African swine fever, high-efficiency expression technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., to achieve the effect of easy purification and high expression

Active Publication Date: 2019-08-02
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no vaccine against African swine fever. How to block the spread of the virus in domestic pigs and prevent the escape of the virus in the body is of great significance to the defense of African swine fever.

Method used

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  • CHO (Chinese hamster ovary) cell strain with high-efficiency expression of CD2V protein of African swine fever (ASF)
  • CHO (Chinese hamster ovary) cell strain with high-efficiency expression of CD2V protein of African swine fever (ASF)
  • CHO (Chinese hamster ovary) cell strain with high-efficiency expression of CD2V protein of African swine fever (ASF)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1, the construction of the recombinant plasmid expressing CD2V gene

[0015] 1.1 Cutting and optimization of CD2V gene

[0016] After analyzing the structural domain of the CD2V gene whose nucleotide sequence is SEQ ID NO: 1 (the amino acid sequence of the encoded protein is SEQ ID NO: 2), the C-terminal 150aa of the protein is cut off, so as to better help The protein folds better into tertiary structure, which exposes its antigenic sites better. For the convenience of protein purification and later detection, 6 His amino acids were added to the C-terminus. Since there are many rare codons in the CD2V nucleotide sequence, the nucleotides were optimized for codons, so that the CD2V protein can be efficiently expressed in CHO cells.

[0017] The nucleotide sequence of the optimized gene is SEQ ID NO: 3, and the amino acid sequence after optimization and modification is SEQ ID NO: 4, which can better express the antigenic site.

[0018] 1.2 Construction of 1...

Embodiment 2

[0071] Example 2: Transfection of CHO-K1 cells

[0072] (1) Preparation: Ultraviolet sterilization in a biosafety cabinet for 30 minutes; F-12 culture solution was placed in a 37°C water bath and preheated to 37°C.

[0073] (2) Add 2.5 μg of the recombinant plasmid DNA and P3000 prepared in Example 1 to 125 μl of OPTI-MEM culture medium, and mix well. Add 3.75 μl lipotectamine3000 to 125 μl OPTI-MEM culture solution and mix well. The liposomes were mixed with the recombinant DNA and allowed to stand at room temperature for 10 min.

[0074] (3) Take out the 6-well plate cells that were plated 24 hours ago from the 37°C incubator, discard the supernatant medium, wash the cells three times with pre-warmed OPTI-MEM culture medium, and discard the OPTI-MEM culture medium.

[0075] (4) Add 2 ml of F-12 culture medium with 10% and 1% double antibody fetal bovine serum to each cell well.

[0076] (5) Gently add the mixture of recombinant DNA and liposomes into each well of cells, m...

Embodiment 3

[0079] Embodiment 3: protein purification and detection

[0080] 4.1 Expression and identification of recombinant CD2V protein in CHO cells

[0081] The cells selected in Example 3 were cultured at 37°C for up to 240 hours, and the supernatant was collected. The collected protein solution was subjected to SDS-PAGE protein analysis. It was found that the CD2V gene before optimization was not expressed in CHO cells, but the optimized CD2V gene was expressed in CHO cells, and its protein content was about 3 mg / L.

[0082] 4.2 Chromatographic purification of recombinant CD2V protein

[0083] The collected CD2V supernatant was subjected to affinity chromatography with Ni Sepharose excel medium of GE. The collected supernatant was centrifuged at 5000rpm for 5min and used as the loading solution.

[0084] 4.2.1 Wash the medium with 5 times column volume of distilled water.

[0085] 4.2.2 Wash the medium with 5 column volumes of equilibration buffer.

[0086] 4.2.3 Sample loadin...

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Abstract

The invention provides CD2V protein of African swine fever (ASF) capable of being expressed with high-efficiency in a CHO (Chinese hamster ovary) cell strain. The amino acid sequence of the CD2V protein is shown in SEQ ID NO:4; a recombinant plasmid constructed by the invention is used for expressing the CD2V protein of an African swine fever virus in CHO cells; the invention further provides a recombinant CHO cell strain prepared by transfecting the CHO cells through the recombinant plasmid, the recombinant CHO cell strain can be used to prepare the CD2V protein, and the prepared protein canbe used for differential diagnosis of the African swine fever. According to the cell strain with the expression of the CD2V protein of the African swine fever, the expression quantity is high, purification is easy, the cell strain can be used for the differential diagnosis, and a solid foundation is laid for the production of subunit vaccines and diagnostic reagents of the African swine fever.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a CHO cell strain highly expressing African swine fever CD2V protein. Background technique [0002] African swine fever (African Swine fever, East African Swine fever, ASF) is an acute, febrile and highly contagious viral disease caused by a viral virus, characterized by a short course of disease, but a mortality rate of up to 100%. , clinical manifestations of fever, skin cyanosis, lymph nodes, kidneys, gastrointestinal mucosal bleeding. The disease was first reported in Kenya in 1909 and has always existed in sub-Saharan African countries. It spread to Western Europe and Latin American countries in 1957, and most of them were extinguished in time. However, there are still cases in Portugal, southwestern Spain and Sardinia in Italy. Popularity. Since 2007, African swine fever has occurred, spread, and spread in many countries around the world...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/34C12N5/10G01N33/569A61K39/12A61P31/20C12R1/91
CPCC07K14/005C12N5/0682G01N33/56983A61K39/12A61P31/20C12N2710/12022C12N2710/12034C12N2510/00A61K2039/552
Inventor 郭伟伟刘大卫向银辉王玉红范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO
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