Chinese hamster ovary (CHO) cell line capable of expressing Rta albumen of Elzatein-Barn (EB) virus stably and efficiently, creation method and application thereof and cell base built by CHO cell line

A technology of stable expression of Epstein-Barr virus, applied in the field of cell bank and CHO cell line

Active Publication Date: 2013-06-05
同昕生物技术(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no recombinant CHO that efficiently and stably expresses EBV-Rta after screening an...

Method used

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  • Chinese hamster ovary (CHO) cell line capable of expressing Rta albumen of Elzatein-Barn (EB) virus stably and efficiently, creation method and application thereof and cell base built by CHO cell line
  • Chinese hamster ovary (CHO) cell line capable of expressing Rta albumen of Elzatein-Barn (EB) virus stably and efficiently, creation method and application thereof and cell base built by CHO cell line
  • Chinese hamster ovary (CHO) cell line capable of expressing Rta albumen of Elzatein-Barn (EB) virus stably and efficiently, creation method and application thereof and cell base built by CHO cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1. CHO / RTA cell line cultivation preparation

[0058] (1) Cell culture

[0059] Dihydrofolate reductase-deficient Chinese hamster ovary cells (CHO / dhfr - ) is a product of American Type Culture Collection (ATCC) company (SCO610). The complete culture medium of the cells must be supplemented with 0.1mmol / L hypoxanthine and 0.016mmol / L thymine, 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Co., Ltd. Ltd.) in a 37°C cell incubator.

[0060] (2) Construction of eukaryotic expression plasmids

[0061] The full-length sequence cDNA of BRLF1 obtained by the method disclosed in the Chinese patent application number CN200610113403 was cloned into the eukaryotic expression vector pCDNA3.1(+) to construct the mammalian cell expression vector pCDNA3.1(+)-EBV-Rta.

[0062] (3) Transfection and screening of positive clones

[0063] Mix the constructed plasmids pCDNA3.1(+)-EBV-Rta and pSV2-dhfr at a molar ratio of 5:1, and co-transfect CHO / dhfr with c...

Embodiment 2

[0077] Example 2, verification of the use of the protein expressed by the cells of the present invention

[0078] Step 1: Prepare microtiter plate

[0079] Dilute the above Epstein-Barr virus Rta protein 1:10000 with coating buffer (pH value 9.6, 0.1mol / L carbonate buffer), add to the wells of the microplate, 100 μl per well, and react at 37°C for 2 hours , shake off the coating solution, pat dry, add 200 μl of blocking solution (containing phosphate buffer with a final concentration of 2% bovine serum albumin) to each well, react at 37°C for 2 hours, shake off the blocking solution, pat dry, and dry , stored in vacuum packaging in aluminum foil bags.

[0080] Step 2: Prepare reagents:

[0081] 1) Preparation of working concentration enzyme solution

[0082] HRP-labeled goat anti-human IgG needs to be purified by affinity chromatography, with strong species specificity, a purity greater than 95%, and a titer of not less than 1:2000. Choose a commercial enzyme conjugate dil...

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Abstract

The invention relates to a Chinese hamster ovary (CHO) cell line capable of showing Rta albumen of Elzatein-Barn (EB) virus stably and efficiently, a creation method and application thereof and a cell base built by the CHO cell line, and belongs to the technical field of biology. Preservation number of the recombination cell line CHO/RTA is CGMCC6955, the CHO cell base expressing the Rta albumen of the EB virus can be used for practical production and formed by the recombination cell line CHO/RTA. The invention further provides a method for creating the CHO cell line. Dihydrofolate reductase defect type Chinese hamster ovary cells (CHO/dhfr-) serve as host cells, and the cell line capable of expressing the Rta albumen of the EB virus stably and efficiently is obtained after screening and amplification of the host cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a CHO cell strain stably and highly expressing Epstein-Barr virus Rta protein, its establishment method, application and cell bank established therefrom. Background technique [0002] Epstein-Barr virus is a gamma herpes virus that infects approximately 95% of adults worldwide. Epstein-Barr virus infection can be divided into two states: latent infection period and lytic replication period. After the initial infection, the virus can establish a lifelong latent infection in the host. Epstein-Barr virus infection from a latent state to a lytic replication state is related to the occurrence of a series of human malignant tumors. In the International Cancer Research Center (IARC) classification standard for carcinogens in 1999, Epstein-Barr virus has been clearly listed as one of the human carcinogenic factors. [0003] The Rta protein expressed by BRLF1 gene is the necessary activatio...

Claims

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Application Information

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IPC IPC(8): C12N5/10C40B40/02C12N15/85
Inventor 贾凤芹李全焦守恕
Owner 同昕生物技术(北京)有限公司
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