Bhk-21 cell line stably expressing map3k8 protein and its construction and application

A BHK-21, stable expression technology, applied in BHK-21 cell line and its construction and application fields, can solve the problems of time-consuming and labor-intensive stable expression, and achieve the effect of improving virus yield and promoting virus replication.

Active Publication Date: 2020-08-28
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that due to the steps of resistance selection and even pressurized amplification, stable expression is relatively time-consuming and labor-intensive

Method used

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  • Bhk-21 cell line stably expressing map3k8 protein and its construction and application
  • Bhk-21 cell line stably expressing map3k8 protein and its construction and application
  • Bhk-21 cell line stably expressing map3k8 protein and its construction and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction process of BHK-21 cell line BHK-21 / MAP3K8 stably expressing MAP3K8

[0039] 1.1 Construction of lentiviral vector and virus packaging

[0040] Specifically include the following steps:

[0041] Step 1: Construction of the recombinant lentiviral vector Lv-MAP3K8: Synthesize the CDS sequence of the Cricetulus griseus (Chinese hamster) MAP3K8 gene by chemical synthesis. The CDS sequence of the MAP3K8 gene refers to the sequence whose accession number is XM_007616553.2 on NCBI, and the CDS sequence of the MAP3K8 gene Recognition sites of Xba I and BamH I were introduced at the 5' end and 3' end of the DNA respectively, and the target gene and the lentiviral vector Lv-pCDH were digested with Xba I and BamH I, and then ligated to obtain the recombinant lentiviral vector Lv-MAP3K8 .

[0042] Step 2: Packaging lentivirus:

[0043] (1) Pack lentivirus Lv-pCDH and Lv-MAP3K8 virus liquids respectively with 293FT cells;

[0044] ① Digestion and inoculatio...

Embodiment 2

[0063] 2.1qRT-PCR verification cell line BHK-21 / MAP3K8 promotes FMDV replication

[0064] Include the following steps:

[0065] (1) Dilute the FMDV venom to a concentration of 1:500 with serum-free DMEM.

[0066] (2) Digest the BHK-21 / MAP3K8 cells and spread them on cell plates, and place them at 37°C, 5% CO 2 In the incubator, when the cells grow to 70%-90%, the cells are washed twice with serum-free DMEM to remove the residual serum in the cells.

[0067] (3) Add the diluted venom to the cells according to an appropriate volume (add 4ul of FMDV venom per 2ml of cell culture medium), and place at 37°C, 5% CO 2 After incubating in the incubator for 1 hour, the venom was discarded, replaced with 2% FBS DMEM maintenance solution, and the culture was continued. Samples were collected at 0, 12h, and 24h after inoculation.

[0068] (4) extract sample RNA and do reverse transcription, then carry out qRT-PCR, the primer (SEQ ID NO:7~8) of the 3D protein of used amplification FMDV...

Embodiment 3

[0084] 3.1qRT-PCR verification cell line BHK-21 / MAP3K8 promotes SVV replication

[0085] Include the following steps:

[0086] (1) Dilute SVV venom to a concentration of 1:500 with serum-free DMEM.

[0087] (2) Digest the BHK-21 / MAP3K8 cells and spread them on cell plates, and place them at 37°C, 5% CO 2 In the incubator, when the cells grow to 70%-90%, the cells are washed twice with serum-free DMEM to remove the residual serum in the cells.

[0088] (3) Add the diluted venom to the cells according to an appropriate volume (2ml of cell culture medium plus 4ul of SVV venom), and place at 37°C, 5% CO 2 After incubating in the incubator for 1 hour, the venom was discarded, replaced with 2% FBS DMEM maintenance solution, and the culture was continued. Samples were collected at 0, 12h, and 24h after inoculation.

[0089] (4) Extract sample RNA for reverse transcription, and then perform qRT-PCR. The absolute quantitative primers (SEQ ID NO: 9-10) used to amplify the conserved...

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Abstract

The present invention relates to a BHK-21 cell strain stably expressing MAP3K8 proteins, and a construction and an application thereof. The cell line is named as BHK-21 / MAP3K8 and a preservation number is CCTCC NO:C2018261. Preparation of the cell line is as follows: synthesizing a target gene MAP3K8, constructing a recombinant lentiviral vector Lv-MAP3K8, packaging the lentivirus expressing the MAP3K8, infecting BHK-21 cells by the lentivirus, and conducting screening, culture and identification to obtain the BHK-21 cell line stably expressing the MAP3K8 proteins. Compared with the BHK-21, the BHK-21 / MAP3K8 cell line is more conducive to FMDV or SVV replication and more suitable for development and production of FMDV or SVV vaccine, and at the same time provides a reliable material for studying gene functions of the MAP3K8.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a BHK-21 cell line stably expressing MAP3K8 protein and its construction and application. Background technique [0002] Stable expression means that the vector enters the host cell and is selectively cultured. The vector DNA is stably present in the cell and can be transcribed, expressed and passed down with the cell. The expression of the target protein in the cell is durable and stable. Generally, the exogenous DNA is cloned into a carrier with a certain resistance or selection gene, and the vector is transfected into the host cell and integrated into the chromosome, but after the exogenous gene enters the cell, part of it can enter the nucleus through the cytoplasm, according to Cell types where up to 80% of foreign DNA that enters the nucleus is transiently expressed. In rare cases, the exogenous DNA entering the cell undergoes a series of non-homologous intermolecul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12N7/00C12R1/91
CPCC12N7/00C12N9/1241C12N15/86C12N2740/15043C12N2770/32051C12N2770/32151C12Y207/11024
Inventor 郑海学张克山郝军红田宏茹毅李丹朱紫祥杨帆刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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