sgRNA of ID1 (Inhibitor of Differentiation 1) genes, knock-out method for ID1 genes, BHK-21 cell line and application thereof

A technology of BHK-21 and cell lines, applied in the biological field, can solve problems such as economic losses, low virus titers, and complex O-type foot-and-mouth disease, and achieve the effect of increasing virus titers and reducing production costs

Active Publication Date: 2018-10-02
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

In 2010, the O-type Mya-98 strain was introduced into my country, causing a severe epidemic, which made the O-type foot-and-mouth disease prevalent in my country more complicated; the A-type SEA-97G1 strain and G2 strain were introduced successively since 2009 and 2013 In my country, the epidemic has caused outbreaks in many provinces in my country, causing huge economic losses
[0005] At present, wild-type BHK-21 cell line is used in the production process of FMD vaccine, and the virus titer in wild-type BHK-21 cell line is low, which makes the production cost of FMD vaccine higher

Method used

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  • sgRNA of ID1 (Inhibitor of Differentiation 1) genes, knock-out method for ID1 genes, BHK-21 cell line and application thereof
  • sgRNA of ID1 (Inhibitor of Differentiation 1) genes, knock-out method for ID1 genes, BHK-21 cell line and application thereof
  • sgRNA of ID1 (Inhibitor of Differentiation 1) genes, knock-out method for ID1 genes, BHK-21 cell line and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A method for knocking out the ID1 gene in BHK-21 cells, comprising the steps of:

[0041]Find the gene sequence of ID1 in GenBank (NW_004801816.1 and XM_021223030), and use the sgRNA design website http: / / crispr.mit.edu / to obtain the sgRNA of the ID1 gene; the sgRNA sequence is shown in SEQ ID NO: 1-2;

[0042] Forward: SEQ ID NO: 1: caccgGGCGCGGGCGAGGTTGTGCT;

[0043] Reverse: SEQ ID NO: 2: aaacAGCACAACCTCGCCCGCGCCc.

[0044] Construct the sgRNA sequence in step (1) into the pspCas9(BB)-2A plasmid (available for purchase) to obtain a positive plasmid for future use;

[0045] It should be noted that since the construction methods in this step are relatively common methods in the field, they will not be described here one by one.

[0046] Cultivate BHK-21 cells, transfect when the cell density of BHK-21 reaches 60%-80%, and set aside;

[0047] Specifically, add 5-10 mL of sterilized PBS solution to BHK-21 cells, shake gently, rinse the cells, discard the PBS solution...

Embodiment 2

[0063] A method for knocking out the ID1 gene in BHK-21 cells, comprising the steps of:

[0064] (1) Find the gene sequence of ID1 in GenBank (NW_004801816.1 and XM_021223030), and then design the sgRNA of ID1 gene from http: / / crispr.mit.edu / website; the sequence of sgRNA is shown as SEQ ID NO: 3-4 shown;

[0065] Forward: SEQ ID NO: 3: caccgCGCCCTGCTGGACGAGCAGC;

[0066] Reverse: SEQ ID NO: 4: aaacGCTGCTCGTCCAGCAGGGCGc.

[0067] (2) Construct the sgRNA sequence in step (1) into the pspCas9(BB)-2A plasmid (available for purchase) to obtain a positive plasmid for future use;

[0068] It should be noted that since the construction methods in this step are relatively common methods in the field, they will not be described here one by one.

[0069] (3) Cultivate BHK-21 cells, transfect when the cell density of BHK-21 reaches 60%-80%, and set aside;

[0070] Specifically, add 5-10 mL of sterilized PBS solution to BHK-21 cells, shake gently, rinse the cells, discard the PBS sol...

Embodiment 3

[0085] Screening and identification of BHK-21 cell line with CRISPR-Cas9 targeted knockout of ID1, the specific steps are as follows:

[0086] Get the BHK-21 cells that knock out the ID1 gene in the first embodiment of the present invention, add puromycin at a final concentration of 3 μg / mL to screen for 5 to 7 days (the gene resistant to puromycin in the positive cells transfected into the plasmid will express, and will not be killed after adding puromycin; and the negative cells that have not been transferred to the plasmid do not express puromycin, and will be killed, and the final survivors obtained after screening are positive cells), and the cells are counted, 100 and 300 cells were plated respectively. After a single cell clone was formed one week later, a single clone was picked with a cloning ring, transferred to a 24-well plate, and expanded for culture. Genomic DNA and protein were extracted from the cells for identification, and then the positive cloned cell line w...

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Abstract

The invention relates to the technical field of biology and discloses sgRNA of ID1 (Inhibitor of Differentiation 1) genes, a knock-out method for ID1 genes, a BHK-21 cell line and the application thereof. Sequences of the sgRNA are shown as any pair of SEQ ID NO:1-2 or SEQ ID NO:3-4. Because the ID1 is capable of inhibiting foot and mouth disease virus multiplication, by knocking-out the ID1 genesin BHK-21 cells, the virus titer of an ID1 knock-out cell line is increased, so that the production cost of the foot and mouth disease virus is reduced.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a sgRNA of ID1 gene, a knockout method of ID1 gene, BHK-21 cell line and application thereof. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious infectious disease infecting cloven-hoofed animals such as pigs, cattle and sheep caused by foot-and-mouth disease virus (FMDV). FMDV has 7 different serotypes including A, O, Asia1, C, SAT1, SAT2 and SAT3, and there is no cross-protection between serotypes. [0003] Over the years, two serotypes of foot-and-mouth disease, O-type and A-type, have seriously endangered my country's breeding industry. After the vaccine strain of Asia1 type FMD was replaced in 2007, the epidemic situation was quickly brought under control. There was no clinical report after 2009, and it has tended to disappear. In 2010, the O-type Mya-98 strain was introduced into my country, causing a severe epidemic, wh...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N15/12C12N5/10C12N7/00C12R1/93
CPCC07K14/4703C12N5/0686C12N7/00C12N15/113C12N15/85C12N15/907C12N2310/10C12N2510/00C12N2770/32121C12N2770/32151C12N2800/107C12N2810/10
Inventor 孙跃峰张永光任亭亭陈豪泰祁林林
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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