Application of calreticulin CALR to disease resistance of pigs

Abstract

The invention provides an application of calreticulin (CALR). The calreticulin (CALR) is used for restraining duplication of viruses especially Japanese encephalitis viruses (Japanese encephalitis virus, JEV) in host cells, so that diseases caused by virus duplication of JEV can be resisted, and disease resistance in accordance with epidemic encephalitis B is provided. The invention also providesa cell strain for resisting virus infection and a non-human mammal animal model for resisting epidemic encephalitis B, and a preparation method of the non-human mammal animal model for resisting epidemic encephalitis B. The CALR is highly conservative in mammals, so that the CALR can be widely applied to prevention and treatment of anthropozoonosis, besides, the CALR can be used as a potential target point for gene editing animal design, drug abuse is reduced, generation of epidemic situation is avoided, and the CALR has high economic value.

Description

technical field

[0001] The invention relates to the field of animal disease resistance breeding, in particular to the application of calreticulin CALR in pig disease resistance. Background technique

[0002] Epidemic Japanese encephalitis (referred to as Japanese encephalitis) is a zoonotic disease caused by Japanese encephalitis virus (JEV) infection, which is also known as Japanese encephalitis virus and belongs to the second category of animal diseases in the country. . Japanese encephalitis is a neurological infectious disease that seriously endangers the health of humans and animals. Mosquitoes are the intermediate vectors of the disease, and they spread the disease mainly through the biting of sick pigs by infected mosquitoes, and are widely spread in pig farms in the form of mosquitoes, pigs, and mosquitoes. Therefore, swine encephalitis B has a typical seasonality, mostly occurs in summer and autumn when there are many mosquitoes and reproduces in large numbers, an...

Examples

Embodiment 1

[0144] Example 1: Construction of porcine CALR gene knockout cell line using CRISPR / Cas9 lentivirus strategy

[0145] 1.1 Design of specific sgRNA targeting CALR gene and construction of expression vector

[0146] In order to study whether the CALR gene is involved in mediating JEV infection of pig PK-15 cells, the CALR gene (accession number: ENSSSCT00000015020) and the whole genome sequence of the pig (version number: Sus_scrofa.Sscrofa11) were downloaded from the Ensemble database (www.ensembl.org). .1). Then, use sgRNAcas9 software (https: / / sourceforge.net / projects / sgrnacas9 / ) to design sgRNA, and then select the sgRNA targeting the porcine CALR gene exon according to the specificity evaluation results. The sequence of the selected sgRNA is:

[0147] CALR-sgRNA 5'-GTCTGACCCTTGTTGCTGAA-3'

[0148] Among them, the PAM sequence for recognizing the target is "GGG". Genome-wide off-target assessment found no off-target sites with 1, 2 and 3 base mismatches ( ...

Embodiment 2

[0164]Example 2: It was found that CALR knockout cells can significantly inhibit the replication of JEV in pig PK-15 cells

[0165] 2.1 Plaque assay was used to detect the effect of CALR on JEV replication

[0166] The effect of CALR knockout cells on JEV replication was investigated using virus plaque assay and absolute quantification assay, respectively. The procedure of the virus plaque experiment is the low-melting point agarose method, and the operation is as follows: BHK cells are inoculated into a 6-well cell culture plate and cultivated to a monolayer. The cells were grown to a confluence of more than 50% for inoculation; preparation of virus dilution: take 100 μL of virus stock solution in a 1.5mL EP tube, and use high-glucose medium DMEM to carry out serial 10-fold dilutions (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 ), gently pipette to mix, and the number of pipettes should be the same for each tube. Be careful not to extend the tip of t...

Embodiment 3

[0176] Example 3: The NS3 gene encoded by JEV cannot be expressed in CALR gene knockout cells

[0177] The expression of NS3 gene after JEV infection in CALR gene knockout and control cells was further compared by immunofluorescence technique. The general experimental procedure is as follows: (1) Solution preparation: 0.3% TritonX-100 (ready to use); blocking solution: final concentration 3% BSA, 0.3% TritonX-100, 10% FBS, stored at 4°C. Antibody diluent: 3% BSA, 0.3% TritonX-100, stored at 4°C. (2) Operation process: Cell fixation: In a 12-well plate, when the cell density reaches 70%-80%, wash the cells twice with pre-cooled PBS, and then fix with pre-cooled 4% paraformaldehyde at room temperature 15min. Remove paraformaldehyde, rinse the cells twice with pre-cooled PBS, add pre-cooled 0.3% TritonX-100, and remove TritonX-100 after standing at room temperature for 10 minutes. Rinse cells: add pre-cooled PBS, incubate on a shaker at room temperature for 5 minutes, repeat t...

technical field

[0001] The invention relates to the field of animal disease resistance breeding, in particular to the application of calreticulin CALR in pig disease resistance. Background technique

[0002] Epidemic Japanese encephalitis (referred to as Japanese encephalitis) is a zoonotic disease caused by Japanese encephalitis virus (JEV) infection, which is also known as Japanese encephalitis virus and belongs to the second category of animal diseases in the country. . Japanese encephalitis is a neurological infectious disease that seriously endangers the health of humans and animals. Mosquitoes are the intermediate vectors of the disease, and they spread the disease mainly through the biting of sick pigs by infected mosquitoes, and are widely spread in pig farms in the form of mosquitoes, pigs, and mosquitoes. Therefore, swine encephalitis B has a typical seasonality, mostly occurs in summer and autumn when there are many mosquitoes and reproduces in large numbers, an...

Examples

Embodiment 1

[0144] Example 1: Construction of porcine CALR gene knockout cell line using CRISPR / Cas9 lentivirus strategy

[0145] 1.1 Design of specific sgRNA targeting CALR gene and construction of expression vector

[0146] In order to study whether the CALR gene is involved in mediating JEV infection of pig PK-15 cells, the CALR gene (accession number: ENSSSCT00000015020) and the whole genome sequence of the pig (version number: Sus_scrofa.Sscrofa11) were downloaded from the Ensemble database (www.ensembl.org). .1). Then, use sgRNAcas9 software (https: / / sourceforge.net / projects / sgrnacas9 / ) to design sgRNA, and then select the sgRNA targeting the porcine CALR gene exon according to the specificity evaluation results. The sequence of the selected sgRNA is:

[0147] CALR-sgRNA 5'-GTCTGACCCTTGTTGCTGAA-3'

[0148] Among them, the PAM sequence for recognizing the target is "GGG". Genome-wide off-target assessment found no off-target sites with 1, 2 and 3 base mismatches ( ...

Embodiment 2

[0164]Example 2: It was found that CALR knockout cells can significantly inhibit the replication of JEV in pig PK-15 cells

[0165] 2.1 Plaque assay was used to detect the effect of CALR on JEV replication

[0166] The effect of CALR knockout cells on JEV replication was investigated using virus plaque assay and absolute quantification assay, respectively. The procedure of the virus plaque experiment is the low-melting point agarose method, and the operation is as follows: BHK cells are inoculated into a 6-well cell culture plate and cultivated to a monolayer. The cells were grown to a confluence of more than 50% for inoculation; preparation of virus dilution: take 100 μL of virus stock solution in a 1.5mL EP tube, and use high-glucose medium DMEM to carry out serial 10-fold dilutions (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 ), gently pipette to mix, and the number of pipettes should be the same for each tube. Be careful not to extend the tip of t...

Embodiment 3

[0176] Example 3: The NS3 gene encoded by JEV cannot be expressed in CALR gene knockout cells

[0177] The expression of NS3 gene after JEV infection in CALR gene knockout and control cells was further compared by immunofluorescence technique. The general experimental procedure is as follows: (1) Solution preparation: 0.3% TritonX-100 (ready to use); blocking solution: final concentration 3% BSA, 0.3% TritonX-100, 10% FBS, stored at 4°C. Antibody diluent: 3% BSA, 0.3% TritonX-100, stored at 4°C. (2) Operation process: Cell fixation: In a 12-well plate, when the cell density reaches 70%-80%, wash the cells twice with pre-cooled PBS, and then fix with pre-cooled 4% paraformaldehyde at room temperature 15min. Remove paraformaldehyde, rinse the cells twice with pre-cooled PBS, add pre-cooled 0.3% TritonX-100, and remove TritonX-100 after standing at room temperature for 10 minutes. Rinse cells: add pre-cooled PBS, incubate on a shaker at room temperature for 5 minutes, repeat t...

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