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Application of porcine SOD1 gene in PRRS (Porcine Reproductive and Respiratory Syndrome) virus resistance

A gene and virus technology, applied in the application field of pig SOD1 gene in anti-PRRS virus, can solve the problems of unable to produce neutralizing antibodies in time, unable to effectively control, persistent infection, etc.

Inactive Publication Date: 2015-01-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PRRSV mainly infects alveolar macrophages of pigs. Now studies have shown that after PRRSV infection, PRRSV infection in innate immunity cannot elicit an effective type I interferon response, humoral immunity cannot produce effective neutralizing antibodies in time, and antibody-dependent Virus-enhancing effects, ultimately leading to immunosuppression and persistent infection
Due to the extremely high mutation rate of the virus, traditional vaccine methods cannot effectively control the disease, and many studies are devoted to finding new methods such as RNAi and interferon to target the virus to make up for the shortcomings of traditional methods

Method used

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  • Application of porcine SOD1 gene in PRRS (Porcine Reproductive and Respiratory Syndrome) virus resistance
  • Application of porcine SOD1 gene in PRRS (Porcine Reproductive and Respiratory Syndrome) virus resistance
  • Application of porcine SOD1 gene in PRRS (Porcine Reproductive and Respiratory Syndrome) virus resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Discovery of a new gene SOD1 resistant to PRRSV infection by analyzing transcriptome data

[0024] Due to differences in the genetic background of different pig breeds and individuals, the resistance to PRRS is also different. Studies have shown that in terms of clinical symptoms and physiological and biochemical indicators, domestic pig breeds such as Tongcheng pigs and Meishan pigs are compared with foreign Landrace pigs, Large white pigs have strong resistance after being infected with highly pathogenic PRRSV. Through the method of high-throughput sequencing, the differential gene expression analysis was performed on the tissues of Landrace pigs and Tongcheng pigs before and after PRRSV infection, and there were significant differences in the expression of some immune-related genes between the two breeds before and after infection. Through the analysis of gene expression differences caused by PRRSV infection among different varieties, not only the infection...

Embodiment 2

[0027] Example 2 Verification of SOD1 Gene Anti-PRRSV Infection Using Cell Biology Research Methods

[0028] 1. Design primers for the SOD1 gene (GenBank accession number is NM_001190422), use reverse transcription PCR technology, reverse transcribe the mRNA and amplify the CDS sequence of the SOD1 gene, the primer sequences are as follows:

[0029] Upstream primer SOD1-F: 5′-GCGTCGAC CATGGCGACGAAGGCCGTGT-3′

[0030] Downstream primer SOD1-R: 5′-CCCTCGAG TTACTGGGTGATCCCAATTAC-3′

[0031] The CDS sequence (SEQ ID No.1) of the amplified SOD1 gene obtained was introduced into the original vector pCMV-Myc-N (purchased from Clonetech Company, and the vector map is as follows: figure 2 shown), construct pCMV-SOD1-Myc vector. Since pCMV-Myc-N has a strong CMV promoter, the SOD1 gene introduced into it can be overexpressed.

[0032] The construction method of pCMV-SOD1-Myc vector is as follows: extract the total RNA of porcine lung tissue, carry out inversion to form cDNA, and use...

Embodiment 3

[0046] Example 3 Preparation method of anti-PRRSV transgenic pigs overexpressing SOD1 gene

[0047] 1. Recovery of donor cells (pig fetal fibroblasts). Take out the cryopreservation tube from the liquid nitrogen, quickly put it into a 37°C water bath, and shake the cryopreservation tube rapidly continuously. After the freezing solution is completely thawed, transfer it to a centrifuge tube, dilute it 3 times with fresh culture medium, centrifuge at 1000rpm for 5 minutes, remove the supernatant, suspend the cell pellet with fresh culture medium, dilute the cells to the required concentration, and inoculate into cultured in a petri dish.

[0048] 2. Using the method of liposome transfection, the target plasmid linked with the SOD1 gene CDS sequence is transferred into the donor cell. The cells were digested by conventional methods, and 0.8 μg of the plasmid pCMV-SOD1-Myc to be transfected and 2 μl of liposome (lipofectamine) were diluted with 100 μl of serum-free medium DMEM. ...

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Abstract

The invention relates to a new use of a porcine SOD1 gene in PRRS (Porcine Reproductive and Respiratory Syndrome) virus resistance and specifically relates to application of the porcine SOD1 gene in PRRS virus resistance. The duplication of the PRRS virus in cells is inhibited by virtue of overexpression of the CDS sequence of the porcine SOD1 gene in the cells. The invention also provides a method for breeding an anti-PRRS virus transgenic pig, and a method for screening anti-PRRS virus pigs according to the expression quantity of the SOD1 gene.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the application of pig SOD1 gene in anti-PRRS virus. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as "PRRS", is caused by porcine reproductive and respiratory syndrome virus (PRRSV), a member of the family Arteriviridae of the order Nidoviridae. There are equine arteritis virus, murine lactate dehydrogenase virus, and simian hemorrhagic fever virus. The disease mainly causes reproductive and respiratory symptoms of pigs, manifested as interstitial pneumonia and mummified sow abortion, which causes huge economic losses to the pig industry all over the world every year. A high fever outbreak occurred in China in 2007. The disease spread rapidly across the country. More than 2 million pigs were infected and 400,000 pigs died. The disease was finally confirmed to be caused by a highly pathogenic PRRS virus. of. PRRSV mainly i...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/85C12N5/10A01K67/027
Inventor 李宁李佳任立明胡晓湘杨倩
Owner CHINA AGRI UNIV
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