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Application of porcine BTG2 (B-cell translocation gene-2) gene in anti-PRRS (porcine reproductive and respiratory syndrome) virus

A gene and virus technology, applied in the field of new functions of the pig BTG2 gene in anti-PRRS virus, can solve the problems of inability to produce neutralizing antibodies in time, high mutation rate, persistent infection, etc., and achieve the effect of inhibiting the replication of PRRS virus

Inactive Publication Date: 2014-04-30
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. Many studies are devoted to finding new methods such as RNAi and interferon to target the virus and make up for the shortcomings of traditional methods.

Method used

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  • Application of porcine BTG2 (B-cell translocation gene-2) gene in anti-PRRS (porcine reproductive and respiratory syndrome) virus
  • Application of porcine BTG2 (B-cell translocation gene-2) gene in anti-PRRS (porcine reproductive and respiratory syndrome) virus
  • Application of porcine BTG2 (B-cell translocation gene-2) gene in anti-PRRS (porcine reproductive and respiratory syndrome) virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 Utilizes the analysis transcriptome data method to discover the gene of new anti-PRRSV infection

[0022] BTG2

[0023] 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 varietie...

Embodiment 2

[0026] Embodiment 2 utilizes cell experiment method to verify BTG2 gene anti-PRRSV infection

[0027] 1. Design primers for the BTG2 gene (GenBank accession number EU255256.1), use reverse transcription PCR technology to reverse transcribe the mRNA and amplify the CDS sequence of the BTG2 gene. The primer sequences are as follows:

[0028] Target gene BTG2:

[0029] Upstream primer BTG2-F:

[0030] 5'-GCGTCGACCATGAGCCAGGCCCGCTGGAC-3';

[0031] Downstream primer BTG2-R:

[0032] 5'-CCCTCGAGACTAACTGGAGACCGCCATGACGTAG-3'.

[0033] The CDS sequence of the amplified BTG2 gene (as shown in SEQ ID No.1) was introduced into the original vector pCMV-Myc-N (purchased from Clonetech, the vector map is as follows figure 2 shown), construct pCMV-BTG2-Myc vector. Since pCMV-Myc-N has a strong CMV promoter, the BTG2 gene introduced into it can be overexpressed.

[0034] 2. Verification of BTG2 gene overexpression

[0035] The constructed pCMV-BTG2 vector connected with the CDS sequen...

Embodiment 3

[0061] Example 3 Utilize BTG2 gene overexpression to prepare transgenic pigs resistant to PRRSV

[0062] 1. Recovery of donor cells. Take out the cryovial from the liquid nitrogen, quickly put it into a 37-degree water bath, and shake the cryovial rapidly. 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.

[0063] 2. Using the method of liposome transfection, the target plasmid is transferred into the donor cells. Digest the cells in a conventional method, dilute 0.8ug of the plasmid to be transfected and 2ul of liposome (lipofectamine) with 100ul serum-free medium DMEM TM 2000). After incubating at room temperature for 20 minutes, add 100ul of the above liquid to each well of the 24-well cultu...

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Abstract

The invention relates to a new function of a porcine BTG2 (B-cell translocation gene-2) in anti-PRRS (porcine reproductive and respiratory syndrome) virus, and in particular provides application of porcine BTG2 gene in anti-PRRS virus. The application is as follows: inhibiting replication of PRRS virus in cells by over-expression of BTG2 gene in cells. The invention also provides a method for screening an anti-PRRS virus pig by utilizing the expression quantity of BTG2 gene, and a method for preparing a cloned embryo of over-expressed BTG2.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the new function of pig BTG2 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 ...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P35/00C12N15/63C12N5/10C12Q1/68
Inventor 李宁李佳任立明李骏蔚
Owner CHINA AGRI UNIV
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