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Application of porcine β-defensin 2 gene in anti-swine influenza

A swine flu virus and gene technology, applied in the field of animal infectious diseases and transgenics, can solve problems such as PBD-2 and its genes that have not yet been seen

Active Publication Date: 2020-10-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] So far, there have been no domestic and foreign research reports on the application of PBD-2 and its genes in anti-animal virus diseases, especially swine flu

Method used

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  • Application of porcine β-defensin 2 gene in anti-swine influenza
  • Application of porcine β-defensin 2 gene in anti-swine influenza
  • Application of porcine β-defensin 2 gene in anti-swine influenza

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Preparation of transgenic pigs overexpressing the PBD-2 gene:

[0044] 1.1 Construction and linearization of pcDNA3.1(+) / pCA-PBD2 transgene vector:

[0045] Total RNA was extracted from porcine liver tissue (Yang et al.2015), and cDNA was obtained after reverse transcription. Using the CDS sequence (GenBank NM_214442.1) of the PBD-2 gene published in the NCBI database, design primers containing EcoRI and XhoI restriction endonuclease sites (the design of the primer pair below contains EcoRI and XhoI restriction endonuclease site):

[0046] Forward primer PBD2 01: CCGGAATTCATGTGGGCCCTCTGCTTG,

[0047] Reverse primer PBD2 02: CCGCTCGAGTCAGGGTCAGCGGATGCA;

[0048] 通过普通PCR方法扩增获得PBD-2基因的CDS序列,其大小为210bp,该CDS序列具体为:ATGAGGGCCCTCTGCTTGCTGCTGCTGACTGTCTGCCTCCTCTCTTCCCAGCTGGCTGCAGGTATTAACCTGCTTACGGGTCTTGGCCAGAGGTCCGACCACTACATATGTGCCAAGAAAGGGGGGACCTGCAACTTCTCCCCCTGCCCGCTCTTCAACAGGATTGAAGGGACCTGTTACAGTGGCAAGGCCAAGTGCTGCATCCGCTGA

[0049] The obtained PBD-2 CDS sequence was comb...

Embodiment 2

[0059] Example 2: Application of PBD-2 Gene in Pigs' Resistance to Swine Influenza Virus Infection

[0060] (1) Transgenic pigs overexpressing PBD-2 had milder pulmonary and ocular lesions after infection with swine influenza than littermate wild-type (non-transgenic) pigs.

[0061] After the H1N1 virus (A / Swine / Nanchang / F9 / 2010, implementing the present invention is not limited to this strain) to 4 40 day old wild-type (ie non-transgenic) pigs (control) tracheal injection of 2mL 5 * 107EID50 / mL and Six 40-day-old transgenic pigs and six 40-day-old littermate wild-type (non-transgenic) pigs were housed and raised together. Clinical symptoms were observed every morning and evening, rectal body temperature was measured with a veterinary thermometer, and the body temperature curve was drawn. Figure 8 The statistical results showed that the body temperature of both transgenic pigs and non-transgenic pigs increased after infection with influenza virus, and the body temperature of ...

Embodiment 3

[0070] Embodiment 3: the application of the artificially synthesized PBD-2 gene in suppressing swine influenza virus replication:

[0071] MDCK cells (purchased from China. Wuhan. Wuhan University, China Center for Type Culture Collection, i.e. CCTCC. Strain number CCTCC: GDC401) were inoculated into 24-well cell culture plates. Wash the cells 2-3 times with sterile PBS buffer (commonly used or commercially available). Then the TCID 50 for 10 4 / 100 μL of H1N1 swine influenza virus solution was diluted 100 times and inoculated with cells, 200 μL per well, and incubated in a 37°C incubator for 1 hour. Discard the liquid in the well, wash twice with PBS buffer, add 500 μL of PBD-2 gene diluted with DMEM cell culture medium (common medium) to each well, the concentration is 12.5, 25, 50, 100, 150 μg / mL respectively , add 4 wells for each dilution, take the supernatant after culturing in an incubator at 37°C for 24 hours, use the NP gene (gene accession number: JF275928.1) as t...

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Abstract

The invention belongs to the technical fields of zoonosis and animal transgenes, and particularly relates application of a PBD (porcine beta-defensins 2) gene in resisting of swine influenza. The application has the advantages that the segments of the PBD-2 gene are excessively expressed in a swine body, so as to obviously improve the resistance ability of the transgenic swine on the swine influenza, namely that the excessively expressed PBD-2 can obviously reduce the virus titer of swine influenza virus in lung tissues of the infected swine and the lesion degree of swine lung due to infectionby the swine influenza virus; by synthesizing or recombining the PBD-2 gene, the duplication of the swine influenza virus can be effectively inhibited; the polypeptide medicines or feed additives canbe prepared by in-vitro expression or synthesizing of the PBD-2 gene.

Description

technical field [0001] The invention belongs to the technical field of animal infectious diseases and transgenes, and in particular relates to the application of porcine beta-defensin 2 gene (PBD-2 gene for short) in anti-swine influenza. The overexpression of the PBD-2 gene in pigs significantly improves the resistance of pigs to swine influenza virus. The artificial synthesis or recombinant PBD-2 gene can also effectively inhibit the replication of swine influenza virus. Background technique [0002] Swine flu is an acute respiratory infectious disease of pigs caused by influenza A virus. Its main clinical symptoms are fever, sneezing, depression, loss of appetite and even extinction. Strong sex, brought great economic losses to the pig industry. In addition, pigs are an important intermediate host for gene reassortment and cross-species transmission of avian influenza and human influenza viruses (also known as the "mixer" of influenza viruses), and swine influenza virus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/85A01K67/027
CPCA01K67/0276A01K2227/108A01K2267/02C07K14/4723C12N15/85
Inventor 周锐黎璐刘逍杨希黄超黄靖周红波
Owner HUAZHONG AGRI UNIV
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