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Screening, cultivation method and application of cell line stably expressing porcine host restriction factor a3z2 molecule

A technology of host restriction factor, A3Z2, applied in microorganism-based methods, cells modified by the introduction of foreign genetic material, applications, etc., can solve the problems of different, limited research, functional differences, etc.

Active Publication Date: 2022-02-18
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But humans and animals are only similar, not identical. Although they are homologous genes, subtle differences will have huge differences in functions and cause different problems.
Current studies on the interaction between porcine epidemic diarrhea virus (PEDV) and porcine host restriction factor A3Z2 (poA3Z2) are limited

Method used

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  • Screening, cultivation method and application of cell line stably expressing porcine host restriction factor a3z2 molecule
  • Screening, cultivation method and application of cell line stably expressing porcine host restriction factor a3z2 molecule
  • Screening, cultivation method and application of cell line stably expressing porcine host restriction factor a3z2 molecule

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Embodiment 1

[0059] Materials and Methods

[0060] Strains and cells, pcDNA3.1-3XFlag-A3Z2 vector was purchased from Clontech Company, Escherichia coli Top 10 strains were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., IPEC-J2 cells were purchased from China Center for Type Culture Collection (Wuhan University) .

[0061]Reagents, FuGene HD was purchased from Promega Company; RNA extraction kit was purchased from Axygen Company; PrimeScript RT Master Mix Perfect Real time kit, Premix Ex TaqTMⅡ (TliRNaseH Plus) kit, 2000bp DNA Marker, Ex-Taq DNA polymerase, T4 DNA ligase and endonuclease were purchased from Takara Company; M-PER Mammalian Protein Extraction Reagent was purchased from Thermo Company. Flag mouse monoclonal antibody was purchased from Beyontian Biotechnology Company.

[0062] Construction of porcine A3Z2 eukaryotic expression vector:

[0063] The porcine-derived A3Z2 gene sequence is shown in Table 1:

[0064] ATGGATCCTCAGCGCCTGAGACAATGGCCAGGGCCGGGACCA...

Embodiment 2

[0075] Construction of porcine A3Z2 eukaryotic expression vector:

[0076] Such as figure 1 , figure 2 As shown in Table 1, the primers A3Z2-F and A3Z2-R for amplifying the full length of the porcine A3Z2 gene were designed, and the PCR product containing the full length of the porcine A3Z2 gene was obtained by one-step RT-PCR (reaction system and conditions) amplification , recovered by gel, connected to the pMD-18T vector, and successfully constructed the pMD-18T-poA3Z2 vector. After the sequence was correct, the pMD-18T-poA3Z2 vector was used as a template, and PCR was performed with primers H1 and H2 containing EcoR I restriction sites. The one-step cloning kit was used to construct and obtain the pcDNA3.1-3XFlag-poA3Z2 vector through PCR amplification, enzyme digestion, ligation, transformation and sequencing identification according to the following reaction system and reaction conditions.

[0077] Screening of IPEC-J2 cell line stably expressing porcine A3Z2:

[007...

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Abstract

The invention discloses a screening, cultivation method and application of a cell line stably expressing porcine host restriction factor A3Z2 molecule, 1) construction of porcine A3Z2 eukaryotic expression vector; 2) stable expression of porcine host restriction factor A3Z2 Screening of cell lines; 3) observation, detection and analysis; 4) virus inoculation; 5) data collection; the IPEC-J2 cell line expressing pig-derived A3Z2 molecules successfully constructed by the present invention, after being inoculated with porcine epidemic diarrhea virus (PEDV), can The cytopathic effect can be significantly inhibited within 48 hours; this cell line can not only be used for the preparation of porcine antiviral protein A3Z2, but also can be used to study the molecular mechanism of the interaction between PEDV and A3Z2, and use this as a target for the screening of new anti-PEDV drugs Provide ideas.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a screening, cultivation method and application of a cell line stably expressing a porcine host restriction factor A3Z2 molecule. Background technique [0002] During the interaction between the virus and the host cell, the cell will express a series of different proteins to inhibit the replication of the virus by exerting different functions in a cell-autonomous manner. These proteins are called host restriction factors or natural resistance factors, which are innate immune factors. Component of the antiviral response, which provides the body with an early line of defense against infection. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide protein 3 (APOBEC3) is a class of host restriction factors with antiviral activity. Members of the APOBEC3 protein family include A3A-H, among which A3G (APOBEC3G), which has been well-studied, is an interferon-stimulated gene (ISG). A3G...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/50C12N5/10C12Q1/6851G01N33/68G01N33/569A61P31/14C12R1/91
CPCC12N15/85C07K14/005C12Q1/6851G01N33/68G01N33/56983A61P31/14G01N2333/165G01N2333/47
Inventor 沈海燕张春红刘志成孙俊颖张建峰
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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