CN110592085B

CN110592085B - H9N2 subtype avian influenza virus inhibitor and application thereof

Info

Publication number: CN110592085B
Application number: CN201910874416.9A
Authority: CN
Inventors: 孟凯; 于江; 王友令; 袁小远; 张玉霞; 杨金兴
Original assignee: Poultry Research Institute Shandong Academy of Agricultural Sciences
Current assignee: Jinan Baiming Biological Pharmaceutical Co ltd
Priority date: 2019-09-17
Filing date: 2019-09-17
Publication date: 2022-05-27
Anticipated expiration: 2039-09-17
Also published as: CN110592085A

Abstract

The invention relates to the technical field of animal immunity and biomedicine, and particularly relates to a chicken gga-miR-30a-3p serving as an H9N2 subtype avian influenza virus inhibitor and application thereof. The inhibitor is gga-miR-30a-3p mimics, and the sequence is shown in SEQ ID NO. 1. The invention provides a design target point for research and development of novel prevention and control medicines for the H9N2 subtype avian influenza virus. The artificially synthesized gga-miR-30a-3p can be used for preparing a medicament or a reagent for preventing and treating H9N2 subtype avian influenza virus.

Description

H9N2 subtype avian influenza virus inhibitor and application thereof

Technical Field

The invention relates to the technical field of animal immunity and biomedicine, and particularly relates to a chicken gga-miR-30a-3p serving as an H9N2 subtype avian influenza virus inhibitor and application thereof.

Background

Avian influenza is a disease syndrome caused by Avian Influenza Virus (AIV) infection of birds, including diseases of respiratory tract, digestive tract, decreased egg production or immunosuppression, and can be divided into three types, i.e., highly pathogenic Avian influenza, low pathogenic Avian influenza and nonpathogenic Avian influenza. Since H9N2 subtype avian influenza viruses (H9N 2 subtype avian influenza viruses, H9N2 subtype AIV) were first isolated in Guangdong province in 1994, H9N2 subtype avian influenza viruses still remain the main low-pathogenicity avian influenza subtype in chicken flocks in China at present. The H9N2 subtype avian influenza virus brings great harm to poultry industry, the virus not only causes respiratory disease of birds, but also attacks the reproductive system of poultry, causes the laying rate of laying birds to be reduced, the growth of meat birds is slow, and simultaneously, the virus is easy to be mixed with other pathogenic microorganisms to be infected and cause death, and the H9N2 subtype avian influenza virus has a wide host range including human beings, pigs and poultry. The recent outbreak of highly pathogenic avian influenza in china with H7N9 resulted in many deaths, with six internal genes (PA, NS, PB2, PB1, NP, M) of the H7N9 strain all from the H9N2 subtype avian influenza virus. In addition, domestic cats and dogs are reported to be susceptible to H9N2 subtype avian influenza, and as these species are more closely related to humans in developed and developing countries, they may pose a greater risk to human health, H9N2 subtype avian influenza will become a reservoir of zoonotic infectious viruses. The most effective means for preventing and controlling avian influenza is immunization, but because the H9N2 subtype avian influenza virus has the characteristics of frequent mutation and recombination, the virus is difficult to eradicate, and the virus is still one of the main infectious diseases which endanger the chicken industry at present. The H9N2 subtype avian influenza needs to explore a new path.

miRNA (micro RNA) is an endogenous, non-coding, single-stranded RNA consisting of about 21-25 nucleotides that, by complementary binding to mRNA, regulates the expression of target genes at the post-transcriptional level. mirnas play a key role in various physiological and pathological processes such as cell differentiation, apoptosis, control of development timing, cell proliferation, metabolism, tumorigenesis, immune response, hormone secretion and the like. Studies have shown that mirnas play an important role in regulating viral replication and gene expression.

More than 800 mirnas have been found in chickens, but relatively few studies have been made on the function of these mirnas.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an inhibitor of H9N2 subtype avian influenza virus, which is prepared into a kit or a medicament for application as an inhibitor of H9N2 subtype avian influenza virus by synthesizing a chicken endogenous miRNA gga-miR-30a-3p sequence and utilizing the sequence to inhibit the replication and proliferation of the H9N2 subtype avian influenza virus in cells.

The invention also provides application of the H9N2 subtype avian influenza virus.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides an inhibitor of H9N2 subtype avian influenza virus, wherein the sequence of the inhibitor gga-miR-30a-3p mimics is shown in SEQ ID NO. 1.

The invention also provides application of the H9N2 subtype avian influenza virus inhibitor in drugs or reagents for inhibiting subtype avian influenza viruses.

The invention provides an application of an H9N2 subtype avian influenza virus inhibitor in a medicament or a reagent for inhibiting subtype avian influenza virus, which specifically comprises the following steps:

(1) the artificially synthesized gga-miR-30a-3p inhibitor shown in SEQ ID NO.1 is transfected into DF-1 cells by using a Lipofectamine 3000 transfection reagent, and the intracellular expression condition of gga-miR-30a-3p is detected by using fluorescent quantitative PCR (polymerase chain reaction) so as to determine that the gga-miR-30a-3p is successfully transferred into the DF-1 cells;

(2) inoculating H9N2 subtype avian influenza virus 24H after transfection, collecting cells 24H after virus infection, extracting H9N2 subtype avian influenza virus RNA according to the instruction of a virus RNA extraction kit, and detecting the RNA content of the H9N2 subtype avian influenza virus in DF-1 cells by using fluorescent quantitative RNA.

According to the invention, artificially synthesized gga-miR-30a-3p mimics is used for transfecting DF-1 cells in vitro, the DF-1 cells are infected by H9N2 subtype avian influenza virus after being transfected for 24H, the cells are collected after 24H, the RNA content of the H9N2 subtype avian influenza virus in the DF-1 cells is detected by a fluorescence quantitative PCR method, and the fact that the gga-miR-30a-3p can obviously inhibit the replication and proliferation of the H9N2 subtype avian influenza virus in the infected cells is found. The invention analyzes miRNAs differentially expressed by chicken embryo fibroblast (DF-1) before and after the infection of H9N2 subtype avian influenza virus by using a high-throughput sequencing technology, finds that gga-miR-30a-3p is obviously reduced and expressed after the infection of the H9N2 subtype avian influenza virus, further researches show that the miRNAs can obviously inhibit the proliferation of the H9N2 subtype avian influenza virus in DF-1, and provides a design target for the research and development of novel prevention and control medicaments of the H9N2 subtype avian influenza virus.

The invention has the beneficial effects that: the invention provides a design target point for research and development of novel prevention and control medicines for the H9N2 subtype avian influenza virus. The artificially synthesized gga-miR-30a-3p can be used for preparing a medicament or a reagent for preventing and treating H9N2 subtype avian influenza virus.

Drawings

FIG. 1 shows the expression of gga-miR-30a-3P at different time points after DF-1 cells are infected by H9N2 subtype avian influenza virus, and P is less than 0.05.

FIG. 2 shows the expression of gga-miR-30a-3P 24H after DF-1 cells are infected by H9N2 subtype avian influenza virus with different infection doses, and P is less than 0.05.

FIG. 3 shows the fluorescence quantitative detection result of virus RNA after DF-1 cells are infected by H9N2 subtype avian influenza virus for 24H under different gga-miR-30a-3p transfection concentrations. In the figure, miR-30a NC represents transfection negative control miRNA mimics, and 10 nm, 20 nm, 40 nm and 80 nm represent gga-miR-30a-3P mimics with transfection concentrations of 10 nm, 20 nm, 40 nm and 80 nm respectively, wherein P is less than 0.05.

Detailed Description

The technical solution of the present invention is further explained and illustrated by the following specific examples.

Example 1.

Expression detection of gga-miR-30a-3p in H9N2 subtype avian influenza virus infected DF-1 cell

(1) After H9N2 subtype avian influenza virus infects DF-1 cells with MOI =0.5, collecting DF-1 cells at different time points (0H, 6H, 12H, 24H and 36H), extracting total RNA, detecting the expression condition of gga-miR-30a-3p by fluorescence quantitative PCR, and the result shows that the expression level of gga-miR-30a-3p is continuously reduced along with the extension of infection time and the expression reaches the minimum level 24H after infection (figure 1).

(2) After H9N2 subtype avian influenza virus infects DF-1 cells at different doses (MOI =0.1, 0.5, 1, 1.5), collecting DF-1 cells, extracting total RNA, detecting the expression condition of gga-miR-30a-3p by fluorescence quantitative PCR, the result shows that the expression level of gga-miR-30a-3p is continuously reduced along with the increase of virus infection dose (figure 2)

Example 2

Method for inhibiting proliferation of H9N2 subtype avian influenza virus in DF-1 cell by utilizing gga-miR-30a-3p

(1) Synthesizing mimics (mimics) of gga-miR-30a-3p and negative control NC, transfecting DF-1 cells with the mimics/NC of the gga-miR-30a-3p with different concentrations (10 nm, 20 nm, 40 nm and 80 nm) respectively, after transfecting for 24 h, collecting the cells, extracting total RNA of the cells by using a Trizol reagent (Invitrogen company), performing reverse transcription by using a Thermo reverse transcription kit to synthesize cDNA, and detecting the expression level of the gga-miR-30a-3p by using fluorescent quantitative PCR;

(2) before H9N2 subtype avian influenza virus is inoculated (separation can be carried out by a conventional method), a virus stock solution is placed on ice to be slowly melted, DF-1 cells are transfected for 24H, H9N2 subtype avian influenza virus with MOI =0.5 is used for infecting the cells, the cells are collected after being infected for 24H, Trizol reagent is used for extracting total RNA of the cells, a Thermo reverse transcription kit is used for reverse transcription to synthesize cDNA, and the expression level of the virus RNA is detected by fluorescence quantitative PCR. The result shows that compared with a negative control NC group, after 40 nm concentration gga-miR-30a-3p mimics is transfected, the content of H9N2 subtype avian influenza virus in DF-1 cells can be reduced by 41% (figure 3), which shows that the gga-miR-30a-3p has good antiviral effect.

The invention provides a new design target point for the research and development of novel H9N2 subtype avian influenza prevention and control medicines. The miRNA sequence can be prepared into a pharmaceutical composition or a reagent for preventing and treating H9N2 subtype avian influenza.

Nucleotide sequence listing

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Claims

1. An application of an inhibitor of H9N2 subtype avian influenza virus in preparation of a medicament or a reagent for inhibiting H9N2 subtype avian influenza virus is characterized in that the inhibitor gga-miR-30a-3p mimics has a sequence shown in SEQ ID NO. 1.