CN109022437B

CN109022437B - Target site sequence for inhibiting goat parainfluenza virus type 3 replication and application thereof

Info

Publication number: CN109022437B
Application number: CN201810862854.9A
Authority: CN
Inventors: 李基棕; 钟纯燕; 毛立; 李文良; 郝飞; 孙敏; 刘茂军; 杨蕾蕾; 张纹纹
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2018-08-01
Filing date: 2018-08-01
Publication date: 2021-09-14
Anticipated expiration: 2038-08-01
Also published as: CN109022437A

Abstract

The invention relates to a target site sequence for inhibiting replication of goat parainfluenza virus type 3 (CPIV 3) and application thereof in preparation of anti-goat parainfluenza virus type 3 medicines. The target site sequence for inhibiting the 3-type replication of the goat parainfluenza virus is positioned in the 3' UTR of an interferon regulatory gene 2(IRF2), the nucleotide sequence of the target site sequence is shown as SEQ ID NO:19, and the target site sequence is directly targeted by a bta-miR-222 sequence, so that mRNA of IRF2 is degraded, the expression quantity of IRF2 protein is reduced, the expression level of the I-type interferon is improved, and CPIV3 proliferation is inhibited. The target site sequence provides a new drug target for developing new antiviral drugs for treating diseases caused by CPIV 3. In addition, the method can also be applied to the aspects of evaluating bta-miR-222 to regulate the transcription level of IRF2 and breeding anti-goat parainfluenza virus type 3 transgenic sheep.

Description

Target site sequence for inhibiting goat parainfluenza virus type 3 replication and application thereof

Technical Field

The invention belongs to the fields of molecular biology and antiviral science, and particularly relates to a target site sequence for inhibiting goat parainfluenza virus type 3 replication and application thereof in preparation of an anti-goat parainfluenza virus type 3 medicament.

Background

Goat parainfluenza virus type 3 (CPIV 3) is a novel member of the genus Respirovirus of the family Paramyxoviridae, a enveloped, single-stranded negative-strand RNA virus. Members of the genus also include human parainfluenza virus types 1 and 3 (HPIV1, HPIV3), Sendai Virus (SV), and bovine parainfluenza virus type 3 (BPIV 3). The pathogen is detected in goat farms in Jiangsu, Anhui and other places from 2013, a CPIV3 infected sheep can cause respiratory diseases of different degrees, and the homology of the genes with HPIV1 and HPIV3 is only 76.9-83.5 percent as found by RT-PCR amplification sequencing of N, M, F and HN genes. And then, the CPIV3 which is separated and identified in the laboratory is subjected to a goat pathogenicity test, and the result shows that the goat infected by the CPIV3 has clinical symptoms such as cough, rhinorrhea, fever, depression and the like, continuous viremia and toxin expelling are generated, severe pathological damage to the lung and the trachea can be seen through autopsy, and the CPIV3 can be horizontally transmitted through aerosol to enable adjacent healthy goats in the colony house to have respiratory symptoms, detect viremia and expel toxin, thereby bringing a new threat to the healthy development of the sheep industry. Epidemiological investigation in the laboratory shows that the detection rate of the CPIV3 pathogenic nucleic acid reaches 44.7 percent, and the detection rate of the antibody reaches 39.3 percent. Therefore, the hazard of the pathogen to the sheep industry should be paid sufficient attention. However, no report on the drug for treating the disease has been found so far.

Micro ribonucleic acid (miRNA) is a type of endogenous non-coding RNA with the length of about 19-24nt, participates in each link of regulating and controlling cell differentiation, proliferation, metabolism and apoptosis, and is closely related to reproductive development, tumor formation and virus infection process of animal organisms. Mature miRNA is identified and combined by RNA-induced silencing complex (RISC), and is complementarily combined with 5 'UTR, 3' UTR or coding region base of target mRNA, thereby degrading mRNA or inhibiting translation of mRNA into protein, and achieving the effect of regulating function after gene transcription. In recent years, mirnas have been extensively studied in antiviral therapy.

bta-miR-222 is found to be remarkably reduced (7.4 times) in a CPIV3 infected cell sample, but no report is made on the effect of bta-miR-222 in regulating virus proliferation. Based on the above, the bta-miR-222 is analyzed by an overexpression and inhibition expression test, and bta-miR-222 can obviously inhibit the replication of CPIV3 in MDBK cells. Therefore, the invention may provide a new therapeutic method or drug target for treating respiratory diseases caused by CPIV3, which is of great significance.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a target site sequence for inhibiting goat parainfluenza virus type 3 replication and application thereof in preparing a CPIV3 replication-resistant medicament, wherein the sequence is positioned on mRNA of an interferon regulatory gene 2(IRF2) and can be directly targeted by a bta-miR-222 sequence, so that the mRNA of IRF2 is degraded, the expression quantity of IRF2 protein is reduced, the I-type interferon expression level is improved, and CPIV3 proliferation is inhibited.

In order to solve the technical problems, the invention provides a target site sequence for inhibiting the 3-type replication of goat parainfluenza virus, wherein the target site is positioned in the 3' UTR of IRF2, and the nucleotide sequence of the target site is shown as SEQ ID NO. 19.

Preferably, the target site is directly targeted by bta-miR-222 sequence, and the bta-miR-222 nucleotide sequence is shown as SEQ ID NO: 1.

Further, the application of the target site sequence for inhibiting the replication of the goat parainfluenza virus type 3 in preparing a medicament for inhibiting the replication of the goat parainfluenza virus type 3.

bta-miR-222 sequence in the preparation of inhibition goat parainfluenza virus type 3 replication medicine application.

An application of a target site sequence for inhibiting the replication of goat parainfluenza virus type 3 in the preparation of a medicament for treating the goat parainfluenza virus type 3.

Application of bta-miR-222 sequence in preparation of medicine for treating goat parainfluenza virus type 3.

Preferably, the recombinant luciferase reporter vector pGL3-IRF 23' UTR is applied to the aspect of evaluating the transcription level of the bta-miR-222 regulatory interferon regulatory gene 2.

The application of the target site sequence for inhibiting the replication of the goat parainfluenza virus type 3 in breeding transgenic sheep resisting the goat parainfluenza virus type 3.

Compared with the prior art, the invention has the advantages that:

1. the invention provides a target site sequence for inhibiting goat parainfluenza virus type 3 replication, and proves that bta-miR-222 can be combined with the target site sequence to inhibit the goat parainfluenza virus type 3 replication, so that similar reports are not found at home and abroad at present, a new drug target is provided for researching and developing new antiviral drugs for treating diseases caused by CPIV3, and related prompts are provided for researching and developing other antiviral drugs.

2. The bta-miR-222 can inhibit the replication of CPIV3, and because miRNA is generated by the expression of cells, compared with other artificially synthesized medicaments, the miRNA has the advantages of low toxicity to organisms and good effect.

3. The target site sequence for inhibiting the replication of the goat parainfluenza virus 3 is positioned in a 3' non-coding region of an interferon regulatory gene 2, and the interference on the site does not cause obvious damage to receptor cells, so that the safety and feasibility of the anti-goat parainfluenza virus 3 medicament and the cultivation of anti-goat parainfluenza virus 3 transgenic sheep can be improved.

4. The target site sequence for inhibiting the replication of the goat parainfluenza virus type 3 provided by the invention can inhibit the replication of the goat parainfluenza virus type 3 on a molecular level, and can be applied to inhibiting other variants of the goat parainfluenza virus type 3 due to the fact that bta-miR-222 family is widely distributed and highly conserved.

Drawings

For a better illustration of the invention, the following figures are provided, which are provided for illustration of the invention only and are not limiting of the invention:

FIG. 1 shows the inhibitory effect of Bta-miR-222 on CPIV3 proliferation.

FIG. 2 is a schematic representation of base pairing of Bta-miR-222 with its target site.

FIG. 3 shows the regulatory effect of Bta-miR-222 on IRF 2.

FIG. 4 shows Bta-miR-222 regulating the expression changes of IFN-alpha and IFN-beta.

FIG. 5 shows the effect of transfection of si-IRF2 knock-out of the bta-miR-222 target gene on CPIV3 replication.

Detailed Description

The following examples are intended to further illustrate the invention but are not intended to limit it.

Example 1 cell transfection and infection with viruses

(1) Sequence synthesis: bta-miR-222 nucleotide sequence is shown as SEQ ID NO 1; 3 sections of siRNA sequences interfering IRF2, namely Si-IRF2 nucleotide sequences, are respectively shown as SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4. The above-mentioned nucleic acid fragments are each synthesized by Shanghai Jima BioLimited.

(2) Cell transfection: MDBK monolayers were prepared in 24-well plates and, when cells grew to 60% confluence, bta-miR-222 was transfected into MDBK cells, along with a transfection control (NC) set up. The transfection procedure was performed according to Lipofectamine 2000 instructions.

MDBK monolayers were prepared in 24-well plates and 3 fragments of si-IRF2 were transfected into MDBK cells when the cells grew to 60% confluence, along with a transfection control (NC). The transfection procedure was performed according to Lipofectamine 2000 instructions.

(3) Virus inoculation: 24h after transfection, 1000TCID was inoculated per well₅₀CPIV3, after 1.5h, the virus solution was discarded, replaced with 500. mu.L of 2% serum DMEM medium, incubated at 37 ℃ and further inoculated with non-transfected cells as a control. Cell supernatants were harvested at 24h and 48h after inoculation, respectively, and reverse transcription real-time quantitative PCR (RT-qPCR) and TCID were used₅₀The CPIV3 virus content was measured to evaluate the replication capacity of CPIV 3. Both the transfection bta-miR-222 and si-IRF2 were found to be effective in inhibiting CPIV3 proliferation, as shown in FIG. 1 and FIGS. 5C and 5D.

Example 2RT-qPCR technique and TCID₅₀Method for evaluating CPIV3 for its replication capability

Reverse transcription real-time quantitative PCR: total RNA in the cell supernatant was extracted with Transzol UP reagent, and One Step PrimeScript from TaKaRa was used^TMThe RT-PCR Kit detects the content of CPIV3 nucleic acid in the supernatant. Amplification was performed using an ABI Step One fluorescent quantitative PCR instrument, and the reaction system and reaction conditions of RT-qPCR were performed with reference to the kit instructions. The sequences of the upstream primer and the downstream primer used are qCPIV3F shown in SEQ ID NO. 5, qCPIV3R shown in SEQ ID NO. 6 and qCPIV3-probe shown in SEQ ID NO. 7, respectively. The results are shown in FIGS. 1(A) and 5 (C): the transfection bta-miR-222 and si-IRF2 can effectively inhibit the proliferation of CPIV 3.

TCID₅₀And (3) detection: the MDBK cells were seeded into a 96-well plate, the harvested virus solution was diluted 10-fold with a maintenance solution until the cells were grown to 90% confluency, and then the cells were seeded with each of the cell supernatants collected above, 100 μ L per well, 4 wells were repeated, cultured at 37 ℃ for 3d, and then the number of wells in which cytopathic effect occurred was observed for each dilution. Virus TCID calculated according to Reed-Muench method₅₀. The results are shown in FIGS. 1(B) and 5 (D): the transfection bta-miR-222 and si-IRF2 can effectively inhibit the proliferation of CPIV 3.

Example 3Bta-miR-222 target Gene prediction and Dual-luciferase reporter System validation bta-miR-222 target Gene

The bioinformatics software TargetScan is used for predicting the target gene and the binding site of bta-miR-222, and the potential target sequence (IRF 23 'UTR: 977-984bp) of bta-miR-222 exists in the 3' UTR of IRF2 gene, and the complementary site is shown in figure 2. Primers were designed based on the 3' UTR of IRF2 mRNA. The primer sequences are IRF2-3 'UTR-F shown in SEQ ID NO. 8 and IRF 2-3' UTR-R shown in SEQ ID NO. 9 respectively. RT-PCR amplification is carried out, the amplified product is cloned in a luciferase report vector pGL3-control (Promega) through Kpn I and Bgl II enzyme cutting sites, and a recombinant luciferase report vector pGL3-IRF 23' UTR is constructed. Carrying out transfection experiments in three groups, wherein the first group is 293T cells co-transfected by pGL3-IRF 23' UTR, pRL-CMV and bta-miR-222; the second group is 293FT cells co-transfected by pGL3-IRF 23' UTR, pRL-CMV and bta-miR-222-mut (seed site of mutation bta-miR-222); wherein the bta-miR-222-mut nucleotide sequence is shown in SEQ ID NO. 10; the third control group NC was pGL3-IRF 23' UTR, pRL-CMV and NC co-transfected 293FT cells. Changes in luciferase were analyzed using a dual-luciferase reporter assay kit (Promega). A transfection control group (NC) was also established.

Luciferase changes were analyzed using the dual-luciferase reporter assay kit, and the results are shown in FIG. 3A: the luciferase activity of the bta-miR-222 transfected group is reduced by about 55% compared with that of the control group, but the luciferase activity of the bta-miR-222-mut transfected group is not significantly different from that of the control group. The RT-qPCR is used for detecting the transcription level of IRF2mRNA, and the result is shown in figure 3B, the IRF2mRNA amount of the transfected bta-miR-222 group is obviously lower than that of the control group, and is reduced by about 30%. The expression level of the IRF2 protein is analyzed by Western blot, and the result shows that the expression level of the IRF2 protein in the bta-miR-222 group is obviously lower than that in the control group, and the figure is 3C.

Example 4Bta Effect of miR-222 on IRF2 and IFN production

MDBK monolayers were prepared in 24-well plates and, when cells were 60% confluent, bta-miR-222 was transfected into MDBK cells, along with a control of transfected (NC) and a control of non-transfected cells (Mock), according to Lipofectamine 2000. Cells were harvested 24h after transfection. Total RNA from the harvested cells was extracted, and IFN mRNA levels in the cells were detected by reverse transcription and RT-qPCR using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix and EasyScript One-Step RT-PCR SuperMix from Tokyo King. And (3) carrying out amplification by using an ABI Step One fluorescence quantitative PCR instrument, wherein the reaction system and the reaction conditions are carried out according to the kit instruction. The IFN expression changes of the transfected miRNA group and the transfected NC control group are calculated by taking GAPDH as an internal reference, and the result is expressed as-delta Ct. The used primer sequences are qIRF2-F shown in SEQ ID NO. 11, qIRF2-R shown in SEQ ID NO. 12, qIFN-alpha-F shown in SEQ ID NO. 13 and qIFN-alpha-R shown in SEQ ID NO. 14 respectively; qIFN-beta-F shown in SEQ ID NO. 15, qIFN-beta-R shown in SEQ ID NO. 16, GAPDH-F shown in SEQ ID NO. 17, and GAPDH-R shown in SEQ ID NO. 18. The result of fluorescent quantitative RT-PCR shows that the average expression levels of IFN-alpha and IFN-beta in cells are remarkably increased after Bta-miR-222 transfects the cells, and are respectively about 1.8 times and 2.1 times, as shown in figure 4.

Example 5 Effect of transfection of si-IRF2 on the bta-miR-222 target Gene

The 3 si-IRF2 described in example 1 were transfected into MDBK cells simultaneously with the establishment of a transfection control (NC), cells were harvested 24h later, and transcription levels of IRF2 gene were measured by RT-qPCR, the results are shown in fig. 5A: after si-IRF2 transfection, the expression level of IRF2 gene is obviously reduced. Western blot detection of the expression level of IRF2 protein showed that the expression level of IRF2 protein was significantly reduced after si-IRF2 transfection, as shown in FIG. 5B. Simultaneously inoculating CPIV3 virus solution after transfecting si-IRF 224 h, harvesting virus solution 24h after inoculation, RT-qPCR technology and TCID₅₀The virus content is detected, and the result shows that the si is transfectedThe IRF2 group had significantly lower CPIV3 virus content than the control group (NC), and the results are shown in FIGS. 5C and 5D.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

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Claims

1. bta-miR-222 for inhibiting goat parainfluenza virus type 3 replication is combined with a nucleotide sequence which is positioned in the 3' UTR of an interferon regulatory gene 2 and is shown as SEQ ID NO. 19, and the bta-miR-222 nucleotide sequence is shown as SEQ ID NO. 1.

2. The use of bta-miR-222 for inhibiting goat parainfluenza virus type 3 replication according to claim 1 in the preparation of a medicament for inhibiting goat parainfluenza virus type 3 replication.

3. The use of bta-miR-222 for inhibiting goat parainfluenza virus type 3 replication according to claim 1 in the preparation of a medicament for treating goat parainfluenza virus type 3.

4. Use of a recombinant luciferase reporter vector pGL3-IRF 23' UTR in assessing the transcriptional level of the bta-miR-222 regulatory interferon-regulated gene 2 that inhibits goat parainfluenza virus type 3 replication of claim 1.

5. The use of bta-miR-222 for inhibiting the replication of goat parainfluenza virus type 3 in breeding transgenic sheep resistant to goat parainfluenza virus type 3 according to claim 1.