CN113527463B - Application of IFITM3 protein related substance in preparation of medicine for treating diseases caused by avian reovirus - Google Patents

Abstract

The invention relates to an application of a substance for enhancing the activity of IFITM3 protein and/or increasing the expression quantity of the gene of the IFITM3 protein and/or increasing the content of the IFITM3 protein, wherein the application is any one of the following substances: the application of Y1 in preparing a medicament for preventing and/or treating diseases caused by the avian reovirus or infection of the avian reovirus; the application of Y2 in preparing the avian reovirus inhibitor; the application of Y3 in inhibiting the replication of avian reovirus; use of Y4 for inhibiting expression of ARV σ C. The invention discloses a method for inhibiting avian reovirus replication by transferring a gene for coding IFITM3 protein into a chicken embryo fibroblast (DF 1), and finding that the relative expression quantity of ARV sigma C is remarkably reduced after the IFITM3 is over-expressed in the DF1 cell. The invention has application value in the treatment of avian reovirus infection and similar viruses.

Description

Application of IFITM3 protein related substance in preparation of medicine for treating diseases caused by avian reovirus

Technical Field

The invention relates to the technical field of biology, in particular to application of IFITM3 protein-related substances in preparation of medicines for treating diseases caused by avian reoviruses.

Background

Avian Reovirus (ARV) belongs to the family of orthoreoviridae, the genus orthoreovirus, and is a double-stranded RNA virus without a membrane vesicle. The ARV is an important poultry immunosuppressive disease, has strong resistance to the environment, can be horizontally transmitted and vertically transmitted, can cause viral arthritis/tenosynovitis, short and small syndromes and the like in chicken flocks in China, particularly seriously induces immunosuppression, causes the reduction of the immune response capability of organisms to vaccines and the resistance capability of various pathogens, and causes great economic loss for poultry farmers due to other diseases which are complicated or secondary. At present, no effective treatment measures exist, and effective prevention and treatment are mainly carried out through vaccination; a plurality of attenuated vaccines and inactivated vaccines are available on the market, wherein the vaccines which are applied more are mainly two vaccine strains of S1133 and VM 0207.

Disclosure of Invention

The technical problem to be solved by the invention is how to prepare a medicament for preventing and/or treating diseases caused by the avian reovirus or avian reovirus infection, and/or how to prepare an avian reovirus inhibitor.

In order to solve the above technical problems, the present invention provides the following applications of a substance that enhances the activity of the IFITM3 protein and/or increases the expression level of the gene of the IFITM3 protein and/or increases the content of the IFITM3 protein:

the application of U1 in preparing medicaments for preventing and/or treating diseases caused by the avian reovirus or infection of the avian reovirus;

the application of U2 in preparing an avian reovirus inhibitor;

use of U3 for inhibiting expression of ARV σ C;

the IFITM3 protein is the protein of the following A1), A2) or A3):

a1 Protein of which the amino acid sequence is a sequence 1 in a sequence table;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein of A1), has more than 80% of identity with the protein shown in A1) and has the same activity;

a3 A fusion protein obtained by linking a protein tag to the N-terminus or/and C-terminus of A1) or A2).

Wherein, the sequence 1 in the sequence table is composed of 113 amino acid residues.

In the application, the protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above application, the protein-tag refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above application, the identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, perresilute Gap cost, and Lambda ratio to 11,1 and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of the identity can be obtained.

In the above applications, the 80% or greater identity may be at least 80%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the application, the avian reovirus inhibitor has the function of inhibiting the replication of avian reoviruses.

In the above application, the substance for enhancing the activity of the IFITM3 protein and/or increasing the expression level of the gene of the IFITM3 protein and/or increasing the content of the IFITM3 protein is a biological material related to the IFITM3 protein, and is any one of the following B1) to B5):

b1 A nucleic acid molecule encoding said IFITM 3;

b2 An expression cassette comprising the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B1);

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3);

b5 A transgenic animal cell line containing the nucleic acid molecule according to B1), or a transgenic animal cell line containing the expression cassette according to B2), or a transgenic animal cell line containing the recombinant vector according to B3).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above application, the nucleic acid molecule of B1) is a gene represented by B1) or B2) as follows:

b1 The coding sequence of the coding chain is a cDNA molecule or a DNA molecule of the nucleotide of the sequence 2 in the sequence table;

b2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 2 in the sequence table.

Wherein, the sequence 2 in the sequence table consists of 342 nucleotides and codes the protein shown in the sequence 1 in the sequence table.

In the above application, the expression cassette containing said nucleic acid molecule (IFITM 3 gene expression cassette) in B2) refers to a nucleic acid molecule capable of expressing IFITM3 in a host cell, which may include not only a promoter for initiating transcription of IFITM3 gene but also a terminator for terminating transcription of IFITM 3. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.

The recombinant expression vector containing the IFITM3 gene expression cassette can be constructed by using the existing eukaryotic expression vector. The eukaryotic expression vector can be pEF1 alpha-Myc, pcDNA3.1-HA, pEGFP and the like.

In the above application, the animal cell line may be chicken embryo fibroblast (DF 1).

In the application, the diseases caused by the avian reovirus can be avian viral arthritis, short and small syndrome, respiratory diseases, intestinal diseases, malabsorption syndrome and the like.

The invention also protects an avian reovirus inhibitor.

The inhibitor can be a substance which only enhances the activity of the IFITM3 protein and/or increases the expression quantity of the gene of the IFITM3 protein and/or increases the content of the IFITM3 protein, and can also contain a carrier or an excipient.

In the avian reovirus inhibitor, the substance that enhances the activity of the IFITM3 protein and/or increases the expression level of the gene of the IFITM3 protein and/or increases the content of the IFITM3 protein is a biological material related to the IFITM3 protein, and is any one of the following B1) to B5):

b1 A nucleic acid molecule encoding IFITM 3;

b2 An expression cassette comprising the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B1);

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3);

b5 A transgenic animal cell line containing the nucleic acid molecule according to B1), or a transgenic animal cell line containing the expression cassette according to B2), or a transgenic animal cell line containing the recombinant vector according to B3).

In the avian reovirus inhibitor, the nucleic acid molecule in B1) is a gene shown in B1) or B2) as follows:

b1 The coding sequence of the coding chain is a cDNA molecule or a DNA molecule of the nucleotide of the sequence 2 in the sequence table;

b2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 2 in the sequence table.

The invention discloses that the relative expression quantity of ARV sigma C is remarkably reduced after IFITM3 is over-expressed in DF1 cells by transferring the gene for coding the IFITM3 protein into chicken embryo fibroblast (DF 1), and the replication of Avian Reovirus (ARV) is inhibited. The invention has application value in the treatment of avian reovirus infection and similar viruses.

Drawings

FIG. 1 is a diagram of agarose gel electrophoresis of an IFITM3 fragment after PCR amplification in example 1 of the present invention. In the figure, M is DL2000 DNA Marker; lane 1 is the PCR amplification product and the arrow indicates the IFITM3 fragment.

FIG. 2 is a photograph of agarose gel electrophoresis of pEF 1. Alpha. -Myc-IFITM3 of example 1 of the present invention, which was verified by double digestion with SalI and Not I. In the figure, M is DL5000 DNA Marker;1 is pEF1 alpha-Myc-IFITM 3 plasmid; 2 is pEF1 alpha-Myc-IFITM 3 double enzyme cutting product, and an arrow indicates the IFITM3 gene fragment.

FIG. 3 is a graph showing the results of indirect immunofluorescence assay of IFITM3 eukaryotic expression in example 1 of the present invention. In the figure, A is a negative control group transfected by pEF1 alpha-Myc, and B is an experimental group transfected by pEF1 alpha-Myc-IFITM 3.

FIG. 4 is a diagram showing the result of Western-blot verifying IFITM3 eukaryotic expression in example 1 of the present invention.

FIG. 5 shows the relative expression level of ARV σ C detected by RT-qPCR in example 1 of the present invention. In the figure, vec is pEF1 alpha-Myc transfection, IFITM3 is pEF1 alpha-Myc-IFITM 3 transfection, data are shown as mean ± standard deviation, repeat number is 3, each group of significance difference is analyzed by t-tests, and represents that the significance analysis result is P <0.001.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.

In the following examples, the endotoxin-removing eukaryotic expression vector pEF 1. Alpha. -Myc is manufactured by Clontech under the accession number 631991.

In the following examples, chick embryo fibroblast (DF 1) and ARV virus S1133 strains are described in non-patent document "Mo Lijun, etc. preliminary analysis of eukaryotic expression and subcellular localization of avian reovirus σ C protein, chinese veterinary science, 2021, 51 (04): 0492-0499", available to the public from the university of Guangxi autonomous region veterinary research institute for the purpose of repeating the experiments of the present application, and not for other uses.

All primer synthesis and sequencing in the examples below was done by rebox biotechnology limited.

The quantitative tests in the following examples, unless otherwise specified, were set up in triplicate and the results averaged.

Example 1

The primers used in this experiment were as follows:

TABLE 1 primer sequences

Note that the underlined sequence is the SalI enzyme recognition site, and the double underlined sequence is the NotI enzyme recognition site.

1 recombinant expression vector pEF1 alpha-Myc-IFITM 3 construction and identification

IFITM3 is a protein whose amino acid sequence is the sequence 1 in the sequence table.

Taking cDNA reverse transcribed from total RNA extracted from chicken embryo fibroblast (DF 1) as a template, amplifying an IFITM3 gene (the nucleotide sequence is shown as a sequence 2 in a sequence table) by PCR (polymerase chain reaction) by using specific primers (shown as F and R aiming at the IFITM3 gene in the table 1), and detecting an amplified fragment with agarose gel electrophoresis, wherein the size of the amplified fragment is about 342bp (shown in the figure 1). The PCR product of the IFITM3 gene was ligated with the SalI and Not I double-digested linearized eukaryotic expression vector pEF1 α -Myc by T4 DNA ligase overnight at 16 ℃ to construct the recombinant plasmid pEF1 α -Myc-IFITM3: pEF1 alpha-Myc-IFITM 3 is a recombinant expression vector of IFITM3 gene obtained by replacing a fragment between SalI and Not I recognition sites of restriction endonucleases of pEF1 alpha-Myc vector (a small fragment including the recognition site of SalI and the recognition site of Not I) with IFITM3 gene of which the coding sequence (CDS) is sequence 2 in the sequence table, and keeping the other sequences of pEF1 alpha-Myc vector unchanged.

pEF1 alpha-Myc-IFITM 3 is transformed into DH5 alpha competent cells, pEF1 alpha-Myc-IFITM 3 recombinant bacteria are obtained by screening positive colonies, and SalI and Not I restriction enzyme double digestion verification and sequencing verification are carried out on the recombinant bacteria. The result of the double restriction enzyme digestion is shown in FIG. 2, and it can be seen from the figure that pEF1 alpha-Myc-IFITM 3 obtains two target bands after double restriction enzyme digestion, the sizes of the two target bands are consistent with those of the expected bands, and the carrier band is about 4622bp and the IFITM3 gene band is 342bp, which respectively shows that the IFITM3 gene is correctly inserted into the expression vector pEF1 alpha-Myc. The sequencing result also proves that the pEF1 alpha-Myc-IFITM 3 contains a nucleotide sequence shown in the sequence 2 in the sequence table, and the construction success of the eukaryotic expression vector pEF1 alpha-Myc-IFITM 3 is shown.

2 overexpression of the IFITM3 Gene in DF1 cells

Extracting pEF1 alpha-Myc-IFITM 3 recombinant plasmid and eukaryotic expression vector pEF1 alpha-Myc plasmid by using an endotoxin-free plasmid extraction kit.

DF1 cells are cultured in a 6-well plate, and when the cell density reaches about 80%, the DF1 cells are transfected by pEF1 alpha-Myc-IFITM 3 (experimental group) and pEF1 alpha-Myc (control group) respectively according to the using instruction of a Lipofectamine 3000, and are cultured in a 37 ℃ constant temperature cell incubator.

Indirect immunofluorescence and Western-blot validation of 3 IFITM3 protein expression

3.1 Indirect immunofluorescence assay

After transfection of DF1 cells for 48 hours in step 2 (pEF 1. Alpha. -Myc-IFITM3 (experimental group) and pEF 1. Alpha. -Myc (control group), respectively), the culture solution was discarded, washed three times with PBS, fixed 30min at 4 ℃ with 4% tissue cell fixative, 0.1% Triton-X-100 permeabilized 15min, blocked for 1 hour with 5 BSA blocking solution, anti-Myc tag Mouse mAb (product of ABCam Co.) (1 diluted 1000) was added as a primary antibody, incubated for 2h at 37 ℃ and washed three times with PBS, FITC-labeled goat Anti-Mouse IgG (product of Invitrogen Co.) (1 diluted 1000) was added as a secondary antibody, incubated for 1h at 37 ℃ in the dark and washed three times with PBS, and photographed by fluorescent microscope observation.

The results are shown in FIG. 3, green fluorescence can be observed in DF1 cells transfected by eukaryotic expression recombinant plasmid pEF1 alpha-Myc-IFITM 3, while green fluorescence is not observed in cells of a negative control group transfected by pEF1 alpha-Myc. Indicating that DF1 cells transfected by pEF1 alpha-Myc-IFITM 3 express IFITM3 protein, and DF1 cells transfected by pEF1 alpha-Myc plasmid do not express IFITM3 protein.

3.2 Western-blot verification

After transfection of DF1 cells for 48h in step 2 (pEF 1 α -Myc-IFITM3 (experimental group) and pEF1 α -Myc (control group), respectively), the culture solution was discarded, PBS washed three times, cells were sufficiently lysed with xTractor Buffer cell lysate to obtain a protein sample, expression of IFITM3 protein was verified by Western-Blot, anti-Myc tag Mouse mAb (ABCam product) (1. The results are shown in FIG. 4, a specific band of Myc-IFITM3 fusion protein can be detected in DF1 cells transfected by eukaryotic expression recombinant plasmid pEF1 alpha-Myc-IFITM 3, the size is about 12kDa, while a negative control group transfected by pEF1 alpha-Myc has no target band, which indicates that DF1 cells transfected by pEF1 alpha-Myc-IFITM 3 express IFITM3 protein, and DF1 cells transfected by pEF1 alpha-Myc plasmid do not express IFITM3 protein.

4 RT-qPCR detection of the effect of IFITM3 on ARV viral replication

Culturing DF1 cells in a 6-well plate, when the cell density reaches about 80%, transfecting pEF1 alpha-Myc-IFITM 3 into the cells by adopting a liposome transfection method to overexpress an IFITM3 gene, infecting an ARV virus S1133 strain after 24h, MOI =1, collecting cell samples 24h after infection, extracting RNA and reversely transcribing the RNA into cDNA, detecting the expression quantity of an ARV sigma C gene by a fluorescence quantitative PCR method, and evaluating the influence of overexpression of IFITM on the replication of the ARV virus by taking GADPH as an internal reference gene and pEF1 alpha-Myc transfection as a control. Primers for ARV σ C are shown in table 1 for F and R of the ARV σ C gene, and primers for internal reference GAPDH are shown in table 1 for F and R of the GAPDH gene. 3 sets of replicates were set and each set was analyzed for significant differences in t-tests.

The results are shown in fig. 5, and the relative expression amount of ARV σ C in the experimental group is lower than that in the control group, and the difference is very significant. Research finds that the sigma C protein participates in the outer capsid of the virus, is adsorbed to a receptor on the surface of a susceptible cell in the form of a homotrimer, plays an important role in the virus infection process, and can be used as an ARV detection index. Therefore, the inhibitor can be used for inhibiting ARV virus replication after IFITM3 is over-expressed in DF1 cells, can be used for preparing an avian reovirus inhibitor, and can be used for preventing and/or treating diseases caused by the avian reovirus.

The invention provides a new idea for the clinical treatment of ARV infection and has important significance for diagnosing and treating diseases caused by ARV infection.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> Guangxi Zhuang nationality autonomous region veterinary research institute

Application of <120> IFITM3 protein related substance in preparation of medicine for treating diseases caused by avian reovirus

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Claims (2)

1. The application of a substance for enhancing the activity of the IFITM3 protein and/or increasing the expression quantity of the gene of the IFITM3 protein and/or increasing the content of the IFITM3 protein, wherein the application is the application of inhibiting ARV sigma C expression for non-therapeutic purposes;

the IFITM3 protein is the protein of the following A1) or A2):

a1 Protein of which the amino acid sequence is a sequence 1 in a sequence table;

a2 A fusion protein obtained by connecting a protein tag to the N-terminus or/and the C-terminus of A1);

the substance for enhancing the activity of the IFITM3 protein and/or increasing the expression quantity of the gene of the IFITM3 protein and/or increasing the content of the IFITM3 protein is a biological material related to the IFITM3 protein, and is any one of the following B1) to B5):

b1 A nucleic acid molecule encoding said IFITM 3;

b2 An expression cassette comprising the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3);

b5 A transgenic animal cell line containing the nucleic acid molecule according to B1), or a transgenic animal cell line containing the expression cassette according to B2), or a transgenic animal cell line containing the recombinant vector according to B3).

2. Use according to claim 1, characterized in that: b1 The nucleic acid molecule is a gene shown in the following b 1) or b 2):

b1 The coding sequence of the coding chain is a cDNA molecule or a DNA molecule of the nucleotide of the sequence 2 in the sequence table;

b2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 2 in the sequence table.

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