Micropeptide coded by long-chain non-coding RNA MALAT1 and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a micropeptide coded by long-chain non-coding RNA MALAT1 and application of the micropeptide, and the micropeptide is specifically a human-derived micropeptide named as miPEP5-P52 and is applied to development of a novel vaccine as a replication enhancer. The test proves that the micropeptide miPEP5-P52 coded by the human MALAT1 gene can promote the replication of the influenza virus. Moreover, the miPEP5-P52 is highly expressed in the mouse body, the virus load is enhanced after the influenza virus stimulation, and the death of the mouse is promoted. The invention reveals that the micropeptide miPEP5-P52 coded by the human MALAT1 gene can significantly enhance influenza virus replication on both cell and animal levels, can be used as a replication enhancer of influenza virus vaccines, and provides a new choice for development of influenza virus vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a micropeptide encoded by long-chain non-coding RNA MALAT1 and an application thereof. Background Art

[0002] Influenza virus, the full name of influenza virus, belongs to the genus influenza virus of the family Orthomyxoviridae according to species classification. Influenza virus is a single-stranded negative-sense RNA virus, which is divided into four subtypes: A, B, C, and D. Among them, influenza A virus has the widest range of transmission and can infect a variety of mammals and poultry. Wild waterfowl are its natural hosts. It is also the pathogen of human influenza pandemic and seasonal influenza. Compared with the other three types of influenza viruses, influenza A virus (IAV) has a wide geographical range, strong pathogenicity, and many transmission hosts. According to genetic evolutionary tree analysis, influenza A virus can be divided into four types: classical, human-like, avian-like, and recombinant. Horses, marine animals, and even tigers can be infected with influenza A virus and spread between the same species.

[0003] In human history, influenza A virus has caused four pandemics, which not only caused huge losses to the global economy, but also caused tens of millions of deaths. The origin of the human influenza outbreak was the Spanish flu in 1918, which caused three major outbreaks in 1918 alone, resulting in the loss of 50 million lives worldwide. At present, vaccination is the main method for the prevention and treatment of influenza A in society, whether for humans or animals, but the demand for it is increasing, especially the demand for vaccines. However, the virus replication does not require the participation of RNA proofreading enzymes and has typical easy mutation characteristics; and in vaccine research and development, the viral load it produces is low, which is far from meeting the development and growth needs of IAV vaccines by relevant companies and unit researchers in various rapidly developing research. The relevant products currently on the market that increase viral load have obvious technical defects in their working principles; their effects cannot achieve the purpose of significantly increasing viral load. In addition, there is no good solution to the problem of how to increase viral load in vaccine research and development on the market. Therefore, how to obtain an effective and safe IAV replication enhancer to solve the problem of low viral yield currently encountered in production and practice has become one of the problems to be solved in the prior art.

[0004] With the continuous innovation of biotechnology and the deepening of high-throughput sequencing, researchers have found that small open reading frames (sORFs) on long non-coding RNA gene sequences can encode small proteins of less than 100 amino acids, which are called micropeptides. A large number of experiments have confirmed that processed and modified micropeptides can play corresponding functions, and some micropeptides processed by long non-coding RNA can act on themselves to promote or inhibit the expression of their own long non-coding RNA. In addition, some researchers have reported on the research and application of enhancers. For example, the cell cycle protein CCND1 can be used as an enhancer in the production of avian retroviruses, and can inhibit the replication of viruses when CCND1 is knocked down; there are also reports that CTHRC1 can be used as a replication enhancer of J subgroup avian leukosis virus, and high expression of CTHRC1 can effectively promote the replication of the virus; in addition, it has been found that host kinase, double cortex adrenergic-like kinase 1 (dclk1) can also promote the replication of J subgroup avian leukosis virus as its replication enhancer. Summary of the invention

[0005] Based on the above, the object of the present invention is to provide a micropeptide miPEP5-P52 encoded by long non-coding RNA MALAT1 and its application.

[0006] To achieve the above objectives, the present invention provides a micropeptide miPEP5-P52, wherein the micropeptide miPEP5-P52 is encoded by the long non-coding RNA MALAT1, and the amino acid sequence of the miPEP5-P52 is shown in SEQ ID No.2.

[0007] Furthermore, the base sequence encoding the micropeptide miPEP5-P52 is shown in SEQ ID No.1.

[0008] The micropeptide miPEP5-P52 can be used to prepare influenza virus replication enhancers or influenza virus vaccine replication enhancers.

[0009] Experiments have shown that by synthesizing miPEP5-P52 in vitro and adding it to cells, miPEP5-P52 can promote influenza virus replication and increase the replication load of influenza viruses compared with the control group after influenza virus infection. miPEP5-P52 was injected into mice through the tail vein, and after influenza virus infection, it was able to enhance the replication load of influenza viruses and promote the death of mice. In summary, the present invention provides a micropeptide miPEP5-P52 that can enhance the replication load of influenza viruses, providing a new option for the preparation of influenza virus vaccine replication enhancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0011] Figure 1 The effect of micropeptide miPEP5-P52 on influenza virus replication at the cellular level. * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).

[0012] Figure 2 The effect of micropeptide miPEP5-P52 on influenza virus replication in mice. ** indicates extremely significant difference (P<0.01); *** indicates extremely significant difference (P<0.001). DETAILED DESCRIPTION

[0013] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods and devices in the following examples are conventional methods and devices unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The determination of the base sequences in the following examples is determined by sequencing by conventional sequencing companies. In order to make the purpose, technical solutions and advantages of the present invention clearer, the specific embodiments of the present invention are described in detail below in conjunction with the specific embodiments. Examples of these preferred embodiments are illustrated in the specific embodiments.

[0014] It should also be noted that, in order to avoid obscuring the technical solution of the present invention due to unnecessary details, only the technical solutions and / or processing steps closely related to the solution of the present invention are shown in the embodiments, while other details that are not very relevant are omitted.

[0015] 293T cells (human embryonic kidney cell line): American Type Culture Collection (ATCC), number CRL-3216.

[0016] Example 1. Micropeptide miPEP5-P52 can enhance the replication load of influenza virus

[0017] 1. Acquisition of miPEP5-P52 gene fragment

[0018] In the present invention, the miPEP5-P52 nucleotide sequence (nucleotide sequence as shown in SEQ ID No. 1) in human MALAT1 (GenBank accession number: NR_002819.4) is used as a template, and the micropeptide miPEP5-P52 (amino acid sequence as shown in SEQ ID No. 2) is synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. After the micropeptide is synthesized, it is verified according to the mass spectrometry results and then a subsequent cell test is carried out.

[0019] 2. Micropeptide miPEP5-P52 promotes influenza virus replication

[0020] 293T cells were cultured in DMEM (Gibco) medium supplemented with 100 units / mL penicillin, 100 units / mL streptomycin, and 10% fetal bovine serum (Gibco) at a final concentration of 100 units / mL and placed at 37°C with 5% CO 2 When the confluence of cells in the 6-well plate reached 80-90%, the micropeptide miPEP5-P52 was added to the 293T cells at a final concentration of 5 μM. After 12 hours, the complete culture medium was discarded and the cells were infected with influenza virus WSN (100 HAU / ml) for 1 hour. The cells were then washed three times with PBS and the virus maintenance solution was added. The supernatant was collected for 10-24 hours for hemagglutination and plaque assays. Figure 1 As shown, the micropeptide miPEP5-P52 can significantly promote the replication of influenza virus. According to the results of the plaque assay, the addition of the micropeptide miPEP5-P52 can enhance the replication load of influenza virus.

[0021] Example 2. Micropeptide miPEP5-P52 can enhance influenza virus replication and promote mouse death

[0022] Take out SPF mice of appropriate age from the mouse room. Before the experiment, clip the toes of the mice and number them, and divide them into blank group, control group and experimental group. The blank group, control group and experimental group were injected with miPEP5-P52 through the tail vein 12h and 0h before influenza virus infection, respectively. After the three groups were pretreated, take out the virus diluted with PBS, ether, and the glass bottle containing cotton. Add 2-3mL of ether to the bottle. Take out the mouse and put it in a glass bottle containing ether. After it is anesthetized, it is infected with mouse virus through nasal mucosal infection. Half of the mice in the three groups were infected with the virus (WSN), and the other half of the mice were added with an equal volume of PBS. The survival rate of the mice was counted every day, and the lung tissue of the infected mice was taken for plaque test. As Figure 2 As shown, the experiment showed that at the animal level, the micropeptide miPEP5-P52 could significantly promote the replication of influenza virus and promote the death of mice. The plaque experiment showed that miPEP5-P52 could enhance the viral load of influenza virus in mice.

[0023] Based on the results of the above two examples, it can be proved that the micropeptide miPEP5-P52 has the function of promoting influenza virus replication and enhancing the influenza virus replication load.

[0024] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A micropeptide miPEP5-P52, characterized in that: The micropeptide miPEP5-P52 is encoded by the long non-coding RNA MALAT1, and the amino acid sequence of the micropeptide miPEP5-P52 is shown in SEQ ID No.

2.

2. The micropeptide miPEP5-P52 according to claim 1, characterized in that The nucleotide sequence encoding the micropeptide miPEP5-P52 is shown in SEQ ID No.

1.

3. Use of the micropeptide miPEP5-P52 as claimed in claim 1 in the preparation of an influenza virus replication enhancer.

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