阅读说明：本技术 lncRNA NONSUST0067151在抑制乙脑病毒增殖中的应用 (Application of lncRNA NONSUST0067151 in inhibition of encephalitis B virus proliferation ) 是由 周晓龙 杜程涛 汪涵 杨松柏 李向臣 赵阿勇 于 2019-10-17 设计创作，主要内容包括：lncRNA NONSUST006715.1在抑制乙脑病毒增殖中的应用,属于生物技术领域。本申请公开了lncRNA NONSUST006715.1在抑制乙脑病毒增殖中的应用。本发明提出了一种具有抑制JEV增殖功能的宿主lncRNA,对于防治乙脑病具有重要意义。(An application of lncRNA NONSUST006715.1 in inhibiting the proliferation of Japanese encephalitis virus belongs to the technical field of biology. The application discloses application of lncRNA NONSUST006715.1 in inhibiting the proliferation of encephalitis B virus. The invention provides host lncRNA with the function of inhibiting JEV proliferation, which has important significance for preventing and treating the encephalopathy.)

Use of lncRNA nonossut 006715.1 for inhibiting the proliferation of encephalitis b virus.

Application of lncRNA NONSUST006715.1 in preparation of medicines for inhibiting encephalitis B virus proliferation.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of lncRNA NONSUST006715.1 in inhibition of Japanese encephalitis virus proliferation.

Background

Epidemic encephalitis B is a mosquito-borne zoonosis caused by Japanese Encephalitis Virus (JEV), occurring mainly in Asia and Pacific countries. The pig is the most main natural proliferation and overwintering host of the JEV, plays an important role in the transmission of 'pig-mosquito-human', and the JEV infecting the swinery in China mainly exists in the forms of I and III genotypes. For the pig industry, epidemic encephalitis B virus still is one of important factors influencing the reproductive capacity of pigs at present, the pigs are susceptible to various breeds and sexes, the pigs are infected more in 7-9 months every year, the infection rate is high, and the incidence rate is 20-30%. The infection of JEV can cause abortion of pregnant sows, and dead fetus, weak fetus and mummy fetus are produced; the affected boars have orchitis, testicles are shrunk and hardened, even lose the function of producing sperm, and finally lose the capability of mating and breeding and are eliminated; the piglets die from encephalitis caused by JEV, so that the expansion of the number of swinery is influenced, and huge economic loss is caused, thereby seriously influencing the development of the pig industry. Because a large amount of vaccines and traditional veterinary drugs are used to have adverse effects on the disease resistance and the environment of the organism to a certain extent, the search for antiviral molecular agents has very important theoretical and practical significance for preventing and treating JEV.

JEV is an acute infectious virus belonging to the flavivirus genus of the Flaviviridae family. The viral genome is single-stranded positive-strand RNA with the total length of 11 kb, and is sequentially provided with structural protein C, E, M and non-structural proteins NS 1-NS 5 from 5 'to 3', the viral RNA is directly translated into the structural protein and the non-structural proteins as mRNA in cytoplasm of a host cell, and the structural protein and the non-structural proteins are assembled and matured in cytoplasmic rough endoplasmic reticulum and finally released by budding. There has been more systematic study of the molecular mechanisms by which JEV invades host cells. In mouse and rat neuronal cells, scientists have found that entry of JEV into cells is mainly dependent on dynamin and caveolae-like endocytosis, but that entry of JEV into cells such as C6/36, Vero, PK15 is latticed protein dependent. Yang et al found that the cellular protein-mediated endocytosis pathway plays a key role in the process of JEV invading PK15 cells. The study of Zhou et al, which reports for the first time the molecular details of the transport of JEV-infected BHK21 cells to lysosomes via vesicles, found that in BHK21 cells, the latticed proteins mediate the process of the endocytosis of JEV particles into cells, then the transport to circulating vesicles Rab11 via vesicle Rab5 protein and finally the virus to lysosomes. Long non-coding RNAs (lncrnas) are a class of non-coding RNAs greater than 200 nucleotides in length, originally thought to be "noise" of genome transcription, with no biological function. In recent years, the interest of scholars at home and abroad on lncRNA is increasing day by day, and researches show that lncRNA plays an important role in life activities such as transcriptional level regulation, chromosome modification, epigenetic regulation, intranuclear transportation and the like. lncRNA has received much attention in recent years as a functionally diverse non-coding RNA in research relating to breeding for disease resistance. There is a growing body of evidence that the transcription of lncRNA in a host is greatly changed after viral infection, and a plurality of studies prove that lncRNA can play an important role in host reaction, natural immunity and other processes caused by viral infection.

After the JEV infects the porcine PK15 cells, a porcine lncRNA and mRNA transcriptome sequencing and lncRNA related experiment are carried out to screen a porcine lncRNA NONSUST006715.1 which can obviously inhibit the proliferation of the Japanese encephalitis virus, so that an antiviral gene is continuously expressed in an animal body by the transcription activation technology of CRISPR-dCas9-VPR and the like to culture an antiviral new strain of the porcine.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme for application of lncrnannosust006715.1 in inhibition of encephalitis B virus proliferation.

The invention is realized by the following technical scheme:

the lncRNA NONSUST006715.1 is applied to inhibiting the proliferation of the Japanese encephalitis virus.

The lncRNA NONSUST006715.1 is applied to preparation of a medicament for inhibiting the proliferation of the Japanese encephalitis virus.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides host lncRNA with the function of inhibiting JEV proliferation, which has important significance for preventing and treating the encephalopathy.

Drawings

FIG. 1 is an analysis of differentially expressed IncRNA and mRNA transcripts. A, statistics of differentially expressed lncRNA and mRNA transcripts. B, differentially expressed lncRNA volcano plot. C, clustering analysis of differentially expressed lncRNA. D, clustering analysis of differentially expressed mrnas.

FIG. 2 shows the results of fluorescence quantitative RCR of antisense AOS-knocked-down lncRNA NONSUST 006715.1. A, detecting the expression level difference of the target lncRNA after different antisense nucleic acids are transfected by fluorescent quantitative PCR. And B, detecting the knocking-down effect of the lncRNA of different purposes at different times after ASO1 transfection by fluorescent quantitative PCR.

FIG. 3 shows the results of immunofluorescence and western blot experiments for detecting the expression level of JEV virus proteins, wherein A knockdown of lncRNA NONSUST006715.1 has an effect on Japanese encephalitis virus structural protein E, and B knockdown of lncRNA NONSUST006715.1 has an effect on Japanese encephalitis virus nonstructural protein NS 3.

FIG. 4 shows the results of the fluorescent quantitative PCR detection of the expression level of lncRNA NONSUST006715.1 and the western blot detection of the JEV virus non-structural protein NS 3. A, detecting the expression level of lncrnanonsust006715.1 by fluorescent quantitative PCR after different inhibitors are treated, and B, detecting the expression level of the JEV virus non-structural protein NS3 by western blot experiment after different inhibitors are treated.

Detailed Description

The present invention is further described below in conjunction with specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. These examples are merely illustrative and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.