Application of PRKRA gene as target in inhibiting replication of peste des petits ruminants virus

文档序号：16770 发布日期：2021-09-21 浏览：23次中文

阅读说明：本技术 Prkra基因作为靶点在抑制小反刍兽疫病毒复制中的应用 (Application of PRKRA gene as target in inhibiting replication of peste des petits ruminants virus ) 是由郑海学陈淑莹朱紫祥杨帆曹伟军齐晓兰唐闻达马昭张向乐刘湘涛于 2021-07-06 设计创作，主要内容包括：本发明属于生物基因工程技术领域,具体涉及一种PRKRA基因作为靶点在抑制小反刍兽疫病毒复制中的应用。本发明首先发现通过抑制或沉默宿主PRKRA基因,能够抑制小反刍兽疫病毒的复制,可作为靶点用于制备抑制小反刍兽疫病毒复制的药物；其次,以PRKRA基因为靶点,本发明设计了小干扰RNA,所述小干扰RNA能够干扰小反刍兽疫病毒复制,可用于制备抑制小反刍兽疫病毒的复制的药物；并且,本发明提供了一种特异性靶向PRKRA基因的sgRNA,所述的sgRNA能够特异性靶向PRKRA基因,结合CRISPR-Cas9技术实现了PRKRA基因的完全敲除,获得的单克隆细胞系PRKRA-KOs对PPRV具有抗性表型,能够显著抑制PPRV在细胞内的复制,为进一步研究PRKRA基因在细胞内调控病原微生物复制的分子机制提供研究工具和材料。(The invention belongs to the technical field of biological gene engineering, and particularly relates to application of a PRKRA gene as a target spot in inhibiting the replication of peste des petits ruminants virus. The invention firstly discovers that the replication of the peste des petits ruminants virus can be inhibited by inhibiting or silencing a PRKRA gene of a host, and the peste des petits ruminants virus can be used as a target for preparing a medicament for inhibiting the replication of the peste des petits ruminants virus; secondly, with PRKRA gene as a target spot, the invention designs small interfering RNA which can interfere the replication of the peste des petits ruminants virus and can be used for preparing a medicament for inhibiting the replication of the peste des petits ruminants virus; moreover, the sgRNA of the specific targeting PRKRA gene can specifically target the PRKRA gene, the complete knockout of the PRKRA gene is realized by combining the CRISPR-Cas9 technology, the obtained monoclonal cell line PRKRA-KOs has a resistance phenotype to PPRV, the replication of PPRV in cells can be obviously inhibited, and research tools and materials are provided for further researching the molecular mechanism of the PRKRA gene for regulating the replication of pathogenic microorganisms in the cells.)

The application of the PRKRA gene as a target spot in screening of drugs for preventing or treating peste des petits ruminants is characterized in that the drugs use the PRKRA gene as a target spot and inhibit or silence the expression of the PRKRA gene.

2. Use of an agent or medicament for inhibiting PRKRA gene expression in the manufacture of a medicament for the prevention or treatment of peste des petits ruminants virus infection.

3. The use of claim 2, wherein the agent or drug is a small interfering RNA targeted to the PRKRA gene.

4. The use of claim 3, wherein the small interfering RNA has the sequence:

PRKRA-siRNA-F:5’-CCCAGUUUAUGAAUGUGAATT-3’；

PRKRA-siRNA-R:5’-UUCACAUUCAUAAACUGGGTT-3’。

5. the use of claim 2, wherein the agent or drug comprises an mRNA sequence of sgRNA and/or Cas9 protein targeted for knockout of the PRKRA gene.

6. The use of claim 5, wherein the medicament delivers an mRNA sequence carrying a sgRNA and/or Cas9 protein targeting the PRKRA gene via a drug delivery vector to inhibit PRKRA gene expression in the animal.

7. The use of claim 6, wherein the drug delivery vehicle is a liposomal nanoparticle.

8. The use of claim 5, wherein the sgRNA includes any one of PRKRA-sgRNA1 and PRKRA-sgRNA2, and the nucleotide sequence of the sgRNA is:

PRKRA-sgRNA1-F：5’-GCCACTGTCCTCGCGCTCCAG-3’；

PRKRA-sgRNA1-R：5’-CTGGAGCGCGAGGACAGTGGC-3’；

PRKRA-sgRNA2-F：5’-GAGATGATAACAGCTAAGCCA-3’；

PRKRA-sgRNA2-R：5’-TGGCTTAGCTGTTATCATCTC-3’。

9. an sgRNA specifically targeting a PRKRA gene knockout, the sgRNA comprising any one of PRKRA-sgRNA1 and PRKRA-sgRNA2, the sgRNA having the nucleotide sequence:

PRKRA-sgRNA1-F：5’-GCCACTGTCCTCGCGCTCCAG-3’；

PRKRA-sgRNA1-R：5’-CTGGAGCGCGAGGACAGTGGC-3’；

PRKRA-sgRNA2-F：5’-GAGATGATAACAGCTAAGCCA-3’；

PRKRA-sgRNA2-R：5’-TGGCTTAGCTGTTATCATCTC-3’。

10. use of the sgRNA of claim 9 in the preparation of a PRKRA gene knockout cell line.

11. A method for constructing a PRKRA gene knockout cell line, comprising the steps of:

(1) preparing sgRNA of claim 9 specifically targeting PRKRA gene, adding CACC cohesive end at 5 'end of forward sequence of sgRNA fragment and AAAC cohesive end at 5' end of reverse sequence as sgRNA oligonucleotide targeting PRKRA gene;

(2) inserting the double-stranded fragment prepared in the step (1) into a multiple cloning site of a Cas9 expression vector to obtain a recombinant vector for simultaneously expressing a Cas9 protein gene and a targeting sgRNA sequence;

(3) transfecting the recombinant vector prepared in the step (2) into a host cell, selecting a single cell, inoculating and culturing to obtain a PRKRA gene function deletion cell line.

12. A PRKRA knock-out cell line constructed according to the method of claim 11.

Technical Field

The invention belongs to the technical field of biological gene engineering, and particularly relates to application of a PRKRA gene as a target spot in inhibiting the replication of peste des petits ruminants virus.

Background

Peste des petits ruminants virus (PPRV) belongs to the genus Morbillivirus (Paramyxoviridae) and the genus Morbillivirus (Morblinavirus) belongs to the family Paramyxoviridae, and RNA viruses which are single-stranded, negative-strand and non-segmented mainly infect Peste ruminants such as goats and sheep. PPRV mainly attacks lymphatic tissues, epithelial cells of digestive tracts and respiratory tracts, is clinically mainly manifested by symptoms of acute fever, enteritis, bronchopneumonia, abortion of pregnant ewes and the like, and causes huge economic loss to livestock breeding industries in China and even countries in global development. Currently, vaccination is the most effective method to combat peste des petits ruminants. Compared with inactivated vaccines, the attenuated vaccines (Nigeria 75/1 and Sungri 96) have better immune effect and longer duration. But the research on the aspects of etiology, pathogenic mechanism and the like has not made a great breakthrough. Therefore, the mechanism of host protein for inhibiting PPRV replication is deeply understood, and the construction of a cell line for inhibiting PPRV virus replication by the CRISPR-Cas9 technology has great significance for the production of Peste des petits ruminants vaccines.

The emergence of RNA interference technology provides possibility for selective toxicity and provides a new idea for antiviral research. RNA interference is an RNA sequence-specific post-transcriptional gene silencing phenomenon. Compared with the traditional means of antiviral treatment, the RNA interference mediated antiviral effect has high specificity and almost has no influence on the expression of non-homologous genes, so that the adverse reaction can be reduced to the minimum; RNA interference can effectively inhibit virus replication, and a small amount of siRNA can achieve the effect of reducing virus expression products; RNA interference can act against conserved regions of the viral genome, limiting to some extent the ability of the virus to produce escape mutants. Therefore, designing synthetic siRNAs targeting the peste des petits ruminants viral genome would likely be an effective way to suppress peste des petits ruminants viral infection.

PRKRA (interferon-induced double-stranded RNA-dependent protein kinase activator A) is a PKR activator independent of ds RNA in cells. Previous studies have shown that PRKRA or PACT (PKR-activating protein) can still activate PKR in the absence of viral infection or ds RNA stimulation of cells, leading to phosphorylation of its downstream molecule eIF2 α, thereby inhibiting host cell translation function and ultimately leading to apoptosis. Normally, PRKRA is present as widely in various tissues and cells as PKR, and remains at a low level, and its expression level increases significantly when cells are altered by environmental stimuli. PRKRA plays an important physiological role in the development of mammals, and is involved in the process of cell growth and tumor development, and PRKRA can also interact with Rb protein to alter the progression of the cell cycle and thereby regulate the growth of cells. PRKRA also plays an important role in tumor treatment, and researches find that PRKRA has the function of promoting cancer cell proliferation, so that a new target point is provided for tumor treatment research.

The invention discovers that the replication of PPRV can be inhibited by inhibiting or silencing PRKRA gene of a host, and the PPRV can be used as a target for preparing a medicament for inhibiting the replication of peste des petits ruminants virus. Based on the method, the PRKRA gene is taken as a target spot, and the small interfering RNA is designed, can interfere the replication of PPRV, and can be used for preparing the medicament for inhibiting the replication of PPRV. In order to further research the molecular mechanism of PRKRA gene regulating the replication of pathogenic microorganisms in cells, the invention designs sgRNA of the specific targeting PRKRA gene, the sgRNA can specifically target the PRKRA gene, the complete knockout of the PRKRA gene is realized by combining the CRISPR-Cas9 technology, the obtained monoclonal cell line PRKRA-KOs has a resistance phenotype to PPRV, the replication of PPRV in cells can be obviously inhibited, research tools and materials are provided for researching the molecular mechanism of PRKRA gene regulating the replication of pathogenic microorganisms in cells, and the method can also be used for animal breeding of PPRV resistance.

Disclosure of Invention

Aiming at the technical problems, the invention firstly discovers that the replication of the peste des petits ruminants virus can be inhibited by inhibiting or silencing the PRKRA gene of a host, and the PPRV can be used as a target for preparing a medicament for inhibiting the replication of the peste des petits ruminants virus; secondly, with PRKRA gene as a target spot, the invention designs small interfering RNA which can interfere the replication of the peste des petits ruminants virus and can be used for preparing a medicament for inhibiting the replication of the peste des petits ruminants virus; moreover, the sgRNA of the specific targeting PRKRA gene can specifically target the PRKRA gene, the complete knockout of the PRKRA gene is realized by combining the CRISPR-Cas9 technology, the obtained monoclonal cell line PRKRA-KOs has a resistance phenotype to PPRV, the replication of PPRV in cells can be obviously inhibited, and research tools and materials are provided for further researching the molecular mechanism of the PRKRA gene for regulating the replication of pathogenic microorganisms in the cells. The method specifically comprises the following steps:

in a first aspect, the invention provides a use of PRKRA gene as a target in screening a medicament for preventing or treating peste des petits ruminants, wherein the medicament uses PRKRA gene as a target to inhibit or silence the expression of PRKRA gene.

In a second aspect, the invention provides an agent or medicament for inhibiting PRKRA gene expression for use in the preparation of a medicament for preventing or treating peste des petits ruminants virus infection.

Preferably, the agent or drug is a small interfering RNA designed to target the PRKRA gene.

Preferably, the sequence of the small interfering RNA is:

PRKRA-siRNA-F:5’-CCCAGUUUAUGAAUGUGAATT-3’；

PRKRA-siRNA-R:5’-UUCACAUUCAUAAACUGGGTT-3’。

preferably, the agent or drug comprises an mRNA sequence of sgRNA and/or Cas9 protein targeted for knockout of the PRKRA gene.

Preferably, the drug delivers the mRNA sequence carrying sgRNA and/or Cas9 protein targeting PRKRA gene to inhibit PRKRA gene expression in animals via a drug delivery vector.

Preferably, the drug delivery vehicle is a liposomal nanoparticle.

Preferably, the sgRNA includes any one of PRKRA-sgRNA1, PRKRA-sgRNA2, the nucleotide sequence of the sgRNA being:

PRKRA-sgRNA1-F：5’-GCCACTGTCCTCGCGCTCCAG-3’；

PRKRA-sgRNA1-R：5’-CTGGAGCGCGAGGACAGTGGC-3’；

PRKRA-sgRNA2-F：5’-GAGATGATAACAGCTAAGCCA-3’；

PRKRA-sgRNA2-R：5’-TGGCTTAGCTGTTATCATCTC-3’。

in a third aspect, the invention provides an sgRNA specifically targeting to knock out PRKRA gene, the sgRNA including any one of PRKRA-sgRNA1 and PRKRA-sgRNA2, the nucleotide sequence of the sgRNA being:

PRKRA-sgRNA1-F：5’-GCCACTGTCCTCGCGCTCCAG-3’；

PRKRA-sgRNA1-R：5’-CTGGAGCGCGAGGACAGTGGC-3’；

PRKRA-sgRNA2-F：5’-GAGATGATAACAGCTAAGCCA-3’；

PRKRA-sgRNA2-R：5’-TGGCTTAGCTGTTATCATCTC-3’。

in a fourth aspect, the invention provides a use of the sgRNA of the third aspect in preparing a PRKRA knockout cell line.

In a fifth aspect, the invention provides a method for constructing a PRKRA gene knockout cell line, wherein the PRKRA gene encoding protein is disabled in a host cell by gene targeting technology.

Preferably, the method is a CRISPR-Cas9 technique.

Preferably, the method comprises the steps of:

(1) preparing sgRNA of the third aspect specifically targeting PRKRA gene, adding CACC cohesive end at the 5 'end of the forward sequence of the sgRNA fragment, and AAAC cohesive end at the 5' end of the reverse sequence to serve as sgRNA oligonucleotide of the targeting PRKRA gene;

(3) transfecting the recombinant vector prepared in the step (2) into a host cell, selecting a single cell, inoculating and culturing to obtain a PRKRA gene function deletion cell line.

In a sixth aspect, the present invention provides a PRKRA knock-out cell line constructed according to the method of the fifth aspect.

In a seventh aspect, the invention provides a use of the PRKRA knockout cell line of the sixth aspect for detection of non-diagnostic therapeutic purposes.

In an eighth aspect, the present invention provides an application of the PRKRA gene-encoded protein loss-of-function cell line in breeding against peste des petits ruminants virus in animals according to the sixth aspect.

The invention has the beneficial effects that: the invention finds that the replication of the peste des petits ruminants virus can be inhibited by inhibiting or silencing the PRKRA gene of a host, and the peste des petits ruminants virus can be used as a target for preparing a medicament for inhibiting the replication of the peste des petits ruminants virus; the PRKRA gene is taken as a target spot, and the small interfering RNA is designed, can interfere the replication of the peste des petits ruminants virus and can be used for preparing a medicament for inhibiting the replication of the peste des petits ruminants virus; the invention provides a sgRNA of a targeting PRKRA gene, which can specifically target an RPSA gene and can realize the complete knockout of the PRKRA gene in a host cell by combining with a CRISPR-Cas9 technology; the sgRNA of the targeting PRKRA gene and the mRNA sequence of the Cas9 protein are delivered into an animal body through a drug carrier, so that the PRKRA gene can be inhibited, and the replication of peste des petits ruminants virus can be inhibited; the invention provides a method for transfecting sgRNA to host cells through a CRISPR-Cas9 technology to construct a cell line with PRKRA gene coding protein loss function, and the cell line with PPRV resistance phenotype is obtained by losing the function of PRKRA gene coding protein, so that the duplication of PPRV can be obviously inhibited, research tools and materials are provided for further researching the molecular mechanism of PRKRA gene regulating and controlling the duplication of pathogenic microorganisms in cells, and the method can also be used for animal breeding of PPRV resistance.

Drawings

FIG. 1 shows the result of PRKRA gene expression in Vero cells after small interfering RNA interference;

FIG. 2 shows the result of PPRV virus expression in Vero cells after small interfering RNA interference;

FIG. 3 shows sequencing alignment results of PX459-PRKRA-sgRNA recombinant plasmid construction;

FIG. 4 shows the result of PCR amplification of Vero cell DNA check primers after PX459-PRKRA-sgRNA plasmid is transfected;

FIG. 5 sequencing map of amplified fragment of Vero cell DNA check primer after PX459-PRKRA-sgRNA1 plasmid transfection;

FIG. 6 Western blotting detection result of PRKRA protein of Vero alternative cell knocked out by PRKRA gene;

FIG. 7 is a Western blotting detection result of intracellular viral protein replication level of a PRKRA gene knockout Vero cell after PPRV challenge;

FIG. 8 shows the qPCR detection result of intracellular viral mRNA level of PRKRA gene knockout Vero cells after PPRV challenge.

FIG. 9 shows the intracellular viral protein staining results of PRKRA gene knockout Vero cells after PPRV challenge.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, it will be appreciated by those of ordinary skill in the art that in various embodiments of the invention, numerous technical details are set forth in order to provide a better understanding of the present application. However, the technical solution claimed in the present application can be implemented without these technical details and various changes and modifications based on the following embodiments.

Definition of

The term "gene silencing" refers to the phenomenon of making a gene under-or not under-expressed without damaging the original DNA, and mainly includes two aspects, namely, gene silencing at the transcription level due to DNA methylation, heterochromatosis, position effect and the like; the second is post-transcriptional gene silencing, which is the inactivation of genes by specific inhibition of target RNA at the post-transcriptional level of the gene, including antisense RNA, co-suppression, gene suppression, RNA interference, and microrna-mediated translation suppression. The invention can inhibit the expression of PRKRA gene in host cells through a gene silencing technology, thereby inhibiting the virus replication after PPRV infection and being used for preventing or treating PPRV infection.

The term "gene targeting" refers to a directional transgenic technology for directionally changing the genetic information of cells or biological individuals by using DNA site-directed homologous recombination, and mainly comprises gene knockout, gene inactivation, gene knock-in, point mutation, deletion of large segments of chromosome groups and the like. Wherein "gene knockout" refers to inactivation of a specific target gene by homologous recombination. According to the invention, through a gene knockout technology, PRKRA gene in host cells is knocked out, and the obtained monoclonal cell line with PRKRA gene coding protein loss of function can inhibit virus replication after PPRV infection; the invention also can successfully construct a monoclonal cell line with the function loss of PRKRA gene coding protein by mutating the PRKRA gene in the host cell or deleting gene segments to cause the code shift mutation of PRKRA gene coding protein.

The term "sgRNA" is a guide RNA that directs the insertion or deletion of uridine residues into the kinetoplast (kinetoplastid) during RNA editing, and is a small non-coding RNA.

The sgRNA of the targeting PRKRA gene is artificially synthesized, a CACC cohesive end is further added to the 5 'end of a forward sequence of a sgRNA fragment, an AAAC cohesive end is added to the 5' end of a reverse sequence to prepare sgRNA oligonucleotide of the targeting PRKRA gene, and the sgRNA oligonucleotide is annealed into a double-stranded fragment;

on the basis of direct target splicing of the PRKRA gene, the PRKRA gene is knocked out by using a CRISPR/Cas9 combined specificity method, taking Vero of Vero monkey kidney cells as an example (the amino acid sequence of the PRKRA gene is shown as SEQ ID No.1, and the nucleotide sequence is shown as SEQ ID No. 2), and a strategy is provided for the prevention or treatment of PPRV. Although only the PRKRA gene in Vero of the Vero cell of the African green monkey is knocked out to obtain a gene knocked-out cell with PPRV resistance, the method can be deduced and expanded to knock out the PRKRA gene in other animal cells to construct the gene knocked-out cell with PPRV resistance.

The CRISPR/Cas9 system realizes the directional recognition and shearing of genes by sgRNA and Cas9, and the sgRNA determines the targeting property of Cas9 and also determines the cutting activity of Cas 9. The invention aims to realize accurate and efficient knockout of PRKRA gene by screening sgRNA sequences aiming at PRKRA gene in vitro and in vivo by using CRISPR/Cas9 gene editing technology, and obtain a PRKRA gene knockout monoclonal cell line capable of inhibiting PPRV virus replication, thereby providing a new strategy for prevention or treatment of PPRV infection.

By using CRISPR/Cas9 gene editing technology, the sgRNA of the targeting PRKRA gene guides Cas9 protein to be combined with the specific sequence position of the PRKRA gene to cut the DNA double strand, so that the gene double strand is broken, random mutation is generated under the action of a cell self-repair mechanism, the reading frame of the gene is changed due to mutation such as nucleotide deletion or insertion, the purpose of losing the function of the gene coding protein is finally achieved, and the gene coding protein function losing cell line is obtained.

The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the following examples were all purchased from conventional biochemicals, unless otherwise specified.

The plasmid sources referred to in the following examples: purchased from the vast plasmid platform.

Cell culture: vero cells are derived from animals of the family monkey (Cercopithecacidae); DMEM medium containing 5% Fetal Bovine Serum (FBS) and 1% double antibody was placed in a medium containing 5% CO₂The culture was carried out in an incubator (37 ℃).

The virus source is as follows: PPRV Nigeria 75/1 vaccine strain is preserved in the foot-and-mouth disease and new disease epidemiology team of Lanzhou veterinary research institute of Chinese academy of agricultural sciences and the national foot-and-mouth disease reference laboratory.

Example 1 PPRV Virus replication results following PRKRA Gene silencing

1. Design of Small interfering RNAs (siRNAs)

The RNA interference target sequences PRKRA siRNA (SEQ ID NO.3-4) and NC siRNA (SEQ ID NO.5-6) of the designed PRKRA gene are shown, and the specific sequences are as follows:

PRKRA-siRNA-F:5’-CCCAGUUUAUGAAUGUGAATT-3’(SEQ ID NO.3)；

PRKRA-siRNA-R:5’-UUCACAUUCAUAAACUGGGTT-3’(SEQ ID NO.4)；

NC siRNA-F:5’-UUCUCCGAACGUGUCACGUTT-3’(SEQ ID NO.5)；

NC siRNA-R:5’-ACGUGACACGUUCGGAGAATT-3’(SEQ ID NO.6)。

construction of PRKRA Gene-silenced cell lines:

(1) preparation of PRKRA gene silencing siRNA Oligo: sending the designed interference RNA sequence to Gima corporation for synthesis to obtain corresponding siRNA Oligo, and using DEPC H₂O resuspend 1OD siRNA to a final concentration of 20 μm. Centrifuging at 10000rpm for 2min before dissolving, slowly opening the tube cover, adding enough DEPC water during dissolving, and fully oscillating to dissolve. Control siRNA (NC) was dissolved in the same manner for use.

(2) Construction of PRKRA gene silencing cell line: the Vero cells are paved in a six-hole plate after being counted, and when the cell fusion degree reaches 70-80 percent, the dissolved siR is pavedNA 6. mu.L was added to 100. mu.L of Opti-MEM with Lipofectamine2000, 6. mu.L, and the two were mixed after resting for 5 min. The liposome-siRNA mixture was left to stand for 20min and added directly to the cell culture medium. The cells were again incubated at 37 ℃ with 5% CO₂And (3) changing the culture solution after culturing for 6h in the incubator, detecting mRNA expression for 24-72h, and detecting protein expression for 48-96 h.

Detection of PRKRA Gene expression level

The Vero cells are respectively transfected with NC siRNA and PRKRA siRNA, after 24h of transfection, PPRV (MOI ═ 1) is inoculated, after 48h of infection, the cells are washed once by PBS, RNA is extracted from the collected cells, and after reverse transcription, the PRKRA mRNA expression level is detected. The detection results are shown in fig. 1, compared with NC siRNA, the PRKRA gene expression in Vero cells after PRKRA siRNA transfection is basically not detected, which indicates that PRKRA siRNA can inhibit PRKRA gene expression.

PPRV Virus results after PPRV Virus infection

Cell transfection and infection methods As described in 3 above, after infecting PPRV for 48h, RNA was extracted from the cells and the expression level of PPRV virus was detected by fluorescent quantitative PCR. The results are shown in fig. 2, compared with Vero cells transfected by NC siRNA, Vero cells transfected by PRKRA siRNA can significantly inhibit the expression of PPRV N protein after PPRV infection.

The results show that the small interfering RNA related to the PRKRA serving as the target spot can obviously inhibit the expression of the PRKRA gene, and further inhibit the replication of PPRV virus. Therefore, PRKRA as a target can be used for screening or designing a medicament for inhibiting PPRV virus replication, and further can be used for preventing or treating Peste des petits ruminants.

Example 2 PRKRA Gene knockout Vero cell line

1. Design of sgRNA targeting PRKRA gene

And (3) querying a PRKRA gene sequence by using an Ensemble database, and positioning a first exon subsection of different transcript overlapping regions of the PRKRA in a genome for target design.

Logging in a CRISPR online design website http:// crispor.tefor.net/designing sgRNA according to a CRISPR/Cas9 design principle, and respectively naming the steps as follows: PRKRA-sgRNAsp1, PRKRA-sgRNAsp 2; a CACC cohesive end was added to the 5 'end of the forward sequence and an AAAC cohesive end was added to the 5' end of the reverse sequence of the sgRNA fragment as a sgRNA oligonucleotide targeting the PRKRA gene (sgRNA 1-oligo). The sgRNA1-oligo was synthesized by Kingzhi Biotechnology, Inc., and the detailed sequence is shown in Table 1.

TABLE 1 sgRNA oligonucleotides targeting the PRKRA gene

Note: the underlined sequences denote the added cleavage sites, and the non-underlined sequences denote the sgRNA sequences (SEQ ID NO. 7-10).

Construction of sgRNA recombinant plasmid PX459-sgRNA

Obtaining double-stranded sgRNA-oligo: the synthesized sgRNA-oligo was diluted to 100. mu. mol/L to formulate a total of 10. mu.L reaction system: 4.5 mu L of upstream primer; downstream primer, 4.5 μ L; 10 × LA PCR Buffer, 1 μ L, gently mix. And (3) annealing procedure: at 95 ℃ for 10 min; and taking out from the PCR instrument for natural cooling.

Enzyme digestion of PX459 vector plasmid: utilizing Bbs I restriction enzyme to cut PX459 vector, and preparing 20 mu L of enzyme cutting system as follows: PX459 vector, 5 μ L; BbsI, 1 μ L; 10 × Buffer, 2 μ L; ddH₂O, 12. mu.L. The mixture was incubated at 37 ℃ for 3 hours for cleavage. Then, nucleic acid electrophoresis was performed, and the linearized PX459 vector fragment containing a sticky end was purified and recovered by using a DNA purification and recovery kit from Promega.

Construction of PX459-sgRNA recombinant plasmid: performing a connection reaction on the purified and recovered PX459 linearization fragment product and a double-chain sgRNA-oligo, wherein the reaction system comprises: t4Ligase, 0.5. mu.L; 10 XT 4Ligase Buffer, 0.5. mu.L; PX459 enzyme digestion purified fragment, 0.5 mu L; double stranded sgRNA-oligo, 3.5. mu.L, for a 5. mu.L system. The ligation product was transformed into Trans 5. alpha. E.coli competent cells at 16 ℃ overnight, and the recombinant plasmid was clonally amplified. Transformation procedure: 50 μ L of Trans5 α competent cells were mixed with 500ng of ligation product and placed on ice for 30 min. The heat shock in the water bath was carried out at 42 ℃ for 45 seconds, and the ice bath was taken out for 2 minutes. To the mixture was added 500mL of non-resistant LB medium, and the mixture was shaken at 37 ℃ and 220rpm for 60 minutes. And centrifuging the recovered bacterial liquid at 4000rpm for 5min at room temperature. After sucking 400. mu.L of the supernatant, the remaining supernatant and the precipitated cells were sufficiently suspended, and the transformed E.coli was spread on an LB plate having ampicillin resistance by means of a smear stick, incubated at 37 ℃ in an incubator for 12 hours, and the growth was observed.

Picking monoclonal colony, shaking with LB liquid culture medium containing ampicillin resistance for 12h, and usingThe plasmid extraction kit extracts and performs sequencing verification, the sequencing result is shown in figure 3, and the sequence obtained by detecting the constructed plasmid by using the TRC universal primer is compared with the original vector and is consistent with the expected result. Plasmid demonstrating expression of sgRNA

PX459-PRKRA-sgRNA1, PX459-PRKRA-sgRNA2 were successfully constructed.

3. Cell transfection

Resuscitating Vero cells in a T25 cell bottle before transfection, culturing by using DMEM medium containing 5% FBS and 1% double antibody, when the cell passage is stable and the state is good for 2-3 times, digesting the cells, then paving the cells in a six-well cell plate, adding 2 mu g of successfully constructed recombinant plasmid and Lipofectamine2000 and 4 mu L (according to the proportion of 1 mu g: 2 mu L) into 50 mu L of Opti-MEM when the cell fusion degree is 70% -80%, and mixing the two after standing for 5 min. The liposome-plasmid DNA mixture was allowed to stand for 20min and added directly to the cell culture medium. The cells were again incubated at 37 ℃ with 5% CO₂After 24 hours of incubation in an incubator, cells were treated with puromycin at a concentration of 8. mu.g/mL for 2-3 days and cells positive for transfection were selected. Subsequently, 100 positive cells were plated in a 96-well plate by cell counting to obtain a single cell clone.

(1) Extracting cell DNA and detecting gene targeting efficiency:

extracting the genome of the single cell clone according to the operation instruction of the trace DNA extraction kit, and further amplifying the PRKRA gene segment containing the targeting site segment by using DNA check primers which are:

PRKRA-check-F：GAACGCAAGCAGGAGGAGGGGGAGT(SEQ ID NO.15)

PRKRA-check-R：GGGTATGAAAAGAGTCGTGCAGGAT(SEQ ID NO.16)。

the results are shown in FIG. 4, and all single cell clones amplified to a size of about 1100bp, consistent with the expected designed fragment size, using wild type Vero cells as controls. And then, the nucleic acid gel at the target position is subjected to gel cutting, purification and recovery, and then is sent to be sequenced, a sequencing map is shown in figure 5, and a large number of nested peaks appear in a sequencing peak map, which indicates that effective gene editing occurs in an amplified fragment.

(2) PRKRA-KOs Western blotting verification

Vero cells (wild-type cells) without gene editing were used as negative controls. Culturing wild type cell (WT) strain and 3 knock-out candidate cell strains (KO 1-KO3), collecting cells, and placing on ice. Adding a proper amount of 1 xSDS loading Buffer, fully stirring and cracking, sucking the cells into an EP tube after the cells completely fall off, and marking; after denaturation in a metal bath for 15 minutes and centrifugation, the supernatant was subjected to SDS-PAGE. Transferring to an NC membrane by a wet transfer method after electrophoresis, sealing by using 5% skimmed milk powder after transfer printing, detecting an antigen-antibody complex by using a purchased 35kDa PRKRA mouse antibody as a primary antibody and a rabbit anti-mouse IgG (IgG-HRP) as a secondary antibody, and verifying the protein expression level of the Vero monoclonal cell line knocked out by the PRKRA gene.

The experimental result is shown in fig. 6, the WT detects a clear PRKRA protein band, and no PRKRA protein band is detected in 3 candidate cell strains, which indicates that the PRKRA gene in three candidate strains of KO1, KO2 and KO3 is successfully targeted, and indicates that the sgRNA provided by the invention and the CRISPR-Cas9 technology are used to completely knock out the PRKRA gene in the host cell.

Example 3 Effect of PRKRA Gene knockout Vero cell line on PPRV replication

Knocking out Vero cells and wild Vero cells by PRKRA genes, after normal subculture, paving the cells on a 35mm cell culture dish, inoculating PPRV Nigeria 75/1 virus, and detecting the PPRV replication condition by Western blotting and qPCR methods respectively.

Western blotting detection results are shown in FIG. 7, and the results show that the N protein expression of PPRV cannot be basically detected in Vero cells knocked out by PRKRA genes.

The qPCR detection result is shown in FIG. 8, and the mRNA levels of the N protein and the P protein after PPRV inoculation of the PRKRA gene knockout Vero cells are obviously reduced compared with wild cells. Indirect immunofluorescence results are shown in fig. 9, the viral capsid protein in wild-type cells increased with the time of viral infection, while PPRV capsid protein expression was barely detectable in PRKRA knockout cells. Wherein blue is the result of nuclear staining and green is the result of PPRV capsid protein staining.

The results show that the PRKRA gene knockout Vero cell obtained by the gene editing technology can obviously inhibit the replication of PPRV and has PPRV resistance. Therefore, the constructed function-losing cells of the PRKRA gene coding protein can be used for breeding animals against PPRV virus.

In conclusion, the cell line with the function loss of the PRKRA gene coding protein is successfully constructed by the CRISPR-Cas9 technology. However, the invention is not limited to the CRISPR-Cas9 technology, and on the basis of the invention, the cell line with the function of the PRKRA gene encoding protein lost can also be obtained by losing the function of the PRKRA gene encoding protein through other technical means. Other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principles of the invention are intended to be included within the scope of the invention.

Sequence listing

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

Application of <120> PRKRA gene as target in inhibiting replication of peste des petits ruminants virus

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 313

<212> PRT

<213> African green monkey (African green monkey)

<400> 1

Met Ser Gly Ser Ala His Ala Ala Gly Ala Pro Pro Leu Gly Ala Gly

1 5 10 15

Ala Ser Gly Thr Pro Ser Leu Gly Leu Met Ile Thr Ala Leu Pro Gly

20 25 30

Leu Thr Pro Ile Gly Val Leu His Gly Thr Gly Met Leu Thr Leu Ala

35 40 45

Ile Pro Val Thr Gly Cys Gly Ala Ser Ala Val Gly Ile His Val Pro

50 55 60

Thr Pro Thr Pro Ala Val Thr Val Gly Ala Ile Thr Cys Thr Gly Gly

65 70 75 80

Gly Thr Ser Leu Leu Leu Ala Leu His Ala Ala Ala Gly Ala Ala Ile

85 90 95

Ala Ile Leu Leu Ala Ala Ala Ser Ile Cys Pro Ala Val Pro Ala Pro

100 105 110

Leu Met Pro Ala Pro Ser Leu Gly Pro Leu Ala Gly Leu Ala Pro Ile

115 120 125

Gly Ser Leu Gly Gly Leu Ala Ile His His Gly Thr Ala Leu Pro Gly

130 135 140

Thr Thr Leu Ser Gly Gly Gly Gly Pro Ala His Leu Ala Gly Thr Thr

145 150 155 160

Thr Ile Cys Ala Leu Gly Ser Pro Met Gly Thr Gly Leu Gly Ala Ser

165 170 175

Leu Leu Gly Ala Leu Ala Ala Ala Ala Gly Leu Pro Leu Ala Leu Pro

180 185 190

Ser Ala Ile Ser Pro Gly Ala His Ile Ser Leu Thr Ala Val Val Gly

195 200 205

His Ser Leu Gly Cys Thr Thr His Ser Leu Ala Ala Ser Pro Gly Gly

210 215 220

Leu Ile Ala Leu Leu Leu Ala Ser Leu Leu Ser Ile Pro Ala Thr Ala

225 230 235 240

Thr Ile Gly Leu Leu Ser Gly Ile Ala Leu Gly Gly Gly Pro Ala Ile

245 250 255

Thr Thr Leu Ala Ile Ala Gly Leu Ser Ala Ala Gly Gly Thr Gly Cys

260 265 270

Leu Ala Gly Leu Ser Thr Ser Pro Ile Thr Val Cys His Gly Ser Gly

275 280 285

Ile Ser Cys Gly Ala Ala Gly Ser Ala Ala Ala His Ala Ala Leu Gly

290 295 300

Thr Leu Leu Ile Ile Ala Gly Ala Leu

305 310

<210> 2

<211> 942

<212> DNA

<213> African green monkey (African green monkey)

<400> 2

atgtcccaga gcaggcaccg cgccgaggcc ccgccgctgg agcgcgagga cagtgggacc 60

ttcagtttgg ggaagatgat aacagctaag ccagggaaaa caccgattca ggtattacac 120

gaatacggca tgaagaccaa gaacatccca gtttatgaat gtgaaagatc tgatgtgcaa 180

atacatgtgc ccacattcac cttcagagta accgttggtg acataacctg cacaggtgaa 240

ggtacaagta agaagctggc gaaacataga gctgcagagg ctgccataaa cattttgaaa 300

gccaatgcaa gtatttgctt tgcagttcct gatcccttaa tgcctgaccc ttccaagcaa 360

ccaaagaacc agcttaatcc tattggttca ttacaggaat tggctattca tcatggctgg 420

agacttcctg aatataccct ttcccaggag ggaggacctg ctcataagag agaatatacc 480

acaatttgca ggctagagtc atttatggaa actggaaagg gggcatcaaa aaagcaagcc 540

aaaaggaatg ctgctgagaa atttcttgcc aaatttagta atatttctcc agagaaccac 600

atttctttaa caaatgtagt aggacattct ttaggatgta cttggcattc cttgaggaat 660

tctcctggtg aaaagatcaa cttactgaaa agaagcctcc ttagtattcc aaatacagat 720

tacatccagc tgcttagtga aattgccaag gaacaaggtt ttaatataac atatttggat 780

atagatgaac tgagcgccaa tggacagtat caatgtcttg ctgaactgtc caccagcccc 840

atcacggtct gtcatggctc cggtatctcc tgtggcaatg cacaaagtga tgcagctcac 900

aatgctttgc agtatttaaa gataatagca gaaagaaagt aa 942

<210> 3

<211> 21

<212> DNA/RNA