Application of Rab1a protein as a target in inhibiting Japanese encephalitis virus infection

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of Rab1a protein as a target in inhibition of Japanese encephalitis virus infection. The application firstly finds that inhibition or silencing of Rab1a protein expression can inhibit Japanese encephalitis virus infection, that is, Rab1a protein can be used as a target for screening drugs for inhibiting Japanese encephalitis virus infection; secondly, the application provides small interfering RNA for interfering Japanese encephalitis virus infection, which can inhibit Japanese encephalitis virus infection. The application provides a new target and treatment scheme for clinical prevention and treatment of Japanese encephalitis virus infection.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of Rab1a protein as a target in inhibiting Japanese encephalitis virus infection. Background Technology

[0002] Japanese encephalitis virus (JEV), also known as Japanese encephalitis virus, belongs to the genus Flaviviridae in the family Flaviviridae. It is an enveloped, single-stranded, positive-sense RNA virus. JEV is the pathogen of Japanese encephalitis (JE), primarily transmitted by mosquitoes. It causes permanent neurological deficits in some survivors, including seizures, paralysis, and intellectual disability. It can also cause abortion in pregnant sows, piglet mortality, and orchitis in boars, leading to various pathological damages that reduce pig productivity, posing a significant threat to human health and the healthy development of animal husbandry. Currently, there is an urgent need to find an effective specific drug treatment for JE, through early intervention with antibodies or drugs to minimize the sequelae of patients.

[0003] The emergence of RNA interference (RNA interference) technology has made selective toxicity possible, providing new insights for antiviral research. RNA interference is a sequence-specific post-transcriptional gene silencing phenomenon. Compared with previous antiviral therapies, RNA interference-mediated antiviral effects are highly specific, having almost no effect on the expression of non-homologous genes, thus minimizing adverse reactions. RNA interference can efficiently inhibit viral replication; a small amount of siRNA can reduce viral expression products. RNA interference can target conserved regions of the viral genome, thereby limiting the virus's ability to generate evasive mutants to some extent. Therefore, the design and synthesis of siRNAs may become an effective way to inhibit Japanese encephalitis virus infection.

[0004] Rab1a protein is a small GTPase primarily located on the endoplasmic reticulum and Golgi apparatus membranes. It is a component of the intracellular vesicle transport system and plays a crucial role in Golgi apparatus homeostasis and transmembrane transport of substances from the endoplasmic reticulum to the Golgi apparatus. The Rab1a protein complex also mediates the initiation of autophagy in host cells. Rab1A is widely overexpressed in human diseases such as cancer and cardiomyopathy, leading to potential pathogenesis mechanisms; the role of Rab1a protein in the infection process varies depending on the pathogen.

[0005] This invention discovers that inhibiting or silencing the Rab1a protein can suppress the production of infectious viral particles of Japanese encephalitis virus (JEV), making it a potential target for the preparation of drugs that inhibit JEV infection. Based on this, this invention designs a small interfering RNA (SRNA) targeting the Rab1a gene. This SRNA can interfere with the production of infectious JEV particles and can be used to prepare drugs that inhibit JEV infection. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention first discovers that inhibiting or silencing the Rab1a protein can suppress the production of infectious viral particles of Japanese encephalitis virus (JEV), making it a target for the preparation of drugs that inhibit JEV infection. Secondly, using the Rab1a protein as a target, this invention designs a small interfering RNA (SRNA), which can interfere with JEV replication and the production of infectious viral particles, and can be used to prepare drugs that inhibit JEV infection. Specifically, it includes the following:

[0007] In a first aspect, the present invention provides the application of Rab1a gene / protein as a target in screening drugs for the prevention or treatment of Japanese encephalitis virus infection, wherein the drug targets Rab1a gene / protein to inhibit or downregulate the expression of Rab1a gene / protein.

[0008] Secondly, the present invention provides the application of a reagent for inhibiting or downregulating Rab1a gene / protein expression in the preparation of drugs for the prevention or treatment of Japanese encephalitis virus infection.

[0009] Preferably, the reagent for inhibiting or downregulating Rab1a gene / protein expression is siRNA or shRNA that specifically interferes with Rab1a gene / protein expression, or a recombinant vector containing siRNA or shRNA.

[0010] Preferably, the siRNA is AB232583.1-mus-288 and / or AB232583.1-mus-420;

[0011] The sequence of AB232583.1-mus-288 is as follows:

[0012] F:5'-GGAGUCCUUCAAUAACGUUTT-3',

[0013] R: 5'-AACGUUAUUGAAGGACUCCTT-3';

[0014] The sequence of AB232583.1-mus-420 is as follows:

[0015] F: 5'-GGAAUUUGCAGAUUCCCUUTT-3',

[0016] R: 5'-AAGGGAAUCUGCAAAUUCCTT-3'.

[0017] Preferably, the reagent or drug comprises sgRNA that targets and knocks out the Rab1a gene, or sgRNA that targets and knocks out the Rab1a gene and the mRNA sequence of the Cas9 protein.

[0018] Preferably, the drug is delivered into the animal via a drug delivery vector, carrying an mRNA sequence of sgRNA targeting the Rab1a gene and the Cas9 protein, to inhibit Rab1a gene expression.

[0019] Preferably, the drug delivery carrier is a liposome nanoparticle.

[0020] Thirdly, the present invention provides an siRNA for treating Japanese encephalitis virus infection, wherein the siRNA is AB232583.1-mus-288 and / or AB232583.1-mus-420;

[0021] The sequence of AB232583.1-mus-288 is as follows:

[0022] F:5'-GGAGUCCUUCAAUAACGUUTT-3',

[0023] R: 5'-AACGUUAUUGAAGGACUCCTT-3';

[0024] The sequence of AB232583.1-mus-420 is as follows:

[0025] F: 5'-GGAAUUUGCAGAUUCCCUUTT-3',

[0026] R: 5'-AAGGGAAUCUGCAAAUUCCTT-3'.

[0027] Fourthly, the present invention provides the use of the siRNA described in the third aspect above in the preparation of a drug for treating Japanese encephalitis virus infection.

[0028] Fifthly, the present invention provides a pharmaceutical composition for treating Japanese encephalitis virus infection, said pharmaceutical composition comprising the siRNA described in the fourth aspect above.

[0029] Preferably, the pharmaceutical composition further includes one or more of a small molecule compound against Japanese encephalitis virus, a Japanese encephalitis virus vaccine, or a monoclonal antibody against Japanese encephalitis virus.

[0030] In a sixth aspect, the present invention provides the use of the pharmaceutical composition described in the fifth aspect above in the preparation of a medicament for treating Japanese encephalitis virus infection.

[0031] The beneficial effects of this invention are: ① This invention discovers that by inhibiting or downregulating the host Rab1a gene, the replication of Japanese encephalitis virus and the production of infectious viral particles can be suppressed, which can be used as a target for the preparation of drugs that inhibit Japanese encephalitis virus infection; ② This invention uses the Rab1a gene as a target and designs a small interfering RNA. The small interfering RNA can not only interfere with the expression of Rab1a, but also inhibit the replication of Japanese encephalitis virus and the production of infectious viral particles. It can be used to prepare drugs that inhibit Japanese encephalitis virus infection, providing a new target and treatment plan for the clinical prevention and treatment of Japanese encephalitis virus infection. Attached Figure Description

[0032] Figure 1 The effects of wild-type, activated, and inactivated Rab1a on the production of infectious viral particles, where Rab1a is wild-type, Q70L is activated, EV is empty vector, and N124I and S25N are inactivated.

[0033] Figure 2 Rab1a knockdown and its effect on cell viability;

[0034] Figure 3 Effects of Rab1a knockdown on JEV RNA, protein, and viral yield. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the various embodiments of this invention will be described in detail below with reference to the accompanying drawings. However, those skilled in the art will understand that many technical details have been provided in the embodiments of this invention to facilitate a better understanding of this application. However, the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0037] Example 1: Effects of Rab1a wild-type, activated, and inactivated types on the production of infectious viral particles.

[0038] 1. Construction and functional validation of wild-type Rab1a and its mutant recombinant expression vectors

[0039] Using the primers in Table 1, Rab1a and its mutant genes were obtained by PCR amplification and overlap PCR amplification techniques. These genes were cloned into the PCMV-3×Flag eukaryotic expression vector via double restriction enzyme digestion sites (Sal I and BamHI). The resulting recombinant expression vectors were named pCMV-Rab1a-3Tag-1, pcDNA-Rab1a-Q70L-3Tag-1 (Rab1a activated form), pcDNA-Rab1a-N124I-3Tag-1, and pcDNA-Rab1a-S25N-3Tag-1 (Rab1a inactivated form), respectively. After transfecting BHK-21 cells with the constructed recombinant plasmids for 24 hours, the cells were infected with JEV at an MOI of 0.1. Forty-eight hours after infection, the cell culture supernatant was collected, and the infectious virus yield of each experimental group was determined using viral plaque assay. The collected cell pellet was used for validation of wild-type Rab1a and its mutant protein expression.

[0040] Table 1 Primer Comparison Table

[0041]

[0042] The results are as follows Figure 1 As shown in the results, immunoblotting analysis indicated that the constructed wild-type Rab1a and its mutants were correctly expressed in BHK-21 cells. Furthermore, the infectious virus yield of BHK-21 cells overexpressing wild-type Rab1a and the activated mutant was significantly higher than that of the blank vector control group, while the infectious virus yield of BHK-21 cells overexpressing the inactivated Rab1a mutant was significantly reduced, with an inhibition rate exceeding 50%. These results suggest that Rab1a protein plays a role in promoting the production of infectious viral particles during JEV infection of host cells.

[0043] The above results indicate that by inhibiting or downregulating the host Rab1a gene, the replication of Japanese encephalitis virus and the production of infectious viral particles can be suppressed, and this gene can be used as a target for the preparation of drugs that inhibit Japanese encephalitis virus infection.

[0044] Example 2: Rab1a siRNA and its effect on cell viability

[0045] Three siRNAs targeting the Rab1a gene were designed and synthesized by Gima Biotech. The naming and sequence information of the siRNAs are as follows:

[0046] AB232583.1-mus-125 (siRNA-125): F: 5'-GCACAAUUGGUGUGGAUUUTT-3' (shown in SEQ ID NO.1), R: 5'-AAAUCCACACCAAUUGUGCTT-3' (shown in SEQ ID NO.2);

[0047] AB232583.1-mus-288 (siRNA-288): F: 5'-GGAGUCCUUCAAUAACGUUTT-3' (shown in SEQ ID NO.3), R: 5'-AACGUUAUUGAAGGACUCCTT-3' (shown in SEQ ID NO.4);

[0048] AB232583.1-mus-420 (siRNA-420): F: 5'-GGAAUUUGCAGAUUCCCUUTT-3' (shown in SEQ ID NO.5), R: 5'-AAGGGAAUCUGCAAUUCCTT-3' (shown in SEQ ID NO.6);

[0049] Control siRNA (siRNA-NC): F: 5'-UUCUCCGAACGUGUCACGUTT-3' (shown in SEQ ID NO.7), R: 5'-ACGUGACACGUUCGGAGAATT-3' (shown in SEQ ID NO.8).

[0050] To further verify the role of the Rab1a gene during JEV infection of host cells, BHK-21 cells were transfected with three Rab1a gene siRNAs (siRNA-125, siRNA-288, and siRNA-420) synthesized by Gima Biotech, as well as the control siRNA-NC. Twenty-four hours after transfection, the knockdown effect of the three Rab1a gene siRNAs on Rab1a gene transcription was detected using real-time quantitative PCR with Rab1a gene-specific primers listed in Table 1. Cell viability of each transfected experimental group was measured using the CCK-8 assay. The siRNA420 experimental group, which showed the best transcriptional knockdown effect, was selected for protein knockdown validation.

[0051] Real-time quantitative PCR results as follows Figure 2 As shown in Figures A and B, siRNA-125 had virtually no effect on the transcriptional level of the endogenous Rab1a gene; siRNA-288 and siRNA-420 significantly reduced the transcriptional level of the endogenous Rab1a gene, and compared with the control siRNA-NC experimental group, the three siRNAs of the Rab1a gene had little effect on the viability of transfected cells; the immunoblotting results are as follows. Figure 2 As shown in the figure, the siRNA-420 described in this application can significantly reduce the expression of endogenous Rab1a protein.

[0052] Example 3: Effects of Rab1a siRNA on JEV RNA, protein, and viral yield

[0053] BHK-21 cells were transfected with siRNA-420 and control siRNA-NC, respectively. Twenty-four hours after transfection, BHK-21 cells were infected with JEV at an MOI of 0.1. Forty-eight hours after infection, cell pellets and cell culture supernatants were collected. The effect of Rab1a gene knockdown on JEV RNA production was detected by RT-qPCR. The effect of Rab1a gene knockdown on the expression levels of JEV-encoded non-structural proteins NS5 and capsid protein C was verified by Western blotting. The viral titer of the collected cell culture supernatants was determined by viral plaque assay to analyze the infectious viral particle yield in the Rab1a gene knockdown experimental group and the control group.

[0054] Real-time quantitative PCR results as follows Figure 3 As shown in Figure A, compared with the control siRNA-NC, siRNA-420 significantly reduced JEV RNA production; grayscale immunoblotting results are shown below. Figure 3 As shown in Figure B, compared with the control siRNA-NC, siRNA-420 significantly inhibited the expression of JEV capsid protein C and the non-structural protein NS5; the yield of infectious viral particles in the collected cell culture supernatant was determined by viral plaque assay, as shown in Figure B. Figure 3 As shown in Figure C, compared with the control siRNA-NC, siRNA-420 can significantly reduce the yield of infectious JEVs, with an inhibition rate of over 50%.

[0055] The above results indicate that the specific siRNA described in this application can not only significantly inhibit the expression of Rab1a protein, but also has significant anti-JEV activity, and can be used as a drug for treating Japanese encephalitis virus infection, showing good application prospects.

Claims

1. Inhibit or downregulate Rab1a The application of gene / protein expression reagents in the preparation of drugs for the prevention or treatment of Japanese encephalitis virus infection, characterized in that, The inhibition or downregulation Rab1a The reagents for gene / protein expression are specific interferences. Rab1a The siRNA for gene / protein expression, wherein the siRNA is AB232583.1-mus-288 and / or AB232583.1-mus-420; The sequence of AB232583.1-mus-288 is as follows: F:5'-GGAGUCCUUCAAUAACGUUTT-3', R: 5'-AACGUUAUUGAAGGACUCCTT-3'; The sequence of AB232583.1-mus-420 is as follows: F: 5'-GGAAUUUGCAGAUUCCCUUTT-3', R: 5'-AAGGGAAUCUGCAAAUUCCTT-3'.

2. A siRNA for treating Japanese encephalitis virus infection, characterized in that, The siRNA is AB232583.1-mus-420; The sequence of AB232583.1-mus-420 is as follows: F: 5'-GGAAUUUGCAGAUUCCCUUTT-3', R: 5'-AAGGGAAUCUGCAAAUUCCTT-3'.

3. A pharmaceutical composition for treating Japanese encephalitis virus infection, characterized in that, The pharmaceutical composition comprises the siRNA of claim 2.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition further includes one or more of the following: a small molecule compound against Japanese encephalitis virus, a Japanese encephalitis virus vaccine, or a monoclonal antibody against Japanese encephalitis virus.

5. The use of the pharmaceutical composition as described in claim 3 or 4 in the preparation of a medicament for treating Japanese encephalitis virus infection.

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