A hepatitis E virus ORF3 protein mutant, gene and recombinant adenovirus

Abstract

The present invention discloses a hepatitis E virus ORF3 protein mutant, gene, and recombinant adenovirus. The hepatitis E virus ORF3 protein mutant of the present invention is obtained by deleting the 56-76aa segment of the hepatitis E virus ORF3 protein, and the amino acid sequence is shown in SEQ ID No. 3. The hepatitis E virus ORF3 protein mutant of the present invention is obtained by deleting the 56-76aa segment of the ORF3 amino acid sequence. This mutant loses the ability to co-localize with mitochondria but retains the immunogenicity of ORF3. When prepared as a vaccine, it can avoid the health risks caused by the co-localization of ORF3 with mitochondria.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a hepatitis E virus ORF3 protein mutant, a gene and a recombinant adenovirus. Background Art

[0002] Hepatitis E virus (HEV) is an important pathogen that causes acute or chronic hepatitis. It is prone to cause chronic infection and liver failure in immunocompromised people and pregnant women.

[0003] HEV encodes three proteins, of which ORF1 is a nonstructural protein consisting of 1766 amino acids and encompasses multiple functional regions, including a methyltransferase domain (Met), a Y domain, a papain-like cysteine ​​protease domain (PCP), a hypervariable region (HVR), a macro domain (X), a helicase domain (Hel), and an RNA-dependent RNA polymerase domain (RdRp). ORF2, consisting of 660 amino acids, is the viral capsid protein. The truncated ORF2 protein p239 of HEV genotype 1 can form virus-like particles (VLPs) with a diameter of 23 nm. Currently, a vaccine against HEV subtype 1 using p239 as an antigen is available.

[0004] The ORF3 protein, composed of 113 amino acids, is the smallest protein encoded by the HEV genome and is present on the surface of viral particles. Studies have shown that immunizing animals with the ORF3 protein as an immunogen can reduce the viral titer in animals (PMID: 36548837, Application of ORF3 Subunit Vaccine for Avian Hepatitis E Virus). Furthermore, peptides designed based on the ORF3 protein can prevent HEV infection in primates after immunization (PMID: 19763777, Immunogenicity and efficacy of a bacterially expressed HEVORF3 peptide, assessed by experimental infection of primates). Using adeno-associated virus (AAV)-mediated expression of ORF3 in mouse muscle, results showed that ORF3 can induce the production of HEV-specific antibodies in mouse serum (PMID: 35215859, Induction of Hepatitis E Virus Anti-ORF3 Antibodies from Systemic Administration of a Muscle-Specific Adeno-Associated Virus (AAV) Vector). The above data indicate that ORF3 can induce specific antibodies against HEV.

[0005] However, studies have reported that ORF3 regulates the host's innate immune response and interferes with host signaling pathways (PMID: 24850742, Enhancement of interferon induction by ORF3 product of hepatitis E virus; PMID: 29872132, HEV ORF3 downregulates TLR7 to inhibit the generation of type I interferon via impairment of multiple signaling pathways.). At the same time, studies have shown that HEV infection can lead to the loss of mitochondrial cristae and mitochondrial swelling (PMID: 32456826, Identification and pathogenicity of a novel genotype avian hepatitis E virus from silkie fowl (gallus gallus)). Mitochondria are the source of cellular energy, and abnormal mitochondrial morphology can affect the cell's energy supply and even lead to cell apoptosis.

[0006] While the intact ORF3 protein has been shown to induce good immunogenicity and induce protective antibodies, our research found that the intact ORF3 protein colocalizes with mitochondria and causes mitochondrial swelling, posing a potential risk to human health as an immunogen. Designing ORF3 protein mutants that retain the immunogenicity of the ORF3 protein while avoiding colocalization with other mitochondria is of great significance. Summary of the Invention

[0007] The present invention addresses the aforementioned deficiencies in the prior art and provides a hepatitis E virus ORF3 protein mutant, gene, and recombinant adenovirus. A mutant (del 56-76aa) was designed that retains ORF3 immunogenicity while preventing colocalization with mitochondria and affecting mitochondrial morphology. This is of great significance for the application of ORF3 as an immunogen for HEV.

[0008] The present invention first provides a hepatitis E virus ORF3 protein mutant, which is a mutant obtained by deleting 56-76aa of the hepatitis E virus ORF3 protein, and the amino acid sequence is shown in SEQ ID No. 3.

[0009] The present invention further provides a gene encoding the hepatitis E virus ORF3 protein mutant. Preferably, the gene sequence is shown as SEQ ID No. 4.

[0010] The present invention further provides a sequence for recombinant adenovirus expression, comprising a shuttle plasmid and an adenovirus backbone plasmid, wherein the gene is inserted into the shuttle plasmid.

[0011] Preferably, the vector used for the shuttle plasmid is pDC315, into which the gene is inserted; and the adenovirus backbone plasmid is pBHGloxΔE1,3Cre plasmid.

[0012] The present invention also provides a method for preparing a recombinant adenovirus, wherein the sequence for expressing the recombinant adenovirus is transfected into competent cells for expression to obtain the recombinant adenovirus.

[0013] The present invention also provides the recombinant adenovirus prepared by the preparation method.

[0014] The present invention also provides the use of the recombinant adenovirus in preparing a hepatitis E vaccine.

[0015] The hepatitis E virus ORF3 protein mutant of the present invention is a mutant obtained by deleting the fragment 56-76aa in the amino acid sequence of ORF3. The mutant loses the ability to co-localize with mitochondria but retains the immunogenicity of ORF3. When prepared into a vaccine, it can avoid the health risks caused by the co-localization of ORF3 with mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of fluorescence confocal microscopy.

[0017] Figure 2 Results for identifying the key amino acid sequence that determines the co-localization of ORF3 with mitochondria.

[0018] Figure 3 This is the result of identifying the co-localization of ORF3 (del-56-76aa) and mitochondria.

[0019] Figure 4 This is a graph showing the results of neutralizing antibody titer detection against HEV induced by ORF3 and ORF3 (del-56-76aa) recombinant adenovirus. DETAILED DESCRIPTION

[0020] Example 1

[0021] It was identified that ORF3 protein can co-localize with mitochondria and cause abnormal mitochondrial morphology.

[0022] ORF3 of HEV genotype 1 was used, wherein the amino acid sequence of ORF3 of genotype 1 (shown in SEQ ID No. 1) is:

[0023] MGSRPCALGLFCCCSSCFCLCCPRHRPVSRLAAVVGGAAAVPAVVS GVTGLILSPSQSPIFIQPTPSPPMSPLRPGLDLVFANPPDHSAPLGVTRPS APPLPHVVDLPQLGPRR.

[0024] The nucleotide sequence of genotype 1 ORF3 (shown in SEQ ID No. 2) is:

[0025] atgggttcgcgaccatgcgccctcggcctattttgctgttgctcctcatgtttctgcctatgctgcccgcgccaccgcccggtca gccgtctggccgccgtcgtgggcggcgcagcggcggttccggcggtggtttctggggtgaccgggttgattctcagcccttcgcaa tcccctatattcatccaaccaaccccttcgcccccgatgtcaccgctgcggccggggctggacctcgtgttcgccaacccgcccgaccactcggctccgcttggcgtgaccaggcccagcgccccgccgctgcctcacgtcgtagacctaccacagctggggccgcgccgc.

[0026] The Ad5 recombinant adenovirus expression system was used to construct a recombinant adenovirus shuttle plasmid expressing GFP and ORF3 and to prepare adenovirus (hereinafter referred to as genotype 1 ORF3).

[0027] In this study, recombinant adenoviral shuttle plasmids (pDC315-GFP and pDC315-ORF3) were successfully constructed by inserting the GFP and ORF3 gene fragments into the shuttle plasmid vector pDC315 using molecular cloning technology mediated by the site-specific enzymes EcoR I and BamH I. Subsequently, the constructed recombinant adenoviral shuttle plasmids and the adenoviral backbone plasmid (i.e., pBHGloxΔE1, 3Cre) were co-transfected into 293 cells (cell density reached 80%). On day 10 after transfection, cells were harvested using a cell scraper, centrifuged, and resuspended in PBS. Subsequently, the cells were subjected to three repeated freeze-thaw cycles and high-speed centrifugation at 12,000 rpm to remove cell debris, thereby collecting pure recombinant adenoviral supernatant.

[0028] In order to increase the yield of recombinant adenovirus, we amplified the two recombinant adenoviruses obtained above in 293 cells. After obvious pathological changes were observed in the cells, three freeze-thaw cycles were repeated, and the cell debris was removed by high-speed centrifugation at 12,000 rpm, and the recombinant adenovirus supernatant was further collected. In order to accurately evaluate the yield of recombinant adenovirus, we titered it, and the titer was expressed in the number of virus particles / ml (vp / mL). After diluting the titer of GFP and ORF3 recombinant adenovirus to 8×1010 vp / mL, we aliquoted the prepared viruses and stored them at ultra-low temperature of -80°C to ensure their long-term stability and experimental effects. The unit of virus titer is the number of virus particles / ml (vp / mL).

[0029] Furthermore, we used recombinant adenovirus Ad5-G1-ORF3 (genotype 1 ORF3) to mediate the expression of ORF3 in S10-3 (human hepatoma cells), using Ad5-GFP as a control. We used the mitochondrial membrane protein Tom20 as a mitochondrial marker and demonstrated that ORF3 can co-localize with mitochondria. Compared with the GFP-expressing control cells, the mitochondria in the ORF3-expressing cells were significantly enlarged and showed abnormal mitochondrial morphology ( Figure 1 ).

[0030] Example 2

[0031] Identify the key amino acid sequence that determines the colocalization of ORF3 protein with mitochondria.

[0032] In Example 1, we identified that ORF3 protein co-localized with mitochondria, leading to abnormal mitochondrial morphology.

[0033] The genotype 1 ORF3 protein consists of 114 amino acids. We identified the key region that determines the colocalization of the ORF3 protein with mitochondria, which helped us design ORF3 protein mutants that do not colocalize with mitochondria.

[0034] The genes of ORF3(1-55aa)-GFP (referring to the gene after the gene sequence encoding ORF3 1-55aa is connected to the GFP expression gene sequence, and other subsequent truncations are expressed in the same form), ORF3(1-76aa)-GFP, ORF3(39-114aa)-GFP and full-length ORF3-GFP were cloned into the pBoBi plasmid vector and co-transfected with the lentiviral packaging plasmid into 293t cells to obtain lentiviruses expressing ORF3(1-55aa)-GFP, ORF3(1-76aa)-GFP, ORF3(39-114aa)-GFP and full-length ORF3-GFP.

[0035] HeLa cells were then infected with the lentivirus, and cell lines stably expressing ORF3(1-55aa)-GFP, ORF3(1-76aa)-GFP, ORF3(39-114aa)-GFP, and full-length ORF3-GFP were selected using puromycin. After a period of screening and culture, the surviving cells were identified as cells stably expressing ORF3(1-55aa)-GFP, ORF3(1-76aa)-GFP, ORF3(39-114aa)-GFP, and full-length ORF3-GFP.

[0036] We identified the co-localization of these three ORF3 protein truncations, full-length ORF3 proteins and mitochondria in HeLa cell stable cell lines expressing ORF3(1-55aa)-GFP, ORF3(1-76aa)-GFP, ORF3(39-114aa)-GFP and full-length ORF3-GFP. In this study, the mitochondrial membrane protein Tom20 was used as a marker to label mitochondria in stable cell lines expressing GFP (as a control) and these truncations and full-length ORF3 proteins.

[0037] The results showed that the full-length ORF3 protein (complete sequence) and the truncated protein ORF3 (1-76aa) could co-localize significantly with mitochondria in stable cell lines, while the truncated protein ORF3 (1-55aa) and the truncated protein ORF3 (39-114aa) could not co-localize with mitochondria ( Figure 2 Therefore, we inferred that the amino acid sequence of 56-76aa of ORF3 protein is the key region that determines the localization of ORF3 protein to mitochondria.

[0038] Example 3

[0039] The co-localization of ORF3 (del-56-76aa) (ORF3 protein mutant after deletion of the 56-76aa fragment, the amino acid sequence is shown in SEQ ID No. 3, the gene sequence is shown in SEQ ID No. 4) with mitochondria was identified.

[0040] To determine whether ORF3(del-56-76aa) colocalizes with mitochondria, we constructed stable cell lines expressing ORF3(del-56-76aa)-GFP in HeLa cells. The ORF3(del-56-76aa)-GFP gene was cloned into the pBoBi plasmid vector and co-transfected with the lentiviral packaging plasmid into 293t cells to generate lentivirus expressing ORF3(del-56-76aa)-GFP. HeLa cells were then infected with the lentivirus, and cell lines stably expressing ORF3(del-56-76aa)-GFP were selected with puromycin. After a period of selection and culture, the surviving cells were designated as cells stably expressing ORF3(del-56-76aa)-GFP. We used the mitochondrial membrane protein Tom20 as a marker to localize the mitochondria.

[0041] Through super-resolution microscopy, we found that the full-length ORF3 protein can co-localize with mitochondria, while ORF3 (del-56-76aa) cannot co-localize with mitochondria. Compared with the GFP control group, the mitochondria in the full-length ORF3 protein stable cell line were enlarged and the mitochondrial morphology was abnormal; while the mitochondrial morphology in the ORF3 (del-56-76aa) stable cell line was similar to the GFP control group. Although ORF3 (del-56-76aa), ORF3 (1-55aa) and ORF3 (39-114aa) cannot co-localize with mitochondria, compared with the full-length ORF3, ORF3 (del-56-76aa) has fewer deleted amino acid sequences and therefore retains more antigenic epitopes ( Figure 3 ).

[0042] Example 4

[0043] Construction of recombinant adenoviral shuttle plasmids expressing GFP, ORF3 and ORF3(del-56-76aa) and preparation of adenovirus.

[0044] In this study, recombinant adenoviral shuttle plasmids (pDC315-GFP, pDC315-ORF3, and pDC315-ORF3(del-56-76aa)) were successfully constructed using molecular cloning techniques mediated by the site-specific enzymes EcoR I and BamH I. These recombinant adenoviral shuttle plasmids were then co-transfected with an adenoviral backbone plasmid (pBHGloxΔE1, 3Cre) into 293 cells (at a cell density of 80%). Ten days after transfection, cells were harvested using a cell scraper, centrifuged, and resuspended in PBS. Subsequently, the cells were subjected to three freeze-thaw cycles and high-speed centrifugation at 12,000 rpm to remove cell debris, resulting in the collection of pure recombinant adenoviral supernatant.

[0045] In order to increase the yield of recombinant adenovirus, we amplified the three recombinant adenoviruses obtained above in 293 cells. After the cells showed obvious pathological changes, three freeze-thaw cycles were repeated again, and the cell debris was removed by high-speed centrifugation at 12000rpm, and the recombinant adenovirus supernatant was further collected. In order to accurately evaluate the yield of recombinant adenovirus, we titered it, and the titer was measured in virus particles / ml (vp / mL). The titer of GFP, ORF3 and ORF3 (del-56-76aa) recombinant adenovirus was diluted to 8×10 10 After the titer is determined, we aliquot the prepared virus and store it at -80°C to ensure its long-term stability and experimental results. The unit of virus titer is virus particles per milliliter (vp / mL).

[0046] Example 5

[0047] The neutralizing antibody titers against HEV induced by ORF3 and ORF3(del-56-76aa) recombinant adenovirus were compared.

[0048] In this study, 6-week-old SPF-grade gerbils were randomly divided into three groups: control group 1, experimental group 1, and experimental group 2, with 6 gerbils in each group, for a total of 18 gerbils. Among them, the gerbils in control group 1 were inoculated with Ad5-GFP by intraperitoneal injection, the gerbils in experimental group 1 were inoculated with Ad5-ORF3, and the gerbils in experimental group 2 were inoculated with Ad5-ORF3 (del-56-76aa). The volume of adenovirus inoculated intraperitoneally in each gerbil was 0.5 mL, and the virus titer was uniformly set at 8×10 10 To enhance the immune effect, the above vaccination procedure was repeated for all groups of gerbils three weeks after the initial vaccination.

[0049] Two weeks after the booster immunization, serum samples were collected from all gerbils. Subsequently, the level of HEV neutralizing antibodies in the serum of each group of gerbils was measured using an in vitro HEV neutralization assay. During the experiment, the serum samples were appropriately diluted and incubated with HEV at 37°C for 2 hours. The incubated mixture was then used to infect S10-3 cells. 48 hours after the cells were infected, immunofluorescence staining was used for detection. In this process, the primary antibody used was a rabbit polyclonal antibody against the HEV ORF2 protein. Finally, the inhibitory effect of neutralizing antibodies in the serum on viral infection was evaluated by fluorescence counting. The lower the fluorescence count value, the stronger the antibody's ability to inhibit HEV infection.

[0050] The experimental results showed that there were significant differences in the levels of HEV neutralizing antibodies between experimental group 1, experimental group 2 and control group 1 (e.g. Figure 4 There was no significant difference in the HEV neutralizing antibody levels between experimental group 1 and experimental group 2. This result indicates that Ad5-ORF3(del-56-76aa) can induce HEV neutralizing antibody levels similar to those of Ad5-ORF3.

Claims

1. A hepatitis E virus ORF3 protein mutant, characterized in that: This is a mutant obtained by deleting 56-76aa of the ORF3 protein of the hepatitis E virus, and its amino acid sequence is shown in SEQ ID No.

3.

2. A gene encoding the hepatitis E virus ORF3 protein mutant according to claim 1.

3. The gene according to claim 2, characterized in that The gene sequence is shown in SEQ ID No.

4.

4. A nucleic acid for recombinant adenovirus expression, characterized in that: It comprises a shuttle plasmid and an adenovirus backbone plasmid, wherein the gene according to claim 2 or 3 is inserted into the shuttle plasmid.

5. The nucleic acid for recombinant adenovirus expression according to claim 4, characterized in that: The vector used for the shuttle plasmid is pDC315, into which the gene according to claim 2 or 3 is inserted; the adenovirus backbone plasmid is pBHGloxΔE1, 3Cre plasmid.

6. A method for preparing a recombinant adenovirus, characterized in that: The nucleic acid for expressing the recombinant adenovirus according to claim 4 or 5 is transfected into competent cells for expression to obtain the recombinant adenovirus.

7. The recombinant adenovirus prepared by the preparation method according to claim 6.

8. Use of the recombinant adenovirus according to claim 7 in the preparation of a hepatitis E vaccine.

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