Hepatitis E virus ORF3 protein mutant, gene and recombinant adenovirus

Abstract

The invention discloses a hepatitis E virus ORF3 protein mutant, a gene and a recombinant adenovirus. The hepatitis E virus ORF3 protein mutant disclosed by the invention is a mutant obtained by deleting 56-76aa of a hepatitis E virus ORF3 protein, and the amino acid sequence of the mutant is shown as SEQ ID No.3. The hepatitis E virus ORF3 protein mutant is obtained by deleting the 56th-76aa fragment in the amino acid sequence of the ORF3, the mutant loses the capability of co-localization with mitochondria, but retains the immunogenicity of the ORF3, and when the hepatitis E virus ORF3 protein mutant is prepared into a vaccine, the health risk caused by co-localization of the ORF3 and the mitochondria can be avoided.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically 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 easy to cause chronic infection and liver failure in immunocompromised people and pregnant women.

[0003] HEV encodes three proteins, of which ORF1 protein is a non-structural protein containing 1766 amino acids and multiple functional regions, including: methyltransferase domain (Met), Y domain, papain-like cysteine ​​protease domain (PCP), hypervariable region domain (HVR), Macro domain (X), helicase domain (Hel) and RNA-dependent RNA polymerase domain (RdRp). ORF2 protein consists of 660 amino acids and is the capsid protein of the virus. The truncated ORF2 protein p239 of genotype 1 HEV can form virus-like particles with a diameter of 23nm. At present, the hepatitis E vaccine against HEV subtype 1 with p239 as the antigen is already on the market.

[0004] ORF3 protein is composed of 113 amino acids and is the smallest protein encoded by the HEV genome. It exists on the surface of virus particles. Studies have shown that immunizing animals with ORF3 protein as an immunogen can reduce the infection titer of the virus in animals (PMID: 36548837, Application of ORF3 Subunit Vaccine for Avian Hepatitis E Virus). Moreover, peptides designed based on ORF3 protein can also prevent HEV infection after immunizing primates (PMID: 19763777, Immunogenicity and efficacy of a bacterially expressed HEVORF3 peptide, assessed by experimental infection of primates). Adeno-associated virus (AAV) was used to mediate the expression of ORF3 in mouse muscle. The results showed that ORF3 can induce the production of specific antibodies against HEV 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, some studies have reported that ORF3 plays a role in regulating the host's innate immune response and interfering with the host's signaling pathways (PMID: 24850742, Enhancement of interferon induction by ORF3 productof 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, some 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. Abnormal mitochondrial morphology can affect the cell's energy supply and even lead to cell apoptosis.

[0006] Although the complete ORF3 protein has been proven to induce good immunogenicity and induce antibodies to protect the body, our study found that the complete ORF3 protein co-localizes with mitochondria and causes mitochondrial swelling, which has the potential risk of affecting human health as an immunogen. It is of great significance to design an ORF3 protein mutant that retains the immunogenicity of the ORF3 protein and avoids co-localization with other mitochondria. Summary of the invention

[0007] The present invention aims at the above-mentioned deficiencies in the prior art and provides a hepatitis E virus ORF3 protein mutant, gene and recombinant adenovirus. An ORF3 (del 56-76aa) mutant is designed that can retain the immunogenicity of ORF3 while avoiding co-localization with mitochondria and affecting mitochondrial morphology, which 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 in 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 a sequence for expressing the recombinant adenovirus is transfected into competent cells for expression to obtain the recombinant adenovirus.

[0013] The present invention also provides a 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 56-76aa fragment 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 and mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0018] Figure 3 This is a diagram showing the co-localization results 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 genotype 1 HEV was used, wherein the amino acid sequence of genotype 1 ORF3 (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 ORF3 of genotype 1).

[0027] In this study, the gene fragments of GFP and ORF3 were inserted into the shuttle plasmid vector pDC315 by molecular cloning technology mediated by the restriction site-specific enzymes EcoR I and BamH I, and recombinant adenovirus shuttle plasmids (pDC315-GFP and pDC315-ORF3) were successfully constructed. Subsequently, the constructed recombinant adenovirus shuttle plasmid and the adenovirus backbone plasmid (i.e., pBHGloxΔE1, 3Cre) were introduced into 293 cells (cell density reached 80%) by co-transfection technology. On the 10th day after transfection, the cells were collected using a cell scraper, centrifuged, and resuspended in PBS. Then, through three repeated freeze-thaw cycles and high-speed centrifugation at 12000rpm, cell debris was removed, and pure recombinant adenovirus supernatant was collected.

[0028] In order to increase the yield of recombinant adenovirus, we amplified the two recombinant adenoviruses obtained above in 293 cells. After obvious lesions were observed in the cells, three freeze-thaw cycles were repeated again, and 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 units of virus particles / ml (vp / mL). After diluting the titer of GFP and ORF3 recombinant adenovirus to 8×1010vp / mL, we packaged 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 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 liver cancer cells), and used Ad5-GFP as a control. We used the mitochondrial membrane protein Tom20 as a mitochondrial marker to prove that ORF3 can co-localize with mitochondria. Compared with the control group cells expressing GFP, the mitochondria in the cells expressing ORF3 were significantly enlarged and showed abnormal mitochondrial morphology ( Figure 1 ).

[0030] Example 2

[0031] Identification of the key amino acid sequence that determines the co-localization of ORF3 protein with mitochondria.

[0032] In Example 1, we identified that the co-localization of ORF3 protein with mitochondria resulted in abnormal mitochondrial morphology.

[0033] The genotype 1 ORF3 protein contains 114 amino acids. The key region that determines the co-localization of the ORF3 protein with mitochondria was identified, which helped us design an ORF3 protein mutant that does not co-localize 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 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 lentivirus expressing ORF3(1-55aa)-GFP, ORF3(1-76aa)-GFP, ORF3(39-114aa)-GFP and full-length ORF3-GFP.

[0035] Then, the lentivirus was used to infect HeLa cells, and the cell lines stably expressing ORF3(1-55aa)-GFP, ORF3(1-76aa)-GFP, ORF3(39-114aa)-GFP and full-length ORF3-GFP were selected with puromycin. After a period of screening and culture, the surviving cells were 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 mark mitochondria in stable cell lines expressing GFP (as a control) and these truncated and full-length ORF3 proteins.

[0037] The results showed that through confocal microscopy, it was found 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 56-76aa amino acid sequence of the ORF3 protein is the key region that determines the localization of the ORF3 protein to mitochondria.

[0038] Example 3

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

[0040] In order to identify whether ORF3 (del-56-76aa) co-localizes with mitochondria, we constructed stable cell lines expressing ORF3 (del-56-76aa)-GFP in HeLa cells. The gene of ORF3 (del-56-76aa)-GFP was cloned into the pBoBi plasmid vector and co-transfected with the lentiviral packaging plasmid into 293t cells to obtain lentivirus expressing ORF3 (del-56-76aa)-GFP. Then the lentivirus infected HeLa cells, and the cell lines stably expressing ORF3 (del-56-76aa)-GFP were selected with puromycin. After a period of screening and culture, the surviving cells were cells stably expressing ORF3 (del-56-76aa)-GFP. We used the mitochondrial membrane protein Tom20 as a marker to locate mitochondria.

[0041] Through ultra-high 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, ORF3(del-56-76aa) has fewer deleted amino acid sequences than the full-length ORF3, and therefore retains more antigenic epitopes accordingly ( Figure 3 ).

[0042] Example 4

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

[0044] In this study, the gene fragments of GFP, ORF3 and ORF3 (del-56-76aa) were inserted into the shuttle plasmid vector by molecular cloning technology mediated by the restriction site-specific enzymes EcoR I and BamH I, and recombinant adenovirus shuttle plasmids (pDC315-GFP, pDC315-ORF3, pDC315-ORF3 (del-56-76aa)) were successfully constructed. Subsequently, the constructed recombinant adenovirus shuttle plasmid and the adenovirus backbone plasmid (i.e., pBHGloxΔE1, 3Cre) were introduced into 293 cells (cell density reached 80%) by co-transfection technology. On the 10th day after transfection, the cells were collected with a cell scraper, centrifuged, and resuspended in PBS. Then, through three repeated freeze-thaw cycles and high-speed centrifugation at 12000rpm, cell debris was removed, and pure recombinant adenovirus supernatant was collected.

[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 vp / mL, we will package the prepared virus and store it at -80℃ ultra-low temperature to ensure its long-term stability and experimental effect. The unit of virus titer is the number of virus particles / ml (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 gerbils were randomly divided into three groups: control group 1, experimental group 1, and experimental group 2, each containing 6 gerbils, 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 was repeated for all groups of gerbils three weeks after the initial vaccination.

[0049] Two weeks after the booster immunization, serum samples from all gerbils were collected. Subsequently, the level of neutralizing antibodies against HEV in the serum of each group of gerbils was measured by an in vitro neutralization experiment of HEV. During the experiment, the serum samples were appropriately diluted and incubated with HEV at 37°C for 2 hours. After that, the incubated mixture was used to infect S10-3 cells. After 48 hours of cell infection, immunofluorescence staining was used for detection. In this process, the primary antibody used was a rabbit polyclonal antibody against the ORF2 protein of HEV. Finally, the inhibitory effect of neutralizing antibodies in serum on viral infection was evaluated by fluorescence counting. The lower the fluorescence count value, the stronger the inhibitory ability of the antibody on 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 ), while there was no significant difference in the level of HEV neutralizing antibodies 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: It is a mutant obtained by deleting 56-76aa of the ORF3 protein of 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 sequence 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 sequence for recombinant adenovirus expression according to claim 4, characterized in that: The vector used for the shuttle plasmid is pDC315, into which the gene of 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 sequence for expression of 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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