WO2023231149A1 - Recombinant orthohepevirus genotype c1 orf2 protein and preparation method therefor and use thereof - Google Patents

Recombinant orthohepevirus genotype c1 orf2 protein and preparation method therefor and use thereof Download PDF

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WO2023231149A1
WO2023231149A1 PCT/CN2022/105382 CN2022105382W WO2023231149A1 WO 2023231149 A1 WO2023231149 A1 WO 2023231149A1 CN 2022105382 W CN2022105382 W CN 2022105382W WO 2023231149 A1 WO2023231149 A1 WO 2023231149A1
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hev
orf2
recombinant
rat
protein
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French (fr)
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Wenshi Wang
Hongbo Guo
Renxian TANG
Kuiyang ZHENG
Hongjuan YOU
Xiaohui DING
Jikai Zhang
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Xuzhou Medical University
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Definitions

  • the present invention belongs to the technical field of hepatitis E virus, and particularly relates to a recombinant Orthohepevirus genotype C1 ORF2 protein and a preparation method therefor and use thereof.
  • Hepatitis E Virus is a non-enveloped, positive single-stranded RNA virus that has been classified into the Orthohepevirus of the family Hepeviridae (Smith DB, Simmonds P, Members Of The International Committee On The Taxonomy Of Viruses Study G, Jameel S, Emerson SU, Harrison TJ, et al. Consensus proposals for classification of the family Hepeviridae. J Gen Virol. 2014 Oct; 95 (Pt 10) : 2223-32) .
  • the genus Orthohepevirus includes four types of HEV-A to HEV-D (Smith DB, Simmonds P, Izopet J, Oliveira-Filho EF, Ulrich RG, Johne R, et al. Proposed reference sequences for hepatitis E virus subtypes. J Gen Virol. 2016 Mar; 97 (3) : 537-42) , wherein the HEV-A primarily infects humans and causes hepatitis E infection in humans, while HEV-C1 primarily infects rats (Reuter G, Boros A, Pankovics P. Review of Hepatitis E Virus in Rats: Evident Risk of Species Orthohepevirus C to Human Zoonotic Infection and Disease. Viruses.
  • rat HEV rat HEV
  • the rat HEV was first found in wild rats in Germany (Johne R, Heckel G, Plenge-Bonig A, Kindler E, Maresch C, Reetz J, et al. Novel hepatitis E virus genotype in Norway rats, Germany. Emerg Infect Dis. 2010 Sep; 16 (9) : 1452-5) , and then found by isolation in Europe, Asia and the United States (Johne R, Dremsek P, Reetz J, Heckel G, Hess M, Ulrich RG. Hepeviridae: an expanding family of vertebrate viruses. Infect Genet Evol. 2014 Oct; 27: 212-29) .
  • Rat HEV can infect humans and is zoonotic.
  • Rat HEV like other HEV viruses, contains three Open Reading Frames (ORFs) encoding ORF1, ORF2 and ORF3 proteins, respectively.
  • ORF1 is a non-structural protein and is involved in viral replication.
  • ORF2 gene encodes the viral capsid protein ORF2, which is primarily involved in assembly of viral particles and binding to a host cell and inducing production of neutralizing antibodies by the host.
  • ORF3 gene encodes multifunctional phosphoprotein ORF3, and is primarily involved in the release of virions.
  • Rat HEV is capable of encoding ORF4, and its C-terminus overlaps that of ORF1 and its function is unknown.
  • ORF2 has three different forms, namely infectious ORF2, glycosylated ORF2 and truncated ORF2, wherein the infectious ORF2 is mainly present in the viral particles, and the glycosylated ORF2 and truncated ORF2 are the major secreted forms of ORF2 (Yin X, Ying D, L Subscribe S, Tang Z, Walker CM, Xia N, et al. Origin, antigenicity, and function of a secreted form of ORF2 in hepatitis E virus infection. Proc Natl Acad Sci U S A. 2018 May 1; 115 (18) : 4773-8) .
  • ORF2 capsid protein is highly conserved and has high immunogenicity.
  • ORF2 is capable of stimulating the body to generate humoral immune responses, and is also a primary target for cellular immune responses. The corresponding antibodies generated by ORF2 stimulation can be detected in the serum of the patient.
  • the HEV-A and HEV-C1 have a distant genetic relationship, and the homology of ORF2 of the two is only 50%–60%. This suggests that the difference between the immunogenicity of human ORF2 and that of rat ORF2 is large, and thus the cross reactivity between the ORF2 protein of the human HEV and an ORF2-targeted antibody generated after the rat HEV infects a human body is low, and the ORF2 protein of the human HEV can neither be used for detecting a serum sample of a patient contracting the rat HEV nor be used for developing a vaccine against the rat HEV.
  • the research and development of the recombinant rat HEV ORF2 protein is of great importance to such prevention and treatment strategies as detection means of rat HEV and vaccine preparation.
  • the present invention aims to provide a recombinant Orthohepevirus genotype C1 ORF2 protein and a preparation method therefor and use thereof.
  • the recombinant rat HEV-C1 ORF2 protein is a consensus amino acid sequence designed according to the known ORF2 of rat HEV, and can retain immunogenicity of the rat HEV to the utmost extent; the recombinant protein can stimulate different species of animals to produce specific antibodies.
  • the present invention provides a recombinant rat HEV-C1 ORF2 protein, wherein an amino acid sequence of the recombinant protein is set forth in SEQ ID No. 1;
  • amino acid sequence of the recombinant protein has at least 90%identity to a sequence set forth in SEQ ID No. 1.
  • the present invention provides a gene encoding the recombinant rat HEV-C1 ORF2 protein, wherein a nucleotide sequence of the gene is set forth in SEQ ID No. 2.
  • the present invention also provides an expression vector of a recombinant rat HEV-C1 ORF2 protein comprising a plasmid backbone and the gene.
  • the gene is inserted into a multiple cloning site of the plasmid backbone.
  • the present invention also provides a recombinant bacterial strain expressing the recombinant rat HEV-C1 ORF2 protein, wherein the recombinant bacterial strain is transformed with the expression vector.
  • E. coli BL21 is used as host bacteria for the recombinant bacterial strain.
  • the present invention provides use of the recombinant rat HEV-C1 ORF2 protein in preparing a vaccine for preventing human infection of rat HEV.
  • the vaccine is a subunit vaccine.
  • the present invention also provides use of the recombinant rat HEV-C1 ORF2 protein in preparing a reagent for detecting human infection of rat HEV.
  • the present invention provides a monoclonal antibody and/or a polyclonal antibody obtained after immunization of an animal with the recombinant rat HEV-C1 ORF2 protein.
  • the present invention provides use of the monoclonal antibody and/or the polyclonal antibody in preparing a reagent for detecting rat HEV.
  • the present invention provides use of the monoclonal antibody and/or the polyclonal antibody in preparing a vaccine for preventing human infection of rat HEV.
  • the present invention has the following beneficial effects: the recombinant Orthohepevirus genotype C1 ORF2 protein provided in the present invention is a consensus amino acid sequence designed according to the known ORF2 of rat HEV, and can retain immunogenicity of the rat HEV to the utmost extent; the recombinant protein can stimulate different species of animals to produce specific antibodies.
  • the recombinant protein can detect antibody levels in different species (rabbits, rats and mice) after immunization with rat HEV-ORF2, and can effectively distinguish the difference in antigen-antibody interactions after immunization with an HEV-A (human HEV, hHEV) ORF2 and the HEV-C1 ORF2.
  • HEV-C1 ORF2-targeted specific antibodies generated in serum of rats immunized with HEV-C1 ORF2 protein can effectively block specific binding of HEV-C1 ORF2 virus-like particles to rat hepatoma cells RH-35, while serum of unimmunized rats and that of rats immunized with the HEV-AORF2 protein show no blocking effect, and thus the recombinant protein is proved to be capable of being used for preparing HEV-C1 vaccines.
  • FIG. 1 shows the homology alignment of HEV-C1 ORF2 amino acid sequences and the generation of a consensus sequence
  • FIG. 2 shows the genetic relationship of HEV-C1 ORF2
  • FIG. 3 shows the plasmid map of pET-21a (+) -Rat ORF2 357-595;
  • FIG. 4 shows the PCR product of HEV-C1 ORF2
  • FIG. 5 shows the identification of the purification of HEV-C1 ORF2 with SDS-PAGE Coomassie staining, where the samples in the lanes from left to right are Marker, sample before passing through the column, sample after passing through the column, sample eluted with 10 mL of wash buffer, sample eluted with 5 mL of elution buffer, sample before dialysis, and sample after dialysis;
  • FIG. 6 shows the identification of denatured and non-denatured recombinant HEV-C1 ORF2 proteins using Western blot
  • FIG. 7 shows that the recombinant HEV-C1 ORF2 protein can form virus-like particles according to observation with an electron microscope
  • FIG. 8 shows the antibody levels of mice, New Zealand white rabbits and rats immunized with HEV-C1 ORF2, where A, B and C are mice, New Zealand white rabbits and rats, respectively;
  • FIG. 9 shows the results of the specific interaction of antibody generated by immunization with recombinant HEV-C1 ORF2 protein with HEV-C1 ORF2, HEV-A ORF2 and HEV-A ORF3, where A is rat 1 and B is rat 2;
  • FIG. 10 shows the analysis of the interaction between serum from a HEV infected patient and HEV-A ORF2 and HEV-C1 ORF2;
  • FIG. 11 shows the results of the detection of HEV-C1 ORF2 protein using the polyclonal antibody prepared by immunization of a rabbit with HEV-C1 ORF2 protein;
  • FIG. 12 shows that HEV-C1 ORF2 can form virus-like particles, which can specifically bind to cell membrane of rat hepatoma cell RH-35 and can fully simulate the process of adsorption of rat HEV virus on the surface of susceptible cells, where A shows the binding of the control to the surface of rat hepatoma cell RH-35, and B shows the binding of HEV-C1 ORF2 to the surface of rat hepatoma cell RH-35;
  • FIG. 13 shows that the HEV-C1 ORF2-targeted specific antibody generated in the serum of a rat immunized with the HEV-C1 ORF2 protein can effectively block the specific binding of HEV-C1 ORF2 virus-like particles to RH-35 cells, while the serum of unimmunized rat and the serum of a rat immunized with HEV-A ORF2 protein have no blocking effect, where A is the immunofluorescence patterns of the negative control groups in which RH-35 cells are not incubated with rHEV-ORF2, and only anti-hHEV-ORF2 and rHEV-ORF2 are added, and B is the immunofluorescence patterns showing the interaction of the RH-35 cells with the rHEV-ORF2, the rHEV-ORF2 and the anti-hHEV-ORF2, and the rHEV-ORF2 and the anti-rHEV-ORF2, respectively.
  • A is the immunofluorescence patterns of the negative control groups in which R
  • the present invention provides a recombinant rat HEV-C1 ORF2 protein, wherein an amino acid sequence of the recombinant protein is set forth in SEQ ID No. 1, which is specifically as follows:
  • the recombinant HEV-C1 ORF2 protein provided by the present invention consists of 240 amino acids and has a molecular weight of 26 kDa.
  • the amino acid sequence of the recombinant HEV-C1 ORF2 protein is a consensus sequence obtained by aligning with the amino acid sequence of HEV-C1 ORF2 in NCBI database and constructing based on functional analysis of Vector NTI Advance 11.5AlignX.
  • the present invention also provides a recombinant rat HEV-C1 ORF2 protein, wherein the amino acid sequence of the recombinant protein has at least 90%identity to the sequence set forth in SEQ ID No. 1, and preferably has at least 95%identity.
  • the sequence set forth in SEQ ID No. 1 is a consensus sequence constructed based on artificial analysis by utilizing the characteristic of consensus sequences, so that a sequence having at least 90%identity to the sequence set forth in SEQ ID No. 1 is equivalent to the sequence set forth in SEQ ID No. 1 in terms of performance and is able to obtain test effect equivalent to that of the sequence set forth in SEQ ID No. 1.
  • Sequence identity is typically measured in percentage identity (or similarity or homology) ; the higher the percentage, the more similar the two sequences.
  • Methods for aligning and comparing sequences are well known in the art. Various programs and alignment algorithms are described below: Smith T, Waterman M. Comparison of biosequences. Advances in Applied Mathematics. 1981 Dec, 2 (4) : 482-489; Needleman S, Wunsch CA general method applicable to the search for similarities in the amino acid sequence of two proteins, J Mol Biol. 1970 Mar; 48 (3) : 443-53; Pearson W, Lipman D. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr; 85 (8) : 2444-8; Higgins D, Sharp P.
  • CLUSTAL a package for performing multiple sequence alignment on a microcomputer. Gene. 1988 Dec 15; 73 (1) : 237-44; Higgins D, Sharp P. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl Biosci. 1989 Apr; 5 (2) : 151-3; Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988 Nov 25; 16 (22) : 10881-10890; Huang X, Miller W, Schwartz S, Hardison R. Parallelization of a local similarity algorithm. Comput Appl Biosci. 1992 Apr; 8 (2) : 155-65; Pearson W. Using the FASTA program to search protein and DNA sequence databases. Methods Mol Biol.
  • the alignment tool ALIGN Myers E and Miller W. Optimal Alignments in Linear Space. CABIOS 4, 1988, 11-17
  • LFASTA Nearson W, Lipman D. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr; 85 (8) : 2444-8
  • ALIGN compares entire sequences
  • LFASTA compares local regions for similarity.
  • these alignment tools and their respective tutorials are available from the website of the National Center for Supercomputer Applications (NCSA) on the Internet.
  • the Blast 2 sequence function can be performed using the default BLOSUM62 matrix set to default parameters (gap existence penalty of 11, and per residue gap penalty of 1) .
  • the alignment should be performed using Blast 2 sequence function, employing PAM30 matrix set to default parameters (gap opening penalty of 9, and gap extension penalty of 1) .
  • BLAST sequence comparison systems are available, for example, from the NCBI website. (Altschul S, Gish W, Miller W, Myers E, Lipman D. Basic local alignment search tool. JMol Biol. 1990 Oct 5; 215 (3) : 403-10; Gish W, States D.
  • the present invention provides a gene encoding the recombinant rat HEV-C1 ORF2 protein, wherein a nucleotide sequence of the gene is set forth in SEQ ID No. 2, which is specifically as follows:
  • the present invention also provides an expression vector of a recombinant rat HEV-C1 ORF2 protein comprising a plasmid backbone and the gene.
  • a plasmid backbone there is no particular limitation for the specific type of the plasmid backbone in the present invention, and conventional commercially available expression vectors, such as expression plasmids including the eukaryotic expression plasmid pcDNA3.1 and the like and related expression plasmids of slow virus/adenovirus vectors (such as pWPI and pAAV) , can be used.
  • the plasmid backbone is preferably pET-21a (+) .
  • the gene is preferably inserted between restriction enzyme digestion sites Nde I and Xho I of the plasmid backbone.
  • restriction enzyme digestion sites Nde I and Xho I of the plasmid backbone There is no particular limitation for the method for constructing the expression vector of the recombinant rat HEV-C1 ORF2 protein, and a conventional method for constructing a recombinant vector in the art can be used.
  • the recombinant expression vector is constructed by using double restriction enzyme digestion and ligation.
  • the present invention also provides a recombinant bacterial strain expressing the recombinant rat HEV-C1 ORF2 protein, wherein the recombinant bacterial strain is transformed with the expression vector.
  • E. coli BL21 is used as host bacteria for the recombinant bacterial strain; there is no particular limitation for the method for the transformation in the present invention, and a conventional method for transformation of an expression vector in the art can be used.
  • the present invention provides use of the recombinant rat HEV-C1 ORF2 protein in preparing a vaccine for preventing human infection of rat HEV.
  • the vaccine is preferably a subunit vaccine.
  • the vaccine comprises the recombinant rat HEV-C1 ORF2 protein and Freund’s complete adjuvant; a concentration of the recombinant rat HEV-C1 ORF2 protein in the vaccine is preferably 0.8–1.2 mg/mL, and more preferably 1 mg/mL.
  • antibodies with relatively high titer can be obtained after immunization of rats, mice and New Zealand white rabbits with the vaccine.
  • the present invention also provides use of the recombinant rat HEV-C1 ORF2 protein in preparing a reagent for detecting human infection of rat HEV.
  • the present invention has found, through researches, that the antibody generated in HEV-A infected patients specifically interacts with HEV-A ORF2, but not with the recombinant rat HEV-C1 ORF2 protein.
  • the recombinant rat HEV-C1 ORF2 protein cannot detect HEV-A infected patients, and the recombinant rat HEV-C1 ORF2 protein can be used for establishing an ELISA method to realize specific diagnosis of human infection of HEV-C1 virus.
  • the present invention provides a monoclonal antibody and/or a polyclonal antibody obtained after immunization of an animal with the recombinant rat HEV-C1 ORF2 protein.
  • a monoclonal antibody and/or a polyclonal antibody obtained after immunization of an animal with the recombinant rat HEV-C1 ORF2 protein.
  • There is no particular limitation for the method for separating and purifying the monoclonal antibody and/or the polyclonal antibody and a conventional method for separating and purifying a monoclonal antibody and/or a polyclonal antibody in the art can be used.
  • the present invention also provides use of the monoclonal antibody and/or the polyclonal antibody in preparing a reagent for detecting rat HEV.
  • the present invention also provides use of the monoclonal antibody and/or the polyclonal antibody in preparing a vaccine for preventing human infection of rat HEV.
  • HEV-C1 ORF2 Sequences of HEV-C1 ORF2 were downloaded from NCBI and sequence numbers were: HEV-C1HEV (AB847305) , HEV-C1 HEV (LC145325) , HEV-C1 HEV (AB847306) , HEV-C1 HEV (AB847309) , HEV-C1 HEV (AB847308) , HEV-C1 HEV(GU345042) , HEV-C1 HEV (GU345043) , HEV-C1 HEV (JN167537) , HEV-C1 HEV (KM516906) , HEV-C1 HEV (JN167538) , HEV-C1 HEV (MK050105) , HEV-C1 HEV (LC225389) , HEV-C1 HEV (AB847307) , HEV-C1 HEV (JX120573) , HEV-C1 HEV (MG813927) and H
  • a nucleotide sequence was designed according to the consensus amino acid sequence obtained by alignment of ORF2 sequences, codon optimization was carried out according to the host E. coli, and a gene encoding the ORF2 sequence was obtained through gene synthesis.
  • the gene was amplified by PCR, restriction enzyme digestion sites NdeI and XhoI were added at two ends of a primer, respectively, the gene encoding the truncated ORF2 (357-595aa) fragment and a pET21a vector were subjected to double restriction enzyme digestion with NdeI and XhoI and gel extraction, and then were ligated using a T4 ligase to construct a recombinant expression plasmid pET-21a (+) -Rat ORF2357-595 (the plasmid map is shown in FIG. 3) .
  • the recombinant expression plasmid was provided with an initiation codon and a His tag, so that the protein of interest and the His tag were expressed in
  • a gene of the HEV-C1 ORF2 (357-595aa) consensus sequence was artificially synthesized.
  • the length of the gene fragment was 720 bp
  • the gene sequence was set forth in SEQ ID No. 2
  • the fragment was synthesized by Suzhou Genewiz Biological Technology Co., Ltd., and specific steps are as follows:
  • ORF2 PCR primers were used for amplification, the length of the gene fragment was 730 bp, restriction enzyme digestion sites NdeI and XhoI were added at the 5’ end and the 3’ end, respectively, and the PCR amplification primers are as follows:
  • the PCR reaction system is shown in Table 1.
  • composition 25 ⁇ L of system 5X Q5 Reaction Buffer 5 ⁇ L 10 mM dNTPs 0.5 ⁇ L 10 ⁇ M Forward Primer 1.25 ⁇ L 10 ⁇ M Reverse Primer 1.25 ⁇ L Template DNA 1 ng Q5 High-Fidelity DNA Polymerase 0.25 ⁇ L Nuclease-Free Water to 25 ⁇ L
  • the size of the PCR product was identified by agarose electrophoresis, and the results are shown in FIG. 4, and the sequence length was as expected.
  • the fragment of interest and the His-tagged vector pET-21a (+) were each enzyme digested with Nde I and Xho I (see Table 2 for restriction enzyme digestion system) , and then were ligated with a T4 ligase (see Table 3 for ligation system) overnight.
  • composition 25 ⁇ L of system DNA (PCR/plasmids) 0.5 ⁇ g 10X rCutSmart Buffer 2.5 ⁇ L (1X) Nde I 0.5 ⁇ L (20 units) Xho I 0.5 ⁇ L (20 units) Nuclease-free Water to 25 ⁇ L
  • plasmid was extracted by using a TIANprep Mini Plasmid Kit (DP103) and sent to Suzhou Genewiz Biological Technology Co., Ltd. for sequencing identification, and the result was correct and the plasmid was named pET-HEV-C1-ORF2-His.
  • the recombinant expression plasmid pET-21a (+) -Rat ORF2 357-595 was transformed into E. coli expression bacteria BL21, isopropyl- ⁇ -D-thiopyran and galactoside were added to induce expression, and finally bacterial liquid was collected, lysed by ultrasound, centrifuged and purified, and the expression of protein was determined by SDS-PAGE and Western blot. Specific steps are as follows:
  • the bacterial cells were collected in 50 mL centrifuge tubes (centrifuging at 4000 rpm for 10 min to collect the precipitate) . After centrifugation for several times, the precipitate of bacterial cells was collected in a 50 mL centrifuge tube. The tubes were cryopreserved at-20°C.
  • the storage buffer was removed by washing with 4 mL of deionized water, and the column was equilibrated using at least 5 mL of lysis buffer.
  • 2SDS-PAGE electrophoresis 12.5%separation gel and 4%concentration gel were prepared. Firstly, the separation gel was prepared, mixed well and then filled into the gel plate, absolute ethanol was used for levelling the gel surface, and 40 min later, the absolute ethanol on the upper layer was poured out, and residual liquid was adsorbed with absorbent paper; then the concentration gel was prepared, mixed well and then filled into the upper layer, and after a comb was inserted, the gel was left to stand for about 40 min at room temperature. After the gel was fully solidified, it was put into a refrigerator at 4°C and stored for later use. See Table 6 for preparation of electrophoresis buffer.
  • the gel plate was put into an electrophoresis frame, and the electrophoresis buffer was added and then the comb was removed. 15 ⁇ L of protein sample was added into each gel hole except for one, which was added with a protein Marker.
  • the electrophoresis frame was put into an electrophoresis tank, and the electrophoresis was started after the electrophoresis buffer in the inner tank and the outer tank was replenished. Line pressing was performed for 30 min at 70 V, and then the voltage was changed to 120 V when the sample was electrophoresed to the boundary line between the concentration gel and the separation gel, and the electrophoresis was stopped when bromophenol blue moved to the bottom of the separation gel.
  • a PVDF membrane with the same size as the gel block of interest was obtained by cutting, soaked in methanol for 30 s to be fully activated, and then placed in recycled wet transfer buffer to be soaked for 3–5 min.
  • a white filter pad with the same size as the gel block was obtained by cutting, fully soaked, and then put on a wet transfer clamp, and the membrane transfer was performed in an electroporator for wet transfer.
  • the sequence was as follows: white plate-sponge pad-white filter paper-PVDF membrane-gel-white filter paper-sponge pad-black plate. The power was connected and the voltage was stabilized at 100 V, and wet transfer was performed for 100 min.
  • the membrane was soaked in a blocking solution (5%skimmed milk powder) and put in a shaker for 2 h of blocking at room temperature. After the blocking was finished, the PVDF membrane was cleaned simply, cut to be consistent with the protein of interest in terms of size and placed into an antibody incubation box.
  • the His antibody serum was subjected to 5000-fold dilution by using 1%BSA and then added into the incubation box. The mixture was incubated on a shaker at 4°C overnight. The antibody incubation box was taken out the next day. After rewarming on the shaker for 30 min at room temperature, the primary antibody was pipetted out and washed 3 times (10 min each time) with TBST wash buffer in a shaking manner.
  • the PVDF membrane was placed in the antibody incubation box, and the diluted anti-rabbit IgG antibody was added (the dilution ratio of 1: 5000, the diluent being 1%BSA) .
  • the mixture was incubated for 2 h on a shaker at room temperature, and then the secondary antibody was pipetted out and washed 3 times (10 min each time) with TBST wash buffer in a shaking manner.
  • ECL was used for chromogenic reaction in Western blot.
  • Recombinant HEV-C1 ORF2 protein forms virus-like particles capable of simulating the structure of HEV-C1 virus and having strong immunogenicity and is a candidate strain of HEV-C1 subunit vaccine
  • the rat needed to be immunized six times. Two weeks after the final immunization, all the blood of the rat was obtained from the heart and the rat was sacrificed, and the serum titer after the sixth immunization was detected.
  • mice 200 ⁇ L of mouse blood was obtained from the inner canthus using capillary pipette before immunization, and serum was separated to be used as a negative control.
  • the purified recombinant HEV-C1 ORF2 protein was diluted to 50 ⁇ L (100 ⁇ g) and emulsified with equal volume of Freund’s complete adjuvant, and subcutaneous injection was performed for immunization.
  • One week after the immunization blood was taken from the inner canthus of the mouse to obtain 200 ⁇ L of mouse serum after the first immunization, and the serum titer was detected.
  • the mouse needed to be immunized six times.
  • One week after the final immunization all the blood of the rat was obtained from the eyeball and the rat was sacrificed by cervical dislocation, and the serum titer after the sixth immunization was detected.
  • An ELISA plate was coated with 200 ng of the recombinant HEV-C1 ORF2 protein, and another plate was coated with ORF3, also with His-tag, as a negative control;
  • IgG-HRP secondary antibodies (1: 500 dilution) against different animal sera (anti-rat, anti-mouse and anti-rabbit) were each added, incubated for 1 h at 37°C and then discarded, and the plate was washed with PBST three times;
  • TMB color developing solution was added for color developing for 2 min, H 2 SO 4 was added to stop the reaction, and an ELISA spectrophotometer plate reader was used for detecting the results at450 nm.
  • the reagents used were:
  • the detection results showed that after the mouse was immunized with the recombinant HEV-C1 ORF2 protein for 6 times, the antibody titer reached a very high level, and the antibody still had very high antibody titer after being subjected to 1000-fold to 32000-fold dilution (A in FIG. 8) ; after the New Zealand white rabbit was immunized, its serum still stayed in the platform stage of ELISA reaction after 16000-fold dilution, and it could still have good interaction with the recombinant HEV-C1 ORF2 protein after 128000-fold dilution (B in FIG. 8) ; after 5 immunizations of the mouse, its serum was still able to interact with the ORF2 protein after 4000-fold dilution.
  • the ELISA method featuring coating with the antigen can detect HEV-C1 ORF2 serum antibody of different species after immunization, and the results show antibody concentration dependence, so that the ELISA method can be established by using the recombinant HEV-C1 ORF2 protein as the antigen for diagnosing HEV-C1 infected patients, and the immune experiments of different animals above show that the HEV-C1 ORF2 protein has very good immunogenicity.
  • the recombinant HEV-C1 ORF2 protein and proteins of HEV-A ORF2 and HEV-A ORF3 were each used to coat an ELISA plate, and the serum of the rat immunized with the recombinant HEV-C1 ORF2 protein five times was subjected to fold dilution, and the diluted serum was incubated with the three different antigens.
  • the method is as follows:
  • ELISA plates were coated with 200 ng of antigen proteins (HEV-C1 ORF2, HEV-A ORF2 and HEV-A ORF3) , respectively;
  • Anti-rat IgG-HRP secondary antibody (1: 500 dilution) was added, and the mixture was incubated at 37°C for 1 h; the secondary antibody was then discarded, and the plate was washed with PBST three times;
  • TMB color developing solution was added for color developing for 2 min, H 2 SO 4 was added to stop the reaction, and an ELISA spectrophotometer plate reader was used for detecting the results at 450 nm.
  • the serum titers of two immunized rats were determined, and it was found that the antibody generated after the immunization with the recombinant HEV-C1 ORF2 protein could specifically interact with HEV-C1 ORF2, but could not interact with the proteins of HEV-A ORF2 and HEV-A ORF3.
  • the results above show that the level of the cross-immune reaction between the HEV-A ORF2 protein and the HEV-C1 immune antibody is very low, so that the ELISA detection method established with the HEV-A ORF2 cannot be used for diagnosing rat HEV infection, and therefore, a diagnostic kit established with the HEV-C1 is very important.
  • the recombinant HEV-C1 ORF2 protein and the protein of HEV-A ORF2 were each used to coat an ELISA plate, and the serum of an HEV-A infected patient was subjected to fold dilution, and the diluted serum was incubated with the two different antigens.
  • the experimental process is as follows:
  • Anti-rat IgG-HRP secondary antibody (1: 1000 dilution) was added, and the mixture was incubated at 37°C for 1 h; the secondary antibody was then discarded, and the plate was washed with PBST three times;
  • TMB color developing solution was added for color developing for 2 min, H 2 SO 4 was added to stop the reaction, and an ELISA spectrophotometer plate reader was used for detecting the results at 450 nm.
  • the polyclonal antibody generated due to immunization with the recombinant HEV-C1 ORF2 protein can be used as a means for experimental detection of HEV-C1.
  • the length of the ORF2 gene sequence was 730 bp.
  • the rHEV strain LCK-3110 (GenBank: MG813927.1) was used as a template, the pcDNA3.1-Flag was used as a vector, and restriction enzyme digestion sites Xho I and EcoRI were added at the upper stream and the down stream, respectively, to design a primer for amplifying the gene sequence.
  • Upstream primer 5’-aacctcgagatgtctgtcgttgtcgtgctcgtgctcg-3’ (SEQ ID No. 5)
  • Downstream primer 5’-aacgaattcgacactgtcggctgctacggct-3’ (SEQ ID No. 6)
  • the DNA agarose solution was transferred to an adsorption column, left to stand for 2 min, and centrifuged at 12000 rpm for 75 s. (the adsorption column could only accommodate 700 ⁇ L of solution each time, and if the solution could not be added into the adsorption column one time, the rest part could be added into the adsorption column after centrifugation)
  • Waste liquid was discarded, and centrifugation at 10000 rpm was performed for 2 min without adding another material.
  • the recombinant plasmid was sent for sequencing identification, and the result was correct and the plasmid was pcDNA3.1-ORF2-FLAG.
  • HEK 293T cells were transfected with the pcDNA3.1-ORF2-flag plasmid constructed above.
  • the cells were seeded into a 6-well plate at 6 ⁇ 10 5 per well, and 2 mL of DMEM high-sugar culture medium containing 10%fetal bovine serum was added, and after being well mixed, the mixture was incubated in an incubator at 37°C and 5%CO 2 for culture, and 124 h later, the cell confluence reached 80%.
  • the DNA-liposome mixture was added into the six-well plate at 200 ⁇ L per well, and then the mixture was well mixed and incubated in an incubator at 37°C and 5%CO 2 , and the medium was changed into a DMEM high-sugar culture medium containing 10%fetal bovine serum 5 h later, thus completing cell transfection.
  • Triton was discarded, and the plate was washed twice with PBS added along the side wall.
  • Blocking solution (5%skimmed milk powder) was added to the wells, and the mixture was left to stand at room temperature for 60 min.
  • Polyclonal antibody (rabbit antibody) generated by recombinant HEV-C1 ORF2 protein was diluted at 1: 2500 and Flag antibody (mouse antibody) was diluted at 1: 5000, and they were incubated together at 37°C for 1 h.
  • Polyclonal antibody generated by immunization with recombinant HEV-C1 ORF2 protein can block the binding of virus-like particles to the cell surface, and the recombinant protein can be used as the main component of HEV-C1 subunit vaccine.
  • RH-35 rat hepatoma cells purchased from Procell Life Science&Technology Co., Ltd. ) were plated on a 12-well plate at a density of 2 ⁇ 10 5 cells/well and incubated overnight in an incubator at 37°C and 5%CO 2 saturation.
  • HEV-C1 ORF2 purified protein (afinal concentration of 20 ⁇ g/mL per well) was mixed with PBS, and a total of 300 ⁇ L of the two was mixed in a 1.5 mL EP tube, and the control group was PBS; the tubes were incubated at 37°C for 1 h.
  • 1%Casein prepared with PBST was added at 300 ⁇ L per well as a blocking solution, and the cells were blocked at room temperature for 1 h.
  • HEV-C1 ORF2 rabbit polyclonal antibody immune serum was added, diluted with 1%BSA at 1: 3000 and incubated for 1 h at room temperature, and the cells were washed three times with PBS.
  • the rabbit fluorescent secondary antibody and Hoechst were diluted with 1%BSA at 1: 500 and incubated for 1 h at room temperature, and the cells were washed three times with PBS.
  • virus-like particles formed by recombinant HEV-C1 ORF2 protein can specifically bind to the cell surface of RH35, and can simulate the biological characteristics of the binding of rat HEV to surface receptor of host cells.
  • RH-35 rat hepatoma cells were plated on a 96-well plate at a density of 1 ⁇ 10 4 cells/well and incubated overnight in an incubator at 37°C and 5%CO 2 saturation.
  • HEV-C1 ORF2 and HEV-A ORF2 purified proteins were mixed with rat immune serum, and there were three groups: rat negative serum diluted with PBS at a final concentration of 1: 20+20 ⁇ g/mL HEV-C1 ORF2 protein; HEV-C1 ORF2 rat immune serum diluted with PBS at a final concentration of 1: 20+20 ⁇ g/mL HEV-C1 ORF2 protein; HEV-A ORF2 rat immune serum diluted with PBS at a final concentration of 1: 20+20 ⁇ g/mL HEV-C1 ORF2 protein; the mixture was incubate for 1 h at 37°C.
  • 1%Casein prepared with PBST was added at 100 ⁇ L per well as a blocking solution, and the cells were blocked at room temperature for 1 h.
  • HEV-C1 ORF2 rabbit immune serum was added, diluted with 1%BSA at 1: 3000 and incubated for 1 h at room temperature, and the cells were washed three times with PBS.
  • the rabbit fluorescent secondary antibody and Hoechst were diluted with 1%BSA at 1: 500 and incubated for 1 h at room temperature, and the cells were washed three times with PBS.
  • the experimental results are shown in FIG. 13, and they suggest that the HEV-C1 ORF2-targeted specific antibody generated in the serum of a rat immunized with the recombinant HEV-C1 ORF2 protein can effectively block the specific binding of HEV-C1 ORF2 virus-like particles to RH-35 cells, while the serum of unimmunized rat and the serum of a rat immunized with HEV-A ORF2 protein have no blocking effect.
  • the experiment proves that the antibody with protection activity is generated in the serum of a rat immunized with the recombinant HEV-C1 ORF2 protein, and the HEV-C1 can be prevented from invading host cells.
  • the recombinant HEV-C1 ORF2 protein can be used as an effective candidate vaccine, while the antibody generated in the serum of rats immunized with the HEV-A ORF2 protein has no cross-protection activity for the rat HEV.

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