DNA molecule containing whole genome cDNA of live hepatitis A virus attenuated vaccine strain as well as preparation method and application of DNA molecule

Abstract

The invention relates to the technical field of biology, and discloses a DNA molecule containing whole genome cDNA of a live hepatitis A virus attenuated vaccine strain and a preparation method and application of the DNA molecule. Whole-genome infectious cDNA cloning construction is performed on the hepatitis A H2 strain for the first time, and a recovery virus with biological characteristics similar to those of a parent H2 strain is obtained through cDNA cloning rescue obtained through construction, so that a powerful tool is provided for scientific research work and medical application related to hepatitis A viruses and attenuated live vaccines.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a DNA molecule comprising the full genome cDNA of a live attenuated hepatitis A virus vaccine strain and its preparation method and use. BACKGROUND

[0002] Hepatitis A virus (HAV), also known as hepatitis A virus, belongs to the family of small RNA viruses and the genus of hepatotropic viruses. HAV is spherical with a diameter of about 27 nm and has no envelope. The capsid is composed of 60 capsid particles and has a icosahedral symmetry structure. HAV is a single-stranded positive-sense RNA virus with a full-length genome of about 7.5 Kb, which contains an open reading frame encoding four structural proteins (VP4, VP2, VP3 and VP1-2A) and six non-structural proteins (2B, 2C, 3A, 3B, 3C and 3D). HAV is mainly transmitted through the fecal-oral route and causes hepatitis A (referred to as "hepatitis A" for short). Vaccination is the most effective way to prevent hepatitis A, and the live attenuated hepatitis A vaccine H2 strain is a cell-adapted attenuated strain obtained by continuous passage of the wild pathogenic strain H2w in vitro cell lines. Due to its clear attenuation effect and good immunogenicity, the live attenuated hepatitis A vaccine using the H2 strain as the virus has been approved for marketing in China since 1992, and has shown high immunogenicity and long-term protection effect in vaccinated populations, and has been used until now. However, the attenuation and cell adaptation mechanism of the H2 strain, and even the attenuated cell adaptation site are still unclear.

[0003] The construction of full-length infectious cDNA clones of RNA viruses greatly facilitates the directed modification of viral genome sequences. By comparing the biological property changes of wild-type viruses and directed modification mutant viruses, the key virulence sites of the virus can be determined, and the replication and pathogenic mechanism of the virus can be clarified. Moreover, using viruses derived from infectious cDNA clones as vaccine strains is closer to international standards, and compared with polyclonal vaccines, it is easier to promote and apply in the world and is more easily accepted internationally.

[0004] Therefore, it is urgent to develop and construct a live attenuated hepatitis A virus vaccine infectious cDNA molecule clone, which not only has important significance for HAV research work, but also is more conducive to the international promotion and application of the hepatitis A vaccine using the H2 strain as the vaccine strain. SUMMARY

[0005] The purpose of the present application is to provide a powerful tool for the cell adaptation and attenuation research of the live attenuated hepatitis A vaccine H2 strain, thereby providing a DNA molecule comprising the full genome cDNA of a live attenuated hepatitis A virus vaccine strain and its preparation method and use. For the first time, the present application constructs a full genome infectious cDNA clone of the hepatitis A H2 strain, and rescues a recovered virus with similar properties to the parent H2 strain.

[0006] To achieve the above object, the present application provides a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 1.

[0007] The present application provides a full-length infectious cDNA clone of hepatitis A virus, wherein the recombinant plasmid comprises a DNA fragment with a nucleotide sequence as shown in SEQ ID NO: 1.

[0008] The present application provides a method for constructing a full-length infectious cDNA clone of hepatitis A virus, wherein the method comprises:

[0009] (1) obtaining two or more DNA fragments of hepatitis A virus genome by means of segment amplification, wherein all the DNA fragments are connected together to comprise a nucleotide sequence as shown in SEQ ID NO: 1;

[0010] (2) connecting the DNA fragments of hepatitis A virus genome obtained in step (1) with a vector to obtain a recombinant plasmid, wherein the recombinant plasmid comprises the vector and a nucleotide sequence as shown in SEQ ID NO: 1 inserted into the vector.

[0011] The present application provides a full-length infectious cDNA clone prepared by the method according to the third aspect.

[0012] The present application provides a method for rescuing virus from a full-length infectious cDNA clone of hepatitis A virus, wherein the method comprises in-vitro transcription using the full-length infectious cDNA clone of hepatitis A virus as a template, and infecting a sensitive cell with the obtained transcribed RNA;

[0013] The full-length infectious cDNA clone of hepatitis A virus is the full-length infectious cDNA clone of hepatitis A virus according to the third aspect or the fourth aspect.

[0014] The present application provides a rescued virus prepared by the method according to the fifth aspect.

[0015] The present application provides a use of the DNA molecule according to the first aspect, the full-length infectious cDNA clone of hepatitis A virus according to the second aspect or the fourth aspect, or the rescued virus according to the sixth aspect in the preparation of a medicament for preventing and / or treating a disease caused by hepatitis A virus infection.

[0016] By means of the above technical solution, the present application can achieve at least the following beneficial effects:

[0017] (1) The present application first completes the whole genome infectious cDNA clone construction of HAV H2 strain, and the recovered virus with similar biological characteristics to the parent H2 strain can be obtained by the molecular rescue of virus from the infectious cDNA clone.

[0018] (2) The infectious cDNA clone molecule provided by the present application can not only be used for developing new HAV vaccine which is more easily accepted internationally, but also be used as viral vector for expressing foreign genes, and has important significance in the research work related to HAV. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the electrophoresis result diagram of the segment RT-PCR amplification of the RNA of the parent virus of the live attenuated HAV vaccine H2 strain in Example 1.

[0020] Figure 2 It is the schematic diagram of the infectious cDNA clone construction of the live attenuated HAV vaccine H2 strain in Example 1.

[0021] Figure 3 It is the linear electrophoresis result diagram of the infectious cDNA clone of the live attenuated HAV vaccine H2 strain in Example 2.

[0022] Figure 4 It is the electrophoresis result diagram of the transcript RNA of the live attenuated HAV vaccine H2 strain in Example 2.

[0023] Figure 5 It is the electrophoresis result diagram of the segment RT-PCR amplification of the recovered virus from the infectious cDNA clone in Example 3.

[0024] Figure 6 It is the IFA result diagram of the Huh7.5.1 infected by the recovered virus from the infectious cDNA clone in Example 4.

[0025] Figure 7 It is the proliferation curve diagram of the recovered virus and the parent virus in the Huh7.5.1 cell line in Example 5. DETAILED DESCRIPTION

[0026] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges are provided as example for constructing new ranges from the stated ranges. Each midpoint between the endpoints of the ranges can be also provided as a point value for constructing new ranges from the stated ranges. The stated ranges are not to be construed as being limited by the exact numerical values described. Any numerical value, however, can be expressed or represented as being approximated by such languages as about, substantially, or essentially, and used in the application and claims herein for exemplification.

[0027] Although the attenuated live hepatitis A vaccine with the attenuated strain of hepatitis A H2 as the virus has been put on the market in the 1990s, since the attenuated strain is isolated, no researcher has obtained the full-length infectious cDNA clone of the attenuated live hepatitis A vaccine H2 strain, only part of the fragment is obtained. The inventors of the present application first obtain the infectious full-length cDNA clone of the H2 strain by segment reverse transcription amplification of the full genome of the H2 attenuated strain and connecting the amplified fragments by the In-fusion seamless connection method, and rescue the recovered virus similar to the H2 parent strain from the infectious cDNA clone.

[0028] The first aspect of the present application provides a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 1.

[0029]

[0030] SEQ ID NO: 1 is the full-length genomic cDNA sequence of hepatitis A virus (H2 strain).

[0031] The nucleotide sequence of the DNA molecule provided by the present application can only comprise the nucleotide sequence of SEQ ID NO: 1, or can comprise other nucleotide sequences or have certain mutations on the basis of the nucleotide sequence of SEQ ID NO: 1, without affecting or substantially affecting the biological properties of the DNA molecule having the nucleotide sequence of SEQ ID NO: 1.

[0032] According to some preferred embodiments of the present application, the DNA molecule comprises a nucleotide sequence derived from hepatitis A virus (especially derived from H2 strain) having more than 80% identity with SEQ ID NO: 1. Preferably, the identity is more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, or more than 99.9%.

[0033] According to a preferred embodiment of the present application, the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO: 1 (i.e. having 100% identity with SEQ ID NO: 1).

[0034] The second aspect of the present application provides a full-length infectious cDNA clone of hepatitis A virus, which comprises a DNA fragment having a nucleotide sequence as shown in SEQ ID NO: 1.

[0035] According to preferred embodiments of the present application, the infectious cDNA clone further comprises at least one genetic element selected from the group consisting of a promoter (such as T7 promoter, etc.), a hammerhead ribozyme (such as hepatitis D virus hammerhead ribozyme HDVr, etc.), and an optional specific enzyme cutting sequence.

[0036] In the art, generally, an infectious cDNA clone (recombinant plasmid) comprises a vector and a foreign gene fragment inserted into the vector sequence. According to the present application, the DNA sequence comprising the DNA fragment as shown in SEQ ID NO: 1 can be regarded as a “foreign gene fragment”. In the present application, the type of vector is not particularly limited, and any plasmid that can be used for preparation of recombinant plasmids in the art can be suitable for use in the present application. For example, commercially available finished plasmids such as pT7, pACYC177, etc. can be used as vectors, or suitable plasmids can be designed and constructed according to the prior art and actual needs.

[0037] According to some preferred embodiments of the present application, wherein the infectious cDNA clone comprises a plasmid sequence and a foreign DNA molecule sequence inserted into the plasmid, the foreign DNA molecule sequence can be provided by the DNA molecule of the first aspect. The specific features of this DNA molecule are as previously described and will not be repeated here.

[0038] According to a particularly preferred embodiment of the present application, wherein the infectious cDNA clone comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0039]

[0040] SEQ ID NO:2 is the nucleotide sequence of the recombinant plasmid into which the DNA molecule (SEQ ID NO:1) of the full-length genome cDNA of the hepatitis A virus inserted after the T7 promoter of the pT7 plasmid and the specific sequence for enzyme digestion.

[0041] The third aspect of the present application provides a method for constructing a full-length infectious cDNA clone of hepatitis A virus, which comprises:

[0042] (1) obtaining two or more DNA fragments of the genome of the hepatitis A virus by means of segmental amplification, wherein all the DNA fragments, when linked together, contain the nucleotide sequence shown in SEQ ID NO:1;

[0043] (2) linking the DNA fragments of the genome of the hepatitis A virus obtained in step (1) with a vector to obtain a recombinant plasmid, wherein the recombinant plasmid comprises the vector and the nucleotide sequence shown in SEQ ID NO:1 inserted into the vector.

[0044] The above method provided by the present application is a method for constructing the full-length infectious cDNA clone of hepatitis A virus of the second aspect.

[0045] Since the nucleotide sequence of SEQ ID NO:1 is relatively long, it is difficult to amplify or insert such a long DNA fragment into a vector when constructing a recombinant plasmid. Therefore, the inventors found, after a large amount of research, that by amplifying the nucleotide sequence of SEQ ID NO:1 in multiple segments and then linking the amplified fragments with the vector fragments, a recombinant plasmid with the correct target fragment inserted can be obtained more quickly and efficiently.

[0046] In the present application, the multiple fragments of SEQ ID NO:1 can be amplified and linked based on any method capable of preparing a recombinant plasmid in the above-mentioned manner, and finally a recombinant plasmid of the present application is obtained. Preferably, the In-Fusion method is used to link the DNA fragments to prepare the recombinant plasmid of the present application. This method can avoid the introduction of redundant sequences, thereby obtaining a recombinant plasmid with an exogenous DNA fragment inserted into the plasmid having a 100% identity with SEQ ID NO:1.

[0047] Preferably, step (1) amplifies 1 DNA fragment comprising the complete nucleotide sequence of the vector and a partial nucleotide sequence of SEQ ID NO: 1 (e.g. a fragment of about 15-50 nucleotides), and 1-4 DNA fragments each comprising a partial nucleotide sequence of SEQ ID NO: 1 (each fragment usually comprising 2000-5000 nucleotides). That is, step (1) amplifies 2-5 fragments in total. Since increasing the number of amplified fragments will result in a decrease in the efficiency of ligation in step (2), in order to improve the production efficiency, the number of amplified fragments in step (2) can be appropriately reduced, preferably step (1) amplifies 2-4 fragments in total.

[0048] According to a preferred embodiment of the present application, in step (1), each of the DNA fragments has an overlap of 15-25 nucleotides with the adjacent DNA fragment. The "overlap" refers to a short fragment of the same nucleotide sequence at the end of two adjacent amplified fragments, which is used to ligate each fragment into one complete full-length infectious cDNA clone in step (2).

[0049] According to a particularly preferred embodiment of the present application, in step (1), 3 DNA fragments are amplified, which are designated as H2-up fragment, H2-down fragment and pT7 fragment, respectively, wherein the H2-up fragment consists of nucleotides 21-3926 of the nucleotide sequence of SEQ ID NO: 2, the H2-down fragment consists of nucleotides 3901-7500 of the nucleotide sequence of SEQ ID NO: 2, and the pT7 fragment consists of nucleotides 7486-10325 and 1-35 of the nucleotide sequence of SEQ ID NO: 2.

[0050] According to a preferred embodiment of the present application, in step (2), the In-Fusion method is used to ligate the DNA fragments.

[0051] The In-Fusion method is a "seamless cloning" technology that can perform single-fragment or multi-fragment directional cloning and seamless ligation at any position of the vector and the target insert, which is different from the traditional enzyme digestion and ligation method. The present application does not have particular limitations on the specific operations and reagents used in the process of ligating the DNA fragments by the In-Fusion method. For example, commercially available kits or custom-made kits can be used, and the relevant operations can be performed according to the kit instructions, or suitable operation methods and reagents can be designed by referring to the prior art.

[0052] Generally, when constructing infectious cDNA clone plasmid, the correct ligation product needs to be transferred into competent cells (usually E. coli) and the competent cells are cultured for amplification. The inventors have found in the research that when the competent cells (bacteria) carrying the cloning plasmid are amplified, the culture temperature has certain influence on the replication of the transferred plasmid. If the temperature is too high, base mutation or sequence loss may occur in the amplification. After a large number of researches, the inventors have found that when the competent cells carrying the cloning plasmid are amplified and cultured at a temperature of about 30°C (for example, 30±1°C), the possibility of obtaining correct infectious cDNA clone can be effectively improved, which is beneficial to the subsequent rescue of recovered viruses similar to the parent strain in characteristics.

[0053] Therefore, preferably, the above construction method further comprises transferring the ligation product obtained in step (2) into competent cells, and amplifying and culturing the competent cells at a temperature of 29-31°C.

[0054] The fourth aspect of the present application provides an infectious full-length cDNA clone prepared by the method according to the third aspect.

[0055] The fifth aspect of the present application provides a method for rescuing virus (from hepatitis A virus cDNA infectious cDNA clone), which comprises in vitro transcription using hepatitis A virus cDNA infectious cDNA clone as template, and infecting sensitive cells with the obtained transcribed RNA;

[0056] The hepatitis A virus cDNA infectious cDNA clone is the hepatitis A virus full-length infectious cDNA clone according to the second aspect or the fourth aspect.

[0057] In the present application, the "sensitive cells" refer to cells that can support the hepatitis A virus transcribed RNA to be translated and assembled into virus after the hepatitis A virus transcribed RNA is transfected into the cells, and the assembled virus can proliferate rapidly. Any cell in the art that can be used for virus rescue and has the above characteristics can be applied to the present application.

[0058] According to a preferred embodiment of the present application, the sensitive cells are Huh7.5.1 cells.

[0059] The sixth aspect of the present application provides a rescued virus (or "recovered virus") prepared by the method according to the fifth aspect.

[0060] The seventh aspect of the present application provides the use of the DNA molecule according to the first aspect, the hepatitis A virus full-length infectious cDNA clone according to the second aspect or the fourth aspect, or the rescued virus according to the sixth aspect in the preparation of a medicament for preventing and / or treating diseases caused by hepatitis A virus infection.

[0061] Preferably, the drug comprises a hepatitis A vaccine, preferably a live attenuated hepatitis A vaccine.

[0062] The present application will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the present application, and are not used to limit the present application.

[0063] In the following examples, the reagents and materials used are commercially available products purchased from regular chemical / biological reagent material suppliers, and the reagents are all of analytical purity. Unless otherwise specified, the operating temperature is room temperature (25±5℃).

[0064] Example 1: Construction of full-length cDNA clone of genome of live attenuated hepatitis A virus H2 strain

[0065] (1) Extraction of viral RNA of live attenuated hepatitis A virus H2 strain

[0066] The viral RNA of live attenuated hepatitis A virus H2 strain (Chinese Academy of Medical Sciences) was extracted using an RNA extraction kit (Thermo) according to the instructions of the kit.

[0067] For long-term preservation, the extracted and purified RNA was stored in a -80℃ refrigerator for standby use.

[0068] (2) Acquisition of viral cDNA of live attenuated hepatitis A virus H2 strain

[0069] The viral RNA of live attenuated hepatitis A virus H2 strain obtained in step (1) was used as a template to obtain viral cDNA by reverse transcription. The reverse transcription reaction system and conditions were as follows: 9 μL of template RNA was mixed with 2 μL of random primer, and after 5 min of reaction at 70℃, it was placed on ice for 10 min; then 4 μL of 5×MLV Buffer, 1 μL of 10 mM dNTP, 0.5 μL of M-MLV, 0.5 μL of RRI, and 3 μL of RNase-free water were added to the system, and after 1 h of reaction at 42℃, it was again reacted at 70℃ for 10 min.

[0070] (3) Amplification of full-length genome fragments of live attenuated hepatitis A virus H2 strain

[0071] The viral cDNA of live attenuated hepatitis A virus H2 strain obtained by reverse transcription was used as a template, which was divided into two fragments, and under the action of Q5 high-fidelity DNA polymerase, amplification was carried out using the primers in Table 1 to obtain DNA fragments H2-up, H2-down, and pT7. The PCR system and conditions are shown in Table 2.

[0072] Table 1

[0073]

[0074] Table 2

[0075]

[0076] The three fragments were verified by 1% agarose gel electrophoresis, and the gene fragments were recovered using a nucleic acid gel recovery kit (Promega).

[0077] The gel electrophoresis diagram is shown in Figure 1 Lane M: DNA Markr DL15000, Lane 1: H2-up, Lane 2: H2-down, Lane 3: pT7 vector. It can be seen from the diagram that the amplified fragments are single and the fragment sizes are consistent with the expected.

[0078] (4) Construction and identification of recombinant plasmid

[0079] Referring to Figure 2 The recovered HAV H2 strain PCR amplification fragment of live attenuated vaccine was subjected to seamless ligation with the pT7 vector under the action of In-Fusion enzyme (TaKaRa). 5x In-Fusion enzyme, water, vector and insert were placed in a PCR tube, and the insert H2-up and H2-down were added in a molar ratio of 2:2:1, and the ligation was performed at 50°C for 15 min. The ligation product was transferred into E. coli HB101 competent cells (TaKaRa), and the plate was inverted and cultured overnight at 30°C in an incubator.

[0080] The next day, single colonies were picked and cultured in liquid LB medium containing resistance, and plasmids were extracted for verification of plasmid size. The infectious cDNA clone with correct plasmid size was sent to GenScript Biotech (Shanghai) Co., Ltd. for sequencing. The bacterial liquid with correct sequencing was inoculated into 250 ml of LB medium containing resistance, and the plasmid was sequenced after growing for 16 h. The plasmid contained the T7 promoter and the specific sequence of the HAV H2 strain live attenuated vaccine, and the results were consistent with the expected, indicating that the infectious cDNA clone recombinant plasmid was successfully constructed, and the recombinant plasmid was named pT7-H2 and used for the following virus rescue.

[0081] Example 2: Rescue of HAV H2 strain virus

[0082] (1) Linearization: The pT7-H2 recombinant plasmid was subjected to single enzyme digestion using restriction endonuclease Xmal (NEB), and the reaction system is shown in Table 3.

[0083] Table 3

[0084] Components Volume / μL Recombinant plasmid pT7-H2 30 (30 μg) Xmal 3 10x Cutsmart Buffer 10 ddH2O 57

[0085] 37℃ reaction for 3h, 1% agarose gel electrophoresis to detect whether it was completely cut. Linearization results are shown in Figure 3 , Lane M: DNA Marker DL15000, Lane 1: pT7-H2 recombinant plasmid, Lane 2: linearized plasmid. The full length of HAV genome is about 7485bp, the length of vector is about 2841bp, and the full length of recombinant plasmid is 10326bp. As can be seen from Figure 3 , the size of the plasmid is consistent with the expected, and the linearization is complete.

[0086] Phenol chloroform extraction and purification of linearized products: 200μL of water was added to the enzyme digestion product, then 300μL of DNA extraction solution (the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1) was added, centrifuged at 13000rpm for 10min, and the supernatant was retained; an equal volume (300μL) of nucleic acid extraction solution (chloroform: isoamyl alcohol = 24:1) was added, centrifuged for 5min, and the supernatant was retained; 750μL of anhydrous ethanol was added, precipitated at -80℃ for 30min, centrifuged at 4℃ at 13000rpm for 10min, and the precipitate was retained; 1mL of 75% ethanol was added for washing once, and centrifuged at 13000rpm for 5min; the precipitate was air-dried, dissolved with RNase-free water, and the concentration was detected.

[0087] (2) In vitro transcription: The purified linearized product was used as a template, and T7 in vitro transcription kit (Promega) was used for in vitro transcription. The reaction system is shown in Table 4.

[0088] Table 4

[0089] Components Volume (μL) 5x transcription buffer 6 rNTP (30 mM rATP, rCTP, rUTP, 10 mM rGTP) 6 m7 Cap analogue (10 μM) 1.5 Linearized template 14 (2.5 μg) T7 Enzyme Mix 2.5

[0090] After 6h of reaction at 37℃, 1% agarose gel electrophoresis was used to detect the transcription product, and the results are shown in Figure 4 , Lane M: DNA Marker DL15000, Lane 1: H2 in vitro transcription product. As can be seen from the figure, the in vitro transcription was successful and the band was single.

[0091] 4μl RQI was added to the reaction product to remove the DNA template, and the reaction was carried out at 37℃ for 20min. The in vitro transcription product was purified by LiCl method. After detecting the RNA concentration of the purified transcription, it was stored in the refrigerator at -80℃.

[0092] (3) Transfection: The purified transcription was transfected into Huh7.5.1 cells using liposome lipo3000 (Invitrogen). The transfection method is as follows:

[0093] 5×10 4 / mL of Huh7.5.1 cell suspension was evenly plated into 6-well plates (2 mL per well) the night before, and the cell density was grown to 30%-40% after 12 h incubation at 37 °C in an incubator. 5 μg of purified RNA product from in vitro transcription was mixed with 125 μL of Opti-MEM, 5 μL of liposome reagent was mixed with 125 μL of Opti-MEM, and the mixture of purified transcription product and Opti-MEM was mixed with the mixture of liposome transfection reagent and Opti-MEM. After mixing, the mixture was incubated at room temperature for 15 min to obtain the RNA-liposome mixture. The growth medium in the cell plate was replaced with Opti-MEM medium, and 250 μL of the RNA-liposome mixture was added to each well of the cell plate. The cell plate was incubated at 37 °C in a 5% CO2 cell incubator for 6 h. After incubation, the cell supernatant was discarded, 2 mL of maintenance medium was added to each well, and the cell plate was incubated at 37 °C in a 5% CO2 cell incubator for 6 days. The culture supernatant of P2 generation was collected, and the cells were subcultured to new cells. The culture supernatant of P2 generation at 0 d, 2 d, 4 d, and 6 d after infection was collected for real-time PCR to monitor the change in RNA copy number. The RNA copy number of P2 generation was observed to increase from 10 5.7 RNA copies / ml to 10 6.8 RNA copies / mL, and the proliferation was obvious. The cell supernatant was obtained by freezing and centrifugation, and was inoculated into new cells for virus propagation. The P3 generation was used as the virus seed liquid, and was named Res-H2.

[0094] Example 3: Determination and analysis of the full sequence of the virus genome recovered from the infectious cDNA clone

[0095] The RNA of the virus Res-H2 recovered from the infectious cDNA clone was extracted using a PureLink TM RNA mini kit (Thermo), and was used as a template for amplification of two fragments of the virus genome recovered from the infectious cDNA clone using the primers H2-1F to H2-5R and H2-6F to H2-10R in Table 5, and a one-step reverse transcription kit (TaKaRa). The reaction system (25 μL) and the procedure are shown in Table 6. In Table 5, the primers marked with “*” are used for both sequencing and sequence amplification.

[0096] Table 5

[0097]

[0098] Table 6

[0099]

[0100] The RT-PCR product was sequenced using primers H2-1R, H2-2F, H2-3F, H2-4F, H2-5F, H2-6F, H2-7F, H2-8F, H2-9F, H2-10F.

[0101] The RT-PCR amplification results are shown in Table 1. Figure 5 , lane M: DNA Marker DL15000, lane 1: H2-up, lane 2: H2-down. As can be seen from the figure, the amplified bands are single and the size is consistent with the expected target fragment. The primer pairs used in lanes 1 and 2 are [H2-1F*, H2-5R*], [H2-6F*, H2-10R*], respectively. The results of sequence determination also show that the sequence of the obtained fragment is specific to the attenuated live vaccine H2 strain of hepatitis A virus, which is consistent with the expectation.

[0102] Example 4: Detection of protein expression of the recovered virus Res-H2 of the infectious cDNA clone of the attenuated live vaccine H2 strain of hepatitis A virus in Huh7.5.1 cells

[0103] The virus-specific proteins in the Huh7.5.1 cells infected with the recovered virus were detected by indirect immunofluorescence (IFA) using a monoclonal antibody against the VP1 protein of hepatitis A virus, according to the following method:

[0104] Huh7.5.1 cells were plated in a 48-well plate and incubated at 37°C overnight. The next day, the confluence of the cells was 30%-40%, and the cells were infected with the recovered virus Res-H2 of the infectious cDNA clone obtained in Example 3 at a ratio of 5 RNA copies per cell. After 4 days of infection, the supernatant was discarded, 4% paraformaldehyde was added for fixation at room temperature for 20 min, the fixing solution was discarded, 0.4% Triton X-100 was used to permeabilize the membrane for 10 min, and PBS was used for washing twice, each time for 5 min. Blocking solution containing 1% BSA, 22.52 mg / mL glycine, and PBST (PBS + 0.1% Tween 20) was added, and the cells were incubated at 37°C for 1 h. Then the solution was discarded, 488 fluorescently labeled goat anti-mouse secondary antibody was added at a volume of 200 μL per well, and the cells were incubated at 37°C for 1 h. The solution was discarded, the cells were patted dry, and PBS was used for washing twice, each time for 5 min. DAPI was added for staining the cell nuclei in the dark, and the cells were observed. The results showed that the recovered virus could express the VP1 protein of hepatitis A virus in Huh7.5.1 cells. Figure 6

[0105] Example 5: Detection of the proliferation characteristics of the recovered virus Res-H2 of the infectious cDNA clone of the attenuated live vaccine H2 strain of hepatitis A virus in Huh7.5.1 cells​

[0106] In order to observe the proliferation characteristics of the recovered virus and the parent strain in the Huh7.5.1 cell line, the recovered virus Res-H2 and the parent strain virus (i.e. the live attenuated vaccine H2 strain of hepatitis A virus used in Example 1) were each infected with a ratio of 5 RNA copies / cell according to the method of Example 4, adsorbed in a 37°C, 5% CO2 incubator for 2h, the virus liquid was discarded, and 2% FBS DMEM maintenance liquid was added, and placed in a 37°C, 5% CO2 incubator for culture. The intracellular and culture liquid viruses were collected at 0 days, 2 days, 4 days, and 6 days after inoculation, and a one-step growth curve was plotted. The results are shown in Figure 2. Figure 7 As can be seen from the figure, the recovered virus Res-H2 can proliferate in the Huh7.5.1 cell line, and the replication ability is consistent with that of the parent strain.

[0107] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the present application, and all fall within the protection scope of the present application.

Claims

1. A DNA molecule, characterized in that, The DNA molecule comprises a nucleotide sequence as shown in SEQ ID NO:

1.

2. The DNA molecule of claim 1, wherein, The DNA molecule comprises a nucleotide sequence derived from a hepatitis A virus, which is identical to SEQ ID NO: 1 at more than 80%; Preferably, the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO:

1.

3. A full-length infectious cDNA clone of a hepatitis A virus, characterized in that, The infectious cDNA clone comprises a DNA fragment with a nucleotide sequence as shown in SEQ ID NO:

1.

4. The full-length infectious cDNA clone of hepatitis A virus according to claim 3, wherein, The hepatitis A virus full-length infectious cDNA clone further comprises at least one genetic element selected from the group consisting of a promoter and a hammerhead ribozyme; optionally, the hepatitis A virus full-length infectious cDNA clone further comprises specific restriction sequences. Preferably, the hepatitis A virus full-length infectious cDNA clone comprises a nucleotide sequence as shown in SEQ ID NO:

2.

5. A method of constructing a full-length infectious cDNA clone of hepatitis A virus, characterized by, The construction method comprises: (1) obtaining two or more DNA fragments of hepatitis A virus genome by means of segmental amplification, wherein all the DNA fragments, after being ligated together, comprise a nucleotide sequence as shown in SEQ ID NO: 1; (2) ligating the DNA fragments of hepatitis A virus genome obtained in step (1) with a vector to obtain a recombinant plasmid, wherein the recombinant plasmid comprises the vector and a nucleotide sequence as shown in SEQ ID NO: 1 inserted into the vector.

6. The method of claim 5, wherein, In step (1), each of the DNA fragments has an overlap of 15-25 nucleotides; Preferably, in step (1), one DNA fragment comprises a complete nucleotide sequence of the vector and a partial nucleotide sequence of SEQ ID NO: 1, and 1-4 DNA fragments each comprising a partial nucleotide sequence of SEQ ID NO: 1 are amplified; In step (2), the vector plasmid is selected from pT7 plasmids; Preferably, in step (2), the DNA fragments are ligated by means of In-Fusion seamless cloning.

7. The full-length infectious cDNA clone prepared by the method of claim 5 or 6.

8. A method of rescuing a hepatitis A virus, characterized by, The method comprises in vitro transcription using the hepatitis A virus infectious cDNA clone as a template, and transfecting sensitive cells with the obtained transcribed RNA; The hepatitis A virus infectious cDNA clone is the hepatitis A virus full-length infectious cDNA clone of claim 3, 4 or 7.

9. The rescue virus prepared by the method of claim 8.

10. Use of the DNA molecule of claim 1 or 2, the hepatitis A virus full-length infectious cDNA clone of claim 3, 4 or 7, or the rescue virus of claim 9 in the preparation of a medicament for preventing and / or treating diseases caused by hepatitis A virus infection.

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