CN114940706B

CN114940706B - Human rotavirus strain, isolated culture method, identification and application

Info

Publication number: CN114940706B
Application number: CN202210419301.2A
Authority: CN
Inventors: 李金松; 段招军
Original assignee: National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention
Current assignee: National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention
Priority date: 2022-04-20
Filing date: 2022-04-20
Publication date: 2023-08-15
Anticipated expiration: 2042-04-20
Also published as: CN114940706A

Abstract

The invention discloses a human rotavirus strain, a separation culture method, identification and application, wherein the human rotavirus strain is a group A G2P4 virus strain, and the preservation number is CGMCC NO.45085; comprises amino acid sequences shown as SEQ ID NO. 1 to SEQ ID NO. 11; comprising the nucleotide sequences shown as SEQ ID NO. 12 to SEQ ID NO. 22. In the isolation and culture method of human rotavirus strain, when rotavirus activation is carried out, the concentration of pancreatin is 15 mug/mL; the concentration of pancreatin in the maintenance solution is 15 mug/mL-20 mug/mL. The invention separates a G2P4 rotavirus epidemic strain, lays a foundation for further researching biological characteristics, and can be used for preparing vaccines or medicines or diagnostic reagents for preventing and/or treating diseases induced by rotavirus.

Description

Human rotavirus strain, isolated culture method, identification and application

Technical Field

The invention belongs to the field of rotaviruses, and in particular relates to a human rotavirus strain, a separation culture method, identification and application.

Background

Rotaviruses (RV) are one of the main causes of severe diarrhea in humans and animals, and it is statistically about 1.11 hundred million individuals worldwide per year in patients with rotavirus-related diarrhea. Human rotaviruses (human rotaviruses, HRV) belong to the genus rotavirus of the reoviridae, which are icosahedral when viewed under the mirror, are non-enveloped, 11-segment, double-stranded positive strand RNA viruses, encoding 6 structural proteins (VP 1, VP2, VP3, VP4, VP6 and VP 7) and 5 non-structural proteins (NSP 1-NSP 5). Human rotaviruses can be divided into nine groups a-J based on VP6 sequences, with group a rotaviruses (group A rotaviruses, RVA) mainly causing diarrhea in infants (under 5 years).

Currently, the global group A rotavirus epidemics are mainly G9P 8, G2P4, G1P 8 and G3P 8, and further G4P 8 and G12P 8. In different periods of China, the main epidemic strains are also continuously changed. In 1998-2000, the G2P4 type was not high in the detection rate in China, and although the G2P4 type rotavirus has been mainly diffused in China in recent years, the G2P4 type has been widely popular in Russian, india and African countries as a main epidemic strain on the whole world. The detection rate of Russian G2P4 rotavirus has been reported to rise from 5.9% in 2016 to 39.1% in 2019. At present, there is no G2P4 type isolated strain in China.

The present invention has been made in view of this.

Disclosure of Invention

The invention aims to solve the technical problems of overcoming the defects of the prior art and providing a human rotavirus strain, a separation culture method, identification and application. The invention separates the clinical specimen into the epidemic strain of G2P4 rotavirus, which lays the foundation for further researching the biological property, and can be used for preparing and evaluating vaccine or medicine or diagnostic reagent.

In order to solve the technical problems, the invention adopts the basic conception of the technical scheme that:

it is a first object of the present invention to provide a polypeptide or a combination of polypeptides comprising a polypeptide as set forth in SEQ ID NO:2-4,7,9-11, or a combination of any one or more of the amino acids shown in the figures;

alternatively, the polypeptide combination comprises a polypeptide as set forth in SEQ ID NO:1 to SEQ ID NO:11, and a polypeptide comprising the amino acid sequence described in seq id no.

As set forth in SEQ ID NO:2 contains 4 mutations in the amino acid sequence shown in seq id no: k41X, M385L, V A, R654K; as set forth in SEQ ID NO:3 contains 2 mutations in the amino acid sequence shown in: DT810-811TR; as set forth in SEQ ID NO:4 comprises 1 mutation in the amino acid sequence shown in figure 4: I106V: as set forth in SEQ ID NO:7 contains 4 mutations in the amino acid sequence: a2T, FK-5 LL, N385T; as set forth in SEQ ID NO:9 contains 3 mutations in the amino acid sequence: R48X, DD259-260XX; as set forth in SEQ ID NO:10 contains 2 mutations in the amino acid sequence shown in seq id no: d140G, V a; as set forth in SEQ ID NO:11 contains 1 mutation in the amino acid sequence shown in seq id no: E171G.

A second object of the present invention is to protect a nucleic acid molecule encoding a combination of polypeptides as described above, comprising a nucleic acid sequence as set forth in SEQ ID NO:12 to SEQ ID NO:22, encoding a nucleotide sequence as set forth in SEQ ID NO:1 to SEQ ID NO:11, and a polypeptide comprising the amino acid sequence described in seq id no.

It is a third object of the present invention to provide a human rotavirus strain comprising a polypeptide or combination of polypeptides as described above, or comprising a nucleic acid molecule as described above.

In a further scheme, the human rotavirus strain is a group A G2P4 virus strain, and the preservation number of the human rotavirus strain is CGMCC NO.45085.

The separation and culture of partial rotavirus types are difficult, and the successful separation of G2P4 rotavirus is not reported in China. The invention successfully separates a strain of G2P4 virus, can be used for preparing vaccines or medicines or diagnostic reagents, and can also provide a basic tool for evaluating research of rotaviruses and research and development of related detection reagents and vaccines in China.

Further aspects, among 11 gene segments of group a G2P4 virus strains:

VP1 has the amino acid sequence shown in SEQ ID NO:1, the nucleotide sequence is shown as SEQ ID NO:12, the amino acid sequence of VP2 is shown as SEQ ID NO:2, the nucleotide sequence is shown as SEQ ID NO:13, the amino acid sequence of VP3 is shown as SEQ ID NO:3, the nucleotide sequence is shown as SEQ ID NO:14, the amino acid sequence of VP4 is shown as SEQ ID NO:4, the nucleotide sequence is shown as SEQ ID NO:15, the amino acid sequence of VP6 is shown as SEQ ID NO:5, the nucleotide sequence is shown as SEQ ID NO:16, the amino acid sequence of VP7 is shown as SEQ ID NO:6, the nucleotide sequence is shown as SEQ ID NO:17, the amino acid sequence of NSP1 is shown as SEQ ID NO:7, the nucleotide sequence is shown as SEQ ID NO:18, the amino acid sequence of NSP2 is shown in SEQ ID NO:8, the nucleotide sequence is shown as SEQ ID NO:19, the amino acid sequence of NSP3 is shown in SEQ ID NO:9, the nucleotide sequence is shown as SEQ ID NO:20, the amino acid sequence of NSP4 is shown as SEQ ID NO:10, the nucleotide sequence is shown as SEQ ID NO:21, the amino acid sequence of NSP5 is shown as SEQ ID NO:11, a nucleotide sequence such as SEO ID NO: shown at 22.

A fourth object of the present invention is to provide a method for isolated culture of human rotavirus strain as described above, comprising:

(1) Obtaining a viral diarrhea faecal sample;

(2) Screening rotavirus positive samples, and carrying out RT-PCR typing detection and sequencing confirmation type on all positive samples;

(3) Adding pancreatin into 10% suspension of selected rotavirus feces for activating, adding activated rotavirus liquid into cultured MA-104 cells, adding pancreatin-containing maintenance liquid, and adding 5% CO at 37deg.C ₂ The cytopathy is cultivated and observed in an incubator, and the culture is harvested after the cytopathy appears.

Further, the concentration of pancreatin upon activation was 15. Mu.g/mL.

Further, the concentration of pancreatin in the solution is maintained at 15 μg/mL-20 μg/mL; preferably, the pancreatin concentration in the maintenance solution is 20. Mu.g/mL.

In the rotavirus culture process, the use of pancreatin has a large influence on successful separation of viruses, and the rotavirus separation rate can be improved by setting different pancreatin concentration gradients on the maintenance solution and searching the most suitable activation and incubation conditions of the rotavirus. In selecting different cells and different rotavirus strains, the difference in tolerance of the selected cells to pancreatin is taken into account, and the concentration of pancreatin required to fuze the best activated virus is important for the successful isolation of the virus. The components in the feces are complex and may interfere with the effectiveness of pancreatin. Thus, the concentration of pancreatin may be different for different specimens, requiring fumbling. The invention finds that the best effect is achieved when the concentration of activated pancreatin is 15 mug/mL and the concentration of pancreatin in the liquid is maintained at 20 mug/mL by groping.

It is a fifth object of the present invention to provide a cell containing a human rotavirus strain as described above.

It is a sixth object of the present invention to provide a vaccine or medicament comprising a polypeptide or combination of polypeptides as described above, and/or a nucleic acid molecule as described above, and/or a human rotavirus strain as described above.

A seventh object of the present invention is to provide a polypeptide or combination of polypeptides as described above, and/or a nucleic acid molecule as described above, and/or the use of a human rotavirus strain as described above for the preparation of a vaccine and/or medicament or diagnostic agent for the prevention and/or treatment of a disease;

preferably, the disease is induced by rotavirus.

By adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects.

1. The invention obtains a G2P4 rotavirus epidemic strain by separating and culturing, and carries out preliminary identification, lays foundation for further researching biological characteristics, and can be used for preparing and evaluating vaccine or medicine or diagnostic reagent.

2. In the isolation culture method of the invention, the concentration of activated pancreatin is 15 mug/mL, the effect of maintaining the concentration of liquid pancreatin at 20 mug/mL is optimal, and the isolation culture condition is optimized.

The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. It is evident that the drawings in the following description are only examples, from which other drawings can be obtained by a person skilled in the art without the inventive effort. In the drawings:

FIG. 1 is a microscopic view (20X) of the process of the MA-104 cell-by-G2P 4 rotavirus fifth generation cytopathy;

FIG. 2 is an indirect immunofluorescence assay for hRV in MA-104 cells;

FIG. 3 shows the growth of the plaque in the G2P4 rotavirus plaque assay;

FIG. 4 is a nucleic acid phase profile of a G2P4 rotavirus isolate of the present invention;

FIG. 5 is a graph of the kinetics of growth of lesions of G2P4 rotavirus with virus challenge 1d-4 d;

FIG. 6 is a rotavirus morphology image under an electron microscope; the arrow in figure a shows mature viral particles (i.e., three-layer viral particles), and figure B shows bilayer viral particles;

FIG. 7 shows the results of an evolutionary analysis of VP1 of the G2P 4-type rotavirus;

FIG. 8 shows the results of an evolutionary analysis of VP2 of the G2P 4-type rotavirus;

FIG. 9 shows the results of an evolutionary analysis of VP3 of the G2P 4-type rotavirus;

FIG. 10 shows the results of an evolutionary analysis of VP4 of the G2P 4-type rotavirus;

FIG. 11 shows the results of an evolutionary analysis of VP6 of the G2P 4-type rotavirus;

FIG. 12 shows the results of an evolutionary analysis of VP7 of the G2P 4-type rotavirus;

FIG. 13 is the results of an evolutionary analysis of NSP1 from a G2P4 rotavirus;

FIG. 14 is the results of an evolutionary analysis of NSP2 from a G2P [4] rotavirus;

FIG. 15 is the results of an evolutionary analysis of NSP3 from a G2P [4] rotavirus;

FIG. 16 is the results of an evolutionary analysis of NSP4 from a G2P [4] rotavirus;

FIG. 17 is the results of an evolutionary analysis of NSP5 from a G2P4 rotavirus;

it should be noted that these drawings and the written description are not intended to limit the scope of the inventive concept in any way, but to illustrate the inventive concept to those skilled in the art by referring to the specific embodiments.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments will be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention, and the following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

EXAMPLE 1 isolation and cultivation

1 specimen

And 2021, collecting 56 infant viral diarrhea stool samples at a monitoring point of a certain Beijing hospital, storing in a refrigerator at-80 ℃, and storing MA-104 cells required by experiments in a diarrhea room (hereinafter referred to as the present room) of a viral disease institute of China center for disease prevention and control.

2 reagents and consumables

DMEM high sugar Medium (GIBCO, U.S. cat# C11995500 BT), low melting agarose (MACKLIN/Michelin, china cat# A800343-5 g), pancreatin (containing EDTA) (Hyclone/sea clone, U.S. cat# SH 30042.01), pancreatin (containing no EDTA) (GIBCO, U.S. cat# 15050065), goat anti-rabbit IgG/FITC tag (China fir gold bridge, china, cat# ZF-0311), 2XMEM (Invitrogen, U.S. cat# 11935046), SDS-PAGE gel preparation kit (Solarbio, china, cat# P1200), rotavirus colloidal gold assay diagnostic kit (Kang Hua, china, cat# 3401864), viral Nucleic Acid Extraction Kit II (Genaid, china, VR 100), I-5 ^TM 2XHigh-Fidelity Master Mix (TSINGKE, USA, cat# TP 001), QIAGEN OneStep RT-PCR kit (Qiagen, germany, cat# 210212).

MA-104 cell culture Medium: 90% dmem+i0% fbs+i% ps; HRV virus-receiving cell maintenance fluid: dmem+pancreatin (EDTA-free); the final concentration of pancreatin in the solution was maintained at 20. Mu.g/mL.

3 method

3.1 sample treatment samples are retrieved from a refrigerator at the temperature of minus 20 ℃ in a hospital and are stored in the refrigerator at the temperature of minus 80 ℃ in the hospital, after thawing, soybean particles (or 100 mu l) are directly taken into an EP tube with the size of 1.5mL, 900 mu l PBS is added into the tube, after full shaking and uniform mixing, the mixture is centrifuged for 10min at 14000r/min, supernatant is sucked by a sterile needle with the concentration of 1mL, the supernatant is sequentially filtered by a filter with the concentration of 0.45 mu m and a filter with the concentration of 0.22 mu m, and the supernatant is separated into 200 mu l/tube and is stored in the refrigerator at the temperature of minus 80 ℃.

3.2 rotavirus screening according to rotavirus colloidal gold method diagnostic kit instruction, sucking 50 μl of the treated feces suspension sample of the infant suspected of viral diarrhea, adding into a detection hole, performing antigen preliminary detection, and if the detection area shows double purple bands, the sample is positive, and only the control band is single purple, and is negative. RT-PCR typing detection and sequencing confirmation type were performed on all positive samples, and genotyping was performed according to national viral diarrhea monitoring protocol (2021) edition, using rotavirus typing website https: rotavirus typing was performed by// www.viprbrc.org/brc/rvaganoxyper. Spgmethod=showclearInputPage & decollator= reo. (see example 3 below in particular)

Rotavirus colloidal gold detection results: 56 samples are screened, wherein 46 samples are positive for colloidal gold, and the detection result of rotavirus G/P typing is 34 samples for G9P 8, 3 samples for G1P 8, 7 samples for G8P 8 and 2 samples for G2P 4.

3.3 cell culture resuscitates MA-104 cells stored in the chamber,

resuscitating and culturing MA-104 cells: the frozen MA104 cells were removed and rapidly thawed by shaking in a 37℃water bath. Slowly adding into culture flask containing MEM solution (growth solution) of 10% new born calf serum, shaking gently, immediately standing at 37deg.C, 5% CO ₂ And (5) culturing in an adhesion way in a incubator. After culturing for 24 hours, the cell adhesion was observed, the old growth solution in the flask was discarded, and 10ml of fresh growth solution preheated in a water bath at 37℃was added. The flask was placed at 37℃with 5% CO ₂ Culturing in incubator until the cells grow into compact monolayer.

Passage of MA-104 cells: after the cells grew into a dense monolayer, the growth solution in the flask was discarded, the cell surface was washed 3 times with 5ml of a 37℃preheated PBS solution, and then pancreatin was added at a concentration of 0.25% and 37℃preheated, and the flask was gently shaken horizontally to uniformly distribute pancreatin on the cell surface to digest the cells. After about 45 seconds, the cells were observed under light to be frosted glass-like, and the pancreatin solution in the flask was aspirated off. The cell surface of the culture flask is firmly flapped, and sand-like cells can slide along the wall of the flask. 30ml of growth solution preheated at 37 ℃ is added, dispersed cells are blown with force, 10ml of cell suspension is respectively taken into a new culture flask, and the culture flask is gently shaken. Placing at 37deg.C, 5% CO ₂ Culturing in incubator for 2 or 3 days to obtain compact monolayer.

Activation of rotavirus: filtering the screened rotavirus liquid with a filter membrane, adding pancreatin with a final concentration of 15 mug/ml, and activating for 1h in a constant-temperature water bath at 37 ℃.

Proliferation of rotavirus: when it is necessary to meet the toxin, 2ml of MA-104 cell sap is mixed according to the ratio of 3 multiplied by 10 ⁵ transferring/mL density to a rotary tube for culturing, adding activated rotavirus solution when cells grow to a monolayer, adsorbing at 37deg.C for 90 min, adding pancreatin maintaining solution with final concentration of 20 μg/mL, and culturing at 37deg.C, 5%CO ₂ Culturing in an incubator, and observing cytopathic effect.

3.4, when the concentration of the pancreatic enzyme after inoculation and activation is fumbly, the concentration of the pancreatic enzyme is 15 mug/mL, four gradients of 5, 10, 15 and 20 mug/mL are respectively set in the maintenance solution, the virus is killed after 4 days or when cytopathy reaches more than 90 percent, the virus is frozen and thawed 3 times at-80 ℃, the virus culture solution after the freeze thawing is sucked out for centrifugation for 10min at 5000r/min, the supernatant is collected and filtered, 50 mu l of the virus culture solution is detected by a method in 3.2, positive is confirmed, and the rest culture solution is preserved for continuous passage use. When the cell lesions are obviously formed, selecting the optimal pancreatin maintenance concentration according to the principle of high lesion time period and lesion cell proportion, and using the concentration to develop subsequent virus subculture. After obtaining the optimal concentration of cultivated pancreatin, the isolated G2P4 virus is identified.

Four concentration gradients of the required pancreatin are set through a pre-experiment, the pathological change state of cells after the toxin inoculation of each gradient is observed, and colloidal gold detection is carried out on a sample after toxin collection. The first generation of colloidal gold detection is weak positive, the 2-3 generation of colloidal gold results are weak positive (the concentration of activated pancreatin is 15 mug/mL, the concentration of pancreatin is maintained to be 20 mug/mL), and the rest groups are not detected. Activating pancreatin concentration of 15 mug/mL, maintaining pancreatin concentration of 20 mug/mL in 7 days of the experimental group, wherein no obvious cytopathy is found in the first 2 generations, no weak cytopathy is found in the 3 rd generation, no obvious cytopathy is found in the 4 th generation of the disease is shown in the following table, and no obvious cytopathy is found in the first 4 generations of the rest concentration groups. The optimal conditions were finally determined to be a venom activated pancreatin concentration of 15 μg/mL and a maintenance liquid pancreatin concentration of 20 μg/mL (see table 1).

TABLE 1 fumbling to maintain pancreatin concentration in solutions after challenge

Note that: -indicating that the toxin has been received

G2P4 rotavirus causes cytopathy

MA104 cells are inoculated into a rotary tube, when the cell density is about 90%, the pancreatin concentration is activated to 15 mug/mL, the condition of the pancreatin concentration of 20 mug/mL is maintained, subculture is continued, the cells in a part of regions become round after the cells in a fifth generation are cultured for 3 days, the cells in the part of regions become gradually increased, the cells in the part of regions start to form clusters and fall off, and the 5 th d is completely diseased (the result is shown in figure 1).

Example 2 identification

1. Indirect immunofluorescence assay 5 well cells were plated in 96 well plates and toxic were combined according to the method described in 3.4 of example 1, and after 48h of toxic combination, an indirect immunofluorescence assay was performed using a goat anti-rabbit rotavirus polyclonal antibody with a primary antibody of 1:500 dilution and a FITC-labeled goat anti-rabbit 1gG with a secondary antibody of 1:1000 dilution, and the results were finally observed under a fluorescence microscope.

Results: the cell infected by the obvious virus can be seen by the immunofluorescence method after inoculating the G2P4 type 5 th generation virus for 24 hours, namely the cell is green and bright, and the cell of the control group is non-fluorescent; at 24h, part of the cells had higher intracellular viral protein content and higher fluorescence value, and part of the cells were at the early stage of infection and the viral fluorescence value was weaker, and the results are shown in FIG. 2.

2. The toxin inoculation process of the plaque purification experiment is the same as the method in 3.4 of the example 1, and in the 2h process of toxin inoculation incubation, a 2XMEM culture medium and a 1% low-melting-point agarose solution are properly prepared, and after the agarose solution is prepared, the agarose solution is placed into a water bath kettle with the temperature of 82 ℃ for heating, and after dissolution, the agarose solution is placed into a water bath with the temperature of 42 ℃ for heat preservation. After the cell inoculation is completed, 2ml of melted agarose is added, the mixture is horizontally placed and cooled to be gel, and a 6-pore plate is placed in an incubator at 37 ℃ in an inverted mode. The plaque growth was observed.

Results: rotavirus had become spotted after 24h under agarose gel, as shown in figure 3, visible as irregular shaped virus plaques. Carefully collecting virus spots, placing the virus spots in 200ul PBS, inoculating the virus according to the sample treatment and the virus activation flow, inoculating the virus into 24 holes, gradually amplifying and culturing the virus spots into 6 holes, and confirming that the collected virus culture solution is positive to rotavirus through colloidal gold; amplified VP4 and VP7 fragments are sequenced to confirm that the plaque is G2P4 rotavirus.

3. Polyacrylamide gel electrophoresis and silver staining identification method uses SDS-PAGE gel preparation kit to prepare 10% polyacrylamide as separating gel, 5% polyacrylamide as laminating gel, and the operation method refers to the appendix B.6.2.1 of infectious diarrhea diagnostic Standard (WS 271-2007), and uses rapid silver staining kit to stain silver nitrate after electrophoresis.

Results: the SDS-PAGE silver staining identifies rotavirus as 11-segment double-chain segmented RNA virus, after electrophoresis on a polystyrene amide gel and silver staining, fragments of different segments of rotavirus form different clustering patterns on the gel due to different moving speeds, and the rotavirus is detected and classified in early stage. The nucleic acid band of the G2P4 rotavirus isolate accords with the distribution condition of the G2P4 rotavirus 4-2-3-2 band reported by other people, accords with the electrophoresis pattern of DS-1 rotavirus nucleic acid PAGE, and the SDS-PAGE electrophoresis pattern is shown in figure 4.

4. Rotavirus growth kinetics curve the purified virus culture solution is inoculated onto MA-104 cells in 6-well holes, 200 mu l of supernatant in different cell holes are collected at different time points of 0h, 24h, 48h, 72h, 96h,120h and 144h after infection, the rotavirus copy number of each time node is measured by using Real time-PCR after nucleic acid extraction, and a one-step growth curve is drawn. Respectively diluting quantitative positive plasmids to 1×10 ² -1×10 ⁷ Copy number was used as standard.

Results: rotavirus lesion growth kinetics curve: real-time PCR standard curve with slope of-3.318, R ² 0.996. The CT value of the virus supernatant collected in 0-144h is subjected to copy number conversion to obtain a growth curve, the virus in the supernatant is less in 96h before the virus, the virus load is rapidly increased in 4d, the peak is reached in 144h, and the growth curve is shown in figure 5.

5. Electron microscopic observation the electron microscopic observation was performed by collecting 30 to 50% of infected cells and completely diseased sample wells, respectively.

Results: when the isolated hRV is detected by an electron microscope, the MA-104 cells are internally provided with rotaviruses, the rotaviruses are round, two types of rotavirus particles are visible, one type of rotavirus particles is about 50-60nm, the outline is unclear, and the other type of rotavirus particles is about 70-80 nm; the 70nm particles exhibited a wheel morphology with a clear profile (see figure 6 for results).

Example 3 genotyping

1. Sample and reagent

The sample (2020 BJ strain) was a virus strain isolated from the fecal sample of example 1.

Reagent: viral Nucleic Acid Extraction Kit II (Geneaid, china, VR 100); QIAGEN OneStep RT-PCR kit (Qiagen, germany, 210212); i-5 ^TM 2Xhigh-Fidelity Master Mix (TSINGKE, USA, TP 001).

2. Nucleic acid extraction fecal samples were prepared as 10% fecal suspension using phosphate buffer (PBS, 0.01mol/L, pH 7.2-7.4), centrifuged at 13000r/min for 10min, 200. Mu.L of supernatant was taken, and sample RNA was extracted according to the protocol using Viral Nucleic Acid Extraction Kit II nucleic acid extraction kit.

3.G, P genotyping RT-PCR typing colloid Jin Shai are checked positive, and then VP4 and VP7 genotyping region sequencing is carried out, so that BLASTN is used for comparing sequences, and the classification is determined.

The G/P type of 2020BJ strain of the invention is G2P4, and the whole genome type belongs to G2-P4-I2-R2-C2-M2-A2-N2-T2-E2-H2 (DS-1-like) genotype.

The 2020BJ strain of the present invention was delivered to China general microbiological culture Collection center for culture Collection system (China general microbiological culture Collection center) at 4 months and 14 days 2022, address: the collection number is CGMCC No.45085, which is classified as Human rotavirus G2P4, human rotavirus-G2P 4 strain.

4. Full genome amplification Reverse Transcription (RT) was performed using highly conserved oligonucleotide sequences at the 5 'and 3' ends of each segment of nucleic acid of rotavirus as reverse transcription (F: 5'-GGCTWT-3', R:5 '-GGTCAY-3') primers, adding 10ul of template RNA and 1 ul (20 nm) of each oligonucleotide primer, with the reaction conditions set to: immediately placing on ice at 98 ℃ for 5min, then carrying out transcription and amplification according to a superscript II reverse transcription kit, configuring a reaction system according to instructions by using an I-5TM2XHIgh-Fidelity Master MIX (Tsingke) kit, and carrying out PCR amplification conditions: 94℃for 3min,94℃for 30sec,55℃for 30sec,72℃for X min (Xmin represents time, extension time is 1 min/kb), 45 cycles; the amplification is carried out according to Wang Mengxuan, ma Xin, wei Yuhang, et al, 7min at 72 ℃ and termination at 4 ℃, and the epidemic trend analysis of rotavirus G9P 8-E2 genotype reassortants in China; for the non-amplified fragments, primers were additionally designed (see Table 2) and amplified under the conditions described above. The band size was determined by 2% agarose gel electrophoresis and the sequencing was performed by Beijing, bio-technologies Co., ltd. GP type validation of sequences using BLASTn and https use: is/www.viprbrc.org/brc/rvaganoxyper. Spg? method=showclearinputpage & decollator= reo for rotavirus 11 segment type identification.

TABLE 2 full genome amplification primer complement sequence information

Gene amplification results: a complete genome of 11 gene segments of the 2020BJ strain was obtained, wherein: VP1 has the total length of 3278bp, and the nucleic acid sequence is shown as SEQ ID NO: shown at 12; VP2 has the total length of 2668bp, and the nucleic acid sequence is shown as SEQ ID NO: 13; VP3 has the total length of 2588bp, and the nucleic acid sequence is shown as SEQ ID NO: 14; VP4 has the total length of 2355bp, and the nucleic acid sequence is shown as SEQ ID NO: 15; VP6 has the total length of 1341bp, and the nucleic acid sequence is shown as SEQ ID NO: shown at 16; VP7 has the total length of 1075bp, and the nucleic acid sequence is shown as SEQ ID NO: shown at 17; the total length of NSP1 is 1562bp, and the nucleic acid sequence is shown in SEQ ID NO: shown at 18; the total length of NSP2 is 1059bp, and the nucleic acid sequence is shown in SEQ ID NO: 19; the total length of NSP3 is 1060bp, and the nucleic acid sequence is shown in SEQ ID NO: shown at 20; the total length of NSP4 is 750bp, and the nucleic acid sequence is shown in SEQ ID NO: 21; the total length of NSP5 is 791bp, and the nucleic acid sequence is shown in SEQ ID NO: shown at 22.

VP1 has the amino acid sequence shown in SEQ ID NO:1, the amino acid sequence of VP2 is shown as SEQ ID NO:2, the amino acid sequence of VP3 is shown as SEQ ID NO:3, the amino acid sequence of VP4 is shown as SEQ ID NO:4, the amino acid sequence of VP6 is shown as SEQ ID NO:5, the amino acid sequence of VP7 is shown as SEQ ID NO:6, the amino acid sequence of NSP1 is shown as SEQ ID NO:7, the amino acid sequence of NSP2 is shown as SEQ ID NO:8, the amino acid sequence of NSP3 is shown as SEQ ID NO:9, the amino acid sequence of NSP4 is shown as SEQ ID NO:10, the amino acid sequence of NSP5 is shown as SEQ ID NO: 11.

5. Homology and phylogenetic analysis 44 reference sequences (see evolutionary tree for reference sequence information) from G2P4 RVA in countries such as Japan, india, kennel, america, etc. were downloaded from GenBank, phylogenetic analysis was performed using MEGA-X software, and a evolutionary tree method was constructed using Neighbor-joining (NJ) method, setting Bootstrap to 1000. And selecting one strain with highest nucleic acid homology of evolution VP7 and VP4 as reference sequence, and analyzing the similarity and amino acid variation of each segment of 2020BJ G2P4 strain with its nucleic acid. Five viruses with high similarity to 2020BJ were selected and analyzed for antigenic variation using BioEdit.

Results:

(1) 2020BJ evolution analysis: rotaviruses are typed according to the characteristics of the sequence, and each segment is divided into a plurality of types. The VP7 protein is glycoprotein neutralizing antigen on the surface of the virus, which can be divided into 35 types at present, wherein the G2 type is divided into 4 subtypes, wherein the IV type is divided into 4 branches, 2020BJ and a strain of virus in 2000 in China belong to a3 branch, and the relationship between the 2020BJ and a strain of virus in 2005 in the adjacent China is the latest and the relationship between the 2020BJ and the strain of virus in 2000 in China is a little more distant. VP4 is most commonly classified as P8, 4 and P6, wherein the P4 type is divided into 4 subtypes, the IV type is divided into three branches a, b and c, 2020BJ and strains of Bengladesh and Italy belong to branch a, and one strain in 1996 in China is singly called branch c. Type I2 of VP6 is classified into nine subtypes, of which 2020BJ has the closest relationship with russia and japan, and is not slightly distant from the same relationship as other segments, although belonging to the vii branch as the mendelean strain. The R2 type of VP1 can be divided into nine subtypes, 2020BJ forms a branch with Montana and Italian strains, and forms a type VII. 2020BJ forms the C2 type VI branch of VP2 with rotaviruses in the United states, bengala, etc. The M2 form of VP3 can be divided into a number of branches, where VP3 of 2020BJ forms the V subtype of M2 with Bengala and Italian strains. NSP1 is the closest related to Bengala and Italian strains and belongs to subtype A2 type II together. NSP2 of 2020BJ is the closest related to Italy and Bengala strains and belongs to subtype N2 type II together. NSP3 is the most closely related to both Italian and Bengala strains, and belongs to subtype IV of T2 together. NSP4 has recently been related to Italian and India G10P 4, and also belongs to the E2 type XII subtype together with several other non-G2P 4 strains from India (see FIG. 7-17 for results of evolutionary analysis of the individual gene segments).

(2) Human rotavirus G2P4 type 2020BJ strain homology and mutation analysis 2020BJ is the highest in similarity with G2P4 type in 2018 source Japanese, and 11 segments are 99% -100% similar. The amino acid sequences of some segments are 100% similar, the remaining segment amino acids varying by 1-5 amino acids (see Table 3).

TABLE 3 typing and homology analysis results for each segment of 2020BJ strain

( And (3) injection: the sequence Gc content was calculated by DNAstar as follows: VP1:32.52; VP2:32.30; VP3:30.80; VP4:33.05; VP6:37.21; VP7:32.40; NSP1:27.53; NSP2:35.67; NSP3:30.99; NSP4:40.87; NSP5:31.41 )

(3) Human rotavirus G2[ P4] 2020BJ strain antigenicity analysis: VP7 and VP4 of rotavirus are neutralizing antigens, and VP7 contains three epitopes, and VP4 has four epitopes, which has great influence on rotavirus epidemic and vaccine effect. Four viruses closest to 2020BJ on the evolutionary tree are selected, and the analysis of the amino acids in the epitope shows that the individual amino acids of the individual epitopes are different among the four viruses closest to the evolutionary relationship, such as 130 th site of VP7-1a epitope, wherein the four viruses are N, and EU839925 is D; position 213 of VP7-1b epitope, wherein four strains are D, and EU839923 is Y; at position 194 of the VP4-1 epitope, four strains were I and KC178797 was L (amino acids of the epitope are shown in tables 4-1 to 4-4).

Table 4-1 2020BJ Strain antigenicity analysis results

Table 4-2 2020BJ Strain antigenicity analysis results

Table 4-3 2020BJ Strain antigenicity analysis results

Table 4-4 2020BJ Strain antigenicity analysis results

In conclusion, the strains with relatively close relatedness on the tree of the 2020BJ G2P4 rotavirus are strains from different ages. It shows that rotavirus G2P4 is in different forms and different variants are popular in different areas or are mutated gradually in local areas. The G2 rotavirus is relatively conservative, but reassortment can also occur, but reassortment within the group is more difficult to identify. Each segment of the 2020BJ G2P4 rotavirus has close relativity to strains in areas such as Bengala and Italy except VP6 on the evolutionary branch, VP6 is the most similar strain from Russian and Japanese, and the sequence similarity is 100%, but the VP6 of the virus is difficult to judge to be recombined at present.

Rotaviruses VP7 and VP4 are neutralizing antigens, and can stimulate the generation of neutralizing antibodies in vivo, so that the effects on the protection effect, epidemic trend and the like of vaccines are large. 2020BJ is the most similar strain in 2018 Japanese uploading sequence, each segment is 99-100%, but the amino acid variation of individual segments is up to 4, but no antigenic site variation is found, and the antigenic variation of 2020BJ is probably smaller than that of Japanese strain. But the three amino acid differences appear in the surface antigen epitope of several other strains of virus (source 2005 and 2008) with closer relativity on the evolutionary tree, suggesting that the antigen locus of rotavirus G2P4 also changes slowly.

The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.

Claims

1. A polypeptide is characterized in that the polypeptide comprises any one of amino acids shown as SEQ ID NO. 2-4,7, 9-10.

2. A polypeptide composition is characterized by comprising any two or more than two of amino acids shown as SEQ ID NO. 2-4,7, 9-11.

3. A polypeptide composition comprising an amino acid sequence as set forth in SEQ ID No. 1 to SEQ ID No. 11.

4. A nucleic acid molecule encoding the polypeptide composition of claim 3, comprising the nucleotide sequence set forth in SEQ ID No. 12 to SEQ ID No. 22, and the amino acid sequence set forth in SEQ ID No. 1 to SEQ ID No. 11.

5. A human rotavirus strain comprising the polypeptide of claim 1, or comprising the polypeptide composition of claim 2 or 3, or comprising the nucleic acid molecule of claim 4.

6. The human rotavirus strain of claim 5, wherein the human rotavirus strain is group a G2P4 virus strain with a preservation number of CGMCC No.45085.

7. The human rotavirus strain of claim 5 or 6 wherein the 11 gene segments of group a G2P4 virus strain:

the amino acid sequence of VP1 is shown as SEQ ID NO. 1, the amino acid sequence of VP2 is shown as SEQ ID NO. 2, the amino acid sequence of VP3 is shown as SEQ ID NO. 3, the amino acid sequence of VP4 is shown as SEQ ID NO.4, the amino acid sequence of VP6 is shown as SEQ ID NO. 5, the amino acid sequence of VP7 is shown as SEQ ID NO. 6, the amino acid sequence of NSP1 is shown as SEQ ID NO. 7, the amino acid sequence of NSP2 is shown as SEQ ID NO. 8, the amino acid sequence of NSP3 is shown as SEQ ID NO. 9, the amino acid sequence of NSP4 is shown as SEQ ID NO. 10, and the amino acid sequence of NSP5 is shown as SEQ ID NO. 11.

8. The human rotavirus strain of claim 7 wherein VP1 has the nucleotide sequence shown in SEQ ID NO. 12, VP2 has the nucleotide sequence shown in SEQ ID NO. 13, VP3 has the nucleotide sequence shown in SEQ ID NO. 14, VP4 has the nucleotide sequence shown in SEQ ID NO. 15, VP6 has the nucleotide sequence shown in SEQ ID NO. 16, VP7 has the nucleotide sequence shown in SEQ ID NO. 17, NSP1 has the nucleotide sequence shown in SEQ ID NO. 18, NSP2 has the nucleotide sequence shown in SEQ ID NO. 19, NSP3 has the nucleotide sequence shown in SEQ ID NO. 20, NSP4 has the nucleotide sequence shown in SEQ ID NO. 21, and NSP5 has the nucleotide sequence shown in SEQ ID NO. 22.

9. A method of isolated culture of a human rotavirus strain as claimed in any one of claims 5 to 8 comprising:

(1) Obtaining a viral diarrhea faecal sample;

(3) Adding pancreatin into 10% suspension of selected rotavirus feces for activation, adding activated rotavirus liquid into cultured MA-104 cells, adding maintenance liquid containing pancreatin, culturing in an incubator, observing cytopathy, and harvesting culture after cytopathy appears.

10. The isolated culture method according to claim 9, wherein the concentration of pancreatin is 15 μg/mL when the activation is performed; the concentration of pancreatin in the maintenance solution is 15 mug/mL-20 mug/mL.

11. The isolated culture method of claim 10, wherein the concentration of pancreatin in the maintenance solution is 20 μg/mL.

12. A cell comprising the human rotavirus strain of any one of claims 5-8.

13. A vaccine or medicament or diagnostic agent comprising a polypeptide according to claim 1, and/or a polypeptide composition according to claim 2 or 3, and/or a nucleic acid molecule according to claim 4, and/or a human rotavirus strain according to any one of claims 5 to 8.

14. Use of a polypeptide according to claim 1, or a polypeptide composition according to claim 2 or 3, or a nucleic acid molecule according to claim 4, or a human rotavirus strain according to any one of claims 5 to 8, in the preparation of a vaccine and/or medicament or diagnostic agent for the prevention and/or treatment of a disease.

15. The use according to claim 14, wherein the disease is induced by rotavirus.