CN112961819B

CN112961819B - Method for constructing bocavirus small intestine epithelial organoid infection model

Info

Publication number: CN112961819B
Application number: CN202011384141.XA
Authority: CN
Inventors: 陈喆; 叶智浩
Original assignee: Baoxin Asia Pacific Biotechnology Shenzhen Co ltd
Current assignee: Baoxin Asia Pacific Biotechnology Shenzhen Co ltd
Priority date: 2020-12-01
Filing date: 2020-12-01
Publication date: 2023-06-09
Anticipated expiration: 2040-12-01
Also published as: CN112961819A

Abstract

The invention discloses a method for constructing a bocavirus small intestine epithelial organoid infection model, which comprises the following steps: isolation and culture of small intestine epithelial organoids; bocavirus infects small intestine epithelial organoids. The model solves the problem that the bocavirus has no method for in vitro separation culture, especially the digestive tract infection model, and simultaneously provides a new experimental foundation for rapidly evaluating the infectious property of the bocavirus and providing a pathogenic mechanism of the bocavirus infection intestinal tract.

Description

Method for constructing bocavirus small intestine epithelial organoid infection model

Technical Field

The invention relates to the technical field of biology, in particular to a method for constructing a bocavirus small intestine epithelial organoid infection model.

Background

Human bocavirus (HBoV) is a novel parvovirus found by molecular screening by the sweden scholars Tobias alander, etc., and has an amino acid sequence homology of 43% with the amino acid sequence of canine parvovirus (canine minute virus, CMV) and 42% with the amino acid sequence of Niu Boka virus (bovine parvovirus, BPV). The genome of the non-enveloped single-stranded linear DNA virus belongs to parvoviridae, boka virus is a regular icosahedral non-enveloped single-stranded linear DNA virus, the whole genome is 5.2kb, 3 open reading frames are contained, non-structural proteins NS1 and NP1 and structural proteins VP1/VP2 are respectively encoded, the genome conservation of HBoV is relatively good, and the diversity of VP1/VP2 genes is relatively high.

Human bocavirus, a newly discovered pathogen, is an important complement to the viral pathogen spectrum of art. Because of the proportion of the pathogenic agents in the upper respiratory tract infection, particularly serious lower respiratory tract infection can be caused, and high importance is brought to scientists and clinical workers in various countries, the pathogen is also gradually and deeply researched. Since 2005, there have been reports worldwide of detection of HBoVs DNA in respiratory tract, serum, fecal and urine samples. Because the DNA sequence of the HBoVs is relatively conserved, the detection is mainly carried out by amplifying the DNA fragments by a PCR method, but the common PCR method is time-consuming and labor-consuming, and is urgently needed for detection purposes, the detection speed of the HBoV can be greatly improved by adopting the real-time fluorescence quantification PCR (Real time PCR) with high sensitivity and specificity, and the quantification of the virus DNA is realized.

At present, research reports that the positive detection rate of HBoV in respiratory tract specimens of respiratory tract infection patients is 0.8% -19%, and the detection rate of fecal or urine specimens of gastrointestinal tract diseases patients is 0.8% -9.1%. HBoVs have a higher infection rate in men than in women, 1.5-2.5:1.HBoVs DNA positive patients vary in age distribution from days to 60 years, with the highest rate of children positive from 6 months to 2 years of age, and with relatively low infection rates in infants below 6 months. Currently, HBoVs are less studied in adult patients, and the detection rate of HBoVs DNA in adult specimens is lower by 0% -1.5%. Like respiratory syncytial virus, HBoVs infection is not significantly seasonal, but current reports predominate in winter. HBoVs positive respiratory and gastrointestinal specimens often have co-detection of other pathogens, with a median mixed infection rate of 42.5%, common mixed infectious pathogens including rhinoviruses, influenza viruses, respiratory syncytial viruses, rotaviruses, and the like.

Recently, new HBoV viruses have been reported successively, named human bocavirus 2 (HBoV 2), human bocavirus 3 (HBoV 3) and human bocavirus 4 (HBoV 4), respectively; it has not been established so far whether 4 human bocaviruses each represent a different virion or a different genotype of a single virus. To clearly distinguish the initially discovered human bocavirus by alander et al from the newly discovered HBoV2-4, it has been reported as HBoV1. So far, HBoV1 and HBoV2 are detected in both respiratory tract and intestinal tract specimens, HBoV3 and HBoV4 are the latest species found in boca, and HBoV4 is detected only in intestinal tract specimens.

HBoV1 DNA was originally detected in respiratory specimens of infants suffering from lower respiratory tract infections and higher detection rates than other viruses, were considered to be a new etiology of lower respiratory tract infections. Respiratory diseases related to the traditional Chinese medicine are reported to be pneumonia, bronchitis, rhinitis, sphagitis, asthma and the like, respiratory tract and gastrointestinal bocavirus infections are reported in succession in various places in China, the respiratory tract is mainly HBoV1, the gastrointestinal tract is mainly HBoV2, in recent years, the gastrointestinal tract infection areas mainly HBoV1 are also gradually and frequently reported, and HBoV1-4 is detected. The difference of the detection rate of the bocavirus in diarrhea cases is large and the highest detection rate is up to 25.77%, so that the bocavirus is most likely to become one of the main diarrhea causing viruses after rotavirus, enteroadenovirus and human calicivirus.

Some studies report that HBoV1 is closely related to severe lung disease and childhood wheezing, more than 50% of asthmatics carrying HBoV1 are single HBoV1 infections, while mixed-infected children are less diagnosed with asthma. For this reason, there is no report of death cases associated with HBoVs. Because HBoVs are often co-detected with other pathogens, there is currently no effective in vitro culture system and sensitive animal model, and it is difficult to determine their infectivity and pathogenicity in respiratory disease.

Researchers have thought that the nucleotide sequences of the epidemic HBoV strains in different regions are highly conserved, but the findings of HBoV2-4 indicate that the nucleotide sequences are also highly diverse. The non-structural protein (NS 1 and NP 1) coding region of HBoV3 has 87% amino acid homology to HBoV1, the structural protein coding region is more similar to HBoV2, the amino acid homology is 77%, and it is considered that it is likely that the recombinant strain from which HBoV1 and HBoV2 are recombined. Likewise, HBoV4 is believed to be recombinantly produced from the non-structural protein coding region of HBoV2 and the structural protein coding region of HBoV 3. Recently, fu et al reported that HBoVs have inter-type recombination of HBoV1 and HBoV4 and intra-type recombination of HBoV 2. The research shows that the combined infection phenomenon exists among HBoVs, and the possibility of recombination is further supported. HBoVs recombination can lead to variations in viral virulence and antigenicity, potentially leading to widespread popularity, and thus HBoVs recombination studies are of great significance for epidemiological investigation and disease control of HBoVs. At present, establishing a bocavirus infection model is a key for researching the infectivity and mechanism of bocavirus in human body.

HBoV1-2 is detected in respiratory tract and stool specimens of Chinese patients, while HBoV3 is detected in stool specimens, but recent studies in lan state report that HBoV3 is detected in respiratory tract specimens, but no domestic report that HBoV4 is detected. The research result shows that HBoVs detected in the Chinese respiratory tract specimen are mainly HBoV1, and the fecal specimen is mainly HBoV 2. The positive detection rate of HBoV in China is quite different and often co-detected with other pathogens, the positive detection rates of respiratory tract specimens and fecal specimens are respectively 1.4% -19.3% and 2.1% -25.6%, and the mixed infection rates are respectively 9.5% -55.2% and 56% -100% (the most common mixed infection pathogens are RSV and rotavirus). The current research results show that HBoV infection is mainly carried out on children under 3 years of age, no obvious seasonal distribution exists, and the symptoms of the respiratory tract and gastrointestinal tract diseases of patients are different. HBoV is currently less studied in adult patients domestically, and reported detection rates for adults are lower. Therefore, the relationship between HBoV and adult respiratory diseases in China still needs to be further studied.

There is no robust in vitro model to evaluate the infectivity of bocaviruses in humans.

Disclosure of Invention

The invention provides a method for constructing a bocavirus small intestine epithelial organoid infection model. Whether in a clinical sample with positive detection of respiratory tract nucleic acid or digestive tract nucleic acid, a Boka virus infection model is established by using small intestine epithelial organoids which can be amplified for a long time, differentiation conditions are close to physiological levels, and the small intestine epithelial organoids which simulate human intestinal epithelium in morphology and function can be used for rapidly evaluating the infectivity of novel enteroviruses on human bodies.

Specifically, the invention provides a method for constructing a bocavirus small intestine epithelial organoid infection model, which comprises the following steps: isolation and culture of small intestine epithelial organoids; bocavirus infects small intestine epithelial organoids.

In some embodiments, the isolation and culture of the small intestine epithelial organoids comprises:

(1) Normal intestinal tissue, myometrium was stripped, mucosal epithelial tissue was cut into small tissue pieces, and washed with ice-cold PBS;

(2) Digesting the tissue blocks by Liberase enzyme, and acutely blowing by a gun head during the digestion period;

(3) The remaining fragments are digested and then treated by trypsin or trypsin instead of enzyme;

(4) Collecting supernatant, and centrifuging at low temperature; the cell pellet is resuspended with a basement membrane matrix and dispersed into a culture plate;

(5) The basement membrane matrix is polymerized for 10-15min at 37 ℃, and human intestinal organoid culture medium is added into each hole for covering culture.

(1) Washing normal intestinal tissue, peeling off the muscle layer with a sterile surgical blade, cutting mucosal epithelial tissue into small pieces of about 5mm in size, and washing with ice-cold PBS 10 times;

(2) Digesting the tissue blocks with Liberase enzyme at 37 ℃ for 60min, and acutely blowing with a gun head every 15min;

(3) Digesting the remaining fragments with trypsin enzyme TrypLE Express at 37deg.C for 20min;

(4) Collecting supernatant, and centrifuging at 4deg.C for 3min with 200 g; cell pellets were resuspended in Matrigel and dispersed into 2 48-well plates;

(5) Matrigel was polymerized at 37℃for 10min and 500ul of human intestinal organoid medium was added per well for overlay culture.

In some embodiments, step (5) further comprises: the medium was replaced every 2 days, replaced with fresh basal medium, the organoids and Matrigel were broken up with a pipette tip, transferred to a centrifuge tube, and further mechanically dissociated, the dissociated organoids were washed with basal medium, centrifuged, the supernatant removed, the pellet resuspended with Matrigel, and medium was added.

In some embodiments, the bocavirus infection of the small intestine epithelial organoids comprises: incubating the small intestine epithelial organoids with the bocavirus positive specimens, while shaking the culture plates evenly and gently; the virus solution was then discarded, the cells were washed with 1 XPBS, and human intestinal organoid medium containing 2% FBS was added for further culture.

In some embodiments, the bocavirus infection of the small intestine epithelial organoids comprises: 2mL of diarrhea specimen liquid positive to clinic bocavirus is selected to incubate small intestine epithelial organoids for 2h, and the culture plate is evenly and gently shaken every 30min in the period to facilitate virus adsorption to host cells; the virus solution was then discarded, the cells were washed 3 times with 1 XPBS, and human intestinal organoid medium containing 2% FBS was added and culture continued.

In some embodiments, isolation and culture of the small intestine epithelial organoids is followed by organoid identification, including one or more of whole exon sequencing and copy number analysis, quality control detection and organoid viability analysis, organoid fluorescent staining techniques.

In some embodiments, the bocavirus infection of the small intestine epithelial organoid is followed by one or more of viral infection identification, fluorescent quantitative PCR technique, immunoblotting technique.

In another aspect, the invention provides a human intestinal organoid medium comprising Advanced DMEM/F12, 50% Wnt3a conditioned medium, 20% R-Spondin conditioned medium, 10% noggin conditioned medium, 5ng/ml FGF7, 20ng/ml FGF10, 50ng/ml hEGF, 5nM Heregulin beta-1, 1 XB 27, 1.25mM N-acetylcysteine, 10nM Leu15-Gastrin I, 10mM nicotinamide, 500nM A83-01, 10. Mu.M SB202190, 10nM Y-27632, 10nM prostaglandin E2, 100. Mu.g/ml Primocin, 100. Mu.g/ml penicillin, 100. Mu.g/ml streptomycin, 10mM HEPES, 2mM L-glutamine.

In some embodiments, wherein the final concentration of Wnt3a is 100ng/ml, the final concentration of R-Spondin is 1 μg/ml, and the final concentration of Noggin is 100ng/ml.

The invention has the following advantages:

the establishment of the model solves the problem that no in-vitro separation culture method exists in the existing bocavirus, in particular to a digestive tract infection model; meanwhile, the method is used for rapidly evaluating the infectivity of the novel enterovirus and provides a new experimental basis for the pathogenic mechanism of viral diseases and the high-throughput screening and research and development of antiviral drugs.

Definitions and general terms

Unless otherwise indicated, the terms used in the specification and claims of the present invention have the following definitions.

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. The invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described herein. In the event of one or more of the incorporated references, patents and similar materials differing from or contradictory to the present application (including but not limited to defined terms, term application, described techniques, etc.), the present application controls.

It should further be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety.

The articles "a," "an," and "the" are intended to include "at least one" or "one or more" unless the context clearly dictates otherwise or otherwise. Thus, as used herein, these articles refer to one or to more than one (i.e., to at least one) object. For example, "a component" refers to one or more components, i.e., more than one component is contemplated as being employed or used in embodiments of the described embodiments.

The term "comprising" refers to compositions, methods, and their respective components that are essential to the methods or compositions and remain open to unspecified elements regardless of necessity.

The term "tissue" means biopsy material or explants taken from a patient or animal or tissue produced in vitro.

The term "stem cell" refers to a cell that is undifferentiated and has the ability to differentiate into a desired cell type (e.g., endothelial cells or intestinal epithelial cells).

The term "culture medium" means a growth supporting liquid having nutrients and substances for culturing cells. Examples of suitable media include, but are not limited to, DMEM/F12 and RPMI.

The source of normal intestinal tissue may be intestinal tissue from intestinal surgery.

The myolayer is peeled off, typically using a sterile surgical blade, but the invention is not limited thereto.

Cut into small tissue pieces, which may be 3mm,4mm,5mm,6mm, to facilitate the next step of digestion. Typically 5mm.

"ice-cold" is between 0 and 5℃and is typically 4 ℃.

"Liberase enzyme" in some embodiments, the invention is selected from the company TH class, roche Life Science. The digestion temperature is selected according to the optimum temperature of the enzyme, and the digestion time is 50 minutes, 60 minutes, 70 minutes or 80 minutes.

The digestion period is vigorously blown with a gun head, and can be blown once at intervals of 10 minutes, 15 minutes or 20 minutes as required.

Trypsin or trypsin-substituted enzymes, in some embodiments, the invention selects the trypsin enzyme trypsin Express of Invitrogen, but the invention is not limited thereto.

Centrifugation is carried out at low temperature, in some embodiments, the invention chooses to centrifuge at 200g for 3min at 4 ℃.

Basal membrane matrix, matrigel, growth factor reduced, in some embodiments, the invention was selected from BD Biosciences.

Culture plates, 25 μl Matrigel/well, contained 500 crypts/debris or 1000 single cells.

(3) Digesting the remaining fragments, and treating with pancreatin at 37deg.C for 20min;

Specifically, in some embodiments, the medium is changed every 2 days and organoids are passaged at 1:5 weekly. At passage, the medium was replaced with fresh basal medium, organoids and Matrigel were broken up with a 1000uL gun head and transferred to a 15mL centrifuge tube. Further mechanical dissociation was achieved with a fire-tipped Pasteur pipette, the dissociated organoids were washed with 10mL of basal medium, centrifuged at 200g for 2min, the supernatant removed, the pellet resuspended in Matrigel, and the culture medium added as described above. Organoids can be stored frozen in liquid nitrogen for at least 2 years.

In some embodiments, the human intestinal organoid medium comprises Advanced DMEM/F12, 50% Wnt3a conditioned medium, 20% R-Spondin conditioned medium, 10% noggin conditioned medium, 5ng/ml FGF7, 20ng/ml FGF10, 50ng/ml hEGF, 5nM Heregulin beta-1, 1 XB 27, 1.25mM N-acetylcysteine, 10nM Leu 15-gastin I, 10mM nicotinamide, 500nM A83-01, 10 μM SB202190, 10nM Y-27632, 10nM prostaglandin E2, 100 μg/ml Primocin, 100units/ml penicillin, 100 μg/ml streptomycin, 10mM HEPES, 2mM L-glutamine.

Identification of organoids

(1) Whole exon sequencing and copy number analysis

For each specimen, 250ng of DNA was sheared and subjected to whole exon sequencing, using the Agilent v2 capture probe set and HiSeq2500 sequencing, reading 76bp long. Each sample produces on average a unique sequence of 9.6 Gb. Prior to identifying mutations, insertions, deletions, the sequence data is locally rearranged to increase sensitivity and reduce mismatches. Somatic copy number analysis was performed on segment copy number maps generated from whole exon sequencing using the SegSeq algorithm.

(2) Quality control detection and organoid vitality analysis

Organoid samples were removed from liquid nitrogen storage and resuscitated, and 8 microliters of-7 mg/mL Basement Membrane Extract (BME) was dispersed into 384 well microplates and allowed to polymerize. Before being resuspended in 2% BME/growth medium (15,000-20,000 organoids/mL), the organoids were mechanically isolated by blow-molding, cultured for 6 days, and analyzed for cell viability for 1-6 days using the CCK8 method (ThermoFisher), and the OD at 450nm was measured by a microplate reader. All screening plates were subjected to stringent quality control assays and Z-factor values were calculated in comparison to negative and positive controls.

(3) Organoid fluorescent staining technique

Collecting organoids with a 1.5ml centrifuge tube, washing cells with cold 1 XPBS for 2 times, fixing cells with 4% paraformaldehyde for 30min, discarding the liquid, adding pre-cooled methanol, fixing at-20deg.C for 10min, removing methanol, and washing with PBS for 3 times; 0.25% Trion-X100 was added, treated for 5min at room temperature and washed 3 times with PBS. Tissue embedding was continued in o.c.t. frozen section embedding medium. Slicing, airing the slices, blocking the slices for 30min by 10% BSA, diluting the primary antibodies by using PBS according to a proportion, and respectively adding 24-hole plates for incubation for 1h; recovering the primary antibody and washing with PBS for 3 times, each time for 10min; the secondary antibodies were diluted proportionally with PBS and incubated in 24 well plates for 30min, respectively. Washing with PBS for 3 times and 10min each time; PBS was used according to 1:10000 DAPI dye is diluted and added into the hole, incubated for 10min at room temperature, and washed by PBS for 3 times for 10min each time; sections were dried overnight and the next day were fixed on slides with Anti-fade without DAPI and observed under a fluorescence microscope.

Fluorescent quantitative PCR and whole genome sequence detection of the bocavirus genome in organoid culture supernatants and organoids:

(1) Reference sequence

Screening out different subtypes of HBoV and HBoV1 representing the whole genome sequence from GenBank, including Swedish strains ST1 and ST2, american strain CRD2, japanese strain JPOC07-511, thailand strains CU6 and CU74, taiwan strains TW925_07, TW2715_06 and TW2717_06, and Chinese strains HK1, HK19, WLL-1, WLL-2, CZ643, FZ1, FZ40, BJ3064, BJ3722, GD-HBoV-571, GD-HBoV-594, GD-HBoV-621, GZ2010-1, GZ2010-03, GZ2011-01, GZ2011-04, SH1, SH2, WH, LWK

(2) HBoV detection

Real time PCR reaction system: 10. Mu.l of 2 XiQ Supermix (Bio-Rad), 2. Mu.l of viral DNA, 0.5. Mu.M each of the upstream and downstream primers, and deionized water were made up to 20. Mu.l. Reaction conditions: 95 ℃ for 15min; 15s at 95℃and 1min at 60℃for 45 cycles. HBOV detection primers are shown in Table 1.

(3) Genome-wide sequencing of HBoV

8 pairs of primers covering the whole genome sequence were designed according to GenBank HBoV whole genome reference sequence NC_007455 and HBoV conserved region (example Table 2). Amplifying the extracted nucleic acid sample by adopting a Premix Ex Taq (Takara), and carrying out PCR reaction by using a PCR reaction system: premix Ex Taq 25ul, 2 ul upstream of primer, 2 ul downstream of primer, 2 ul cDNA, ddH ₂ O19. Mu.l, total volume 50. Mu.l, annealing temperature and product size are shown in Table 2. All PCR products are purified by a gel recovery kit (TaKara), non-genome terminal sequence amplified products are recovered and then are directly sequenced, HBoV1 genome terminal amplified sequences are cloned on a pMD18-T vector (TaKara), DH5a competent cells are transformed, and positive clone sequencing is selected.

(4) Recovery and purification of PCR products

1) A1% agarose gel was prepared with 0.5 XTAE buffer, then 50. Mu.l of the PCR reaction product was electrophoresed, the target DNA was excised under an ultraviolet lamp with a clean scalpel, and placed in a 1.5ml centrifuge tube for a short period of time.

2) 300 mu l of gel block melting solution DR-I Buffer is added to 100mg of agarose, and the gel is thoroughly melted after being mixed uniformly in a water bath at 60 ℃ for 10min.

3) And adding the DR-II Buffer with the volume of half of the DR-I Buffer into the melted gum solution, and uniformly mixing.

4) The Spin Column was mounted on a Collection Tube, the solution of the above operation (4.3) was transferred to the Spin Column, centrifuged at 12000rpm for 1min, and the filtrate was discarded.

5) Mu.l of Rinse A was added to Spin Column and centrifuged at 12000rpm for 30s, and the filtrate was discarded.

6) Mu.l of Rinse B was added to Spin Column and centrifuged at 12000rpm for 30s, and the filtrate was discarded.

7) Spin Column was idle for 1min to remove ethanol.

8) The Spin Column is put into another new 1.5ml centrifuge tube, 30 mul of the filtration Buffer is added into the central filter membrane of the Column, and the Column is placed for 2min at room temperature, so that the temperature of the eluent is increased to 55-80 ℃, and the Elution efficiency of DNA is improved.

9) The liquid in the tube, i.e.the recovered DNA fragment, was centrifuged at 12000rpm for 1min and immediately used or stored at-20℃for further use.

(5) Ligation of purified product with T vector

The reaction system:

after gentle mixing, the mixture was connected at 16℃for 30 minutes.

In another aspect, the invention provides a human intestinal organoid medium comprising Advanced DMEM/F12, 50% Wnt3a conditioned medium, 20% R-Spondin conditioned medium, 10% noggin conditioned medium, 5ng/ml FGF7, 20ng/ml FGF10, 50ng/ml hEGF, 5nM Heregulin beta-1, 1 XB 27, 1.25mM N-acetylcysteine, 10nM Leu 15-gastin I, 10mM nicotinamide, 500nM A83-01, 10 μM SB202190, 10nM Y-27632, 10nM prostaglandin E2, 100 μg/ml Primocin, 100units/ml penicillin, 100 μg/ml streptomycin, 10mM HEPES, 2mM L-glutamine.

In some embodiments, hEGF selects Peprotech; b27, invitrogen was selected; n-acetylcysteine Sigma company; leu 15-gateway I selected Sigma Co; a83-01, a TGF-beta receptor blocker, tocres; SB202190, p38 blocker, sigma company; niacinamide, sigma; y27632, tocres; primocin, vivogen.

Drawings

FIG. 1A/B/C the process of culturing and preparing small intestine epithelial organoids (hSIOs).

FIG. 2 identification of small intestine epithelial organoids.

FIG. 3A model of infection of small intestine epithelial organoids by Boka virus.

FIG. 4 genotyping chart of the bocavirus strain:

sequencing results showed that almost all sequenced strains were 5299bp in genomic length, except for the following 3 strains: 32012GZ1185, 2012GZ8500 and 2012GZ5497 (5298, 5298 and 5297bps respectively), average g+c content 42.31%, contains 3 open reading frames, coding regions for proteins NS1, NP1, VP1/VP2 respectively. The genomic structure of all sequenced strains was consistent with the 2 HBoV swiss prototype strains st1 and st2 and the other HBoV strains currently found.

FIG. 5. Evolution analysis of HBoV1 whole gene and NS1, NP1, VP1/2 genes: (a) a whole gene sequence evolution analysis map; (B) NS1 evolution analysis map; (C) NP1 evolutionary analysis plots; (D) VP1/VP2 evolution analysis map:

the results of the whole genome evolution analysis showed (FIG. 5A) and (FIG. 5B-D: the first group contained Swiss prototype strains st1, st2 and other HBoV type 1 isolates, the second group contained Thailand isolate CU74 and taiwan isolate TW 2717-06, and the third group consisted of Guangzhou strains LWK and GZ 9081. 17 HBoV isolated in this experiment had no significant variation in evolution. In the gene tree constructed in 3 open reading frames (FIG. 5B-D), NS1 was highly conserved; VP1/VP2 had a large variation, although the overall variation was less than 2% and 1%, and the results obtained by the evolution analysis based on VP1/VP2 sequences were consistent with those obtained by the whole genome analysis.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. In addition, the technical features of the different embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

Example 1 cultivation of small intestine epithelial organoids

(1) Normal intestinal tissue was washed, the myometrium was peeled off with a sterile surgical blade, mucosal epithelial tissue was cut into small pieces of approximately 5mm in size, and washed 10 times with ice-cold PBS.

(2) The tissue pieces were digested with Liberase (TH grade, roche Life Science Co.) at 37deg.C for 60min, and vigorously blown with a gun head every 15 min.

(3) The remaining chips were digested and treated with TrypLE Express (Invitrogen) at 37℃for 20min.

(4) The supernatant was collected and centrifuged at 200g for 3min at 4 ℃. The cell pellet was resuspended in Matrigel (growth factor reduced, BD Biosciences) and dispersed into 2 48 well plates (25 μl Matrigel/well containing 500 crypts/debris or 1000 single cells).

The formula of the human intestinal organoid culture medium comprises:

advanced DMEM/F12, 50% Wnt3a conditioned medium (Wnt 3a final concentration 100 ng/ml), 20% R-Spondin conditioned medium (Rspondin final concentration 1. Mu.g/ml), 10% Noggin conditioned medium (Noggin final concentration 100 ng/ml), 5ng/ml FGF7 (Peprotection), 20ng/ml FGF10 (Peprotection), 50ng/ml hEGF (Peprotection), 5nM Heregulin beta-1, 1 XB 27 (Invitrogen), 1.25mM N-acetylcysteine (Sigma), 10nM Leu 15-Gastin I (Sigma), 10mM nicotinamide (Sigma), 500nM A83-01 (Tocris), 10. Mu.M SB202190 (Sigma), 10nM Y-27632 (Tocris), 10nM prostaglandin E2, 100. Mu.g/ml Primocin, 100units/ml penicillin, 100. Mu.g/ml streptomycin, 10mM HEPES, 2mM HEPA.

(6) The medium was changed every 2 days and organoids were passaged at 1:5 weekly. At passage, the medium was replaced with fresh basal medium, organoids and Matrigel were broken up with 1000 μl gun tips and transferred to a 15ml centrifuge tube. Further mechanical dissociation was achieved with a fire-tipped Pasteur pipette, the dissociated organoids were washed with 10ml of basal medium, centrifuged at 200g for 2min, the supernatant removed, the pellet resuspended in Matrigel, and the culture medium added as described above. Organoids can be stored frozen in liquid nitrogen for at least 2 years.

(7) Identification of organoids

1) Whole exon sequencing and copy number analysis

2) Quality control detection and organoid vitality analysis

Organoid samples were removed from liquid nitrogen storage and resuscitated, and 8 microliters of-7 mg/ml Basal Membrane Extract (BME) was dispensed into 384 well microplates and allowed to polymerize. Before being resuspended in 2% BME/growth medium (15,000-20,000 organoids/ml), the organoids were mechanically isolated by blow-molding, cultured for 6 days, and analyzed for cell viability for 1-6 days using the CCK8 method (ThermoFisher), and the OD at 450nm wavelength was determined by an microplate reader. All screening plates were subjected to stringent quality control assays and Z-factor values were calculated in comparison to negative and positive controls.

3) Organoid fluorescent staining technique

Organoids were collected using a 1.5ml centrifuge tube, after washing the cells 2 times with cold 1 XPBS, the cells were fixed with 4% paraformaldehyde for 30min, the liquid was discarded, pre-chilled methanol was added, fixed at-20℃for 10min, methanol was removed, and washed 3 times with PBS. 0.25% Trion-X100 was added, treated for 5min at room temperature and washed 3 times with PBS. Tissue embedding was continued in o.c.t. frozen section embedding medium. Slicing, airing the slices, blocking the slices for 30min by 10% BSA, diluting the primary antibodies by using PBS according to a proportion, and respectively adding 24-hole plates for incubation for 1h; the primary antibody was recovered and washed 3 times with PBS for 10min each. The secondary antibodies were diluted proportionally with PBS and incubated in 24 well plates for 30min, respectively. Washing with PBS for 3 times and 10min each time; diluting DAPI dye with PBS according to 1:10000, adding into the hole, incubating for 10min at room temperature, and washing with PBS for 3 times for 10min each time; sections were dried overnight and the next day were fixed on slides with Anti-fade without DAPI and observed under a fluorescence microscope.

Example 2 organoid infection model-Boka Virus infection of Small intestine epithelial organoids

The method is characterized by directly carrying out virus challenge experiments on clinical positive samples and detecting the bocavirus genome in the organoid culture supernatant and organoids by real-time fluorescence quantitative PCR, and comprises the following steps:

(1) Model of organoid infection

Aiming at human intestinal organoids, a diarrhea specimen positive to clinical bocavirus is adopted for infection, and a real-time fluorescent quantitative PCR technology and an immunoblotting technology are applied for virus infection identification.

(2) Virus challenge experiment

The relevant experiments of the Boka virus infection are all carried out in a biosafety secondary laboratory (BSL-2), a Boka virus positive specimen is selected, 2mL of prepared specimen liquid which is packaged and stored is taken for incubation of organoids for 2 hours, and a culture plate is evenly and gently shaken every 30min during the incubation period so as to be beneficial to adsorbing host cells by viruses. The virus solution was then discarded, the cells were washed 3 times with 1 XPBS, and intestinal organoid medium containing 2% FBS was added and culture continued. And collecting the supernatant of the organoid culture solution and the organoid lysate at different times.

(3) Real-time fluorescent quantitative PCR and whole genome sequence detection of organoid culture supernatant and bocavirus genome in organoids

1) Reference sequence

2) HBoV detection

Real time PCR reaction system: 10. Mu.l of 2 XiQ Supermix (Bio-Rad), 2. Mu.l of viral DNA, 0.5. Mu.M each of the upstream and downstream primers, and deionized water were made up to 20. Mu.l. Reaction conditions: 95 ℃ for 15min; 15s at 95℃and 1min at 60℃for 45 cycles.

TABLE 1 detection primers for HBoV

3) Genome-wide sequencing of HBoV

8 pairs of primers covering the whole genome sequence were designed according to GenBank HBoV whole genome reference sequence NC_007455 and HBoV conserved region (Table 2). Amplifying the extracted nucleic acid sample by adopting a Premix Ex Taq (Takara), and carrying out PCR reaction by using a PCR reaction system: premix Ex Taq 25ul, 2 ul upstream of primer, 2 ul downstream of primer, 2 ul cDNA, ddH ₂ O19. Mu.l, total volume 50. Mu.l, annealing temperature and product size are shown in Table 2. All PCR products are purified by a gel recovery kit (TaKara), non-genome terminal sequence amplified products are recovered and then are directly sequenced, HBoV1 genome terminal amplified sequences are cloned on a pMD18-T vector (TaKara), DH5a competent cells are transformed, and positive clone sequencing is selected.

TABLE 2 genome-wide sequencing primers for HBoV

^a According to GenBank accession NC 007455.

4) Recovery and purification of PCR products

4.1 1% agarose gel was prepared with 0.5×TAE buffer, then 50. Mu.l of the PCR reaction product was subjected to electrophoresis, and the target DNA was excised with a clean scalpel under an ultraviolet lamp, placed in a 1.5ml centrifuge tube, and the ultraviolet irradiation time was not too long.

4.2 300. Mu.l of gel block melting solution DR-I Buffer is added to 100mg of agarose, and the gel is thoroughly melted by water bath at 60 ℃ for 10min after uniform mixing.

4.3 Adding DR-II Buffer with one half volume of DR-I Buffer into the melted gum solution, and uniformly mixing.

4.4 Placement of Spin Column on Collection Tube, transfer the solution of operation (4.3) above to Spin Column, centrifuge at 12000rpm for 1min, discard the filtrate.

4.5 500. Mu.l of RinseA was added to Spin Column and centrifuged at 12000rpm for 30s, and the filtrate was discarded.

4.6 700. Mu.l of Rinse B was added to Spin Column, centrifuged at 12000rpm for 30s, and the filtrate was discarded.

4.7 Spin Column idle for 1min to remove ethanol.

4.8 Spin Column is put into another new 1.5ml centrifuge tube, 30 μl of the filtration Buffer is added into the central filter membrane of the Column, and the Column is placed for 2min at room temperature, so that the temperature of the eluent is increased to 55-80 ℃, and the Elution efficiency of DNA is improved.

4.9 12000rpm for 1min, and the liquid in the tube, i.e., the recovered DNA fragment, was used immediately or stored at-20℃for further use.

5) Ligation of purified product with T vector

The reaction system:

after gentle mixing, the mixture was connected at 16℃for 30 minutes.

Example 3 effect test on organoid built model.

The experimental result shows that the antiviral drug can inhibit the replication of virus particles in cells, and the effects of different concentrations and different types of drugs show different inhibition effects. This shows that the method can be used for successfully constructing the in vitro infection model of the bocavirus and can be used for screening and evaluating the effect of anti-bocavirus medicaments.

In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "other embodiments," "examples," "particular examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that changes, modifications, substitutions and variations may be made therein by those of ordinary skill in the art without departing from the spirit and scope of the invention, which is defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> Baozhen Enhance biotechnology (Shenzhen Co., ltd.)

<120> method for constructing Boka virus small intestine epithelial organoid (hSIOs) infection model

<130> 2020.11

<160> 21

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213> Synthesis

<400> 1

gagctctgta agtactatta c 21

<210> 2

<211> 21

<212> DNA

<213> Synthesis

<400> 2

ctctgtgttg actgaataca g 21

<210> 3

<211> 20

<212> DNA

<213> Synthesis

<400> 3

agaggctcgg gctcatatca 20

<210> 4

<211> 25

<212> DNA

<213> Synthesis

<400> 4

tcttcatcac ttggtctgag gtctt 25

<210> 5

<211> 27

<212> DNA

<213> Synthesis

<400> 5

aggaacaccc aatcarccac ctatcgt 27

<210> 6

<211> 20

<212> DNA

<213> Synthesis

<400> 6

gccggcagac atattggatt 20

<210> 7

<211> 22

<212> DNA

<213> Synthesis

<400> 7

ggacgtgtag ccagaagaga tt 22

<210> 8

<211> 21

<212> DNA

<213> Synthesis

<400> 8

gggagaagga ctaagcaaga g 21

<210> 9

<211> 21

<212> DNA

<213> Synthesis

<400> 9

gtcaagcgaa gtaacaggat g 21

<210> 10

<211> 21

<212> DNA

<213> Synthesis

<400> 10

ggcacgcctt atagaacaag t 21

<210> 11

<211> 19

<212> DNA

<213> Synthesis

<400> 11

ggtaaccacc gcaatcagt 19

<210> 12

<211> 21

<212> DNA

<213> Synthesis

<400> 12

gagattgcag ctcttctaca g 21

<210> 13

<211> 20

<212> DNA

<213> Synthesis

<400> 13

cctcgtcttc atcacttggt 20

<210> 14

<211> 20

<212> DNA

<213> Synthesis

<400> 14

aagacaagca tcgctcctac 20

<210> 15

<211> 21

<212> DNA

<213> Synthesis

<400> 15

acaggttcac cgttatcaag t 21

<210> 16

<211> 22

<212> DNA

<213> Synthesis

<400> 16

cacagacaga agcagacgag at 22

<210> 17

<211> 23

<212> DNA

<213> Synthesis

<400> 17

ggtgagaagt gacagctgta ttg 23

<210> 18

<211> 21

<212> DNA

<213> Synthesis

<400> 18

tggtcacctc tacaaaacag a 21

<210> 19

<211> 22

<212> DNA

<213> Synthesis

<400> 19

gtctgatgac tgtggtccta ct 22

<210> 20

<211> 21

<212> DNA

<213> Synthesis

<400> 20

tcccaacaag aagagttcag t 21

<210> 21

<211> 24

<212> DNA

<213> Synthesis

<400> 21

tgtacaacaa caacacatta aaag 24

Claims

1.A method for constructing a bocavirus small intestine epithelial organoid infection model, wherein the method comprises the following steps: isolation and culture of small intestine epithelial organoids; bocavirus infects small intestine epithelial organoids;

small intestinal epithelial organoids were obtained using human intestinal organoid medium including Advanced DMEM/F12, 50% Wnt3a conditioned medium, 20% R-Spondin conditioned medium, 10% Noggin conditioned medium, 5ng/ml FGF7, 20ng/ml FGF10, 50ng/ml hEGF, 5nM Heregulin beta-1, 1 xb 27, 1.25mM N-acetylcysteine, 10nM leu15-gas I, 10nM mM niacinamide, 500nM a83-01, 10 μm SB202190, 10nM Y-27632, 10nM nM prostaglandin E2, 100 μg/ml Primocin, 100units/ml penicillin, 100 μg/ml streptomycin, 10mM HEPES, 2mM L-glutamine;

wherein, the final concentration of Wnt3a is 100ng/ml, the final concentration of R-Spondin is 1 ug/ml, and the final concentration of Noggin is 100 ng/ml;

wherein, the separation and culture of the small intestine epithelial organoids comprises:

2. The method of claim 1, wherein the isolation and culture of small intestine epithelial organoids comprises:

(1) Washing normal intestinal tissue, removing muscle layer with sterile surgical blade, cutting mucosal epithelial tissue into 5mm size pieces, and washing with ice-cold PBS for 10 times;

(4) Collecting supernatant, and centrifuging at 4deg.C for 3min at 200 g; cell pellets were resuspended in Matrigel and dispersed into 2 48-well plates;

(5) Matrigel was polymerized at 37℃for 10min and 500. Mu.l of human intestinal organoid medium was added to each well for overlay culture.

3. The construction method according to claim 1 or 2, wherein after step (5), further comprising: the human intestinal organoid medium was replaced every 2 days, the medium was replaced with fresh basal medium, organoids and Matrigel were broken up with a pipette tip, transferred to a centrifuge tube, and further mechanically dissociated, the dissociated organoids were washed with basal medium, centrifuged, the supernatant removed, the pellet resuspended with Matrigel, and medium was added.

4. The method of claim 1, wherein the bocavirus infection of the small intestine epithelial organoids comprises: incubating the small intestine epithelial organoids with the bocavirus positive specimens, while shaking the culture plates evenly and gently; the virus solution was then discarded, the cells were washed with 1 XPBS, and human intestinal organoid medium containing 2% FBS was added for further culture.

5. The method of claim 1, wherein the bocavirus infection of the small intestine epithelial organoids comprises: selecting a diarrhea specimen liquid 2ml positive with clinical bocavirus to incubate a small intestine epithelial organoid 2h, and uniformly and gently shaking the culture plate every 30min during the incubation period so as to facilitate virus adsorption to host cells; the virus solution was then discarded, the cells were washed 3 times with 1 XPBS, and human intestinal organoid medium containing 2% FBS was added and culture continued.

6. The method of claim 1, wherein the isolation and culture of the small intestine epithelial organoids further comprises organoid identification, including one or more of whole exon sequencing and copy number analysis, quality control detection and organoid viability analysis, organoid fluorescent staining techniques.

7. The method of claim 1, wherein the method further comprises one or more of virus infection identification, fluorescent quantitative PCR technique and immunoblotting technique after infection of the small intestine epithelial organoid with bocavirus.

8. Use of human intestinal organoid medium in constructing a bocavirus small intestine epithelial organoid infection model comprising Advanced DMEM/F12, 50% Wnt3a conditioned medium, 20% R-Spondin conditioned medium, 10% Noggin conditioned medium, 5ng/ml FGF7, 20ng/ml FGF10, 50ng/ml hEGF, 5nM Heregulin beta-1, 1 xb 27, N-acetylcysteine of 1.25mM, 10nM leu 15-gastin I, 10mM niacinamide, 500nM a83-01, 10 μm SB202190, 10nM Y-27632, 10nM nM prostaglandin E2, 100 μg/ml Primocin, 100units/ml penicillin, 100 μg/ml streptomycin, 10mM HEPES, 2mM L-amide;

wherein, the final concentration of Wnt3a is 100ng/ml, the final concentration of R-Spondin is 1 ug/ml, and the final concentration of Noggin is 100ng/ml.