CN107881152A - A kind of cell line and its construction method and application for being used to detect hepatitis A virus titre - Google Patents

A kind of cell line and its construction method and application for being used to detect hepatitis A virus titre Download PDF

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CN107881152A
CN107881152A CN201711406305.2A CN201711406305A CN107881152A CN 107881152 A CN107881152 A CN 107881152A CN 201711406305 A CN201711406305 A CN 201711406305A CN 107881152 A CN107881152 A CN 107881152A
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cell line
hav
hepatitis
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mavs
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CN107881152B (en
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寸韡
毕研伟
龙琼
肖红剑
李育中
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Institute of Medical Biology of CAMS and PUMC
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Abstract

The present invention relates to a kind of cell line and its construction method and application for being used to detect hepatitis A virus titre.The present invention utilizes the property that 3ABC protease can be mutual with MAVS, by MAVS C-terminals and fluorescin construction of fusion protein, and it is packaged into lentiviral particle, after infection cell, the fusion protein of expression can be anchored on the mitochondria in cytoplasm into spot distribution, under puromycin screening, the stable cell lines of expressed fusion protein can be obtained stablizing, fluorescin meeting disperse in whole cell or is navigated in nucleus after this cell line is infected by HAV, in the presence of methylcellulose, fluorescence plaque can be observed, HAV infection titer is can detect by calculating plaque number.Compared with conventional method, the inventive method detection HAV titres only need 14 days, the Viral diagnosis cycle that significantly shorten, and it is simple to operate, sensitivity is higher, HAV course of infection, application easy to spread can be observed directly.

Description

It is a kind of be used for detect hepatitis A virus titre cell line and its construction method with Using
Technical field
The invention belongs to Medical Biology detection technique field, and in particular to one kind is used for qualitative and quantitatively detects A type liver The cell line and its construction method of scorching virus titer and application.
Background technology
Hepatitis A virus (Hepatitis A virus, HAV) infection is considered as a very important public health Problem.In development or developed country, HAV be cause the main pathogens of oxyhepatitis and part of Acute liver failure it One.According to statistics, the whole world about 1,400,000 hepatitis A virus infections every year.Because attenuated live vaccine for hepatitis A is with going out Live vaccine sequentially with present, the propagation of hepatitis A virus has obtained effective control.
Hepatitis A virus is a kind of positive chain RNA virus, belongs to picornaviridae hepatovirus.After HAV infection cells Cytopathic effect will not be produced, this just brings certain difficulty to observe and detecting HAV infection.At present, general use is exempted from The methods of epidemic disease fluorescence, ELISA, RT-PCR, tissue cultures median infective dose (CCID50), enters to the HAV in HAV titres and food Row detection.ELSA methods predominantly detect HAV antigen, it is impossible to embody HAV live virus granule numbers completely, and sensitivity is also poor； Tissue cultures median infective dose (CCID50) can detect live virus number, but detection cycle is longer needs 21-28 days；RT-PCR methods By carrying out augmentation detection to the target fragment of HAV genes, there is the characteristics of simple and high sensitivity, but such a method can not Distinguish infectious and non-infectious virus granule number.Therefore how overcome the deficiencies in the prior art is current medical science detection technique neck The problem of domain urgent need to resolve.
The content of the invention
It is of the invention to solve the problems such as detection of hepatitis A virus infection titre is time-consuming longer, complex operation is cumbersome Purpose is to provide a kind of quick, simple, accurately qualitative and quantitatively detecting hepatitis A virus titre cell line and its structure Method and application.
To achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of construction method for being used to detect the cell line of hepatitis A virus titre, comprises the following steps：
Step (1), plasmid construction：
Using the total serum IgE in Huh7.0 cells or tree shrew liver as template, MAVS C-terminal gene sequences are expanded using RT-PCR MAVS is arranged, and double digestion is carried out to amplified production and Lentiviral, then by T4 ligases to being expanded after double digestion Product and Lentiviral are attached by T4 ligases, obtain recombinant plasmid LV- fluorescin-MAVS (C- terminal)-IRES-PURO-WPRE；
Described MAVS C-terminals amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2；
Described Lentiviral is LV- fluorescin-IRES-PURO-WPRE carriers；
Step (2), slow virus packaging：
The method cotransfection of liposome is used together with pMD2.G, SPAX2 plasmid for the recombinant plasmid that step (1) is obtained In 293T cells, slow virus is made；
Step (3), stable cell lines structure：
It is then that the Huh7.0 of expresses fluorescent fusion protein is thin by slow-virus infection Huh7.0 cells made from step (2) Born of the same parents are to expand culture, screen the Huh7.0 cell lines of expresses fluorescent fusion protein by puromycin afterwards, obtain being used to detect The cell line of hepatitis A virus.
It is further preferred that described fluorescin is EGFP or mCherry.
It is further preferred that using the total serum IgE of Huh7.0 cells as template, using LV-EGFP-IRES-PURO-WPRE During carrier, RT-PCR expands the primer as shown in SEQ ID NO.3 and SEQ ID NO.4；Using the total serum IgE of Huh7.0 cells as Template, during using LV-mCherry-IRES-PURO-WPRE carriers, RT-PCR amplification the primers such as SEQ ID NO.4 and SEQ Shown in ID NO.5；Using the total serum IgE in tree shrew liver as template, during with LV-EGFP-IRES-PURO-WPRE carriers, RT-PCR expands Increase the primer as shown in SEQ ID NO.6 and SEQ ID NO.7.
It is further preferred that amplification program is 98 DEG C of pre-degeneration 3min；30 circulations are carried out again, and circulation follows bar Part is 98 DEG C of denaturation 10s, 62 DEG C of annealing 30s, 72 DEG C of extension 20s；Finally using 72 DEG C of extension 5min.
It is further preferred that in step (2), recombinant plasmid that step (1) obtains and pMD2.G and, SPAX2 plasmids Mass ratio is 5:2:3.
As an alternative, the plasmid construction of step (1) can also be：Synthesize the MAVS C-terminal gene sequences of rhesus macaque Row, afterwards, are inserted into Lentiviral construction recombination plasmid by the MAVS C-terminal gene orders of rhesus macaque, are recombinated Plasmid LV- fluorescins-MAVS (C-terminal)-IRES-PURO-WPRE；
The MAVS terminal genes sequence such as SEQ ID NO.9 of described rhesus macaque；
Described Lentiviral is LV- fluorescin-IRES-PURO-WPRE carriers.
The fluorescence that the present invention provides the above-mentioned construction method structure for being used to detect the cell line of hepatitis A virus titre melts Hop protein.The composition of described fluorescent fusion protein：(1), one section of transmembrane region (TM areas) that can be positioned on mitochondria, one section can By the sequence of HAV3ABC protease specificities identification cutting, sequence signature is：Such as SEQ ID NO.1, SEQ ID NO.2, SEQ Shown in ID NO.9.(2), fluorescin.(3), add SV40 nuclear localization signals between fluorescin and MAVS or be not added with SV40 and appraise and decide Position signal
The present invention also provides the cell of the above-mentioned construction method structure for being used to detect the cell line of hepatitis A virus titre System.
Present invention simultaneously provides the above-mentioned cell line for being used to detect hepatitis A virus titre in detection hepatitis A virus Application in titre.
The present invention provides a kind of method for detecting hepatitis A virus titre, is used to detect hepatitis A virus using above-mentioned The cell line of the construction method structure of the cell line of titre, comprises the following steps：
Obtained cell line is inoculated in Tissue Culture Plate；Contain in described Tissue Culture Plate containing 10%FBS's DMEM；
Second day, HAV is taken, it is 10 to press 10 times of gradient dilutions with the DMEM containing 2%FBS-1~10-8, obtain viral dilution Liquid；
The culture medium abandoned in Tissue Culture Plate is inhaled afterwards, and viral dilution liquid is added in Tissue Culture Plate by 100 μ L/ holes, 37 DEG C, 5%CO are placed in after shaking up2Incubator is cultivated, and is rocked Tissue Culture Plate every 1h, is inhaled after 4h and abandon viral dilution liquid, The DMEM containing 1%~2% methylcellulose, 2%FBS is added by 250 μ L/ holes, or containing 0.4%~1% agar, 2%FBS DMEM, in 37 DEG C, 5%CO23-5d is cultivated in incubator；The number of fluorescence microscopy Microscopic observation fluorescence plaque, afterwards by such as Lower formula calculates HAV titres：
HAV titres (FFU/ml)=fluorescence plaque number/inoculation volume * extension rates.
It is further preferred that inoculum concentration when cell line is inoculated in Tissue Culture Plate is 5000/hole.
It is further preferred that when viral dilution liquid is added in Tissue Culture Plate by 100 μ L/ holes, each dilution factor is set Fixed 3 multiple holes.
Present inventive concept：
1. plasmid construction
Using the total serum IgE in Huh7.0 cells or tree shrew liver as template, carry out expanding MAVS C-terminal bases using RT-PCR Because of sequence MAVS (cleavage site containing HAV 3ABC protease and the transmembrane region for being positioned at mitochondria), through inscribe ferment treatment Afterwards, it is inserted into the Lentiviral LV- fluorescin-IRES-PURO-WPRE carriers treated through same restriction endonuclease, Build LV- fluorescins-MAVS (C-terminal)-IRES-PURO-WPRE, the fluorescin-MAVS (C- of expression Terminal)-IRES-PURO-WPRE structure such as Fig. 1.
Using the total serum IgE in Huh7.0 cells as template, carry out expanding MAVS C-terminal gene orders MAVS using RT-PCR (cleavage site and transmembrane region containing HAV 3ABC protease), the core of SV40 nuclear localization signals (NLS) is contained wherein in primer Acid sequence (SEQ ID NO.8), after inscribe ferment treatment, it is inserted into the Lentiviral treated through same restriction endonuclease In LV- fluorescins-IRES-PURO-WPRE, fluorescin-NLS-MAVS (C-terminal)-IRES-PURO- of structure WPRE, its structure such as Fig. 2.
Or the MAVS C-terminal gene orders of synthesis rhesus macaque, construction recombination plasmid LV- fluorescin-MAVS (C- terminal)-IRES-PURO-WPRE。
2. slow virus is packed
Correct recombinant plasmid will be sequenced and mix (recombinant plasmid in proportion with packaging plasmid respectively：pMD2.G：PSPAX2= 5:2:3), transfected using the method for liposome, preparation expression fluorescin-MAVS (C-terminal), fluorescin- NLS-MAVS (C-terminal) slow virus, is stored in -80 DEG C of refrigerators after packing.
3. stable cell lines are built and Function detection
By the slow-virus infection Huh7.0 cells of preparation, after infection next day nutrient solution is replaced with containing the high sugar of 10%FBS DMEM, continue to cultivate 48h, in fluorescence microscopy Microscopic observation green fluorescent protein or the expression of red fluorescent protein.By table Expand up to the Huh7.0 cell lines of green fluorescent protein or red fluorescent protein and cultivate, by adding antibiotic-puromycin Screen the Huh7.0 cell lines of expressing green fluorescent protein or red fluorescent protein.
The cell line for taking the infection screening of HAV virus liquids to obtain, micro- sem observation fluorescence disperse situation or fluorescence enter core situation.
4. titre detects
The cell line that screening obtains is inoculated in 48 porocyte culture plates, second day, 10 μ L HAV are taken, with containing 2%FBS DMEM by 10 times of gradient dilutions be 10-1~10-8, the culture medium abandoned in 48 orifice plates is inhaled, viral dilution liquid is light by 100 μ L/ holes Light to add 48 orifice plates, each dilution factor sets 3 multiple holes, 37 DEG C, 5%CO is placed in after shaking up2Incubator is cultivated, every 1h Jiggle 48 orifice plates, inhaled after 4h and abandon viral dilution liquid, added by 250 μ L/ holes containing 1%~2% methylcellulose, 2%FBS DMEM, or the DMEM containing 0.4%~1% agar, 2%FBS, in 37 DEG C, 5%CO21-4d is cultivated in incubator.Fluorescence microscope The number of lower observation fluorescence plaque, so as to calculate HAV titre.Specific formula is as follows：
HAV titres (FFU/ml)=fluorescence plaque number/inoculation volume * extension rates.
Meanwhile HAV viruses are done 10 times and are serially diluted, take 10-6、10-7、10-8The virus liquid 1ml of dilution factor, is inoculated with respectively In human diploid cell-KMB17, each dilution factor does 5 repetitions, is placed in 35 DEG C and cultivates 21 days, hepatitis A virus is extracted after harvest, Detected with ELISA, HAV titre (CCID is calculated by Reed-Muench methods50).(with reference to 2015 editions《Chinese people's republicanism State's pharmacopeia》3rd)
By comparing the titre testing result of two methods, show that two methods measure the proportionate relationship of titre.
For the method for simplifying and accurately detecting HAV infection, the present invention utilizes the original of HAV 3ABC and MAVS interactions Reason constructs a kind of fluorescin alignment system directly perceived, simple, quick and comes qualitative and quantitatively detect the infection of HAV viruses and its sense Contaminate power.
During HAV infection cells, nonstructural protein 3A BC protease, which is acted in cell, to be positioned on mitochondria The antiviral antiviral signal protein of signal protein-mitochondria (Mitochondrial antiviral signaling Protein, MAVS), after MAVS is cut by 3ABC protease, the N-terminal of MAVS albumen can come off from mitochondria.MAVS is one The antiviral signal protein that kind is positioned on mitochondria, it is the substrate specificity of HAV3ABC protease.MAVS C-terminal contains 3ABC The recognition site and mitochondria positioning signal of protease, 3ABC protease processing sites are MAVS PROTEIN Cs ends, when by 3ABC albumen After cleavage, the N-terminal of MAVS albumen can come off from mitochondria.The present invention utilizes the mutual property that 3ABC protease can be with MAVS Matter, by MAVS C-terminals (containing HAV 3ABC proteolytic cleavage sites and the transmembrane region being positioned on mitochondria) and fluorescin structure Fusion protein is built, and is packaged into lentiviral particle.After slow virus infected cell, the fusion protein of expression can be anchored on cell Into spot distribution on mitochondria in matter, under antibiotic-puromycin screenings, can obtain stablizing the steady of expressed fusion protein Determine cell line, fluorescin is understood disperse and in whole cell or navigated in nucleus after this cell line is infected by HAV.
In order to it is quick, directly perceived, simply HAV titres are detected, with the cell line containing fluorescin reporting system Based on, the semi-solid nutrient mediums such as methylcellulose are covered on the cell monolayer of infection, due to methocel solution Have excellent thickening property, can limit the diffusion of infecting virus particle in cell well, so formed clearly green fluorescence or Red fluorescence plaque, HAV titre can be calculated by observing fluorescence plaque number.Compared with conventional method, fluorescence egg is utilized The method of white relocation system detection HAV titres only needs 1-4 days, the production of vaccine cycle substantially reduced, and operates letter List, sensitivity are higher, can observe directly HAV course of infection.This quick, simple, accurately detection HAV titres side The foundation of method will provide a kind of effective means in real time for HAV basic research and production of vaccine.
Compared with prior art, its advantage is the present invention：
Compared with conventional method, the method that HAV titres are detected using fluorescin relocation system is a kind of quick, letter Method that is single, accurately qualitative and quantitatively detecting HAV infection and its appeal, the method result is good, and repeatability is good, completes The detection of Live Attenuated HAV titre only needs 1-4 days (conventional method needs -28 days 21 days), and the vaccine substantially reduced Production cycle, the quality of vaccine is ensure that, shorten the launch immunity inoculation time, so as to extend the term of validity of vaccine. Due to HAV, cultivation cycle is long in vitro and can also observe directly HAV infection without obvious cytopathy, the method Journey.In addition this fluorescin relocation system can be also used for the detection of HCV titres and course of infection.
Brief description of the drawings
Fig. 1 is fluorescin-MAVS (C-terminal)-IRES-PURO structure charts；
Fig. 2 is fluorescin-NLS-MAVS (C-terminal)-IRES-PURO structure charts；
Fig. 3 is EGFP-hMAVS (C396-540)-IRES-PURO structure charts；
Fig. 4 schemes for fluorescent fiber sem observation Huh7.0-EGFP-hMAVS；
Fig. 5 is that HAV infects Huh7.0-EGFP-hMAVS (C396-540) fluorescence disperse figures afterwards；
Fig. 6 is that HAV infects the fluorescence plaque figure that Huh7.0-EGFP-hMAVS (C396-540) is formed；
Fig. 7 is mcherry-NLS-hMAVS (C396-540)-IRES-PURO structure charts；
Fig. 8 is fluorescent fiber sem observation Huh7.0-mCherry-NLS-MAVS (C396-540) figure；
Fig. 9 is that fluorescence enters core figure to HAV infection Huh7.0-mCherry-NLS-hMAVS (C396-540) afterwards；
Figure 10 is that HAV infects the fluorescence plaque figure that Huh7.0-mCherry-NLS-hMAVS (C396-540) is formed.
Figure 11 is EGFP-tMAVS (C365-503)-IRES-PURO-WPRE structure charts；
Figure 12 is fluorescent fiber sem observation Huh7.0-EGFP-tMAVS (C365-503) figure；
Figure 13 is that HAV infects Huh7.0-EGFP-tMAVS (C365-503) fluorescence disperse figures afterwards；
Figure 14 is that HAV infects the fluorescence plaque figure that Huh7.0-EGFP-tMAVS (C365-503) is formed.
Figure 15 is EGFP-RhMAVS (C397-541)-IRES-PURO-WPRE structure charts；
Figure 16 is fluorescent fiber sem observation Huh7.0-EGFP-RhMAVS (C397-541) figure；
Figure 17 is that HAV infects Huh7.0-EGFP-RhMAVS (C397-541) fluorescence disperse figures afterwards；
Figure 18 is that HAV infects the fluorescence plaque figure that Huh7.0-EGFP-RhMAVS (C397-541) is formed.
Embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair Bright scope.In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art Or carried out according to product description.Material therefor or the unreceipted production firm person of equipment, it is that can be obtained by buying Conventional products.
Usual solid is formulated in liquid in the present invention, and percentage sign is mass percentage concentration, and ratio is mass ratio；Liquid is matched somebody with somebody It is formed in liquid, percentage sign is concentration expressed in percentage by volume, and ratio is volume ratio, unless otherwise indicated.
Embodiment 1
Step (1), plasmid construction：
Using the total serum IgE in Huh7.0 cells as template, using P1, P2 as primer, carry out expanding MAVS C ends using RT-PCR Gene order MAVS (C396-540) is held, after BsrGI/MluI double digestions, the slow virus crossed with same ferment treatment is inserted into and expresses LV-EGFP-MAVS (C396-540)-IRES-PURO-WPRE restructuring is built in carrier LV-EGFP-IRES-PURO-WPRE carriers Plasmid, through sequencing analysis, sequence is correct, carry greatly be stored in -20 DEG C it is standby.
Specific method is as follows：
PCR amplification system and amplification program
1st, cDNA is synthesized
Synthesized using takara PrimeScriptII 1st Strand cDNA synthesis Kit by specifications cDNA：
System：
Volume (ul)
Huh7.0 RNA 2
Primer P2 0.5
dNTP 4
Rnase free ddH2O 6.5ul
Total 13
65 DEG C of 5min, afterwards rapid ice bath 1min.
5*FS buffer are added into above reaction system：4ul, DTT：1ul, RNase inhibitor：1ul, enzyme： 1ul；55℃：1h, 70 DEG C：15min.
2、PCR
PCR amplification system
Volume (ul)
5X Q5 buffer 4
5X Q5 enhancer buffer 4
dNTP 1.6
P1 0.4
P2 0.4
Template cDNA 1
Water 10.2
Q5 enzymes 0.2
PCR amplification programs：
98 DEG C of for 3min-- (98 DEG C of for10s, 62 DEG C of for 30s, 72 DEG C of for 20s) × 30cycle--72 DEG C of for 5min,
3rd, digestion
Digestion system
37 DEG C, digestion is overnight.
4th, connect
Linked system
16 DEG C of connections are overnight.
EGFP-hMAVS (C396-540)-IRES-PURO of structure structural representation such as Fig. 3.
Step (2), slow virus packaging：
The day before transfection, digestion 293T cells (digest 2min) with 0.25% pancreatin room temperature, with 2.0 × 107Individual cell concentration It is inoculated in T175 blake bottles (culture medium is the DMEM containing 10%FBS), when transfection same day cell fusion degree reaches 40-50%, Transfected.By recombinant plasmid LV-EGFP-hMAVS (C396-540)-IRES-PURO-WPRE and packaging plasmid (pMD2.G and PSPAX2 (recombinant plasmid) is mixed in proportion：pMD2.G：PSPAX2=5:2:3), transfected using lipfectmin2000, Wherein, lipfectmin2000 and recombinant plasmid and packaging plasmid total amount ratio are 50ul：20ug, concrete operations by specification Carry out.Transfection is after 37 DEG C, 5%CO212h is incubated in incubator；The DMEM nutrient solutions containing 10% (V/V) hyclone are changed, Continue to cultivate 48h, observe observe expression under fluorescence microscope, collect the cell supernatant containing virion (with inhaling Pipe takes supernatant), and through 3000g, 4 DEG C centrifugation 5min, remove cell fragment, collect vial supernatant, through 24000rpm, 4 DEG C 2h is centrifuged, removes supernatant, adds 200 μ l virus frozen stock solutions, 4 DEG C of dissolvings overnight, after packing, are stored in -80 DEG C of refrigerators.
Step (3), stable cell lines structure：
The previous day is infected, Huh7.0 cells are reached into 6 orifice plates (DMEM that culture medium is the 10%FBS containing 2ml), are put in 37 DEG C, 5%CO2Cultivate in cell culture incubator, when infection same day cell fusion degree reaches 50%, felt with the slow virus of preparation Dye.Before infection, cell culture medium is replaced with the DMEM containing 2%FBS, adds the virus liquid supernatant of harvest, wherein, infection multiplicity MOI=0.1, in 37 DEG C of overnight incubations.Nutrient solution is replaced with the DMEM in high glucose containing 10%FBS by next day, continues to cultivate 48h, in The expression of observe observe under fluorescence microscope.The Huh7.0 cell lines for expressing fluorescin are existed with 0.25% pancreatin Room temperature digests 5min, then proceedes to cultivate in T25 blake bottles, changes culture medium into puromyc containing 5ug/ml in, 10% The Huh7.0 cell lines of FBS DMEM screening expression fluorescins, are named as Huh7.0-EG FP-hMAVS (C396-540).
The previous day is infected, Huh7.0-EGFP-hMAVS (C396-540) cell is reached into 48 orifice plates the (volume of culture medium For 250ul), it is put in 37 DEG C, 5%CO2Cultivated in cell culture incubator.HAV virus liquids are inoculated in above-mentioned cell by MOI=1, Micro- sem observation fluorescence disperse situation.Will be from cytoplasm disperse to whole thin if cell infects green fluorescent protein by HAV In born of the same parents.
Step (4), titre detection：
Obtained cell line will be screened and be inoculated in 48 porocyte culture plates that (culture medium be the DMEM containing 10%FBS, is inoculated with Measure as 5000/hole), second day, 10 μ LHAV are taken, are serially diluted with 2% (V/V) FBS DMEM by 10 times as 10-1~10-6, The culture medium abandoned in 48 orifice plates is inhaled, viral dilution liquid is gently added into 48 orifice plates by 100 μ L/ holes, each dilution factor sets 3 again Hole, 37 DEG C, 5%CO are placed in after shaking up2Incubator is cultivated, and is jiggled 48 orifice plates every 1h, is inhaled after 4h and abandon viral dilution Liquid, the DMEM containing 1% methylcellulose, 2%FBS is added by 250 μ L/ holes, in 37 DEG C, 5%CO23-4d is cultivated in incubator. The number of fluorescence microscopy Microscopic observation fluorescence plaque, so as to calculate HAV titre.
Meanwhile HAV viruses are done 10 times and are serially diluted, take 10-6、10-7、10-8The virus liquid 1ml of dilution factor, is inoculated with respectively In human diploid cell-KMB17, each dilution factor does 5 repetitions, and being placed in 35 DEG C of cultures, (culture medium was containing 5%FBS's in 21 days MEM, incubation change liquid in every 7 days), hepatitis A virus is extracted after harvest, is detected with ELISA, based on Reed-Muench methods Calculate HAV titre (CCID50).(with reference to 2015 editions《Pharmacopoeia of People's Republic of China》3rd)
By comparing the titre testing result of two methods, show that two methods measure the proportionate relationship of titre.
Concrete outcome：
1. obtain the cell line-Huh7.0-EGFP-hMAVS (C396- of stable expression EGFP-hMAVS (C396-540) 540), and fluorescin in cytoplasm into spot distribution such as Fig. 4.
After 2.HAV infection Huh7.0-EGFP-hMAVS (C396-540), fluorescin can be more in disseminated cell (such as Fig. 5).
After 3.HAV infection Huh7.0-EGFP-hMAVS (C396-540), fluorescin more in disseminated cell, can add first After base cellulose, fluorescence " plaque " point (such as Fig. 6) can be formed.
After 4.HAV infection Huh7.0-EGFP-hMAVS (C396-540), fluorescin more in disseminated cell, can add first After base cellulose, fluorescence " plaque " point can be formed, by observing the number of fluorescence " plaque ", calculates HAV titre.Utilize this side Method detects 3 crowdes of HAV virus titer, and as a result well, 3 crowdes of viral LgFFU/ml are 6.94,6.88,6.93 respectively, ELISA method Testing result LgCCID50/ ml is 7.67,7.35,7.53 respectively.Compared with ELISA method, the detection of fluorescence relocation system is utilized HAV virus titers are less than LgCCID50/ ml, about LgCCID50The 90%~95% of/ml, available for Live Attenuated HAV Titre detects.
Embodiment 2
Step (1), plasmid construction：
Using the total serum IgE in Huh7.0 cells as template, using P2, P3 as primer, carry out expanding MAVS C ends using RT-PCR End group is because of sequences h MAVS (C396-540), after BsrGI/MluI double digestions, is inserted into the slow virus crossed with same ferment treatment and expresses LV-mCherry-NLS-hMAVS (C396-540)-IRES- is built in carrier LV-mCherry-IRES-PURO-WPRE carriers PURO-WPRE recombinant plasmids, through sequencing analysis, sequence is correct, carry greatly be stored in -20 DEG C it is standby.The mcherry-NLS- of structure HMAVS (C396-540)-IRES-PURO-WPRE structure is shown in Fig. 7.
1st, cDNA is synthesized
Synthesized using takara PrimeScriptII 1st Strand cDNA synthesis Kit by specifications cDNA：
System：
Volume (ul)
Huh7.0 RNA 2
Primer P2 0.5
dNTP 4
Rnase free ddH2O 6.5ul
Total 13
65 DEG C of 5min, afterwards rapid ice bath 1min.
5*FS buffer are added into above reaction system：4ul, DTT：1ul, RNase inhibitor：1ul, enzyme： 1ul；55℃：1h, 70 DEG C：15min.
2、PCR
PCR amplification system
Volume (ul)
5X Q5 buffer 4
5X Q5 enhancer buffer 4
dNTP 1.6
P3 0.4
P2 0.4
Template cDNA 1
Water 10.2
Q5 enzymes 0.2
PCR amplification programs：
98 DEG C of for 3min-- (98 DEG C of for10s, 62 DEG C of for 30s, 72 DEG C of for 20s) × 30cycle--72 DEG C of for 5min。
3rd, digestion
Digestion system
37 DEG C, digestion is overnight.
4th, connect
Linked system
Volume (ul)
The PCR primer of BsrGI/MluI digestions 1.5
The carrier of BsrGI/MluI digestions 4.5
T4 ligases 1
T4 buffer 1
H2O 2
16 DEG C of connections are overnight.
MCherry-NLS-hMAVS (C396-540)-IRES-PURO of structure structural representation such as Fig. 7.
Step (2), slow virus packaging：
The day before transfection, digestion 293T cells (digest 2min) with 0.25% pancreatin room temperature, with 2.0 × 107Individual cell concentration It is inoculated in T175 blake bottles (culture medium is the DMEM containing 10%FBS), when transfection same day cell fusion degree reaches 40-50%, Transfected.By recombinant plasmid LV-mCherry-NLS-hMAVS (C396-540)-IRES-PURO-WPRE and packaging plasmid (pMD2.G and pSPAX2) mixes (recombinant plasmid in proportion：pMD2.G：PSPAX2=5:2:3), using lipfectmin2000 Transfected, wherein, lipfectmin2000 and recombinant plasmid and packaging plasmid total amount ratio are 50ul：20ug, specific behaviour Make by specification progress.Transfection is after 37 DEG C, 5%CO212h is incubated in incubator；Change containing 10% (V/V) hyclone DMEM nutrient solutions, continue to cultivate 48h, observe observe expression under fluorescence microscope, collect the cell containing virion Supernatant (takes supernatant) with suction pipe, and through 3000g, 4 DEG C of centrifugation 5min, removes cell fragment, collect vial supernatant, warp 24000rpm, 4 DEG C of centrifugation 2h, remove supernatant, add 200 μ l virus frozen stock solutions, and 4 DEG C of dissolvings overnight, after packing, are stored in -80 DEG C refrigerator.
Step (3), stable cell lines structure：
The previous day is infected, Huh7.0 cells are reached into 6 orifice plates (DMEM that culture medium is the 10%FBS containing 2ml), are put in 37 DEG C, 5%CO2Cultivate in cell culture incubator, when infection same day cell fusion degree reaches 50%, felt with the slow virus of preparation Dye.Before infection, cell culture medium is replaced with the DMEM containing 2%FBS, adds the virus liquid supernatant of harvest, wherein, infection multiplicity MOI=0.1, in 37 DEG C of overnight incubations.Nutrient solution is replaced with the DMEM in high glucose containing 10%FBS by next day, continues to cultivate 48h, in The expression of observe observe under fluorescence microscope.The Huh7.0 cell lines for expressing fluorescin are existed with 0.25% pancreatin Room temperature digests 5min, then proceedes to cultivate in T25 blake bottles, changes culture medium into puromycin containing 5ug/ml, 10%FBS DMEM screening expression fluorescin Huh7.0 cell lines, be named as Huh7.0-mCherry-NLS-hMAVS (C396-540) (such as Fig. 8).
The previous day is infected, Huh7.0-mCherry-NLS-hMAVS (C396-540) cell is reached into 48 orifice plates (culture The volume of base is 250ul), it is put in 37 DEG C, 5%CO2Cultivated in cell culture incubator.HAV virus liquids are inoculated in by MOI=1 State in cell, micro- sem observation Fluorescence Fluorescence enters core situation.If cell is infected by HAV, red fluorescent protein will be from cell Matter enters in nucleus (such as Fig. 9).
Step (4), titre detection：
Obtained cell line will be screened and be inoculated in 48 porocyte culture plates that (culture medium be the DMEM containing 10%FBS, is inoculated with Measure as 5000/hole), second day, 10 μ LHAV are taken, are serially diluted with 2% (V/V) FBS DMEM by 10 times as 10-1~10-6, The culture medium abandoned in 48 orifice plates is inhaled, viral dilution liquid is gently added into 48 orifice plates by 100 μ L/ holes, each dilution factor sets 3 again Hole, 37 DEG C, 5%CO are placed in after shaking up2Incubator is cultivated, and is jiggled 48 orifice plates every 1h, is inhaled after 4h and abandon viral dilution Liquid, the DMEM containing 1% methylcellulose, 2%FBS is added by 250 μ L/ holes, in 37 DEG C, 5%CO23-4d is cultivated in incubator. The number of fluorescence microscopy Microscopic observation fluorescence plaque, so as to calculate HAV titre (such as Figure 10).
Meanwhile HAV viruses are done 10 times and are serially diluted, take 10-6、10-7、10-8The virus liquid 1ml of dilution factor, is inoculated with respectively In human diploid cell-KMB17, each dilution factor does 5 repetitions, and being placed in 35 DEG C of cultures, (culture medium was containing 5%FBS's in 21 days MEM, incubation change liquid in every 7 days), hepatitis A virus is extracted after harvest, is detected with ELISA, based on Reed-Muench methods Calculate HAV titre (CCID50).(with reference to 2015 editions《Pharmacopoeia of People's Republic of China》3rd)
By comparing the titre testing result of two methods, show that two methods measure the proportionate relationship of titre.
Concrete outcome：
1. obtain the cell line-Huh7.0-mCherry- of stable expression mCherry-NLS-HMAVS (C396-540) HMAVS (C396-540), and fluorescin in cytoplasm into spot distribution such as Fig. 8.
After 2.HAV infection Huh7.0-mCherry-NLS-hMAVS (C396-540), fluorescin can enter nucleus In (such as Fig. 9).
After 3.HAV infection Huh7.0-mCherry-NLS-hMAVS (C396-540), fluorescin can be entered in cell In core, after adding methylcellulose, fluorescence " plaque " point (such as Figure 10) can be formed.
After 4.HAV infection Huh7.0-mCherry-NLS-hMAVS (C396-540), fluorescin can more in disseminated cell, After adding methylcellulose, fluorescence " plaque " point can be formed, by observing the number of fluorescence " plaque ", calculates HAV titre.Profit 3 crowdes of HAV virus titer is detected with the method, as a result well, 3 crowdes of viral LgFFU/ml are 6.90,6.88,6.96 respectively, ELISA method detection result LgCCID50/ ml is 7.67,7.35,7.53 respectively.Compared with ELISA method, system is relocated using fluorescence The HAV virus titers of system detection are less than LgCCID50/ ml, about LgCCID50The 90%~95% of/ml, it is attenuated available for hepatitis A The titre detection of live vaccine.
Embodiment 3
Step (1), plasmid construction：
Using the total serum IgE of tree shrew liver as template, using P4, P5 as primer, carry out expanding MAVS C-terminal bases using RT-PCR Because of sequence MAVS (C365-503), after BsrGI/MluI double digestions, the Lentiviral crossed with same ferment treatment is inserted into LV-EGFP-tMAVS (C365-503)-IRES-PURO-WPRE restructuring matter is built in LV-EGFP-IRES-PURO-WPRE carriers Grain, through sequencing analysis, sequence is correct, carry greatly be stored in -20 DEG C it is standby.EGFP-tMAVS (C365-503)-IRES- of structure PURO-WPRE structure is shown in Figure 11.
Specific method is as follows：
1st, cDNA is synthesized
Synthesized using takara PrimeScriptII 1st Strand cDNA synthesis Kit by specifications cDNA：
System：
Volume (ul)
Huh7.0 RNA 2
Primer P2 0.5
dNTP 4
Rnase free ddH2O 6.5ul
Total 13
65 DEG C of 5min, afterwards rapid ice bath 1min.
5*FS buffer are added into above reaction system：4ul, DTT：1ul, RNase inhibitor：1ul, enzyme： 1ul；55℃：1h, 70 DEG C：15min.
2、PCR
PCR amplification system
Volume (ul)
5X Q5 buffer 4
5X Q5 enhancer buffer 4
dNTP 1.6
P4 0.4
P5 0.4
Template cDNA 1
Water 10.2
Q5 enzymes 0.2
PCR amplification programs：
98 DEG C of for 3min-- (98 DEG C of for10s, 62 DEG C of for 30s, 72 DEG C of for 20s) × 30cycle--72 DEG C of for 5min。
3rd, digestion
Digestion system
37 DEG C, digestion is overnight.
4th, connect
Linked system
Volume (ul)
The PCR primer of BsrGI/MluI digestions 1.5
The carrier of BsrGI/MluI digestions 4.5
T4 ligases 1
T4 buffer 1
H2O 2
16 DEG C of connections are overnight.
EGFP-tMAVS (C365-503)-IRES-PURO of structure structural representation such as Figure 11.
Step (2), slow virus packaging：
The day before transfection, digestion 293T cells (digest 2min) with 0.25% pancreatin room temperature, with 2.0 × 107Individual cell concentration It is inoculated in T175 blake bottles (culture medium is the DMEM containing 10%FBS), when transfection same day cell fusion degree reaches 40-50%, Transfected.By recombinant plasmid LV-EGFP-tMAVS (C365-503)-IRES-PURO-WPRE and packaging plasmid (pMD2.G and PSPAX2 (recombinant plasmid) is mixed in proportion：pMD2.G：PSPAX2=5:2:3), transfected using lipfectmin2000, Wherein, lipfectmin2000 and recombinant plasmid and packaging plasmid total amount ratio are 50ul：20ug, concrete operations are illustratively Book is carried out.Transfection is after 37 DEG C, 5%CO212h is incubated in incubator；The DMEM containing 10% (V/V) hyclone is changed to cultivate Liquid, continue to cultivate 48h, observe observe expression under fluorescence microscope, collect the cell supernatant containing virion and (use Suction pipe takes supernatant), and through 3000g, 4 DEG C of centrifugation 5min, remove cell fragment, vial supernatant is collected, through 24000rpm, 4 DEG C centrifugation 2h, removes supernatant, adds 200 μ l virus frozen stock solutions, 4 DEG C of dissolvings overnight, after packing, are stored in -80 DEG C of refrigerators.
Step (3), stable cell lines structure：
The previous day is infected, Huh7.0 cells are reached into 6 orifice plates (DMEM that culture medium is the 10%FBS containing 2ml), are put in 37 DEG C, 5%CO2Cultivate in cell culture incubator, when infection same day cell fusion degree reaches 50%, felt with the slow virus of preparation Dye.Before infection, cell culture medium is replaced with the DMEM containing 2%FBS, adds the virus liquid supernatant of harvest, wherein, infection multiplicity MOI=0.1, in 37 DEG C of overnight incubations.Nutrient solution is replaced with the DMEM in high glucose containing 10%FBS by next day, continues to cultivate 48h, in The expression of observe observe under fluorescence microscope.The Huh7.0 cell lines for expressing fluorescin are existed with 0.25% pancreatin Room temperature digests 5min, then proceedes to cultivate in T25 blake bottles, changes culture medium into puromycin containing 5ug/ml, 10%FBS DMEM screening expression fluorescin Huh7.0 cell lines, be named as Huh7.0-EGFP-tMAVS (C365-503) (as scheme 12)。
The previous day is infected, Huh7.0-EGFP-tMAVS (C365-503) cell is reached into 48 orifice plates the (volume of culture medium For 250ul), it is put in 37 DEG C, 5%CO2Cultivated in cell culture incubator.HAV virus liquids are inoculated in above-mentioned cell by MOI=1, Micro- sem observation fluorescence disperse situation.If cell is infected by HAV, green fluorescent protein will be from cytoplasm disperse to whole thin In born of the same parents (such as Figure 13).
Step (4), titre detection：
Obtained cell line will be screened and be inoculated in 48 porocyte culture plates that (culture medium be the DMEM containing 10%FBS, is inoculated with Measure as 5000/hole), second day, 10 μ L HAV are taken, are serially diluted with 2% (V/V) FBS DMEM by 10 times as 10-1~10-6, The culture medium abandoned in 48 orifice plates is inhaled, viral dilution liquid is gently added into 48 orifice plates by 100 μ L/ holes, each dilution factor sets 3 again Hole, 37 DEG C, 5%CO are placed in after shaking up2Incubator is cultivated, and is jiggled 48 orifice plates every 1h, is inhaled after 4h and abandon viral dilution Liquid, the DMEM containing 1% methylcellulose, 2%FBS is added by 250 μ L/ holes, in 37 DEG C, 5%CO23-4d is cultivated in incubator. The number of fluorescence microscopy Microscopic observation fluorescence plaque, so as to calculate HAV titre (such as Figure 14).
Meanwhile HAV viruses are done 10 times and are serially diluted, take 10-6、10-7、10-8The virus liquid 1ml of dilution factor, is inoculated with respectively In human diploid cell-KMB17, each dilution factor does 5 repetitions, and being placed in 35 DEG C of cultures, (culture medium was containing 5%FBS's in 21 days MEM, incubation change liquid in every 7 days), hepatitis A virus is extracted after harvest, is detected with ELISA, based on Reed-Muench methods Calculate HAV titre (CCID50).(with reference to 2015 editions《Pharmacopoeia of People's Republic of China》3rd)
By comparing the titre testing result of two methods, show that two methods measure the proportionate relationship of titre.
Concrete outcome：
1. obtain stable expression EGFP-tMAVS (C365-503) cell line-Huh7.0-EGFP-tMAVS (C365- 503), and fluorescin in cytoplasm into spot distribution such as Figure 12.
After 2.HAV infection Huh7.0-EGFP-tMAVS (C365-503), fluorescin can be permeated into whole cell (such as Figure 13).
After 3.HAV infection Huh7.0-EGFP-tMAVS (C365-503), fluorescin can be permeated into whole cell, added After entering methylcellulose, fluorescence " plaque " point (such as Figure 14) can be formed.
After 4.HAV infection Huh7.0-EGFP-tMAVS (C365-503), fluorescin more in disseminated cell, can add first After base cellulose, fluorescence " plaque " point can be formed, by observing the number of fluorescence " plaque ", calculates HAV titre.Utilize this side Method detects 3 crowdes of HAV virus titer, and as a result well, 3 crowdes of viral LgFFU/ml are 7.02,6.93,6.98 respectively, ELISA method Testing result LgCCID50/ ml is 7.61,7.42,7.64 respectively.Compared with ELISA method, the detection of fluorescence relocation system is utilized HAV virus titers are less than LgCCID50/ ml, about LgCCID50The 90%~95% of/ml, available for Live Attenuated HAV Titre detects.
Embodiment 4
Step (1), synthesize recombinant plasmid：
The MAVS C-terminal gene order RhMAVS (C397-541) of rhesus macaque are synthesized, build LV-EGFP-RhMAVS (C397-541)-IRES-PURO-WPRE recombinant plasmids, through sequencing analysis, sequence is correct, carry greatly be stored in -20 DEG C it is standby.Structure EGFP-RhMAVS (the C397-541)-IRES-PURO-WPRE built structure is shown in Figure 15.
Step (2), slow virus packaging：
The day before transfection, digestion 293T cells (digest 2min) with 0.25% pancreatin room temperature, with 2.0 × 107Individual cell concentration It is inoculated in T175 blake bottles (culture medium is the DMEM containing 10%FBS), when transfection same day cell fusion degree reaches 40-50%, Transfected.By recombinant plasmid LV-EGFP RhMAVS (C397-541)-IRES-PURO-WPRE and packaging plasmid (pMD2.G and PSPAX2 (recombinant plasmid) is mixed in proportion：pMD2.G：PSPAX2=5:2:3), transfected using lipfectmin2000, Wherein, lipfectmin2000 and recombinant plasmid and packaging plasmid total amount ratio are 50ul：20ug, concrete operations are illustratively Book is carried out.Transfection is after 37 DEG C, 5%CO212h is incubated in incubator；The DMEM containing 10% (V/V) hyclone is changed to cultivate Liquid, continue to cultivate 48h, observe observe expression under fluorescence microscope, collect the cell supernatant containing virion and (use Suction pipe takes supernatant), and through 3000g, 4 DEG C of centrifugation 5min, remove cell fragment, vial supernatant is collected, through 24000rpm, 4 DEG C centrifugation 2h, removes supernatant, adds 200 μ l virus frozen stock solutions, 4 DEG C of dissolvings overnight, after packing, are stored in -80 DEG C of refrigerators.
Step (3), stable cell lines structure：
The previous day is infected, Huh7.0 cells are reached into 6 orifice plates (DMEM that culture medium is the 10%FBS containing 2ml), are put in 37 DEG C, 5%CO2Cultivate in cell culture incubator, when infection same day cell fusion degree reaches 50%, felt with the slow virus of preparation Dye.Before infection, cell culture medium is replaced with the DMEM containing 2%FBS, adds the virus liquid supernatant of harvest, wherein, infection multiplicity MOI=0.1, in 37 DEG C of overnight incubations.Nutrient solution is replaced with the DMEM in high glucose containing 10%FBS by next day, continues to cultivate 48h, in The expression of observe observe under fluorescence microscope.The Huh7.0 cell lines for expressing fluorescin are existed with 0.25% pancreatin Room temperature digests 5min, then proceedes to cultivate in T25 blake bottles, changes culture medium into puromycin containing 5ug/ml, 10%FBS DMEM screening expression fluorescin Huh7.0 cell lines, be named as Huh7.0-EGFP-RhMAVS (C397-541) (as scheme 16)。
The previous day is infected, Huh7.0-EGFP-RhMAVS (C397-541) cell is reached into 48 orifice plates the (body of culture medium Product is 250ul), it is put in 37 DEG C, 5%CO2Cultivated in cell culture incubator.HAV virus liquids are inoculated in above-mentioned cell by MOI=1 In, micro- sem observation fluorescence disperse situation.If cell is infected by HAV, green fluorescent protein will be from cytoplasm disperse to whole In individual cell (such as Figure 17).
Step (4), titre detection：
Obtained cell line will be screened and be inoculated in 48 porocyte culture plates that (culture medium be the DMEM containing 10%FBS, is inoculated with Measure as 5000/hole), second day, 10 μ L HAV are taken, are serially diluted with 2% (V/V) FBS DMEM by 10 times as 10-1~10-6, The culture medium abandoned in 48 orifice plates is inhaled, viral dilution liquid is gently added into 48 orifice plates by 100 μ L/ holes, each dilution factor sets 3 again Hole, 37 DEG C, 5%CO are placed in after shaking up2Incubator is cultivated, and is jiggled 48 orifice plates every 1h, is inhaled after 4h and abandon viral dilution Liquid, the DMEM containing 1% methylcellulose, 2%FBS is added by 250 μ L/ holes, in 37 DEG C, 5%CO21-2d is cultivated in incubator. The number of fluorescence microscopy Microscopic observation fluorescence plaque, so as to calculate HAV titre (such as Figure 18).
Meanwhile HAV viruses are done 10 times and are serially diluted, take 10-6、10-7、10-8The virus liquid 1ml of dilution factor, is inoculated with respectively In human diploid cell-KMB17, each dilution factor does 5 repetitions, and being placed in 35 DEG C of cultures, (culture medium was containing 5%FBS's in 21 days MEM, incubation change liquid in every 7 days), hepatitis A virus is extracted after harvest, is detected with ELISA, based on Reed-Muench methods Calculate HAV titre (CCID50).(with reference to 2015 editions《Pharmacopoeia of People's Republic of China》3rd)
By comparing the titre testing result of two methods, show that two methods measure the proportionate relationship of titre.
Concrete outcome：
1. obtain the cell line-Huh7.0-EGFP-RhMAVS of stable expression EGFP-RhMAVS (C397-541) (C397-541), and fluorescin in cytoplasm into spot distribution such as Figure 15.
After 2.HAV infection Huh7.0-EGFP-RhMAVS (C397-541), fluorescin can be permeated into whole cell (such as Figure 16).
After 3.HAV infection Huh7.0-EGFP-RhMAVS (C397-541), fluorescin can be permeated into whole cell, added After entering methylcellulose, fluorescence " plaque " point (such as Figure 17) can be formed.
After 4.HAV infection Huh7.0-EGFP-RhMAVS (C397-541), fluorescin more in disseminated cell, can add first After base cellulose, fluorescence " plaque " point can be formed, by observing the number of fluorescence " plaque ", calculates HAV titre.Utilize this side Method detects 3 crowdes of HAV virus titer, and as a result well, 3 crowdes of viral LgFFU/ml are 7.10,6.89,6.97 respectively, ELISA method Testing result LgCCID50/ ml is 7.43,7.51,7.69 respectively.Compared with ELISA method, the detection of fluorescence relocation system is utilized HAV virus titers are less than LgCCID50/ ml, about LgCCID50The 90%~95% of/ml, available for Live Attenuated HAV Titre detects.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent thereof.
Partial order list is as shown in the table.
Sequence table
<110>China Medical Sciences Academy Medical Biology Institute
<120>A kind of cell line and its construction method and application for being used to detect hepatitis A virus titre
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Claims (10)

1. a kind of construction method for being used to detect the cell line of hepatitis A virus titre, it is characterised in that comprise the following steps：
Step（1）, plasmid construction：
Using the total serum IgE in Huh7.0 cells or tree shrew liver as template, MAVS C-terminal gene orders are expanded using RT-PCR MAVS, and double digestion is carried out to amplified production and Lentiviral, then produced by T4 ligases to being expanded after double digestion Thing and Lentiviral are attached by T4 ligases, obtain recombinant plasmid；
Described MAVS C-terminals amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2；
Described Lentiviral is LV- fluorescin-IRES-PURO-WPRE carriers；
Step（2）, slow virus packaging：
By step（1）The method cotransfection 293T that liposome is used together with pMD2.G, SPAX2 plasmid for obtained recombinant plasmid is thin In born of the same parents, slow virus is made；
Step（3）, stable cell lines structure：
By step（2）Obtained slow-virus infection Huh7.0 cells, then by the Huh7.0 cell lines of expresses fluorescent fusion protein Expand culture, screen the Huh7.0 cell lines of expresses fluorescent fusion protein by puromycin afterwards, obtain being used to detect A type The cell line of hepatitis viruse.
2. the construction method according to claim 1 for being used to detect the cell line of hepatitis A virus titre, its feature exist In：Described fluorescin is EGFP or mCherry.
3. the construction method according to claim 1 for being used to detect the cell line of hepatitis A virus titre, its feature exist In：Using the total serum IgE of Huh7.0 cells as template, during using LV-EGFP-IRES-PURO-WPRE carriers, used in RT-PCR amplifications Primer is as shown in SEQ ID NO.3 and SEQ ID NO.4；Using the total serum IgE of Huh7.0 cells as template, using LV-mCherry- During IRES-PURO-WPRE carriers, RT-PCR expands the primer as shown in SEQ ID NO.4 and SEQ ID NO.5；With tree shrew Total serum IgE in liver is template, during with LV-EGFP-IRES-PURO-WPRE carriers, RT-PCR amplification the primers such as SEQ ID Shown in NO.6 and SEQ ID NO.7.
4. the construction method according to claim 1 for being used to detect the cell line of hepatitis A virus titre, its feature exist In：Amplification program is 98 DEG C of pre-degeneration 3min；Carry out again 30 circulation, circulation follow condition for 98 DEG C be denatured 10s, 62 DEG C Anneal 30s, 72 DEG C of extension 20s；Finally using 72 DEG C of extension 5min；Step（2）In, step（1）Obtained recombinant plasmid with PMD2.G and, the mass ratioes of SPAX2 plasmids be 5:2:3.
5. the construction method according to claim 1 for being used to detect hepatitis A virus titre cell line, it is characterised in that： Step（1）Plasmid construction can also be：The MAVS C-terminal gene orders of rhesus macaque are synthesized, afterwards, by the MAVS of rhesus macaque C-terminal gene order is inserted into Lentiviral construction recombination plasmid, obtains recombinant plasmid LV- fluorescins-MAVS- IRES-PURO-WPRE；
The MAVS terminal genes sequence such as SEQ ID NO.9 of described rhesus macaque；
Described Lentiviral is LV- fluorescin-IRES-PURO-WPRE carriers.
6. in the construction method for the cell line for detecting hepatitis A virus titre described in claim 1-5 any one The fluorescent fusion protein arrived.
7. the construction method structure for being used to detect the cell line of hepatitis A virus titre described in claim 1-5 any one Cell line.
8. the cell line for being used to detect hepatitis A virus titre described in claim 7 is in hepatitis A virus titre is detected Application.
9. it is a kind of detect hepatitis A virus titre method, using described in claim 1-5 any one be used for detect first The cell line of the construction method structure of the cell line of Hepatitis virus titre, it is characterised in that comprise the following steps：
Obtained cell line is inoculated in Tissue Culture Plate；Contain the DMEM containing 10% FBS in described Tissue Culture Plate；
Second day, HAV is taken, it is 10 to press 10 times of gradient dilutions with the DMEM containing 2% FBS-1~10-8, obtain viral dilution liquid；
The culture medium abandoned in Tissue Culture Plate is inhaled afterwards, and viral dilution liquid is added in Tissue Culture Plate by 100 μ L/ holes, shaken up After be placed in 37 DEG C, 5% CO2Incubator is cultivated, and is rocked Tissue Culture Plate every 1h, is inhaled after 4h and abandon viral dilution liquid, by 250 μ L/ holes are added containing 1% ~ 2% methylcellulose, 2%FBS DMEM, or the DMEM containing 0.4% ~ 1% agar, 2%FBS, in 37 DEG C, 5% CO23-5d is cultivated in incubator；The number of fluorescence microscopy Microscopic observation fluorescence plaque, HAV drops are calculated by equation below afterwards Degree：
HAV titres（FFU/ml）=fluorescence plaque number/inoculation volume * extension rates.
10. the method for detection hepatitis A virus titre according to claim 8, it is characterised in that cell line is inoculated in It is 5000/hole to inoculum concentration during Tissue Culture Plate；When viral dilution liquid is added in Tissue Culture Plate by 100 μ L/ holes, often Individual dilution factor sets 3 multiple holes.
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