CN105985934A - Divalent, trivalent or multivalent HPV pseudovirus and application thereof - Google Patents
Divalent, trivalent or multivalent HPV pseudovirus and application thereof Download PDFInfo
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Abstract
The invention relates to the field of immunology and particularly discloses a divalent, trivalent or multivalent HPV pseudovirus, a two-type, three-type or multi-type composition containing the HPV pseudovirus and a method using the divalent, trivalent or multivalent HPV pseudovirus to detect HPV neutralizing antibodies.
Description
Technical field
The present invention relates to field of immunology.More particularly it relates to an divalence, trivalent or multivalence HPV pseudovirus, two kinds containing described HPV pseudovirus, three kinds or multiple type composition and the method utilizing its detection HPV neutralizing antibody.
Background technology
HPV (Human Papillomavirus is called for short HPV) is main passes through human body close contact, such as the virus spreading through sex intercourse, and can cause the mankind's multiple proliferative epithelial lesion, including papilloma (wart) and tumor-like lesion.Specifically, the disease of HPV induction mainly includes 3 big classes, the 1st class: the malignant changes such as the cancer of uterine neck, vagina, female vulva, penis and anus and certain form of head and neck neoplasm.The cervical cancer patient of 100% is all that HPV infection is caused, the cancer of anus of 90%, and the vulva of 40%, vagina and penis, the oropharynx of 12% and the mouth cancers of 3% are owing to HPV infection.2nd class: the benign lesion such as genital wart such as flat wart, condyloma acuminatum, is a kind of sexually transmitted disease, very common in the crowd that sexual behaviour enlivens.Although genital wart will not cause serious consequence as cancer; but pathology would generally cause more painful clinical symptoms such as cusalgia, bleeding and the pain of patient; produce awkward, anxiety and the negative psychoreaction such as feel oneself inferior simultaneously, and the process repeatedly treated wastes substantial amounts of medical resource.Worldwide estimate that the genital wart being caused by non-carcinogenic HPV (mainly 6 and 11 type) has 30,000,000, the pathology of 20～50% wherein also includes the mixed infection of high-risk HPV.3rd class: HPV infection can also cause recurrent respiratory papilloma (RRP), this be a kind of rare, there is potentially fatal disease, occurring mainly in adolescence, sometimes, substantial amounts of papilloma can cause expiratory dyspnea and cause less age death of child.
The Epidemic Scope that HPV (HPV) infects is wide, and the malignant tumour of the related lethal being caused and multiple sexually transmitted disease have serious harmfulness to human health, and exploitation safely and effectively prevention or therapeutic vaccine are significant.The critical index of HPV vaccine research exploitation for the neutralizing antibody level of HPV, hence set up efficient, accurately and easily neutralizing antibody detection method particularly significant.But, owing to HPV has strict host specificity, its virus replication is closely related with the differentiation state of host cell simultaneously, is difficult at present carry out tissue cultures rapidly and efficiently to HPV in vitro, also lacks suitable animal model.Therefore exploration foundation can Rapid identification or screening neutralizing antibody be very necessary with the method assessing vaccine protective to be selected in time.
At present, the HPV neutralizing antibody appraisal procedure of number of different types is had been developed in, such as the appraisal procedure (White based on immunodeficient mouse tissue transplantation model
WI etc., Journal of Virology, 1998, volume 72: 959-964 page), the appraisal procedure (Meyers based on the primary epithelial tissue of Infection in Vitro
C etc., science, 19 92 years, volume 257: 971-973 page), based on HPV viruslike particle (VLP) ELISA method (1 999 years, Semin Cancer
Biol) and blood clotting Inhibition test (Roden RB etc., Journal of Virology, 1 99
6 years, volume 70: 3298-3301 page) etc., but there is the problems such as the neutralization protective effect that operation is complicated, efficiency is low, length experimental period or can not accurately reflect antibody in these methods.Therefore, in the urgent need to development can convenient, effectively detect HPV neutralizing antibody method, at the same time it is wished that new method can be suitable for carrying out high-throughout detection.
Recently, research display HPV VLP have can non-specific parcel plasmid nucleic acid feature, therefore the HPV pseudovirus (HPV pseudovirus) carrying reporter plasmid can be built for external effectively simulation HPV infection process, and can be used for being used for quickly detecting (Buck CB etc. to HPV neutralizing antibody, Journal of Virology, 2004, volume 78: 751-757 page).Compare other conventional methods based on the neutralizing antibody detection method of HPV pseudovirus and there is the advantage more significantly easy, efficiency is high, shorten, for different HPV hypotypes, there is preferable versatility experimental period.At present, increasingly extensive application has been obtained based on the neutralizing antibody detection method of HPV pseudovirus.
However as going deep into of research, having higher requirement the detection of HPV neutralizing antibody, in addition to sensitivity and the degree of accuracy well, requirement simultaneously can be more quick, easy, can objectively quantify detection more accurately to improve repeatability.Be currently based on the neutralizing antibody detection method of HPV pseudovirus: (1) with green fluorescent protein (GFP) as reporter gene, build with the HPV pseudovirus of GFP reporter gene expression element, after infecting 293TT cell with fluorescence microscope range estimation or flow cytometer (
FACS) detect the cell (infected cell) of fluoresced green, by detecting HPV neutralizing antibody, its titer is assessed to the blocking ability that pseudovirus infects；(2) with SEAP (SEAP) as reporter gene, build the HPV pseudovirus with SEAP reporter gene expression element, pass through centrifugal collecting cell culture supernatant after infecting 293TT cell, developed the color by SEAP chemiluminescence detection kit afterwards, obtain result with chemiluminescence ELIASA again, by detecting HPV neutralizing antibody, its titer is assessed to the blocking ability that pseudovirus infects.(3) to be built with the HPV pseudovirus of beta galactosidase reporter plasmid, infect 293FT cell, use spot detection instrument Elispot detection, the i.e. infected cell quantity of the blue cell number being identified by Elispot in each hole.
Finding in the application, the operation of above-mentioned first method is complex time-consumingly, needs to carry out cell dissociation operation, then hole-specifically detects with FACS, and the detection process of 1 96 orifice plate typically requires 2 to 3 hours, time and effort consuming；The operation of second method can use the instrument of similar ELIASA quickly to scan judgement, be conducive to batch detection, but its kit is relatively costly, it predominantly detects enzyme-catalytic chromogenic reaction degree, there is the problem of sensitivity in simultaneously this method, cell SEAP secretory volume when being infected on a small quantity is low will exist the low problem of the male/female value ratio (P/N than) of readings, can affect result and judge therefore to improve P/N ratio, it is accomplished by using more substantial pseudovirus, be unfavorable for improving conventional efficient.
The present invention establishes a kind of HPV neutralizing antibody detection method simple, accurate and sensitive, it is the method for a kind of high flux detection divalence, trivalent or multivalence HPV virucidin, can preferably meet the requirement to two kinds, three kinds or more different type HPV neutralizing antibody test experience simultaneously.
Content of the invention
The present invention seeks to disclose a kind of divalence, trivalent or multivalence pseudovirus detecting HPV neutralizing antibody.
Divalence disclosed by the invention, trivalent or multivalence pseudovirus comprise the reporter plasmid of HPV capsid protein and encoding fluorescent protein.
Divalence disclosed by the invention, trivalent or multivalence pseudovirus, wherein said HPV capsid protein be structural proteins L1, L2 or L1+L2 be wild type full-length or mutant or the albumen truncating.
Divalence disclosed by the invention, trivalent or multivalence pseudovirus, HPV type is the 6th, the 11st, the 16th, the 18th, the 26th, the 30th, the 31st, the 33rd, the 34th, the 35th, the 39th, 45,5l, the 52nd, the 53rd, the 56th, the 58th, the 59th, the 66th, the 67th, the 68th, the 69th, the 70th, the 72nd, the 73rd, the 82nd, 85 or 97 type, preferably the 6th, the 11st, the 16th, the 18th, the 31st, the 33rd, the 45th, the 52nd, 58 type.
Divalence disclosed by the invention, trivalent or multivalence pseudovirus, HPV structural proteins L1 and L2 is for being implemented in same expression plasmid or being implemented in different plasmid respectively.
Divalence disclosed by the invention, trivalent or multivalence pseudovirus, the reporter gene of the reporter plasmid that this pseudovirus comprises is in the different colours such as green fluorescent protein, red fluorescent protein, yellow fluorescence protein or blue fluorescent protein wantonly two kinds, three kinds or multiple.
Divalence disclosed by the invention, trivalent or multivalence pseudovirus, the signal that the reporter plasmid expression product of mediation after target cell infection produces allows for being read plate instrument by flow cytometer or cell imaging type and identifies and detect, and i.e. needs to select its expression product to can result in the reporter gene producing appropriate signals.In the present embodiment, the fluorescent reporter gene of different colours is selected to include but is not limited to following fluorescin reporter gene.
Red fluorescent protein reporter plasmid (report carrier) includes but is not limited to: pEF1a-E2-Crimson, pDsRed1-C1, pDsRed1-N1, pDsRED-Monomer-N1, pDsRED2-ER
、pDsRED2-Mito、pDsRED2-Nuc、pDsRED2-Peroxi、pDsRED-Express-N1 、pLVX-DsRed-Monomer-N1、p8RwB、pRwB、ptwB、pCIR。
Yellow fluorescence protein report carrier includes but is not limited to: pEYFP-C1, pEYFP-N1, pEYFP-Golgi, pEYFP-Mem, pEYFP-ER.
Green fluorescent protein report carrier includes but is not limited to: pEGFP-1, pEGFP-C1, pEFFP-C2, pEGFP-N1, pEGFP-N3, pAcGFP1-1, pEGFP-Actin, pIRES2-EGFP
、pLEGFP-C1、pLVX-IRES-ZsGreen1、pLVX-AcGFP1-N1、
pLEGFP-N1、pUB-GFP、pEF-GFP、 pCAG-GFP、pCMV-GFP、pCIneo-GFP、pCDNA3-GFP。
Divalence disclosed by the invention, trivalent or multivalence pseudovirus, every type pseudovirus contains the expression plasmid of unique color fluorescin respectively, and each type pseudovirus consumption is for adjusting according to the titre measuring pseudovirus to the positive ratio of identical cell infection.The rough schematic of divalence pseudovirus is shown in accompanying drawing 1, and the rough schematic of trivalent pseudovirus is shown in accompanying drawing 2.
Another object of the present invention is the preparation method disclosing a kind of multivalence pseudovirus detecting HPV neutralizing antibody.Disclosed by the inventionDivalence, trivalent or multivalenceThe preparation method of pseudovirus is as follows:
A. by the reporter plasmid cotransfection 293FT cell of the gene containing HPV capsid protein L 1, the gene expression plasmid of L2 and encoding fluorescent protein, express three kinds of different type pseudovirus respectively, gather in the crops pseudovirus；B. the cell number that pseudovirus titre can be infected by every milliliter of cape horn fever venom represents, takes the consumption of every kind of pseudovirus for making various pseudovirus titre be maintained at same level, is mixed to prepare divalence, trivalent or multivalence HPV pseudovirus.
In said method, step A is for by 1) express two kinds of gene expression plasmids of L1, L2, or L1+L2 gene expression plasmid, and 2 respectively) the plasmid co-transfection 293FT cell of gene containing encoding fluorescent protein.
The preferred preparation method of divalence disclosed by the invention, trivalent or multivalence pseudovirus is as follows:
Take the gene reporter plasmid of encoding fluorescent protein, it is dissolved in culture medium by 1-5:1-5 mass ratio with HPVL1L2 plasmid, in transfection to 293FT cell, after 48 to 72 hours, collect cell after transfection, add lysate cell lysis, lysate is centrifuged supernatant and purifies to obtain HPV pseudovirus stoste；Measure each unit price pseudovirus titre, the cell number that i.e. every milliliter cape horn fever venom can infect respectively, take the consumption of every kind of pseudovirus for making various pseudovirus infection cell number be maintained at same level, be mixed to prepare divalence, trivalent or multivalence HPV pseudovirus.
The 3rd purpose of the present invention is the method disclosing the cell that a kind of detection is infected by HPV virus,Concrete grammar is as follows:
(1) making the cell being infected by divalence, trivalent or multivalence HPV pseudovirus with being suitable to fluorescence detection equipment, such as flow cytometer, cell imaging type read the signal of plate instrument detection, but not infected cell is without signal；With
(2) detect this signal by fluorescence detection equipment and detect the cell being infected by different type HPV pseudovirus.
In said method, count respectively for the cell being infected by different shaped other HPV pseudovirus.
Last purpose of the present invention is to disclose a kind of method detecting HPV neutralizing antibody,Concrete grammar is as follows:
Detected sample is contacted in nutrient solution 293FT cell with HPV pseudovirus co-incubation, application fluorescence detection equipment detecting instrument (such as flow cytometer, cell imaging type read plate instrument), by detection with the cell of the signal directly or indirectly being produced by reporter protein, detect the quantity of infected cell；Whether measuring and calculating sample there are HPV neutralizing antibody and/or its titre (titer).
Described detected sample is the biological sample of the separation from individuality to be checked, preferably serum；Or from the biological sample of tissue cultures, the monoclonal antibody etc. of preparation.
Brief description
The rough schematic of Fig. 1 divalence pseudovirus, including the Y type pseudovirus of the other pseudovirus of the X-type of A color fluorescence and B color fluorescence.
Fig. 2 divalence pseudovirus HPV16E2-HPV18GFP is neutralized antibody test and compares 293FT HPV16E2-HPV18GFP.001 including cell controls 293FT control.001 and pseudovirus to positive serum.
Fig. 3 divalence pseudovirus HPV16E2-HPV18GFP is neutralized antibody test to positive serum, and blood serum sample concentration 40 starts the flow cytometer detection figure of 4 times of stepwise dilutions totally 4 gradients (the 40th, the 160th, the 640th, 2560 times) again.
Fig. 4 divalence pseudovirus HPV16E2-HPV18GFP is neutralized antibody test to positive serum, and blood serum sample concentration 10240 starts the flow cytometer detection figure of 4 times of stepwise dilutions totally 4 gradients (the 10240th, the 40960th, the 163840th, 655360 times) again.
Fig. 5 unit price pseudovirus HPV16E2 is neutralized antibody test, the flow cytometer detection figure of cell controls and pseudovirus comparison to positive serum.
Fig. 6 unit price pseudovirus HPV16E2 is neutralized antibody test to positive serum, and blood serum sample concentration 40 starts the flow cytometer detection figure of 4 times of stepwise dilutions totally 4 gradients (the 40th, the 160th, the 640th, 2560 times) again.
Fig. 7 unit price pseudovirus HPV16E2 is neutralized antibody test to positive serum, and blood serum sample concentration 10240 starts the flow cytometer detection figure of 4 times of stepwise dilutions totally 4 gradients (the 10240th, the 40960th, the 163840th, 655360 times) again.
Fig. 8 unit price pseudovirus HPV18GFP is neutralized antibody test, the flow cytometer detection figure of cell controls and pseudovirus comparison to positive serum.
Fig. 9 unit price pseudovirus HPV18GFP is neutralized antibody test to positive serum, and blood serum sample concentration 40 starts the flow cytometer detection figure of 4 times of stepwise dilutions totally 4 gradients (the 40th, the 160th, the 640th, 2560 times) again.
Figure 10 unit price pseudovirus HPV18GFP is neutralized antibody test to positive serum, and blood serum sample concentration 10240 starts the flow cytometer detection figure of 4 times of stepwise dilutions totally 4 gradients (the 10240th, the 40960th, the 163840th, 655360 times) again.
The specific embodiment of the invention is as follows
The structure of embodiment 1.HPV structural proteins expression vector
From HPV disclosed in Genbank
L1 and L2 gene order is chosen the gene of following sequence number: HPV6
The gene order (No. GenbankAF092932.1) of L1, L2, the gene order (Genbank HE574705.1) of HPV11 L1, L2, the gene order (Genbank NC_001526.2) of HPV16 L1, L2, the gene order (Genbank GQ180792.1) of HPV18 L1, L2, the gene order (Genbank M12732.1) of HPV33 L1, L2, the gene order (Genbank KC860271.1) of HPV58 L1, L2.
The method using gene chemical synthesis is respectively synthesized said gene fragment, selects the expression vector establishment plasmid of following mammalian expression systems: pCMVp-NEO-BAN, pSSB-H2, pBI CMV1
Clontech, pBK-CMV, pBudCE4.1, pCAGGS, pCEP4, pCI, pCI-neo, pcDNA3.1/Hygro(+/-), the specific embodiment of the invention chooses pCMVp-NEO-BAN, it is connected with L1, L2 genetic fragment of HPV16, HPV18 and HPV58 type, structure is expressed as carrier: pCMVp-NEO-BAN-16L1L2, pCMVp-NEO-BAN-18L1L2, pCMVp-NEO-BAN-58L1L2；Choose the mammalian expression vector of Invitrogen
PCEP4, is connected with L1, L2 genetic fragment of HPV6, HPV11, HPV33 type, builds and is expressed as carrier: pCEP4-6L1L2, pCEP4-11L1L2, pCEP4-33L1L2.
The preparation of embodiment 2.HPV16L1L2 pseudovirus
1. take the gene reporter plasmid pEF1a-E2-Crimson of encoding fluorescent protein, HPV plasmid pCMVp-NEO-BAN-16L1L2 is dissolved in culture medium by 1:1 mass ratio, add lipofectamine Lipofectamine2000(or other) be dissolved in culture medium, mixing, room temperature stands to obtain mixed liquor；Joining mixed liquor in the 293FT cell that the degree of converging inoculated in advance reaches 50-70%, shaking up, put CO2 incubator, 37 DEG C, 5%CO2 concentration is cultivated.
2. after cultivating 6-24 hour, suck nutrient solution, be slowly added to 37 DEG C of preheated DMEM complete mediums, put back to CO2 incubator and continue to cultivate 24-72 hour.
3. discard culture supernatant, with the PBS washed cell of 37 DEG C of preheatings once, the pancreatin adding 0.05% is cleared up 5 minutes, adds 37 DEG C of complete mediums preheating to terminate, after making cell suspend with pipette piping and druming, proceed to 15ml centrifuge tube, 1000rpm(201xg) centrifuge, supernatant discarded, add DPBS-Mg washed cell once, the centrifugal supernatant that goes, centrifuges after addition DPBS-Mg is resuspended and again removes supernatant.
4.
Add and precipitate isopyknic cell pyrolysis liquid (containing 0.5%Brij58,0.2%Benzonase, 0.2%Plasmid with cell
The DPBS-Mg of Safe, 1xAntibiotic-antimycotic), suspended cell, put 37 DEG C of incubators, hatch 24 hours.
5. after taking out cracking, cell pipe is placed 5 minutes to ice bath, adds the 5M sodium chloride solution (make final concentration to 850mM) of 0.17 times of volume, and ice bath 10-20 minute after mixing, 5000xg centrifuges 10 minutes.
6. careful supernatant of drawing, the centrifuge tube that packing is crossed to 1.5ml silicidation, marked title, batch etc., put-80 DEG C frozen.
The preparation of embodiment 3.HPV other types L1L2 pseudovirus
Take the gene reporter plasmid pEF-GFP of encoding fluorescent protein, HPV plasmid pCMVp-NEO-BAN-18L1L2, prepare green fluorescent protein HPV18 type pseudovirus according to the method for embodiment 2.
Take the gene reporter plasmid pEYFP-Mem of encoding fluorescent protein, HPV plasmid pCMVp-NEO-BAN-58L1L2, prepare yellow fluorescence protein HPV58 type pseudovirus according to the method for embodiment 2.
Take the gene reporter plasmid pEF1a-E2-Crimson of encoding fluorescent protein, HPV plasmid pCEP4-6L1L2, prepare red fluorescent protein HPV6 type pseudovirus according to the method for embodiment 2.
Take the gene reporter plasmid pEF-GFP of encoding fluorescent protein, HPV plasmid pCEP4-11L1L2, prepare green fluorescent protein HPV11 type pseudovirus according to the method for embodiment 2.
Take the gene reporter plasmid pEYFP-Mem of encoding fluorescent protein, HPV plasmid pCEP4-33L1L2, prepare yellow fluorescence protein HPV33 type pseudovirus according to the method for embodiment 2.
Embodiment 4. applies flow cytomery each type HPV pseudovirus titre
1. collect the 293FT cell of exponential phase, count with haemocytometer and use DMEM complete culture solution diluting cells concentration to 1.5 × l05Individual/ml.
2.
In 96 porocyte culture plates, every hole adds 100 μ l 293FT cells, hatches 6 hours in 37 DEG C of 5% CO2 incubator.
3.
Dilution HPV pseudovirus starts dilution, 8 gradients of two-fold dilution from 1000 times, adds pseudovirus dilution according to every hole 100 microlitre, after cultivating 72 hours, by the cell infection rate in the every hole of flow cytomery, do linear regression, calculate infection rate and reach the pseudovirus extension rate of 10-20%.Calculate the cell number of 1 milliliter of pseudovirus infection as pseudovirus titre.
The preparation of embodiment 5. HPV trivalent pseudovirus and detection NAT
1. prepare trivalent pseudovirus: according to pseudovirus titre, take each type pseudovirus of certain volume so that quantity or the ratio of mixing postoperative infection cell are identical, and dilution, mixed preparing obtains trivalent pseudovirus.
2.
Dilution test serum: test serum complete medium is started 8 gradients of 4 times of stepwise dilutions by 40 times, according to every gradient 60 HL serum, do two parallel.
3.
Test serum and the premix of virus: take 96 new porocyte culture plates, every hole adds 60 μ l serum dilutions and 60 μ l pseudovirus dilutions, pats surrounding and mixes, sets complete culture solution alternative serum dilution simultaneouslyPseudovirus control wellsWith serum-free without pseudovirusCell control well。
4.
Test serum and the preincubate of pseudovirus mixed liquor: 4 DEG C of refrigerators of 96 well culture plate are placed 1 hour.
5.
Draw the pseudovirus of 100 μ L and test serum composition adds in the culture plate corresponding aperture having completed cell in advance for 6 hours from the 96 each holes of well culture plate, pat culture plate surrounding and mix.
6.
Tissue Culture Plate is placed in 37 DEG C/5%CO2Incubator is hatched.After 72 hours, from incubator, take out Tissue Culture Plate.
7.
Put into and can detect in cell imaging type polychrome reading plate instrument, read every hole and express the quantity of different fluorecyte.
Or with trypsinization every hole inner cell, be resuspended in PBS solution, by cell infection rate under the different fluorescence channel of the every porocyte of flow cytomery.
8. calculate test serum NAT.Neutralize titre calculation: ask for sample and compare each multiple hole average, withPseudovirus control wellsContrast, sample well Control of Fluorescence reaches the extension rate corresponding to 50%, is the neutralization titre value of this serum.If testing sample is monoclonal antibody, then reach the corresponding monoclonal antibody content (microgram) of 50% using Control of Fluorescence rate as shouldIn antibody and titre。
Embodiment 6. applies flow cytomery divalence pseudovirus to infect the NAT of serum
Take the gene reporter plasmid pEF1a-E2-Crimson of encoding fluorescent protein, HPV plasmid pCI-neo promega
-16L1L2, prepares red fluorescent protein HPV16E2 type pseudovirus according to the method for embodiment 2.
Take the gene reporter plasmid pEF-GFP of encoding fluorescent protein, HPV plasmid pCI-neo promega-16L1L2, prepare green fluorescent protein HPV18GFP type pseudovirus according to the method for embodiment 2.
Collect the 293FT cell of exponential phase, count with haemocytometer and use DMEM complete culture solution diluting cells concentration to 1.5 × l05Individual/ml.In 96 porocyte culture plates, every hole adds 100 μ l 293FT cells, hatches 6 hours in 37 DEG C of 5% CO2 incubator.
Dilution HPV16E2 type and HPV18GFP pseudovirus start dilution from 1000 times, 8 gradients of two-fold dilution, according to every hole 100 microlitre pseudovirus, after cultivating 72 hours, with the cells ratio expressing fluorescin in flow cytometer (Beckman Coulter EPICS XL) detection hole, calculate the cell number of 1 milliliter of pseudovirus infection as pseudovirus titre.
According to pseudovirus titre, take 16 and 18 type pseudovirus so that the cell number of infection is identical；Dilution, mixed preparing obtains divalence pseudovirus 293FT HPV16E2-HPV18GFP.
Dilution test serum: positive serum complete medium is started 8 gradients of 4 times of stepwise dilutions by 40 times, according to every gradient 60 HL serum, does two parallel holes.
Test serum and the premix of virus: take 96 new porocyte culture plates, every hole adds 60 μ l serum dilutions and 60 μ l pseudovirus dilutions, pats surrounding and mixes, sets complete culture solution alternative serum dilution simultaneouslyPseudovirus control wellsWith serum-free without pseudovirusCell control well。
Test serum and the preincubate of pseudovirus mixed liquor: 4 DEG C of refrigerators of 96 well culture plate are placed 1 hour.
Draw the pseudovirus of 100 μ L and test serum composition and comparison from the 96 each holes of well culture plate, adherent be slowly added in the culture plate corresponding aperture having completed cell in advance for 6 hours, pat culture plate surrounding and mix.
Tissue Culture Plate is placed in 37 DEG C/5%CO2 incubator and hatches.After 72 hours, from incubator, take out Tissue Culture Plate.
It by the cell infection rate of different type pseudovirus in flow cytometer (BD FACSCalibur) detection hole, is specifically shown in accompanying drawing.(being to read the cell number that different fluorescence is expressed in every hole when reading the detection of plate instrument by cell imaging type).
Calculate test serum NAT.Neutralize titre calculation: ask for sample and compare each multiple hole infection rate or infection cell number average, contrast pseudovirus control wells calculates sample well Control of Fluorescence rate under each dilution factor, calculate the extension rate reaching corresponding to 50% suppression, be the neutralization titre value of this serum.Two type HPV NAT testing result such as following tables of positive serum to be measured.
Claims (12)
1. one kind is detected the divalence of HPV neutralizing antibody, trivalent or multivalence HPV pseudovirus, it is characterised in that this pseudovirus comprises the reporter plasmid of HPV capsid protein and encoding fluorescent protein.
2. divalence as claimed in claim 1, trivalent or multivalence HPV pseudovirus, it is characterised in that wherein said HPV capsid protein is the wild type full-length of structural proteins L1+L2 or mutant or the albumen truncating.
3. divalence as claimed in claim 1, trivalent or multivalence pseudovirus, it is characterized in that HPV type is the 6th, the 11st, the 16th, the 18th, the 26th, the 30th, the 31st, the 33rd, the 34th, the 35th, the 39th, 45,5l, the 52nd, the 53rd, the 56th, the 58th, the 59th, the 66th, the 67th, the 68th, the 69th, the 70th, the 72nd, the 73rd, the 82nd, 85 or 97 type, preferably the 6th, the 11st, the 16th, the 18th, the 31st, the 33rd, the 45th, the 52nd, 58 type.
4. the divalence as described in claim the 1st, 2 or 3, trivalent or multivalence pseudovirus, it is characterised in that the expression product of described reporter plasmid be green fluorescent protein, red fluorescent protein, yellow fluorescence protein, in blue fluorescent protein wantonly two kinds, three kinds or multiple.
5. divalence as claimed in claim 4, trivalent or multivalence pseudovirus, it is characterised in that preferred green fluorescent protein, red fluorescent protein or yellow fluorescence protein.
6. divalence as claimed in claim 5, trivalent or multivalence pseudovirus, it is characterized in that every type pseudovirus contains the reporter plasmid of unique color fluorescin respectively, each type pseudovirus consumption is for adjusting according to the titre measuring pseudovirus to reaching the positive ratio of identical cell infection.
7. the preparation method of the divalence as described in claim 5 or 6, trivalent or multivalence pseudovirus, it is characterized in that the method is: A. is by the reporter plasmid cotransfection 293FT cell of the gene expression plasmid containing HPV capsid protein and the gene of encoding fluorescent protein, express different type pseudovirus capsid protein respectively, it spontaneously wraps up the plasmid containing reporter and is assembled into virion, prepares pseudovirus；B. the cell number that pseudovirus titre can be infected by every milliliter of cape horn fever venom represents, take the consumption of every kind of pseudovirus for making various pseudovirus infection cell ratio reach same level, the infection proportion of about 10-25%, preferably 15-20%, is mixed to prepare divalence, trivalent or multivalence HPV pseudovirus.
8. preparation method as claimed in claim 7, it is characterised in that in the method, step A is for by 1) two kinds of gene expression plasmids of L1, L2, or L1+L2 gene expression plasmid, and 2) gene co-transfection 293FT cell containing encoding fluorescent protein.
9. a detection is by the method for HPV pseudovirus or virus infected cell, comprising:
Making the cell being infected by divalence, trivalent or multivalence HPV pseudovirus with being suitable to fluorescence detection equipment, such as flow cytometer, cell imaging type read the signal of plate instrument detection, but not infected cell is without signal；With
Detect this signal by fluorescence detection equipment and detect the cell being infected by different type HPV pseudovirus.
10. method as claimed in claim 9, counts respectively for the cell being infected by different shaped other HPV pseudovirus.
11. 1 kinds of methods detecting HPV neutralizing antibody, it is characterised in that the method comprises the steps:
Detected sample gradient dilution liquid is contacted in nutrient solution 293FT cell with HPV pseudovirus co-incubation, application fluorescence detection equipment (such as flow cytometer, cell imaging type read plate instrument), by detection with the cell of the signal directly or indirectly being produced by reporter protein, detect ratio or the quantity of infected cell；Whether measuring and calculating sample there are HPV neutralizing antibody and/or its titre (titer).
12. methods as claimed in claim 11, wherein said detected sample for the biological sample of the separation from individuality to be checked is: serum, monoclonal antibody, or the biological sample from tissue cultures.
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| WO2008085938A2 (en) * | 2007-01-08 | 2008-07-17 | Rosalind Franklin University Of Medicine And Science | Antibody inhibiting infection of papillomavirus |
| CN101551392A (en) * | 2008-01-22 | 2009-10-07 | 厦门大学 | Method for detecting human papilloma virus neutralizing antibody |
| CN102268076A (en) * | 2010-07-02 | 2011-12-07 | 厦门大学 | Truncated human papillomavirus (HPV) type 52 L1 protein |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114716562A (en) * | 2021-01-04 | 2022-07-08 | 中国医学科学院基础医学研究所 | Human papilloma virus 58 type chimeric protein and application thereof |
| CN114716562B (en) * | 2021-01-04 | 2023-11-10 | 中国医学科学院基础医学研究所 | Human papilloma virus 58 chimeric protein and application thereof |
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