CN104634976A - Human cytomegalovirus active infection rapid detection method and reagent kit for detection method - Google Patents
Human cytomegalovirus active infection rapid detection method and reagent kit for detection method Download PDFInfo
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Abstract
The invention discloses a human cytomegalovirus active infection rapid detection method and a reagent kit for the detection method. The human cytomegalovirus active infection rapid detection method and the reagent kit are mainly used for rapidly monitoring and diagnosing of human cytomegalovirus active infection of pregnant women, newborn and people with immunocom promise including transplantation patients, HIV suffers, tumor patients, diabetics patients and the old people with the immune function decreased due to the age factor. The invention also relates to the preparation of a reagent kit utilizing the detection method.
Description
Technical field
The invention belongs to antibody engineering and diagnostic method field, particularly relate to a kind of human cytomegalovirus infection method for quick and the kit for this detection method.
Background technology
The infection of human cytomegalovirus (human cytomegalovirus, HCMV) in crowd is very general, and in worldwide distribution, China about 90% adult can detect HCMV antibody.HCMV is once infect and will hide all the life in host body, " balance " state is at the normal crowd's virus and host of immunologic function, when body causes hypoimmunity due to a variety of causes (HIV, transplanting and aging etc.), this " balance " will be broken and cause HCMV reactivation to infect, and the virus causing reactivation to infect also can derive from other the infecteds from host itself.Primary infection and reactivation infect is referred to as Active infection, and in AIDS patient, transplanting receptor and the elderly, HCMV Active infection can cause various diseases, even causes death; Immunologic function not yet fully-developed fetus be then teratogenesis, cause inborn defect.
Also uncertain in the congenital infection rate of the current HCMV of China, but research shows, and developed country's congenital HCMV infection rate is lower than developing country, and wherein Italy is 0.47% (CI:0.22 – 1.0%); Sweden is 0.46% (CI:0.37 – 0.58%); The congenital infection rate of Gambia HCMV is up to 13.6%; The U.S. is 1%, annual about 40000 neonates infect with congenital HCMV, wherein 10% ~ 15% Late-onset sequelae, is mainly SNHL, dysnoesia, the low inferior nervous system injury disease of learning ability, wherein because SNHL pays the medical bill up to 1,000,000,000 dollars every year.China's newborn population about 1,615 ten thousand in 2011, data according to the U.S. calculate, within 2011, China about has 16.2 ten thousand neonates to there occurs congenital HCMV infection, family and country all will pay high cost for this reason, have scholar to report, actual China congenital HCMV infects 2-3 times that neonate is 16.2 ten thousand person/year.
In HIV patient, HCMV causes patient's PVR and then blind main pathogen, by the end of 2013 the end of the year China report HIV person and AIDS patients totally 43.4 ten thousand examples altogether, and infection conditions is in rising trend.Current ART can extend the time-to-live of HIV person greatly, even access expansion age at death, but blind the urgent need in HIV patient that HCMV virus infections causes solves.
China implements the number of cases of organ transplant also in the trend increased year by year, and 2013 are only kidney transplant one, and China just implements more than 6400 examples.HCMV interstitial pneumonia, GVHD are the most common or the most serious complication of allogeneic bone marrow transplantation, and as there is HCMV pneumonia, case fatality rate can up to 80% ~ 90%.
In the elderly of immunosenescence, HCMV infection can be risk factor especially angiocardiopathy, diabetes and the tumour of multiple chronic disease, directly affects the life-span.A research is by detecting HCMV cellular immune level in HCMV pp65T cell effect investigation over-65s crowd, and to their follow-up investigation survival rate, find the crowd (to HCMV acellular immunization) of mortality ratio far below pp65T cell effect feminine gender of crowd's same time period of the pp65T cell effect positive (having immunization of cell to HCMV), and occurred by the ratio of disease of cardiovascular system death the increase being greater than 2 times in this crowd.Clinically also observed HCMV infection in immunocompromised crowd and can reduce the release of insulin, and beta Cell of islet is the target cell of HCMV, is therefore also lowly the hazards of type-II diabetes to the immune defense of HCMV.In addition, immunosenescence crowd HCMV infects also relevant to the chronic inflammation disease such as tumour, rheumatic arthritis and Crohn disease.
Also there is no the vaccine that HCMV can be prevented to infect at present, but clinical research finds that earlier antiviral treatment can alleviate the damage of congenital infection nervous system in children, saves hearing; Transplant patient's case fatality rate can be reduced, avoid excessive immune to suppress; Prevent AIDS human retina pathology.Therefore, urgent clinical needs are HCMV Active infection detection means fast and accurately, though it is HCMV diagnosis " goldstandard " that conventional viral is separated cultivation, needs just can obtain positive findings in 4 weeks, is difficult to the requirement meeting quick diagnosis.ELISA method detects the diagnosis that HCMV specific IgM antibodies can be used for the normal HCMV Active infection of immunologic function or HCMV disease, but its positive rate is low, suppresses patient can lack antibody response or antibody delay appearance at sever immune.HCMV pp65 is envelope protein, and between the capsid and its coating of this virus, belong to low matrix phosphorylated protein, molecular weight is 65kD, accounts for 15% of viral total protein, is the major viral proteins of excitating organism cellular immunity.Because HCMV mainly hides in human peripheral blood single nucleus cell, there is virus when reactivation infects and first in mononuclearcell, copy propagation, then cause humoral immune reaction with flow propagation.Therefore, the time that in mononuclearcell, pp65 antigen occurs comparatively specific IgM antibodies occur early, and can not express because patient immune function is low and reduce.Detect that pp65 antigenemia not only can illustrate that there is HCMV reactivation infects, alternatively patient's disappearance is for the cellular immunity of HCMV simultaneously.Therefore, be recently by " the new goldstandard " of the clinical detection HCMV Active infection accepted extensively by detecting patient HCMV pp65 antigenemia.
Given this, we establish and utilize monitoring peripheral blood in patients pp65 antigenemia to diagnose the method for HCMV Active infection, and form detection kit.
Summary of the invention
The object of the invention is raiser diagnosing for active human cytomegalovirus infection method for quick and the kit for this detection method.
The present invention adopts following technical scheme to achieve these goals:
A kind of human cytomegalovirus infection method for quick, is characterized in that, comprise the following steps:
(1) by erythrocyte cracked liquid process fresh peripheral blood specimen splitting erythrocyte, regather, wash centrifugation and obtain corresponding leucocyte, the cell smear that preparation detects;
(2) self-control HCMV pp65 monoclonal antibody is adopted to carry out indirect immunofluorescene assay to cell smear; Described self-control HCMV pp65 monoclonal antibody is that purifying obtains from Hybridoma Cell Culture supernatant prepared by HCMV pp65 carrier for expression of eukaryon immune mouse;
(3) in fluorescence microscopy Microscopic observation testing result;
(4) observations needs when positive control and negative control are set up simultaneously just credible, occur in sample that >=1 Positive fluorescence signal can be judged to be that human cytomegalovirus infection is positive, occur that >=50 Positive fluorescence signals can be judged to be human cytomegalovirus infection strong positive;
Described human cytomegalovirus infection method for quick, is characterized in that,
Described self-control HCMV pp65 method for preparing monoclonal antibody is:
The acquisition of A, HCMV pp 65DNA recombinant plasmid
(1) according to the HCMV gene order announced in Genebank, determine HCMV pp 65 gene coded sequence, be designed for the PCR primer of amplification HCMV pp 65, with HCMV AD169 strain RNA for template, reverse transcription becomes cDNA, go out not containing the UL83 gene of introne by pcr amplification again, be cloned on pcDNA3.0 carrier, construction recombination plasmid pcDNA3.0-pp65, transformation of E. coli DH5 α, extract plasmid, obtain the correct pcDNA3.0-pp65 plasmid of sequence by DNA sequencing;
(2) by pcDNA3.0-pp65 plasmid transfection correct for sequence to HEK 293 cell, after 24h, Westernblotting identifies that it whether can at eukaryotic expression HCMV pp65 albumen, and identifying can at the correct pcDNA3.0-pp65 plasmid of the sequence of eukaryotic expression HCMV pp65 albumen;
(3) by the bacterial classification mass propgation containing the pcDNA3.0-pp65 plasmid identified, alkaline lysis cracking thalline is passed through after collecting thalline, again successively through Sepharose 4FF molecular sieve, Plasmid Select affinity chromatography and SOURCE 30Q Anionic column chromatography purifying, obtain the pp65DNA recombinant plasmid after purifying;
B, mouse immune
Pp65DNA recombinant plasmid after purifying is mixed with the ratio of adjuvant in 3:1, multiple spot intramuscular injection is carried out to mouse; Every immunity in 2 weeks once, totally 4 times; After last immunity 1 week, the blood sampling of mouse tail, by Double diffusion in gel testing inspection antibody titer, got the mouse that antibody titer is greater than 1:8, aseptic taking-up spleen after putting to death, the cell of the BALB/c mouse spleen after polishing adaptive immune;
C, Fusion of Cells
Get Sp2/0 myeloma cell to mix in the ratio of 1:10 with the BALB/c mouse splenocyte after immunity, the centrifugal 10min of 1000rpm, abandon supernatant, put 37 DEG C of water-bath preheatings, in 45s, the PEG 1500 of the 1ml concentration 50% being preheated to 37 DEG C is added with 1ml suction pipe, limit edged vibrates gently, then in 90s, add the 1640 incomplete nutrient culture media that 30ml is preheated to 37 DEG C, room temperature leaves standstill 10min, the centrifugal 10min of 1000rpm, abandons supernatant, and it is resuspended containing 1640 nutrient culture media of 20%FCS and HAT to add 20ml, be dispensed on 96 orifice plates of existing feeder cells, in 5%CO
2cultivate in incubator, the upgrowth situation of observation of cell, within 4 ~ 6 days, continue to cultivate with 1640 nutrient culture media containing 20%FCS and HT afterwards, still with there being 1640 nutrient culture media of 20%FCS and HT to continue to cultivate when again changing liquid, 1640 medium culture of 20%FCS are used instead after 4 ~ 6, by the time, when cell colony is greater than 1/4 area bottom 96 orifice plates, after getting supernatant detection fusion, whether hybridoma secretes anti-pp65 antibody; D, indirect immunofluorescence detect pp65 monoclonal antibody in culture supernatant
By 5 × 10
4in/hole inoculation MRC-5 cell to 96 orifice plate, overnight incubation; Press MOI=0.02 next day and inoculate each hole of HCMV AD169 strain virus to 96 orifice plate; Cultivate after 72h and discard nutrient culture media, with PBS washed cell 3 times, each 3min, dries; Every hole adds 100 μ l absolute ethyl alcohols, room temperature effect 15min; Discard ethanol, add PBS and wash 3 times, each 3min; Hybridoma culture supernatant 50 μ l to be checked is added, 4 DEG C of overnight incubation to every hole; Add PBS and wash 3 times, each 3min; Add the goat anti-mouse IgG-FITC two diluted to resist, hatch 30min for 37 DEG C, add PBS and wash 3 times, each 3min; Observe under inverted fluorescence microscope, the hybridoma of the screening immunofluorescence dyeing positive;
E, hybridoma colonized culture
Hybridoma cloning adopts limiting dilution assay, and hybridoma H0105 and F0249 of anti-HCMV pp65 protein monoclonal antibody is produced in final acquisition 2 strains;
The hypotype of F, monoclonal antibody
Test with Mouse monoclonal antibody isotyping kit, the monoclonal antibody hypotype of result display H0105 secretion is IgG2b; The monoclonal antibody hypotype of F0249 secretion is IgG1, and light chain is κ chain.
The kit that a kind of human cytomegalovirus infection method for quick uses, it is characterized in that, this kit is made up of pp65 antibody application liquid, erythrocyte cracked liquid, immobile liquid, rupture of membranes agent, fluorescein-labelled two anti-application liquid, contrast slide, positive control, negative control.
The kit that described human cytomegalovirus infection method for quick uses, is characterized in that: positive control is that the HEK293 cell of lentivirus mediated stable transfection HCMV pp65 mixes gained in proportion with normal human lymphocytes; Negative control is negative human lymphocyte; Erythrocyte cracked liquid is the ammonium chloride solution of 0.83%; Rupture of membranes agent is the phosphate buffer containing 0.05% Triton X-100; Pp65 antibody application liquid is pressed 1:1000 dilution with the PBS containing 10%BSA again after being mixed by H0105 and F0249 hybridoma supematant purified product 3:1 and is obtained; Fluorescein-labelled two anti-application liquid dilute marked by fluorescein isothiocyanate sheep anti-mouse antibody by pressing 2000:1 containing the PBS of 10%BSA obtains.
Described human cytomegalovirus infection method for quick, is characterized in that, detect positive control be the HEK293 cell of the stable transfection HCMV pp65 gene obtained by slow-virus infection with normal human lymphocytes by 1:10000 mixing gained.
The present invention sets up the Active infection detection method of human cytomegalovirus, compared with the conventional method, there is following important effect: quick and precisely can diagnose human cytomegalovirus infection (with virus purification concordance rate up to 96%), with foreign same type Product checking result concordance rate up to 98.43%.
Accompanying drawing explanation
Fig. 1 is pp65 protein expression A in the HEK293 cell of indirect immunofluorescence qualification stable transfection pp65 gene: the DAPI dyeing surely turning HEK293 cell; B: the pp65 monoclonal antibody dyeing surely turning HEK293 cell; The DAPI dyeing of C: untransfected HEK293 cell; The pp65 monoclonal antibody dyeing of D: untransfected HEK293 cell;
Fig. 2 is the cytomegalovirus antigenemia positive findings A that the present invention detects: nuclear targeting; B:pp65 antigenemia kit coloration result.
Embodiment,
The assembling of HCMV pp65 antigenemia detection kit and use
1, the assembling of kit
One, the preparation of slide is contrasted
Collect appropriate healthy human peripheral blood, be separated lymphocyte with lymphocyte separation medium, for subsequent use after cell count; Collect the HEK293 cell of stably express HCMV pp65 gene, carry out cell count, and it is mixed as positive control, using normal human lymphocytes as negative control with normal human lymphocytes by 1:10000.Respectively in the position of microslide mark "+" coating 2 × 10
5individual positive control cell, in the position of mark "-" coating 2 × 10
5individual positive negative control cell.After room temperature is dried, fix 15min by immobile liquid room temperature, seal after freeze drying.
Two, the preparation of anti-HCMV pp65 monoclonal antibody application liquid
By H0105 and F0249 two strain hybridoma produce monoclonal antibody-purified after, the solution of 2mg/ml is prepared into the PBS of 0.1M pH7.4, both equal-volumes are mixed, again mixed liquor is used containing 0.1%Proclin300, it is 1 μ g/ml that the 0.1M pH7.4PBS of 5%BSA is diluted to antibody total concentration, is distributed into 4ml every bottle.
Three, FITC marks the preparation of goat anti-mouse IgG antibodies application liquid
FITC marks goat anti-mouse IgG antibodies purchased from Life Technologies company (Lot:1515529), with containing 0.1%Proclin 300, it is 1 μ g/ml that the 0.1M pH7.4PBS of 5%BSA and 0.02% Evans blue is diluted to antibody total concentration, is distributed into 4ml every bottle.
Four, 10 × erythrocyte cracked liquid preparation
By ammonium chloride 16.04g, sodium bicarbonate 1.68g, EDETATE SODIUM 0.74g, moltenly regulate pH7.2-7.4 with 200ml pure water (resistance value 18.25 megaohm).
Five, 5 × paraformaldehyde immobile liquid preparation
PH7.2-7.4 is regulated with 250ml pure water (resistance value 18.25 megaohm) by molten for 50g paraformaldehyde.
Six, 5 × permeable membrane liquid preparation
6.25ml Triton-100X 0.1M pH7.4PBS is diluted to 250ml.
Seven, each component prepared with said method is assembled into kit after the assay was approved.
2, the using method of the present embodiment kit
1) leukocyte suspension preparation
1) use sterilizing deionized water by 10 × erythrocyte cracked liquid, dilute 10 times, be placed in 4 DEG C of environment more than precooling 1h;
2) 2ml EDTA anticoagulation is got in sterile working, adding aseptic 50ml bores in end centrifuge tube, 1000 × g (2500rpm) centrifugal 2min, draw blood plasma (retaining sallow layer) for antibody test, add 1 × erythrocyte cracked liquid of 30ml4 DEG C of precooling, ice bath or 4 DEG C hatch 10min splitting erythrocyte;
3) 1000 × g (2500rpm) centrifugal 2min, supernatant discarded;
4) if erythrocyte splitting is insufficient, cracking is repeated once;
5) 30ml 0.1M PBS (pH7.4) is added, the cell precipitation at the bottom of resuspended pipe, 1000 × g (2500rpm) centrifugal 2min, supernatant discarded;
6) by 1ml 0.1M PBS (pH7.4) re-suspended cell precipitation;
7) getting 100 μ l cell suspensions joins in 900 μ l PBS, and mixing, carries out cell count;
8) cell concentration to 2.0 × 10 are adjusted
6cells/ml;
9) if cell concentration is lower, can the PBS re-suspended cell of centrifugal suitable volumes again.
(2) film-making
1) get 100 μ l cell suspensions, be applied in the circular scope of microslide diameter 6mm, 600rpm (60 × g) centrifugal 5min, if do not have slide hydro-extractor slide room temperature can be placed, natural evaporate to dryness, makes cell be attached on microslide;
2) every part of sample prepares 3 cell smears;
3) in vent cabinet, on the microslide of evaporate to dryness, drip cell immobile liquid, cover the position scribbling cell completely, incubated at room 15min;
4) wash 3 times with PBS, each 3min, dry;
5) on microslide, drip permeable membrane liquid and cover the position scribbling cell completely, incubated at room 15min;
6) cleansing solution washs 3 times, each 3min, dries; If (slide needs to store, and after available PBS washing once, room temperature is dried, frozen with-80 DEG C of refrigerators.)
(3) indirect IF staining
1) contrast slide PBS is soaked 1min, dry, test together with sample;
2) there is the region of cell to drip the pp65 antibody application liquid of 35 μ l to slide, in 37 DEG C of wet boxes, hatch 60min;
3) cleansing solution washs 3 times, each 3min, dries;
4) have the region of cell to drip the anti-application liquid of fluorescence two of 35 μ l to slide, in 37 DEG C of wet boxes, lucifuge hatches 30min;
5) 0.1% Azo-Blue dye liquor is dripped to covering cell, room temperature lucifuge effect 5min;
6) cleansing solution washs 3 times, each 3min, dries;
7) drip appropriate mountant, use cover glass mounting, immediately fluorescent microscope 400 × read result (also can with 1000 × improve resolution), preservation of taking a picture.
(4) quality control
Positive control should show the dyeing of yellowish green positive cell, and negative control should dye without yellow green.Although program strictly follows quality control, single positive cell likely can occur in negative control, should not be regarded as invalid dyeing.In negative control, observe single positive cell and do not mean that the testing result can not explaining sample.
(5) result is explained
The testing result of sample is qualitatively, and therefore result should only have feminine gender or the positive.Definition according to below:
Positive: to have one or more antigen-positive cell in each double dyeing, positive cell number is being greater than 50 for strong positive.
Negative: in each double dyeing, there is no antigen-positive cell.
The quantity of every part of pigmented section cell can not lower than 5 × 10
4individual cell, otherwise result is invalid.
Table 1 the present embodiment and virus purification and Brite CMV Turbo Kit detect the results contrast of 300 parts of transplant patient's samples simultaneously
The preparation of the HCMV pp65 antibody material used in the present embodiment.
The preparation of 1.HCMV pp65DNA recombinant plasmid
(1) pcr amplification HCMV pp65 gene
According to the HCMV gene order announced in Genebank, determine HCMV pp65 gene coded sequence, the PCR primer being designed for amplification HCMV pp65 is:
HCMV pp65-F：5-’CTCGAG ATGGAGTCGCGCGGTC5-3’
HCMV pp65-R：5-’GGATCC ACCTCGGTGCTTTTT-3’
With HCMV AD169 strain RNA for template, reverse transcription becomes cDNA, then is gone out not containing the UL83 gene of introne by pcr amplification.PCR reaction is carried out in 50 μ l systems, and response parameter is 94 DEG C of sex change 5min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min40s, totally 35 circulations, and last 72 DEG C extend 10min.Amplified production 1% agarose gel electrophoresis detects, and has a specific amplification band at 1600bp place, and molecular weight is consistent with the HCMV pp65 gene of reporting for work.
(2) structure of HCMV pp65 gene eucaryon expression plasmid
By HCMV pp65 gene PCR product BamHI and Hind III digestion, carry out enzyme with identical restriction endonuclease to pcDNA3.0 plasmid to cut simultaneously, digestion products is cut glue and is reclaimed after agarose gel electrophoresis, HCMV pp65 gene PCR product after being cut by enzyme with T4DNA ligase is again connected with pcDNA3.0, transform DH5 α competence bacterium, coat on the solid LB flat board containing ampicillin, 37 DEG C of overnight incubation.From transformation plate, picking list colony inoculation LB liquid medium, extracts plasmid after amplification cultivation, obtains the correct pcDNA3.0-pp65 plasmid of sequence by DNA sequencing;
(2) HCMV pp65 gene eucaryon expression plasmid ability to express detects
By pcDNA3.0-pp65 plasmid transfection correct for sequence to HEK 293 cell, Western after 24h
Blotting identifies that it whether can at eukaryotic expression HCMV pp65 albumen, and identifying can at the correct pcDNA3.0-pp65 plasmid of the sequence of eukaryotic expression HCMV pp65 albumen;
(3) large-scale purification of HCMV pp65 gene eucaryon expression plasmid
By the bacterial classification mass propgation containing the pcDNA3.0-pp65 plasmid identified, alkaline lysis cracking thalline is passed through after collecting thalline, again successively through Sepharose 4 FF molecular sieve, Plasmid Select affinity chromatography and SOURCE 30Q Anionic column chromatography purifying, obtain the pp65DNA recombinant plasmid after purifying;
(4) mouse immune
Mixed with the ratio of Freund's complete adjuvant in 3:1 by pp65DNA recombinant plasmid after purifying, adjustment plasmid concentration to 0.1 μ g/ μ l, injects appropriate dimethyl sulfoxide at plan inoculation position before immune mouse, carries out multiple spot intramuscular injection subsequently to mouse; Every immunity in 2 weeks once, totally 4 times; After last immunity 1 week, the blood sampling of mouse tail, by Double diffusion in gel testing inspection antibody titer, got the mouse that antibody titer is greater than 1:8, aseptic taking-up spleen after putting to death, the cell of the BALB/c mouse spleen after polishing adaptive immune;
(4) Fusion of Cells
Get Sp2/0 myeloma cell to mix in the ratio of 1:10 with the BALB/c mouse splenocyte after immunity, the centrifugal 10min of 1000rpm, abandon supernatant, put 37 DEG C of water-bath preheatings, in 45s, the PEG 1500 of the 1ml concentration 50% being preheated to 37 DEG C is added with 1ml suction pipe, limit edged vibrates gently, then in 90s, add the 1640 incomplete nutrient culture media that 30ml is preheated to 37 DEG C, room temperature leaves standstill 10min, the centrifugal 10min of 1000rpm, abandons supernatant, and it is resuspended containing 1640 nutrient culture media of 20%FCS and HAT to add 20ml, be dispensed on 96 orifice plates of existing feeder cells, in 5%CO
2cultivate in incubator, the upgrowth situation of observation of cell, within 4 ~ 6 days, continue to cultivate with 1640 nutrient culture media containing 20%FCS and HT afterwards, still with there being 1640 nutrient culture media of 20%FCS and HT to continue to cultivate when again changing liquid, 1640 medium culture of 20%FCS are used instead after 4 ~ 6, by the time, when cell colony is greater than 1/4 area bottom 96 orifice plates, after getting supernatant detection fusion, whether hybridoma secretes anti-pp65 antibody; D, indirect immunofluorescence detect pp65 monoclonal antibody in culture supernatant
By 5 × 10
4in/hole inoculation MRC-5 cell to 96 orifice plate, overnight incubation; Press MOI=0.02 next day and inoculate each hole of HCMV AD169 strain virus to 96 orifice plate; Cultivate after 72h and discard nutrient culture media, with PBS washed cell 3 times, each 3min, dries; Every hole adds 100 μ l absolute ethyl alcohols, room temperature effect 15min; Discard ethanol, add PBS and wash 3 times, each 3min; Hybridoma culture supernatant 50 μ l to be checked is added, 4 DEG C of overnight incubation to every hole; Add PBS and wash 3 times, each 3min; Add the goat anti-mouse IgG-FITC two diluted to resist, hatch 30min for 37 DEG C, add PBS and wash 3 times, each 3min; Observe under inverted fluorescence microscope, the hybridoma of the screening immunofluorescence dyeing positive;
E, hybridoma colonized culture
Hybridoma cloning adopts limiting dilution assay, and hybridoma called after H0105 and F0249 of anti-HCMV pp65 protein monoclonal antibody is produced in final acquisition 2 strains;
The hypotype of F, monoclonal antibody
Test with Mouse monoclonal antibody isotyping kit, the monoclonal antibody hypotype of result display H0105 secretion is IgG2b; The monoclonal antibody hypotype of F0249 secretion is IgG1, and light chain is κ chain.
Fig. 1 is pp65 protein expression A in the HEK293 cell of indirect immunofluorescence qualification stable transfection pp65 gene: the DAPI dyeing surely turning HEK293 cell; B: the pp65 monoclonal antibody dyeing surely turning HEK293 cell; The DAPI dyeing of C: untransfected HEK293 cell; The pp65 monoclonal antibody dyeing of D: untransfected HEK293 cell;
Fig. 2 is the cytomegalovirus antigenemia positive findings A that the present embodiment detects: nuclear targeting; B:pp65 antigenemia kit coloration result.
A kind of human cytomegalovirus infection method for quick and the kit for this detection method; Bo Rui bio tech ltd, Wang Mingli Anhui
PCR primer:
HCMV pp65-F：5-’CTCGAG ATGGAGTCGCGCGGTC5-3’
HCMV pp65-R：5-’GGATCC ACCTCGGTGCTTTTT-3’
Claims (5)
1. a human cytomegalovirus infection method for quick, is characterized in that, comprises the following steps:
(1) by erythrocyte cracked liquid process fresh peripheral blood specimen splitting erythrocyte, regather, wash centrifugation and obtain corresponding leucocyte, the cell smear that preparation detects;
(2) self-control HCMV pp65 monoclonal antibody is adopted to carry out indirect immunofluorescene assay to cell smear; Described self-control HCMV pp65 monoclonal antibody is that purifying obtains from Hybridoma Cell Culture supernatant prepared by HCMV pp65 carrier for expression of eukaryon immune mouse;
(3) in fluorescence microscopy Microscopic observation testing result;
(4) observations needs when positive control and negative control are set up simultaneously just credible, occur in sample that >=1 Positive fluorescence signal can be judged to be that human cytomegalovirus infection is positive, occur that >=50 Positive fluorescence signals can be judged to be human cytomegalovirus infection strong positive.
2. human cytomegalovirus infection method for quick according to claim 1, is characterized in that,
Described self-control HCMV pp65 method for preparing monoclonal antibody is:
The acquisition of A, HCMV pp 65 DNA recombinant plasmid
(1) according to the HCMV gene order announced in Genebank, determine HCMV pp 65 gene coded sequence, be designed for the PCR primer of amplification HCMV pp 65, with HCMV AD169 strain RNA for template, reverse transcription becomes cDNA, go out not containing the UL83 gene of introne by pcr amplification again, be cloned on pcDNA3.0 carrier, construction recombination plasmid pcDNA3.0-pp65, transformation of E. coli DH5 α, extract plasmid, obtain the correct pcDNA3.0-pp65 plasmid of sequence by DNA sequencing;
(2) by pcDNA3.0-pp65 plasmid transfection correct for sequence to HEK 293 cell, after 24h, Western blotting identifies that it whether can at eukaryotic expression HCMV pp65 albumen, and identifying can at the correct pcDNA3.0-pp65 plasmid of the sequence of eukaryotic expression HCMV pp65 albumen;
(3) by the bacterial classification mass propgation containing the pcDNA3.0-pp65 plasmid identified, alkaline lysis cracking thalline is passed through after collecting thalline, again successively through Sepharose 4 FF molecular sieve, Plasmid Select affinity chromatography and SOURCE 30Q Anionic column chromatography purifying, obtain the pp65 DNA recombinant plasmid after purifying;
B, mouse immune
Pp65 DNA recombinant plasmid after purifying is mixed with the ratio of adjuvant in 3:1, multiple spot intramuscular injection is carried out to mouse; Every immunity in 2 weeks once, totally 4 times; After last immunity 1 week, the blood sampling of mouse tail, by Double diffusion in gel testing inspection antibody titer, got the mouse that antibody titer is greater than 1:8, aseptic taking-up spleen after putting to death, the cell of the BALB/c mouse spleen after polishing adaptive immune;
C, Fusion of Cells
Get Sp2/0 myeloma cell to mix in the ratio of 1:10 with the BALB/c mouse splenocyte after immunity, the centrifugal 10min of 1000rpm, abandon supernatant, put 37 DEG C of water-bath preheatings, in 45s, the PEG 1500 of the 1ml concentration 50% being preheated to 37 DEG C is added with 1ml suction pipe, limit edged vibrates gently, then in 90s, add the 1640 incomplete nutrient culture media that 30 ml are preheated to 37 DEG C, room temperature leaves standstill 10min, the centrifugal 10min of 1000rpm, abandons supernatant, and it is resuspended containing 1640 nutrient culture media of 20%FCS and HAT to add 20ml, be dispensed on 96 orifice plates of existing feeder cells, in 5% CO
2cultivate in incubator, the upgrowth situation of observation of cell, within 4 ~ 6 days, continue to cultivate with 1640 nutrient culture media containing 20%FCS and HT afterwards, still with there being 1640 nutrient culture media of 20%FCS and HT to continue to cultivate when again changing liquid, 1640 medium culture of 20%FCS are used instead after 4 ~ 6, by the time, when cell colony is greater than 1/4 area bottom 96 orifice plates, after getting supernatant detection fusion, whether hybridoma secretes anti-pp65 antibody;
D, indirect immunofluorescence detect pp65 monoclonal antibody in culture supernatant
By 5 × 10
4in/hole inoculation MRC-5 cell to 96 orifice plate, overnight incubation; Press MOI=0.02 next day and inoculate each hole of HCMV AD169 strain virus to 96 orifice plate; Cultivate after 72h and discard nutrient culture media, with PBS washed cell 3 times, each 3min, dries; Every hole adds 100 μ l absolute ethyl alcohols, room temperature effect 15min; Discard ethanol, add PBS and wash 3 times, each 3min; Hybridoma culture supernatant 50 μ l to be checked is added, 4 DEG C of overnight incubation to every hole; Add PBS and wash 3 times, each 3min; Add the goat anti-mouse IgG-FITC two diluted to resist, hatch 30min for 37 DEG C, add PBS and wash 3 times, each 3min; Observe under inverted fluorescence microscope, the hybridoma of the screening immunofluorescence dyeing positive;
E, hybridoma colonized culture
Hybridoma cloning adopts limiting dilution assay, and hybridoma H0105 and F0249 of anti-HCMV pp65 protein monoclonal antibody is produced in final acquisition 2 strains;
The hypotype of F, monoclonal antibody
Test with Mouse monoclonal antibody isotyping kit, the monoclonal antibody hypotype of result display H0105 secretion is IgG2b; The monoclonal antibody hypotype of F0249 secretion is IgG1, and light chain is κ chain.
3. one kind as described in claim 1 or 2 human cytomegalovirus infection method for quick use kit, it is characterized in that, this kit is made up of pp65 antibody application liquid, erythrocyte cracked liquid, immobile liquid, rupture of membranes agent, fluorescein-labelled two anti-application liquid, contrast slide, positive control, negative control.
4. the kit of human cytomegalovirus infection method for quick use according to claim 3, is characterized in that: positive control is that the HEK293 cell of lentivirus mediated stable transfection HCMV pp65 mixes gained in proportion with normal human lymphocytes; Negative control is negative human lymphocyte; Erythrocyte cracked liquid is the ammonium chloride solution of 0.83%; Rupture of membranes agent is the phosphate buffer containing 0.05% Triton X-100; Pp65 antibody application liquid is pressed 1:1000 dilution with the PBS containing 10% BSA again after being mixed by H0105 and F0249 hybridoma supematant purified product 3:1 and is obtained; Fluorescein-labelled two anti-application liquid dilute marked by fluorescein isothiocyanate sheep anti-mouse antibody by pressing 2000:1 containing the PBS of 10% BSA obtains.
5. the kit of human cytomegalovirus infection method for quick use according to claim 4, it is characterized in that, detect positive control be the HEK293 cell of the stable transfection HCMV pp65 gene obtained by slow-virus infection with normal human lymphocytes by 1:10000 mixing gained.
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