CN104086651A - Preparation method and application of anti-HCMV (human cytomegalovirus) Pp65 protein monoclonal antibody - Google Patents
Preparation method and application of anti-HCMV (human cytomegalovirus) Pp65 protein monoclonal antibody Download PDFInfo
- Publication number
- CN104086651A CN104086651A CN201410313847.5A CN201410313847A CN104086651A CN 104086651 A CN104086651 A CN 104086651A CN 201410313847 A CN201410313847 A CN 201410313847A CN 104086651 A CN104086651 A CN 104086651A
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- hcmv
- antibody
- cell
- cell strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a high-specificity and high-affinity anti-Pp65 monoclonal antibody which is generated by a hybridoma cell strain 8D6 of the anti-Pp65 monoclonal antibody, wherein the cell strain 8D6 is collected in China Center For Type Culture Collection (CCTCC) and has a collection number of CCTCC NO:C201476. The monoclonal antibody secreted by the hybridoma cell strain has good affinity and specificity, and the immunoreactivity and the specificity of the antibody are superior to those of domestic and imported monoclonal antibodies. The anti-Pp65 monoclonal antibody can serve as a key raw material for blood white cell immunofluorescent cytochemical diagnosis and immunofluorescent quantitative PCR (polymerase chain reaction) detection of HCMV (human cytomegalovirus) infection, also can serve as a key raw material for detecting HCMV by using a double antibody sandwich method, has important meaning for establishment of a quick and sensitive clinical diagnosis reagent to HCMV infection, and has important application prospects.
Description
Technical field
The invention belongs to genetically engineered field, field, be specifically related to preparation method and the application thereof of anti-HCMV Pp65 protein monoclonal antibody.
Background technology
Human cytomegalovirus (human cytomegalovirus, HCMV) is the double-stranded DNA virus of a kind of nucleic acid size approximately 235 kb, belongs to herpetoviridae β and belongs to.HCMV infects very general [1], can in immune deficiency individuality and newborn infant, cause serious disease, most the infecteds are without clinical symptom, but mother's infection in pregnancy can cause fetal infection, by blood, can enter various target organs, cause multisystem visceral organ injury (if pneumonia, minority are hepatitis and encephalitis), survivor is accompanied by neural heariing loss, mental retardation and other neural system sequela, and HCMV is infected and to carry out in early days diagnoses and treatment and seem particularly important.HCMV Pp65 albumen is the early stage a kind of structural protein that produce of HCMV, when the HCMV of state of activation infects, Pp65 antigen is expressed in peripheral blood lymphocyte, monocyte, polymorphonuclear leukocyte and vascular endothelial cell in 6～24 h, and this shows an early stage index of Pp65 albumen HCMV Active infection.Thereby HCMV Pp65 antigen is for the detection of HCMV and the monitoring important in inhibiting [5] in therapeutic process.The antigenemic detection of Pp65 is at present internationally recognized standard detecting method [2-3], be immunofluorescence or immunoenzyme chemical staining detection method, utilize the HCMV Pp65 antigen [4] in the monoclonal antibody specific direct-detection peripheral blood cells of HCMV proteins encoded.But, but the method still lacks unified quality control standard at present, and in testing process, be subject to the impact of subjective factor, limited its application clinically.HCMV diagnostic kit quality is uneven at present in addition, causes serious hindrance also to prevention and the diagnosis and treatment of HCMV.
HCMV is the modal a kind of virus that can cause congenital infection in the whole world, China grownup, infects up to 60% ~ 100%, even in developed country, crowd infection rate is also up to 40% ~ 60%.Its infection can make immunologic function patient immature or defect that serious disease occurs, thereby can accelerate its function of immune system infringement as HIV patient infection, causes patient death; It is also to cause Organ Transplantation Patients to occur infecting complication that HCMV infects, and causes the failure of organ transplantation; HCMV is also the main pathogens that causes congenital and perinatal infection, can cause many organs irreversible lesion such as miscarriage, premature labor, fetal intrauterine growth retardation, newborn infant's microcephaly, hepatosplenomegaly.Therefore, HCMV has caused serious public health problem, and when still there is no effective HCMV vaccine listing at present, prevention becomes the Main Means that prevents that HCMV from propagating.
Current research shows, HCMV Pp65 is topmost a kind of in envelope protein, between viral capsid and coating, account for 95% of cortex albumen dense granule, it is a kind of phosphorylated protein [6] of low matrix, belong to immediate early protein, conventionally at virus infected cell 3～4 h, start to synthesize, and in the inherent Peripheral polymorphonuclear leukocytes of 6 ~ 24 h great expression [7-8], HCMV Pp65 has high conservative simultaneously, with other spore exanthema virus without homology, wide expression is in infectious and noninfective virion, and almost all can detect in HCMV infected patient serum, thereby, HCMV Pp65 albumen becomes the important symbol thing of HCMV Infect And Diagnose.It is the prerequisite for the treatment of that early detection infects to reactivity HCMV, and the antigenemic detection of HCMV is at present the important method that quick diagnosis HCMV infects.
The domestic diagnosis that HCMV is infected mainly adopts take the indirect elisa method [9] that totivirus is antigen, can be large due to HCMV molecular weight, and complex structure, and have cross reaction with other simplexviruss, therefore usings the poor specificity of intact virus as antigen.And HCMV Pp65 antigen is the early protein occurring in virus replication, therefore, by detecting Pp65, judge that virus infection is the main method of finding clinically viremia, it is polyclonal antibody [10].And the main dependence on import of critical materials that the HCMV of China's diagnosis at present infects causes diagnostic reagent price high, to patient, caused white elephant.
Reference:
[1]?Simona?M.,?Rosaria?S.,?Stefania?M.,?
et?al.?Comparison?of?real-time?PCR?and?Pp65?antigen?assays?for?monitoring?the?development?of?Cytomegalovirus?disease?in?recipients?of?solid?organ?and?bone?marrow?transplants[J].?New?Microbiol,?2011,?34:157-164.
[2]Moses?S.,?Malathi?J.,
et?al.Determination?of?human?cytomegalovirus?Pp65?antigenemia?among?renal?transplant?patients[J].?Indian?J?Nephrol.?2012,?22(5):?347–352.
[3]Emerson?C.,?Jacyr?P.,?Ana?P.,?
et?al.?Comparison?of?a?rapid?cytomegalovirus?Pp65?antigenemia?assay?revealed?by?immunofluorescence?to?an?in-house?assay?revealed?by?immunoperoxidase?for?diagnosis?in?solid?organ?transplant?recipient?patients[J].?Braz?J?Infect?Dis?2010;14(3):322-324.
[4]Saracino?A.,?Colucci?R.,?Latorraca?A.,?
et?al.?The?effects?of?preemptive?therapy?using?a?very?low?threshold?of?Pp65?antigenemia?to?prevent?cytomegalovirus?disease?in?kidney?transplant?recipients:?a?single-center?experience[J].?Transplant?Proc.?2013,?45(1):182-184.
[5]Gimeno?C.,?Solano?C.,?Latorre?JC.,?
et?al.?Quantification?of?DNA?in?plasma?by?an?automated?real-time?PCR?assay?(cytomegalovirus?PCR?kit)?for?surveillance?of?active?cytomegalovirus?infection?and?guidance?of?preemptive?therapy?for?allogeneic?hematopoietic?stem?cell?transplant?recipients[J].?J?Clin?Microbiol.?2008?,?46(10):3311-8.
[6]?Chevillotte?M.,?Landwehr?S.,?Linta?L.,?
et?al.?Major?tegument?protein?Pp65?of?human?cytomegalovirus?is?required?for?the?incorporation?of?pUL69?and?pUL97?into?the?virus?particle?and?for?viral?growth?in?macrophages[J].?
J?Virol.?2009,?83(6):?2480-2490.
[7]John?PT.?Human?cytomegalovirus?tegument?proteins?(Pp65,?pp71,?pp150,?pp28)[J].?Virol?J,?2012,9:22-23.
[8] Gao Rongbao, Wang Mingli, Chen Jingxian. the progress of human cytomegalic inclusion disease virus latent infection [J]. foreign medical science: virusology fascicle, 2005,12 (3): 373-376.
[9]Kondo?K.,?Kaneshima?H.,?Mocarski?ES.?Human?cytomegalovirus?latent?infection?of?granulocyte-macrophaye?progenitors[J].?Proc?Natl?Acad?Sci?USA,?1994,91(25):?11879-11883。
[10] Li Changgui, Wang Jianfeng, Ying Zhifang, etc. the polyclonal antibody of preparing with recombinant human cytomegalovirus Pp65 is set up the infectious viral particle method [J] that detects. Chinese biological engineering magazine, 2005,25 (10): 47～49.
Summary of the invention
For the deficiencies in the prior art, the present invention is cloning HCMV virus strain Pp65 full-length gene and on the basis of this albumen of escherichia coli expression, further by recombinant protein immune animal, from positive colony, screened the anti-Pp65 monoclonal antibody of a plant height specificity and high-affinity, and the immunoreactivity of this antibody and specificity are better than domestic and monoclonal antibody import, experimental evidence shows, this antibody has as blood leucocyte Immunofluorescence to be diagnosed, immunofluorescence quantitative PCR detects the critical materials that HCMV infects, also can be used as the critical materials that double antibody sandwich method detects HCMV.
Technical scheme provided by the present invention is: the anti-Pp65 monoclonal antibody of a plant height specificity and high-affinity, its hybridoma cell strain 8D6 by anti-Pp65 monoclonal antibody is produced, described cell strain 8D6 is preserved in Chinese Typical Representative culture collection center (CCTCC) on May 21st, 2014, and (preservation address is: Wuhan City, Hubei Province Wuhan University, postcode: 430072), preserving number is CCTCC NO:C201476, after testing survival.
The present invention also provides the hybridoma cell strain 8D6 of secretion high specific and the anti-Pp65 monoclonal antibody of high-affinity, and described cell strain 8D6 is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:C201476.
The present invention also provides a kind of method of preparing the anti-Pp65 monoclonal antibody of high specific and high-affinity, and it take HCMV Pp65 as antigen, prepares hybridoma, obtains monoclonal antibody.
The present invention also provides a kind of human cytomegalovirus's of detection test kit, it is characterized in that: it comprises that anti-Pp65 monoclonal antibody as claimed in claim 1 is as detecting antibody.
The present invention is in order to obtain anti-HCMV Pp65 protein monoclonal antibody, the crucial medical material infecting as clinical immunodetection HCMV, adopt recombinant expressed HCMV Pp65 protein fragments immunity BALB/c mouse, by immune lymph-node cell and sp2/0 cytogamy, adopt indirect ELISA method screening positive clone and titration, with Western blotting, carry out antibodies specific evaluation again, finally by immunofluorescence technique, evaluate its using value.By a large amount of screenings, finally obtained the hybridoma cell strain of 1 strain energy stably excreting high-affinity and the anti-HCMV Pp65 of high specific, called after 8D6.Western blotting and indirect ELISA detect it and tire and reach respectively 1: 4000 and 1: 10
5, immunofluorescence experiment presentation of results, the monoclonal antibody of this hybridoma cell strain secretion has good avidity and specificity, and experimental data shows that the immunoreactivity of this antibody and specificity are better than domestic and monoclonal antibody import.Experimental evidence shows, this antibody has the critical materials that detects HCMV infection as the diagnosis of blood leucocyte Immunofluorescence, immunofluorescence quantitative PCR, also can be used as the critical materials that double antibody sandwich method detects HCMV, to setting up HCMV, to infect the reagent for clinical diagnosis of rapid sensitive significant, for setting up HCMV infection clinical detection method, lay a good foundation, there is important application prospect.
Accompanying drawing explanation
Fig. 1 Fig. 1 fused cell upgrowth situation (100 *).A: the Growth of Hybridoma Cell situation of first day after merging; B: the Growth of Hybridoma Cell situation of the 3rd day after merging; C: the Growth of Hybridoma Cell situation of the tenth day after merging.
The affinitive layer purification of Fig. 2 antibody and SDS-PAGE identify.A: antibody affinity chromatography figure, B: antibody purification SDS-PAGE evaluation (lane M: protein relative molecular mass standard, lane 1: ascites before purifying, lane 2: ammonium sulphate precipitation antibody purification, lane 3:protein A affinity purification antibody).
Monoclonal antibody after Fig. 3 band western blotting relative immunity reaction purification Identification.Ck: negative control, the e. coli bl21 that transforms empty carrier is induced rear total protein; Lane 1: total protein after restructuring Pp65 BL21 bacterial strain inducing; Lane 2: restructuring purifying Pp65 albumen; Lane 3:1 type herpes virus hominis lysate; Lane 4:HCMV employing virus cracking liquid total protein; Purifying Pp65 albumen after lane 5:HCMV virolysis; Lane 6: standard substance Pp65 albumen.
Fig. 4 Western blot detects tiring of monoclonal antibody 8D6.Lane CK: negative control (not adding primary antibodie); Lane 1-8: monoclonal antibody 8D6 is respectively through 1 * 10
2, 5 * 10
2, 1 * 10
3, 2 * 10
3, 4 * 10
3, 8 * 10
3, 1 * 10
4, 2 * 10
4doubly after dilution; Lane 9: positive control (anti-Pp65 monoclonal antibody standard substance).
Fig. 5 indirect ELISA detects tiring of monoclonal antibody.
The immunoreactivity (100 *) of Fig. 6 immunofluorescence check Dispersal risk and HCMV infection.A and a are respectively the negative control (not adding primary antibodie) of B and b; A1 wherein, B1, a1, and the cell of b1 for directly observing under visible ray without laser excitation, A2, B2, a2, and b2 is the result of observing after fluorescence excitation, the primary antibodie of B2 is 8D6, b2 be other antibody of preparing be primary antibodie.
The immunoreactivity (100 *) of Fig. 7 immunofluorescence check Dispersal risk and HCMV infection.A and a are respectively the negative control (not adding primary antibodie) of B and b; A1 wherein, B1, a1, and the cell of b1 for directly observing under visible ray without laser excitation, A2, B2, a2, and b2 is the result of observing after fluorescence excitation, and the primary antibodie of B2 is 8D6, the primary antibodie of b2 is import reference standard antibody.
Embodiment
Detailed description below by embodiment is further illustrated the present invention, but is not limitation of the present invention, only does example explanation.
material and main agents are as follows:
animal and cell strain:sP2/0 murine myeloma cell is so kind as to give by professor Xia Ningshao of HSPH of Xiamen University, and people's lung embryo fibroblast is preserved by this laboratory, and BALB/c female mice is purchased from Xiangya Medical College, Zhongnan Univ.
main agents:pTO-T7, the anti-Pp65 monoclonal antibody of standard by Hu'nan Kangrun Pharmaceutical Co., Ltd. be so kind as to give, standard restructuring Pp65 albumen is purchased from U.S. ProSpec company, IPTG-sheep anti mouse is purchased from Kang Wei reagent company, HRP-sheep anti mouse is so kind as to give by professor Xia Ningshao of HSPH of Xiamen University, protein relative molecular mass standard is purchased from GeneStar company, HRP-DAB substrate colouring reagents box is purchased from TianGen company, PEG 1500, HT and HAT substratum are purchased from Sigma company, and 1640 substratum are purchased from Gibco company.
embodiment mono-: animal immune and serum titer detect: Pp65 albumen foot plate immune mouse
Pp65 albumen and Freund's complete adjuvant after purifying is fully emulsified, adopt foot plate immunization method, with 3 of the dosage immunity of every 100 μ g BALB/c female mice in 6 ~ 8 week age.After two weeks, Pp65 recombinant protein and Freund's incomplete adjuvant is fully emulsified, adopt same dosage and method booster immunization BALB/c mouse.Front 1 d of each immunity gathers mice serum, and indirect ELISA method detects Serum Antibody and tires.Merge the Pp65 recombinant protein immunity foot plate that does not add adjuvant for front 3 d, every dosage is 100 μ g.Institute's blood sampling is diluted in physiological saline at 1: 5, then blood suspension is placed in to 37 ℃ of 2 h, then moves to 4 ℃ and spend the night, centrifugal 10 min of 4000 g after hemagglutination, drawing supernatant liquor is gained antiserum(antisera), and indirect ELISA method detects serum titer, chooses serum titer and is greater than 1 * 10
6mouse carry out cytogamy test.
Mouse, after foot plate immunity three times, is collected respectively immunity front and back serum, then by 1: 10, and 1: 10
2, 1: 10
3, 1: 10
4, 1: 10
5, 1: 10
6carry out gradient dilution, then carry out indirect ELISA mensuration, mouse after initial immunity in body IgG antibody horizontal very low, after the 1st booster immunization, serum is that sun turns (2.1 times with feminine gender value are made as cut off value), illustrated that Pp65 recombinant protein has good immunogenicity, can in Mice Body, cause stronger immune response, after the 3rd immunity, after 1000000 times of dilutions, in serum, IgG tires up to more than 1.0 (table 1), and now the mouse after immunity can be used for follow-up test.
Table 1 indirect ELISA detects antibody titer in mice serum
embodiment bis-: cytogamy, positive hybridoma cell screening and subclone
Select SP2/0 myeloma cell and immune mouse lymph-node cell that growth conditions is good with serum free medium, to wash after 3 times respectively, with mixing in 1: 5 ~ 1: 10, centrifugal 5 min of 1500 g, abandoned supernatant.After jog centrifuge tube makes cell dispersed, slowly add the PEG 1 500 of 37 ℃ of preheatings of 1 mL and mix gently, then adding 20 mL 1640 substratum.After centrifugal 5 min of 800 g, abandon supernatant, add 20% FBS-HAT-1640 substratum suspension cell and mix.
Cell after mixing is evenly laid in 96 orifice plates, is placed in 37 ℃, 5% CO
2incubator in cultivate, every day tracing observation, after merging, 7 ~ 10 d change liquid with 10% FBS-HT-1640 nutrient solution, until clone, grow to 1/3 of hole floorage ~ 1/2 o'clock and detect antibody in hybridoma supernatant by indirect ELISA method, select the hole that positive value is high and cell colony number is few, by limiting dilution assay, carry out cloning.
After sp2/0 cell and immune mouse lymphocyte merge, visible fused cell volume is large, circular and bright, in background, there is the not fused cell that a large amount of volumes are little (Fig. 1 A), after HAT selects to cultivate, the 2nd d just has not fused cell to die off, while cultivating 3 ~ 5 d, there is little clone to occur (Fig. 1 B), during the 10th d, clone has grown to approximately 1/3(Fig. 1 C of hole floorage), every hole is containing 1 or several cell clone, by observation, count and indirect ELISA, show that the fusion rate of hybridoma and the positive rate of secretory antibody are respectively 92.5% and 81.9%.After 3 subclones, from more than 60 positive colonies, screen the hybridoma cell strain of the anti-HCMV Pp65 of 1 strain energy stably excreting monoclonal antibody, called after 8D6.
By CB damping fluid (15 mmol/L Na for the Pp65 albumen of purifying
2cO
3, 35 mmol/L NaHCO
3) to be diluted to concentration be 1 mg/L, coated 96 hole ELISA plates, 4 ℃ are spent the night.Wash after plate 1 time, add Hybridoma Cell Culture supernatant liquor, hatch after 30 min for 37 ℃, wash plate 5 times, add HRP-sheep anti mouse two anti-, hatch 30 min for 37 ℃, wash plate 5 times, finally add TMB substrate developer, 37 ℃ of reaction 10 min, add stop buffer termination reaction, 450 nm/630 nm dual wavelengths detect.Through above-mentioned indirect ELISA, identify, determine in the monoclonal antibody of preparing and be IgG2b hypotype.
embodiment tri-: monoclonal antibody produces and purifying
Adopt in Mice Body and induce ascites method manufacture order clonal antibody.Get 8 ~ 10 w BALB/c mouse, the aseptic paraffin oil of first abdominal injection, every 0.5 mL.The backward intraperitoneal of 3 d injects 8D6 hybridoma cell strain, and adjusting cell density is 1 * 10
6~ 2 * 10
6/ mL, every mouse peritoneal is injected 0.5 mL cell suspension.After inoculating cell 7 ~ 12 d, can obviously see that mouse web portion expands, after collection ascites, carry out purifying.First add saturated ammonium sulphate to carry out preliminary purification at 1: 1 the ascites being collected, 4 ℃, centrifugal 10 min of 12000 g, remove supernatant, PBS damping fluid (20 mmol/L NaCl, 2.68 mmol/L KCl, the 10 mmol/L Na of certain volume for precipitation
2hPO
4, 1.76 mmol/L KH
2pO
4, pH 7.4) dissolve after, the PBS damping fluid dialysis 2 times of putting into 10 times of volumes, each 2 h.4 ℃, centrifugal 10 min of 8 000 g, collect supernatant, then move in the Binding buffer(Protein A Sefinose Kit of dialysis equilibrium to 20 times volume in dialysis tubing and provide), dialysis antibody carries out affinitive layer purification with protein A, the effect of SDS-PAGE purification Identification.
Antibody after dialysis is splined on the Protein A affinity column of binding buffer liquid balance, with the binding buffer liquid of 3 times of column volumes, wash not in conjunction with albumen, there is penetrating peak in about 1 min, use again elution buffer wash-out monoclonal antibody, at 11 min left and right 8D6, be eluted (Fig. 2 A), collect wash-out antibody, carry out SDS-PAGE and identify antibody purity (Fig. 2 B lane 3), after Protein A affinity purification, antibody purity is very high, almost can't see other foreign proteins on SDS-PAGE glue.
embodiment tetra-: the specificity of human cytomegalic inclusion disease virus separation and Culture, monoclonal antibody, the evaluation of tiring
With the DMEM nutrient solution containing 10% foetal calf serum, cultivate human embryonic lung fibroblast in 10 mm culture dish, after treating that cell grows to ware bottom surface area 40% ~ 50%, the HCMV virus strain of clinical separation is infected to human embryonic lung fibroblast, and infective dose is 1000 pfu/ wares, is placed in 5% CO
2, in 37 ℃ of cell culture incubators, hatch after 1 h, use the DMEM cell culture fluid of 2% calf serum instead and cultivate, infect and cultivate after 5 ~ 7 d, until cell, substantially occur after pathology, collect the cell conditioned medium liquid that contains HCMV.Supernatant liquor 10000 g are centrifugal, and 20 min remove cell debris, with 50% saturated ammonium sulphate 1 time, after 20 mmol/L PBS dialysis, obtain and have infective virus.
Cytomegalovirus cracking total protein is carried out to 10% SDS-PAGE, electrotransfer is (200 mA to PVDF film, 1 h), with skimmed milk-TN damping fluid (5% skimmed milk, 50 mmol/L TrisHCl, 150 mmol/L NaCl, pH 7.5) sealing 2 h, TNT damping fluid (50 mmol/L TrisHCl, 150 mmol/L NaCl, 0.05% Tween-20, pH 7.5) wash film 3 times, each 10 min, then add monoclonal antibody (skimmed milk 1:2000 dilution), room temperature reaction 2 h, wash film 3 times, each 10 min, add HRP enzyme mark sheep anti mouse two anti-(skimmed milk 1:3 000 dilution), after room temperature reaction 2 h, TNT washes film 3 times, each 10 min, finally with HRP-DAB substrate colouring reagents box, develop the color.
By CB liquid (15 mmol/L Na for the Pp65 albumen after purifying
2cO
3, 35 mmol/L NaHCO
3) to be diluted to concentration be 1 mg/L, coated ELISA 96 orifice plates, 4 ℃ are spent the night, and wash after plate 1 time, add the monoclonal antibody (1: 10 of gradient dilution
2, 1: 10
3, 1: 10
4, 1: 10
5, 1: 10
6), hatch after 30 min for 37 ℃, wash plate 5 times, add HRP-sheep anti mouse two anti-, hatch 30 min for 37 ℃, wash plate 5 times, finally add TMB substrate developer, 37 ℃ of reaction 10 min, add stop buffer termination reaction, and 450 nm/630 nm dual wavelengths detect.
Intestinal bacteria recombinant bacterial strain is expressed to total protein and carry out 10% SDS-PAGE, electrotransfer is (200 mA, 1 h) to PVDF film, with skimmed milk-TN damping fluid (5% skimmed milk, 50 mmol/L TrisHCl, 150 mmol/L NaCl, pH 7.5) sealing 2 h, TNT damping fluid (50 mmol/L TrisHCl, 150 mmol/L NaCl, 0.05%Tween-20, pH 7.5) wash film 3 times, each 10 min, then add respectively monoclonal antibody (by 1: 10
2, 5: 10
2, 1: 10
3, 2: 10
3, 4: 10
3, 8: 10
3, 1: 10
4, 2: 10
4gradient dilution), room temperature reaction 2 h, wash film 3 times, each 10 min, add HRP enzyme mark sheep anti mouse two anti-(with skimmed milk 1:3 000 dilution), after room temperature reaction 2 h, TNT washes film 3 times, and each 10 min, finally with the colour developing of HRP-DAB substrate colouring reagents box.
Adopt immunoreactivity and the specificity of the check monoclonal antibody purifications such as restructuring Pp65 albumen that this experiment expresses, cell cultures HCMV, 1 type herpes virus hominis, Western blot result shows, in electrophoresis transferring film, carry out after immunoblotting reaction, on each self-corresponding film, all there is specific band to occur (Fig. 3 lane 1,2,4,5), with positive controls in band position consistency (Fig. 3 lane 6).Not only there is specificity with restructuring Pp65 antigen in antibody, and there is strong specific immune response in natural Pp65 albumen, with other albumen in 1 type herpes virus hominis and intestinal bacteria without any cross reaction, and without any band, show (Fig. 3 lane CK in negative control group, lane 3), illustrated that this strain antibody has good immunoreactivity and specificity is anti-, there are the potentiality as the exploitation of immunodiagnosis critical materials.
After monoclonal antibody 8D6 gradient dilution, as primary antibodie, carry out band Western blotting and detect, result demonstration, anti-Pp65 monoclonal antibody still can be seen obvious specific band (Fig. 4 lane 5) when being diluted to 4000 times, through 1 * 10
4doubly after dilution, can also see more weak immune response object band (Fig. 4 lane 7), illustrate that the avidity of antibody is strong.8D6 monoclonal antibody is pressed to the laggard row indirect ELISA of gradient dilution to be detected, result shows that antibody reacts and has high sensitivity with restructuring Pp65 albumen and commercial Pp65 protein immunization, it is tired all up to 1:100000(Fig. 5), further proved that the anti-Pp65 monoclonal antibody of preparation has high avidity to antigen.
embodiment five: 8D6 gram of anti-immunofluorescence experiment detects
Human embryonic lung fibroblast (HEL) is cultured in 24 well culture plates of placing cell climbing sheet with the DMEM nutrient solution of 10% foetal calf serum, cultivate HEL cell, after treating that cell grows to ware bottom surface area 40% ~ 50%, the HCMV virus strain of clinical separation is infected to HEL cell (virus liquid that is about to preserve drops in the culture dish of the HEL that grown), infective dose is 500 pfu/ holes, is placed in 5% CO
2in cell culture incubator, hatch 1 h for 37 ℃, using the DMEM cell culture fluid of 2% calf serum instead cultivates, infect and cultivate after 5 ~ 7 d, until cell, substantially occur after pathology, with tweezers, carefully press from both sides out creep plate, PBS washing creep plate 3 times, each 5 min, then add 4% paraformaldehyde to fix 10 min, then with PBS, wash creep plate 3 times, each 5 min, then adding liquid (2% TritonX-100-PBS) acts on 5 min, after PBS washing, add anti-Pp65 monoclonal antibody (dilution in 1: 1000), hatch 30 min for 25 ℃, after PBS washing, add FITC-sheep anti mouse two anti-, hatch 30 min for 25 ℃, PBS washing 3 times, add anti-quencher to be placed on microscopy under fluorescent microscope.
Getting respectively viral cell and the control cells of infection is fixed on cover glass, with the 8D6 monoclonal antibody screening, 60 strain positive colonies, other antibody except 8D6, the domestic and anti-HCMV Pp65 of the import standard monoclonal antibody of purchase are carried out immunofluorescence dyeing detection again, relatively specificity and the avidity of antibody.When the monoclonal antibody that relatively prepared by the present invention, find, all having there is obvious yellow-green colour positive signal (Fig. 6 B2 and b2) in all antibody, and in the cell contrast of not infecting, does not have positive fluorocyte to occur (Fig. 6 A2 and a2).And in all monoclonal antibodies, the fluorescence that the cell that 8D6 is corresponding sends is the brightest, clear picture and lasting time long (Fig. 6 B2), illustrate that 8D6 has good immunoreactivity and high avidity, has the potentiality of the critical materials infecting as diagnosis HCMV.
8D6 is further carried out to immunofluorescent test with the anti-HCMV Pp65 of import monoclonal antibody, fluorescent reaction when Fig. 7 B2 and Fig. 7 b2 are respectively monoclonal antibody 8D6 and import monoclonal antibody as primary antibodie, from result, can find, the 8D6 monoclonal antibody of this research preparation is during as primary antibodie, while adopting immunofluorescence to detect the detection of HCMV cells infected, its fluorescence intensity and time length are all better than import primary antibodie, and the anti-HCMV Pp65 monoclonal antibody that further illustrates this research preparation has a good application prospect.
Claims (4)
1. the anti-Pp65 monoclonal antibody of a plant height specificity and high-affinity, it is characterized in that: its hybridoma cell strain 8D6 by anti-Pp65 monoclonal antibody is produced, described cell strain 8D6 is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:C201476.
2. secrete the hybridoma cell strain 8D6 of high specific and the anti-Pp65 monoclonal antibody of high-affinity, described cell strain 8D6 is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:C201476.
3. a method of preparing the anti-Pp65 monoclonal antibody of high specific and high-affinity, is characterized in that, it take HCMV Pp65 as antigen, prepares hybridoma, obtains monoclonal antibody.
4. a test kit that detects human cytomegalovirus, is characterized in that: it comprises that anti-Pp65 monoclonal antibody as claimed in claim 1 is as detecting antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410313847.5A CN104086651A (en) | 2014-07-03 | 2014-07-03 | Preparation method and application of anti-HCMV (human cytomegalovirus) Pp65 protein monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410313847.5A CN104086651A (en) | 2014-07-03 | 2014-07-03 | Preparation method and application of anti-HCMV (human cytomegalovirus) Pp65 protein monoclonal antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104086651A true CN104086651A (en) | 2014-10-08 |
Family
ID=51634434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410313847.5A Pending CN104086651A (en) | 2014-07-03 | 2014-07-03 | Preparation method and application of anti-HCMV (human cytomegalovirus) Pp65 protein monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104086651A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357403A (en) * | 2014-11-04 | 2015-02-18 | 北京科兴生物制品有限公司 | Monoclonal antibody for resisting human cytomegalovirus and application |
| CN104634976A (en) * | 2015-01-13 | 2015-05-20 | 王明丽 | Human cytomegalovirus active infection rapid detection method and reagent kit for detection method |
| CN108949698A (en) * | 2018-07-31 | 2018-12-07 | 广东和信健康科技有限公司 | Hybridoma cell strain C11-6F7 and its HCMV monoclonal antibody and application of generation |
| CN113121656A (en) * | 2019-12-31 | 2021-07-16 | 湖南师范大学 | EBV envelope glycoprotein gp350 epitope and monoclonal antibody thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1686541A (en) * | 2005-04-11 | 2005-10-26 | 王明丽 | Preparation method of human macrocell virus pp65 protein vaccine |
| CN103276008A (en) * | 2013-04-25 | 2013-09-04 | 湖南师范大学 | HCMV pp65 prokaryotically-expressed recombinant strain and construction method thereof |
-
2014
- 2014-07-03 CN CN201410313847.5A patent/CN104086651A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1686541A (en) * | 2005-04-11 | 2005-10-26 | 王明丽 | Preparation method of human macrocell virus pp65 protein vaccine |
| CN103276008A (en) * | 2013-04-25 | 2013-09-04 | 湖南师范大学 | HCMV pp65 prokaryotically-expressed recombinant strain and construction method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 姜永忠: "人巨细胞病毒pp65单克隆抗体的制备及其体外中和活性鉴定", 《中国硕士学位论文医药卫生科技辑》 * |
| 宋玉靖: "人巨细胞病毒pp65基因片段的原核表达及鉴定", 《中国生物制品学杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357403A (en) * | 2014-11-04 | 2015-02-18 | 北京科兴生物制品有限公司 | Monoclonal antibody for resisting human cytomegalovirus and application |
| CN104634976A (en) * | 2015-01-13 | 2015-05-20 | 王明丽 | Human cytomegalovirus active infection rapid detection method and reagent kit for detection method |
| CN108949698A (en) * | 2018-07-31 | 2018-12-07 | 广东和信健康科技有限公司 | Hybridoma cell strain C11-6F7 and its HCMV monoclonal antibody and application of generation |
| CN108949698B (en) * | 2018-07-31 | 2019-05-31 | 广东和信健康科技有限公司 | Hybridoma cell strain C11-6F7 and its HCMV monoclonal antibody and application of generation |
| CN113121656A (en) * | 2019-12-31 | 2021-07-16 | 湖南师范大学 | EBV envelope glycoprotein gp350 epitope and monoclonal antibody thereof |
| CN113121656B (en) * | 2019-12-31 | 2022-03-18 | 湖南师范大学 | EBV envelope glycoprotein gp350 epitope and monoclonal antibody thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110964102B (en) | Monoclonal antibody capable of simultaneously combining with canine, feline and mink parvoviruses, variable region sequence thereof, hybridoma cell strain and application | |
| CN105675873B (en) | A kind of detection kit and its application | |
| CN104483490A (en) | Hog cholera virus inhibition ELISA antibody detection kit and application thereof | |
| CN106350533A (en) | Anti-PD-L1-CAR-T, and preparation method and application thereof | |
| CN104086651A (en) | Preparation method and application of anti-HCMV (human cytomegalovirus) Pp65 protein monoclonal antibody | |
| AU2010330349B2 (en) | Antibody that binds to H-1 parvovirus | |
| CN111474357A (en) | Test strip for rapidly detecting African swine fever virus, and preparation method and application thereof | |
| CN114716540A (en) | Canine parvovirus monoclonal antibody and application thereof | |
| CN101597334A (en) | Monoclonal antibody of bluetongue virus (BTV) and preparation method and application | |
| CN101793897A (en) | Method for improving screening efficiency of hybridoma | |
| CN111139224A (en) | Monoclonal cell strain resisting SWP2 protein and application thereof | |
| CN106632676B (en) | Mouse-anti-pig PD-L1 monoclonal antibody and application thereof | |
| CN104965083B (en) | A kind of kit of detection H3N2 hypotype canine influenza virus | |
| CN104911150B (en) | The foundation of monoclonal antibody hybridoma cell strain and its preparation and application of monoclonal antibody of a kind of H3N2 canine influenza virus | |
| CN109280644B (en) | Anti-human IgG monoclonal antibody, hybridoma cell strain and application thereof | |
| CN101671655B (en) | Monoclonal antibody hybridoma cell of HIV P24 and application | |
| Yan et al. | Recombinant Soluble Henipavirus Glycoprotein Preparation | |
| US11767356B1 (en) | Canine parvovirus nanobody CPV-VHH-E3 and application thereof | |
| CN115838421A (en) | Antibody targeting influenza B virus nucleoprotein and application thereof | |
| CN108866008A (en) | The monoclonal antibody and its cell strain of anti-Koi herpesvirus and application | |
| CN109535249B (en) | Monoclonal antibody ZKns3G2 and application thereof | |
| CN111995673A (en) | Dengue virus antigen rapid detection test paper | |
| CN109553680B (en) | Monoclonal antibody ZKns4B8 and application thereof | |
| CN109503712B (en) | Monoclonal antibody ZKns2E11 and application thereof | |
| CN110272497B (en) | High-affinity anti-cyclic citrullinated peptide tetravalent small-molecule antibody and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20141008 |