CN101974086A - Preparation method and applications of anti-latent membrane protein LMP2A monoclonal antibody - Google Patents
Preparation method and applications of anti-latent membrane protein LMP2A monoclonal antibody Download PDFInfo
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Abstract
The invention provides a preparation method of an anti-latent membrane protein LMP2A monoclonal antibody. In the preparation method, the full-length sequence of LMP2A is amplified from EBV-positive nasopharyngeal carcinoma cell strain C666, and a monoclonal antibody preparation method is used for preparing the anti-latent membrane protein LMP2A monoclonal antibody, wherein the primer sequence of the full-length sequence of the amplified LMP2A is SEQ ID NO:1-3. The invention also provides an LMP2A monoclonal antibody obtained by using the preparation method. The invention also provides applications of the monoclonal antibody in preparing detection kits, detecting LMP2A expression in nasopharyngeal carcinoma tissues, neutralizing target antigens and preparing targeting medicines. The invention utilizes the advantages of the monoclonal antibody in diagnosis, treatment and prevention of diseases, has the advantages of strong specificity and high sensitivity, and performs important functions on diagnosis and treatment of nasopharyngeal carcinoma. The invention avoids serious side reactions caused by radiotherapy, chemotherapy and the like which are conventional for tumor treatment.
Description
Technical field
The present invention relates to the Medical Molecular Biology field, be specifically related to anti-EBV latent membrane protein LMP2A MONOCLONAL ANTIBODIES SPECIFIC FOR method and application thereof.
Background technology
(Nasopharyngeal carcinoma NPC) is a kind of more rare malignant tumour to nasopharyngeal carcinoma, and its morbidity has the characteristics of race and areal distribution inequality.According to statistics, annual nearly 80,000 people in the whole world are diagnosed as nasopharyngeal carcinoma, and annual death toll is about 50,000, is positioned at the 23rd of New Development tumour.Nasopharyngeal carcinoma is occurred frequently, and sickness rate is about 25-50/100 in China south and south east asia, 000 people, serious threat human health.Than other neck tumours, the age of onset of nasopharyngeal carcinoma is young relatively, and also easier generation whole body diffusion often is transferred to organs such as bone, lung, liver, and patients with terminal (N2, N3) distant metastasis rate is up to 20%～40%.Early stage nasopharyngeal carcinoma has higher susceptibility to radiotherapy, and curative effect is up to 80-90%, its 5 years total survival rates about about 50%.And the curative effect of advanced NPC is very poor, 5 years survival rates only 8%～10%.Because the nasopharyngeal carcinoma to distant metastasis does not also have a kind of effective method for treatment up to now, so the nasopharyngeal carcinoma prognosis is still very poor.Therefore, although nasopharyngeal carcinoma is relatively more responsive to radiotherapy mostly, shift the main causes of death that remain Most patients.
It is closely related that the outstanding feature of nasopharyngeal carcinoma is that the morbidity of nasopharyngeal carcinoma and EBV infect, and nearly all patient and whole cancer cells all carry EBV nucleic acid and express small part encoding viral gene.The vivo and vitro experiment of our seminar all confirms, the latent membrane protein LMP2A of encoding viral is playing a significant role aspect the transfer of nasopharyngeal carcinoma and the recurrence.
Summary of the invention
In order to overcome above-mentioned technological deficiency, first purpose of the present invention has provided a kind of anti-EBV latent membrane protein LMP2A MONOCLONAL ANTIBODIES SPECIFIC FOR method.This preparation method is the full length sequence that amplifies LMP2A from EBV male human nasopharyngeal epithelioma 1 C666, utilize method for preparing monoclonal antibody to prepare anti-EBV latent membrane protein LMP2A monoclonal antibody, the primer sequence such as the SEQ ID NO:1-3 of the full length sequence of described amplification LMP2A, specific as follows:
up-LMP2A-BglII：5′gaagatctatggggtccctagaaatggt3′；
up-flag-LMP2A-BglII：
5′gaagatctaccatggactacaaggacgacgatgacaaggggtccctagaaatggtgc3′；
down-flag-LMP2A-EcoR?I：
5′cggaattcttacttgtcatcgtcgtccttgtagtctacagtgttgcgatatggg3′
Second purpose of the present invention provided the LMP2A monoclonal antibody of utilizing above-mentioned preparation method to obtain.Utilize this antibody capable to be used to prepare the detection kit aspect.
The present invention also provides monoclonal antibody to detect the application of LMP2A expression aspect in the nasopharyngeal carcinoma tissue, may further comprise the steps:
1) sets up exogenous expression LMP2A stable cell line, extract total cellular protein, identify antibody with Western Bloting method;
2) expression that detects cell with the immunofluorescence dyeing method and organize LMP2A with and inferior location situation;
3) method with immunohistochemical staining detects expression and the inferior location situation that nasopharyngeal carcinoma is organized LMP2A.
The present invention also provide the LMP2A monoclonal antibody in and application aspect the target antigen, be about to LMP2A as target spot, utilize the neutralizing effect of monoclonal antibody specificity, vivo and vitro studies confirm that its therapeutic action to nasopharyngeal carcinoma, specific implementation method is as follows:
(1) influence that the tumour of expressing the LMP2A cell formed ability of vitro detection monoclonal antibody with and reverse the ability of EMT phenomenon.
1) hatches jointly with cell and the cell of monoclonal antibody processing endogenous and exogenous expression LMP2A, utilize mtt assay to detect the propagation situation of cell;
2) utilize plate clone and soft-agar cloning to form the transformation ability of experimental observation after antibody treatment.
3) how much detect side group cell (SP) number with flow cytometer behind the antibody treatment cell.
4) after antibody and cell are hatched for some time jointly, utilize the method for immunofluorescence dyeing to detect the expression of its EMT and stem cell related molecule sign.
5) after antibody and cell are hatched for some time jointly, extract cell total rna and protein, detect its EMT and stem cell related molecule sign respectively and change situation at mRNA level and protein level.
(2) ability of checking monoclonal antibody inhibition tumor growth in the body.
The subcutaneous kind of nude mice is grown human nasopharyngeal epithelioma 1 CNE2-LMP2A and the cellular control unit CNE2-Vector of exogenous expression LMP2A, after the tumour to be formed, injects antibody, observes the tumor growth situation.
Detection method of the present invention is a feature of utilizing the monoclonal antibody high specificity highly sensitive, on the basis that studies confirm that the vital role of LMP2A in the transfer of nasopharyngeal carcinoma and recurrence.So the design primer amplifies the full length sequence of LMP2A from EBV male human nasopharyngeal epithelioma 1 C666, prepare monoclonal antibody, the expression of LMP2A in can the specific detection nasopharyngeal carcinoma, the binding of pathological diagnosis, can predict patient's PD and prognosis, the more important thing is LMP2A and to play an important role aspect the nasopharyngeal carcinoma treatment as target spot.Simultaneously, based on the effect of LMP2A in other EBV related neoplasms,, can detect equally and judging prognosis and the effect of playing treatment such as Hodgkin lymphoma, cancer of the stomach, t cell lymphoma, immunoblastic lymphoma etc.
The present invention utilizes the latent membrane protein LMP2A of EBV coding as target spot, prepare the monoclonal antibody detection kit of anti-LMP2A, the expression of LMP2A in detection and the closely-related nasopharyngeal carcinoma tissue of EBV, in view of studies confirm that LMP2A is in the developing vital role of nasopharyngeal carcinoma, thereby utilize the propagation that has suppressed tumour cell in the monoclonal antibody specificity with LMP2A, reverse its inductive EMT and changed, suppressed the transfer of tumour; Utilize the target characteristic of mediated monoclonal antibody, connect chemotherapeutics and radiotherapeutic sensitizer, reached the purpose of targeted therapy.This invention has utilized monoclonal antibody in the diagnosis of disease and the advantage aspect the treatment prevention, and high specificity is highly sensitive.
Description of drawings
Fig. 1 is the electrophoretic band figure after the amplification of full length sequence of LMP2A;
Fig. 2 is the foundation of the stable cell line of exogenous expression LMP2A, its RNA and protein expression situation;
Fig. 3 observes the Asia location situation of LMP2A in cell with immunofluorescence;
Fig. 4 is the expression that detects LMP2A in the nasopharyngeal carcinoma tissue;
Fig. 5 is that monoclonal antibody suppresses the cell proliferation situation;
Fig. 6 is the growing state that monoclonal antibody can suppress the mouse Subcutaneous tumor.
Embodiment
The present invention is based upon the full length sequence that amplifies LMP2A from EBV male human nasopharyngeal epithelioma 1 C666, the preparation monoclonal antibody, and the expression of detection LMP2A, and with the technology of LMP2A as the target treatment nasopharyngeal carcinoma.
Embodiment 1: the amplification of the design of primers of amplification LMP2A full length sequence and the full length sequence of LMP2A thereof
Utilize software Primer5.0 to design according to the design of primers principle, and pass through the full length sequence that experimental verification can amplify LMP2A, primer sequence is as follows in detail:
up-LMP2A-BglII：
5′gaagatctatggggtccctagaaatggt3′；
up-flag-LMP2A-BglII：
5′gaagatctaccatggactacaaggacgacgatgacaaggggtccctagaaatggtgc3′；down-flag-LMP2A-EcoR?I：
5′cggaattcttacttgtcatcgtcgtccttgtagtctacagtgttgcgatatggg3′
The concrete experimental procedure condition and the electrophorogram of amplification LMP2A full length sequence are as follows:
RT (reverse transcription)
(1) gets C666 cell RNA 2 μ g, press following system application of sample (volume 15 μ l) one by one: RNA 2 μ g, random primer 1 μ l water (μ l) trim to 15 μ l;
(2) put 5min in 70 ℃ of constant temperature PCR instrument, taking-up places on ice immediately afterwards;
(3) add following mixture 10 μ l 1 μ l
/ pipe, operation: mMLV on ice
dNTP?mix 2μl
5×buffer 5μl
dd?H2O 2μl
3)PCR
Above-mentioned cDNA product is diluted 5 times with sterilized water, and the cDNA that gets after 2 μ l dilute carries out the PCR reaction as template, adds reacted constituent one by one according to following system:
10 * buffer (containing MgCl220mM), 2.5 μ l
dNTP?mix(10mM?each) 1.0μl
primer1(sense，10μM) 0.5μl
primer2(anti-sense，10μM)?0.5μl
primer?GAPDH?mixture(10μM)0.5μl
Taq enzyme (5u/ μ l) 0.3 μ l
cDNA 2.0μl
dd?H2O 17.7μl
Cumulative volume 25 μ l
(3) add sample after, the centrifugal several seconds, put and carry out amplified reaction in the PCR instrument;
(4) PCR reaction conditions: 941min ℃, 9430sec ℃, 5030sec 30cycles ℃, 7245sec ℃, 7210min ℃;
(5) PCR product electrophoresis: 1.2% sepharose: claim 1.2g agar Icing Sugar+100ml tbe buffer liquid (0.5 *), be poured on the rubber moulding of having sealed in advance after in microwave oven, being heated to the off-bottom of agarose particle, plug comb, drive bubble away, treat the solid application of sample of gelling after half an hour: the sample solution of 4 μ lPCR product+2 μ l; 80V, the 20min electrophoresis; The back is analyzed with gel Cheng Xiangyi, takes pictures.Electrophoretic band is seen Fig. 1, and bands visible is clear single.
The anti-LMP2A monoclonal antibody that embodiment 2 prepares, the expression of LMP2A in the detection nasopharyngeal carcinoma tissue
1. the evaluation of the foundation of exogenous expression LMP2A stable cell line and LMP2A antibody
The nasopharyngeal carcinoma cell CNE2, the SUNE1 that take the logarithm vegetative period, two strain cells infect Pbabe and Pbabe-LMP2A virus simultaneously respectively, with the substratum screening that contains the 1ug/ml tetracycline 3 days, go down to posterity down.Protein lysate cracking CNE2-Vector, CNE2-LMP2A, SUNE1-Vector, the SUNE1-LMP2A cell, the BCA method is carried out protein quantification.98 ℃ of 10min sex change, last sample 20ug carries out western blotting and detects; Use the Trizol lysing cell simultaneously, take out RNA, carry out RT-PCR and detect.Fig. 2 is the foundation of the stable cell line of exogenous expression LMP2A, its RNA and protein expression situation, thus we as can be seen transfection the cell of LMP2A can detect the expression of LMP2A in transcriptional level and translation skill, and control cells is not expressed.
2. immunofluorescence detects the inferior positioning states of LMP2A in cell and tissue
With behind the cell dissociation in 24 orifice plates of cover glass are housed inoculating cell CNE2-Vector, CNE2-LMP2A, SUNE1-Vector, SUNE1-LMP2A, cultivate and discard substratum after 24 hours, fixed cell carries out the antibody incubation fluorescent dye, paraffin section with the nasopharyngeal carcinoma tissue carries out fluorescent dye equally, takes pictures with the fluorescence co-focusing microscopic examination.As shown in Figure 3, shown the inferior positioning states of cell, we can see that the film location of LMP2A is very clear.
3. immunohistochemical method detects the expression of LMP2A in the nasopharyngeal carcinoma tissue
With the section of nasopharyngeal carcinoma paraffin specimen, use the anti-LMP2A monoclonal antibody of preparation, according to immunohistochemical methods test kit explanation dyeing, just putting microscopic examination and taking pictures.As shown in Figure 4, the Asia location situation of LMP2A in the nasopharyngeal carcinoma sample is film location, and its expression is specific, and normal epithelium cell does not detect the expression of LMP2A.
Utilize the paraffin organization sample that detects nasopharyngeal cancer patient among the present invention at the monoclonal antibody of LMP2A, about 60% nasopharyngeal cancer patient is expressed LMP2A, and mainly is expressed in the aggressive edge of cancer nests, sees Table 1.Table 1 is to utilize monoclonal antibody to detect the positive expression situation of nasopharyngeal cancer patient sample example number; Credit is analysed by statistics, patient's prognosis of high expression level LMP2A is worse than the low verified LMP2A of patient (P=0.001) that expresses and can induces epithelium-mesenchyme to change (EMT), increase stem cell-like cell (side group cell-SP cell) number in the nasopharyngeal carcinoma, and the expression of LMP2A and epithelial molecular marker E-cadherin negative correlation are proportionate with the expression of stem cell molecular marker Bmi-1; And these may be the important mechanisms that causes nasopharyngeal carcinoma to shift and recur.Utilize this monoclonal antibody to detect the paraffin organization sample of other a collection of nasopharyngeal cancer patient equally, credit is analysed by statistics, and the expression of LMP2A and patient's recurrence rate are proportionate.
Table 1
| Case load | LMP2A(+) | LMP2A(-) |
| 150 | 92 | 58 |
The influence that the tumour of expressing the LMP2A cell formed ability of vitro detection monoclonal antibody with and reverse the ability of EMT phenomenon.
1.1 hatch jointly with monoclonal antibody and endogenous and exogenous expression LMP2A cell, utilize mtt assay to detect the propagation situation of cell
With cell C666, CNE2-LMP2A digestion is cultivated with 96 orifice plates, and antibody is added in the cell with finite concentration, and the blank group is set, with mtt assay monitoring 7 days, the propagation situation of analysis of cells.As shown in Figure 5, monoclonal antibody can suppress the propagation of cell.(P＜0.05)
1.2 utilize plate clone and soft-agar cloning to form the transformation ability of experimental observation after antibody treatment
After antibody and cell C666, CNE2-LMP2A hatched a few hours jointly, blank (promptly do not add antibody respectively and add IgG) is set, peptic cell is diluted to certain cell number and spreads into 6 orifice plates and cultivate, and observes size and number situation that the clone forms.
1.3 how much detect side group cell (SP) number with flow cytometer behind the antibody treatment cell
After antibody and cell C666, CNE2-LMP2A hatched a few hours jointly, blank (promptly do not add antibody respectively and add IgG) is set, peptic cell, Hoechst33342 is hatched with DNA fuel, carries out the SP detection with flow cytometer.
1.4 after antibody and cell are hatched for some time jointly, extract cell total rna and protein, detect its EMT and stem cell related molecule sign respectively and change situation at mRNA level and protein level.
After antibody and cell C666, CNE2-LMP2A hatched a few hours jointly, blank is set, the protein lysate lysing cell, the BCA method is carried out protein quantification.98 ℃ of 10min sex change, last sample 20ug carries out western blotting and detects; Use the Trizol lysing cell simultaneously, take out RNA, carry out RT-PCR and detect, (E-cadherin is Fibronectin) at the expression of protein level and mRNA level to analyze EMT related molecular marker thing.
1.5 after antibody and cell are hatched for some time jointly, utilize the method for immunofluorescence dyeing to detect the expression of its EMT and stem cell related molecule sign
Cell C666, CNE2-LMP2A digestion shop is gone into to contain in 24 orifice plates of cover glass, after treating cell attachment, antibody is added, blank (promptly independent antibody and the adding IgG of not adding) is set simultaneously, after a few hours, discards substratum, fixed cell, use an anti-anti-E-cadherin of being, Fibronectin antibody carries out immunofluorescence dyeing, and the fluorescence co-focusing microscopic examination is taken pictures.
The checking monoclonal antibody suppresses the ability of tumor growth in 2 bodies
The subcutaneous kind of nude mice is grown human nasopharyngeal epithelioma 1 CNE2-LMP2A and the cellular control unit CNE2-Vector of exogenous expression LMP2A, after the tumour to be formed, inject antibody, observe the tumor growth situation, tumour grew after one week, according to tumour size average packet, injected monoclonal antibody, after one week, inject antibody group tumour and obviously dwindle.The tumour size cases as shown in Figure 6.
Utilize monoclonal antibody specific, in tumor tissues, detect LMP2A, binding of pathological is learned diagnosis, can predict the development of the nasopharyngeal cancer patient state of an illness, and utilize monoclonal antibody specific recognition and neutralizing effect, suppressed to express the propagation of tumour cell of LMP2A and the EMT that LMP2A causes and changed, reduce the number of stem cell-like cell in the nasopharyngeal carcinoma, suppress the transfer and the recurrence of nasopharyngeal carcinoma, make LMP2A become the novel target spot of nasopharyngeal carcinoma treatment.
Equally, be applied to this monoclonal antibody detection kit relevant and express the detection and the prediction prognosis of the tumour of LMP2A such as Hodgkin lymphoma, cancer of the stomach, t cell lymphoma, immunoblastic lymphoma etc. with EBV, and by in the specificity and LMP2A with and the targeted therapy of mediation, also can play certain therapeutic action.
Claims (5)
1. anti-EBV latent membrane protein LMP2A MONOCLONAL ANTIBODIES SPECIFIC FOR method, it is characterized in that, from EBV male human nasopharyngeal epithelioma 1 C666, amplify the full length sequence of LMP2A, utilize method for preparing monoclonal antibody to prepare anti-EBV latent membrane protein LMP2A monoclonal antibody, the primer sequence of the full length sequence of described amplification LMP2A such as SEQ ID NO:1-3.
2. the LMP2A monoclonal antibody of utilizing the described preparation method of claim 1 to obtain.
3. the application of the described LMP2A monoclonal antibody of claim 2 aspect the monoclonal antibody detection kit of the anti-LMP2A of preparation detection.
4. the described LMP2A monoclonal antibody of claim 2 application aspect the LMP2A expression in detecting the nasopharyngeal carcinoma tissue is characterized in that, may further comprise the steps:
1) sets up exogenous expression LMP2A stable cell line, extract total cellular protein, identify antibody with Western Bloting method;
2) detect cell and expression and the inferior location situation of organizing LMP2A with the immunofluorescence dyeing method;
3) method with immunohistochemical staining detects expression and the inferior location situation that nasopharyngeal carcinoma is organized LMP2A.
The described LMP2A monoclonal antibody of claim 2 in and application aspect the target antigen, it is characterized in that, be target spot with LMP2A.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103102412A (en) * | 2013-01-29 | 2013-05-15 | 陈仁杰 | Humanized anti-nasopharyngeal-cancer LMP2A extracellular region antibody Fab and application thereof |
| CN105153301A (en) * | 2015-05-29 | 2015-12-16 | 南京医科大学 | Monoclonal antibody specifically binding to tandem epitope fusion proteins at second and fifth sites of LMP2A extracellular region and use thereof |
| CN111690617A (en) * | 2020-05-18 | 2020-09-22 | 李欣 | Monoclonal antibody against EB virus LMP2A, cell strain and application thereof |
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| CN1687779A (en) * | 2005-04-15 | 2005-10-26 | 南京医科大学 | Application of latent membrane protein 2A of recombined EB virus for testing nasopharyngeal carcinoma |
| WO2009066824A1 (en) * | 2007-11-19 | 2009-05-28 | Seoul National University Industry Foundation | Vaccine comprising monocyte or immature myeloid cells(imc) which were loaded with the ligand of natural killer t cell and antigen |
| CN101643497A (en) * | 2009-09-15 | 2010-02-10 | 南京医科大学 | HLA-A2 restrictive epitope polypeptide of LMP2A protein source and purpose thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687779A (en) * | 2005-04-15 | 2005-10-26 | 南京医科大学 | Application of latent membrane protein 2A of recombined EB virus for testing nasopharyngeal carcinoma |
| WO2009066824A1 (en) * | 2007-11-19 | 2009-05-28 | Seoul National University Industry Foundation | Vaccine comprising monocyte or immature myeloid cells(imc) which were loaded with the ligand of natural killer t cell and antigen |
| CN101643497A (en) * | 2009-09-15 | 2010-02-10 | 南京医科大学 | HLA-A2 restrictive epitope polypeptide of LMP2A protein source and purpose thereof |
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| 《中国优秀硕士学位论文全文数据库-医药卫生科技辑》 20080415 许文嵘 EB病毒潜伏膜蛋白2A多表位重组基因在大肠杆菌中的表达、纯化、单克隆抗体的制备及其在血清学检测中的应用 第18-40页 1-3、5 , * |
| 《南京医科大学学报》 20020131 彭光勇等 爱泼斯坦-巴尔病毒潜伏期膜蛋白2A cDNA的克隆及序列分析 第4-6、9页 1 第22卷, 第1期 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103102412A (en) * | 2013-01-29 | 2013-05-15 | 陈仁杰 | Humanized anti-nasopharyngeal-cancer LMP2A extracellular region antibody Fab and application thereof |
| CN105153301A (en) * | 2015-05-29 | 2015-12-16 | 南京医科大学 | Monoclonal antibody specifically binding to tandem epitope fusion proteins at second and fifth sites of LMP2A extracellular region and use thereof |
| CN111690617A (en) * | 2020-05-18 | 2020-09-22 | 李欣 | Monoclonal antibody against EB virus LMP2A, cell strain and application thereof |
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