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Preparation method and applications of anti-latent membrane protein LMP2A monoclonal antibody

A monoclonal antibody, latent membrane protein technology, applied in the field of medical molecular biology, can solve the problems of poor curative effect and poor prognosis of nasopharyngeal carcinoma

Inactive Publication Date: 2011-02-16
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the curative effect of advanced nasopharyngeal carcinoma is very poor, and the 5-year survival rate is only 8% to 10%.
The prognosis of nasopharyngeal carcinoma is still poor because there is no effective treatment for distant metastatic nasopharyngeal carcinoma so far

Method used

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  • Preparation method and applications of anti-latent membrane protein LMP2A monoclonal antibody
  • Preparation method and applications of anti-latent membrane protein LMP2A monoclonal antibody
  • Preparation method and applications of anti-latent membrane protein LMP2A monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Design of primers for amplifying the full-length sequence of LMP2A and the amplification of the full-length sequence of LMP2A

[0034] According to the principle of primer design, it is designed using the software Primer5.0, and the full-length sequence of LMP2A can be amplified after experimental verification. The detailed primer sequence is as follows:

[0035] up-LMP2A-BglII:

[0036] 5'gaagatctatggggtccctagaaatggt3';

[0037] up-flag-LMP2A-BglII:

[0038] 5'gaagatctaccatggactacaaggacgacgatgacaaggggtccctagaaatggtgc3';down-flag-LMP2A-EcoR I:

[0039] 5'cggaattcttacttgtcatcgtcgtccttgtagtctacagtgttgcgatatggg3'

[0040] The specific experimental steps, conditions and electropherograms for amplifying the full-length sequence of LMP2A are as follows:

[0041] RT (reverse transcription)

[0042] (1) Take 2 μg of RNA from C666 cells, and add samples one by one according to the following system (volume 15 μl): RNA 2 μg, random primer 1 μl water (μl) to balance...

Embodiment 2

[0063] The anti-LMP2A monoclonal antibody prepared in Example 2 detects the expression of LMP2A in nasopharyngeal carcinoma tissue

[0064] 1. Establishment of stable cell lines exogenously expressing LMP2A and identification of LMP2A antibodies

[0065] Nasopharyngeal carcinoma cells CNE2 and SUNE1 in the logarithmic growth phase were taken, and the two cell lines were infected with Pbabe and Pbabe-LMP2A viruses at the same time, and were screened with a medium containing 1ug / ml puromycin for 3 days, and passed down. CNE2-Vector, CNE2-LMP2A, SUNE1-Vector, SUNE1-LMP2A cells were lysed with protein lysate, and protein quantification was performed by BCA method. Denature at 98°C for 10 minutes, load 20ug of the sample for western blotting detection; at the same time, use Trizol to lyse the cells, extract RNA, and perform RT-PCR detection. figure 2 It is the establishment of a stable cell line exogenously expressing LMP2A, and the expression of its RNA and protein. From this we...

Embodiment 3

[0073] Example 3 LMP2A is used as a target, and monoclonal antibodies are used for specific neutralization to treat nasopharyngeal carcinoma

[0074] 1. In vitro detection of monoclonal antibody on the tumorigenicity of LMP2A-expressing cells and its ability to reverse EMT.

[0075] 1.1 Incubate with monoclonal antibody and endogenous and exogenous expression LMP2A cells, and use MTT method to detect cell proliferation

[0076] The cells C666 and CNE2-LMP2A were digested and cultured in a 96-well plate. Antibodies were added to the cells at a certain concentration, and a blank control group was set. The MTT method was used to monitor for 7 days to analyze the proliferation of the cells. Such as Figure 5 As shown, monoclonal antibodies can inhibit cell proliferation. (P<0.05)

[0077] 1.2 Using plate cloning and soft agar colony formation experiments to observe the transformation ability of cells treated with antibodies

[0078] After incubating the antibody with cells C66...

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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 [0001] The invention relates to the field of medical molecular biology, in particular to a preparation method and application of an anti-EBV latent membrane protein LMP2A monoclonal antibody. Background technique [0002] Nasopharyngeal carcinoma (NPC) is a rare malignant tumor, and its incidence is characterized by uneven racial and geographical distribution. According to statistics, about 80,000 people are diagnosed with nasopharyngeal cancer every year in the world, and the annual death toll is about 50,000, ranking 23rd among new tumors. Nasopharyngeal carcinoma has a high incidence in southern my country and Southeast Asia, with an incidence rate of about 25-50 / 100,000 people, which seriously threatens human health. Compared with other head and neck tumors, the onset age of nasopharyngeal carcinoma is relatively young, and it is more likely to spread throughout the body, often metastasizing to bones, lungs, liver and other organs, and the distant metast...

Claims

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Application Information

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IPC IPC(8): A61P35/00G01N33/574A61P35/04C07K16/08G01N33/577A61K39/42C12N5/09A61P31/20
Inventor 曾木圣刘万里李满枝洪明晃曹京燕胡利娟张华
Owner SUN YAT SEN UNIV
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