ELISA reagent kit for screening, diagnosis and treatment effect forecast of nasopharyngeal carcinoma

A diagnostic reagent, EB virus technology, applied in the medical field, can solve problems such as poor specificity

Active Publication Date: 2008-04-02
同昕生物技术(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, EBNA1 is expressed in both the incubation period and the lysis period of EB virus. Although VCA and EA have a high detection rate in nasopharyngeal carcinoma (NPC) patients, their specificity is not good, and there are still relatively high levels in healthy people. high detection rate

Method used

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  • ELISA reagent kit for screening, diagnosis and treatment effect forecast of nasopharyngeal carcinoma
  • ELISA reagent kit for screening, diagnosis and treatment effect forecast of nasopharyngeal carcinoma
  • ELISA reagent kit for screening, diagnosis and treatment effect forecast of nasopharyngeal carcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1. Obtaining of recombinant plasmids pGEX-R185 and pGEX-R150

[0099] 1. Acquisition of BRLF1 gene

[0100] (1) Design and synthesis of primers: The PCR primer sequences designed according to the known BRLF1 sequence (GenBank No. gi: 94734074) are:

[0101] F: 5'-CCGGAATTCATGAGGCCTAAAAAGGATGGCTT-3';

[0102] R: 5′-TGCTCTAGACTAAAATAAGCTGGTGTCAAAAATAG-3′

[0103] (2) RT-PCR amplification:

[0104] B95-8 cells (ATCC Number: CRL- 10624 TM ) into the lytic phase (Feng.P, Chan.SH, Soo.MY, etc. Antibody response to Epstein-Barr virus Rta protein in patients with nasopharyngeal carcinoma: a new serologic parameter for diagnosis. Cancer 2001 Oct 1; 92( 7): 1872-80.), using RT-PCR method to obtain the cDNA of BRLF1 gene, the specific method is as follows:

[0105] PCR reaction tubes, tips, and amplification water were routinely treated with DEPC.

[0106] Two-step RT-PCR kit from TaKaRa Company ( Item number in the catalog TAK_RR019A), using the total RNA of B95-8...

Embodiment 2

[0178] Example 2, Preparation and Purification of GST-R150 and GST-R185 Proteins

[0179] 1 Induced expression and identification of recombinant fusion protein

[0180] After the above pGEX-R185 and pGEX-R150 recombinant plasmids were transformed into Escherichia coli BL21(DE3) (commercially purchased Promega L1191), single clones were screened on the LB plate, and a small amount of single clones were selected and cultured overnight at 37°C at 150rpm overnight, at a ratio of 1:500 For expansion, 100mg / ml AMP was added at 1:1000, 37°C, 180rpm (desktop high-speed refrigerated centrifuge (Eppendorf Company)), and cultured for 4 hours (full temperature shaking incubator (Harbin Donglian Factory, HZQ-F)). 1mmol / L IPTG (TaKaRa Company) was added to the medium at a ratio of 1:1000, and induced for 4 hours. The bacterial cells before induction, 1h, 2h, 3h, and 4h after induction were centrifuged at 13,000rpm to obtain a precipitate, added to denatured Loading Buffer (commercially ava...

Embodiment 3

[0203] Embodiment 3 application ELISA method detects the antibody of Epstein-Barr virus BRLF1 gene expression antigen in serum

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Abstract

The present invention discloses two antigen protein fragments of protein product Rta of nasopharyngeal specific gene BRLF1, encoding gene, recombinant carrier, fusion protein (GST and GST-R185-R150), nasopharyngeal cancer diagnosis agent containing the compounds and the ELISA agent box, as well as the application of the compounds in detection of EB virus in vitro. The ELISA agent box of the present invention can be used for nasopharyngeal cancer screening, early diagnosis and forecasts of treatment results. The present invention has the advantages of simplicity, sensitivity and specificity, and is suitable for early diagnosis and forecasts of nasopharyngeal carcinoma, and the large-scale screening of high-risk groups. The present invention is easy to be widely promoted and used; the use is safe and clean; the present invention has broad market prospects.

Description

technical field [0001] The present invention relates to the field of medicine. Specifically, the present invention relates to the establishment of a genetically engineered strain containing the partial sequence of the immediate early gene BRLF1 of the Epstein-Barr virus cleavage and replication phase, the purification of the protein expressed by the two recombinant plasmids, the preparation and application of its monoclonal antibody. Background technique [0002] Epstein-Barr virus is a gamma herpes virus that infects approximately 95% of adults worldwide. Epstein-Barr virus infection can be divided into two states: latent infection period and cleavage replication period. After the initial infection, the virus can establish a lifelong latent infection in the host, and persistent EB virus cleavage and replication state infection can lead to a series of human malignant diseases. Tumors (Rickinson AB, Lee SP, Steven NM. Cytotoxic T lymphocyte responses to Epstein-Barr virus. C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/38C12N15/63G01N33/574
Inventor 胡怀忠
Owner 同昕生物技术(北京)有限公司
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