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Preparations of human rhinopharynx tissue specific gene C10RF102 coding protein, polyclonal antibody and monoclonal antibody thereof

A tissue-specific, protein-encoding technology, applied in the direction of anti-animal/human immunoglobulin, microbial-based methods, recombinant DNA technology, etc., can solve the lack of specificity in the diagnosis of nasopharyngeal carcinoma, and the lack of diagnostic indicators for nasopharyngeal carcinoma, etc. problems, to achieve the effects of high sensitivity, early detection, early treatment, and radical treatment

Inactive Publication Date: 2009-08-12
CENT SOUTH UNIV
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Problems solved by technology

For example, Epstein-Barr virus is not only related to the occurrence of nasopharyngeal carcinoma, but also closely related to Burkitt lymphoma and upper respiratory tract infection. Recently, it has also been reported that Epstein-Barr virus DNA was detected in various T-cell lymphomas, and not all nasopharyngeal carcinoma patients The incidence is all related to Epstein-Barr virus. More than 90% of the population has been infected with EBV before adulthood. The detection of EBV in the serum of NPC patients does not prove that EBV is directly related to NPC. Compared with pathological diagnosis of EBV antibodies in patients with nasopharyngeal carcinoma The positive coincidence rate is only 72.6% to 77.3%
Therefore, although the Epstein-Barr virus-related antibody titer has a high correlation with the occurrence and development of nasopharyngeal carcinoma, and its detection method is also superior, it can only be used as a related indicator to prompt and cannot be used as a nasopharyngeal carcinoma. cancer diagnostic markers
Some other tumor markers related to nasopharyngeal carcinoma, such as interleukin, tumor necrosis factor, intercellular adhesion molecule, and telomerase, are widely expressed in various tissues and have certain expression differences in different tumors. Application and diagnosis of nasopharyngeal carcinoma also lack specificity

Method used

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  • Preparations of human rhinopharynx tissue specific gene C10RF102 coding protein, polyclonal antibody and monoclonal antibody thereof
  • Preparations of human rhinopharynx tissue specific gene C10RF102 coding protein, polyclonal antibody and monoclonal antibody thereof
  • Preparations of human rhinopharynx tissue specific gene C10RF102 coding protein, polyclonal antibody and monoclonal antibody thereof

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Experimental program
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Embodiment 1

[0023]1) Prokaryotic expression of His-C1ORF102 fusion protein: ① Design specific primers according to the C1ORF102 gene reference sequence (GenebankAccession NM_145047), C1ORF102-forward: 5'-ATACATATGTCGGTGCGCACGTTACCG-3', C1ORF102-reverse: 5'-CTCGAGCTATAACTCATCCATCAT-3'. The 5' end of the primer sequence has an Nde I restriction site, and the 3' end primer has an Xho I restriction site. ② Using the human fetal brain cDNA library plasmid (product of Clontech) as a template, PCR amplified the coding region of the C1ORF102 gene, and recovered the PCR product band. Nde I and Xho I were used to digest the PCR product and the pET28b vector (commercialized expression vector from Novagen). The gel recovery kit recovered the linear 5.3kb vector and C1ORF102 gene coding region. ③The pET28b vector was connected with the C1ORF102 gene to construct a fusion gene in line with the reading frame. Escherichia coli JM109 was transformed with the pET28b-C1ORF102 plasmid containing the fusion...

Embodiment 2

[0026] Antibody preparation of protein encoded by C1ORF102:

[0027] Preparation of C1ORF102-encoded protein polyclonal antibody: Harvest and purify the fusion protein to immunize rabbits, the first dose is 400ug / kg, boost once on the 3rd day with a dose of 200ug / kg, and continue to boost once on the 28th day with a dose of 200ug / kg, one week Antiserum was obtained after blood collection. The antiserum was first precipitated hemoglobin and albumin with n-octanoic acid, centrifuged to remove the precipitate, and the supernatant was used at 50% saturation (NH4) 2 SO 4 The solution is precipitated, centrifuged, and the supernatant is removed, and the precipitate is the immunoglobulin IgG component, and the obtained is an anti-C1ORF102 polyclonal antibody. Affinity chromatography purification kit from Pierce Company was used for affinity chromatography, and the specific steps were strictly followed the instructions of Pierce Company to purify anti-C1ORF102 IgG, which was then pu...

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Abstract

The invention provides preparation of human nasopharyngeal tissue-specific gene C10RF102 coded protein and an antibody thereof. The invention adopts polymerase chain reaction to clone a protein-coding sequence of a C10RF102 gene, constructs fusion expression plasmid of the gene, introduces the fusion expression plasmid into RosettaBlue (DE3) escherichia coli, induces and purifies C10RF102 protein, and prepares a monoclonal antibody and a polyclonal antibody; and a C10RF102 gene coded product is specifically expressed in human nasopharyngeal tissue and is in down-regulated expression of nasopharyngeal carcinoma. The preparation of the C10RF102 gene coded product and the antibody thereof can provide a novel means for the gene diagnosis and treatment of the nasopharyngeal carcinoma, and can realize the early diagnosis, early detection and early treatment of the nasopharyngeal carcinoma.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to the acquisition of the expression product of human nasopharyngeal tissue-specific gene C1ORF102 and the preparation of antibodies. Background technique [0002] Nasopharyngeal carcinoma is one of the most common malignant tumors in southern China and Southeast Asia. The incidence rate in this region ranks first in the world, seriously threatening people's lives. At present, there are many methods for early detection of nasopharyngeal carcinoma, including the detection of Epstein-Barr virus serological markers, nasopharyngeal carcinoma-related tumor markers such as interleukin, tumor necrosis factor, intercellular adhesion molecule, and telomerase. Although these indicators alone or in combination provide useful information for the diagnosis of NPC, they lack sufficient sensitivity and specificity. For example, Epstein-Barr virus is not only related to the occurrence of nasophary...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P21/02C07K16/18C12P21/08C12R1/19
Inventor 李桂源向波王理李夏雨谭怡忻李小玲张文玲易梅刘玮
Owner CENT SOUTH UNIV
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