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Neural protein NSPc1 having influence on nerve tumor cell differentiation by tretinoin signal pathway

A nervous system and protein technology, applied in anti-tumor drugs, serum immunoglobulins, genetic material components, etc., can solve problems such as unclear possible role of NSPc1 and limitations of NSPc1 application

Inactive Publication Date: 2007-04-25
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the absence of an in-depth understanding of the regulatory mechanism of NSPc1 target genes in vivo, the possible role of NSPc1 in tumorigenesis remains unclear.
Before these details are studied clearly, the application of NSPc1 in molecular typing diagnosis and the next step of gene therapy will be limited

Method used

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  • Neural protein NSPc1 having influence on nerve tumor cell differentiation by tretinoin signal pathway
  • Neural protein NSPc1 having influence on nerve tumor cell differentiation by tretinoin signal pathway
  • Neural protein NSPc1 having influence on nerve tumor cell differentiation by tretinoin signal pathway

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Embodiment 1

[0037] Primer sequences and purposes used in the present invention:

[0038] Primer name

Embodiment 2

[0040]Cloning of the NSPc1 gene:

[0041] HeLa cells were cultured on a 10cm dish in DMEM containing 10% FBS at 37 degrees, 5% CO 2 Cultured until the number of cells reached 2×10 7 Above, cells were harvested to extract total RNA and subjected to reverse transcription. Reverse transcription was carried out using a kit from PROMEGA, and the method is shown in the kit instructions. Using this cDNA as a template, the NSPc1 gene was amplified. The primers used were N-up / dn (see Example 1 for the sequence). The conditions of the PCR reaction are: NSPc1, the annealing temperature is 62 degrees, and the extension is 1.5 minutes. The other conditions are the same as the ordinary PCR conditions, and Pfu enzyme is used.

[0042] After the PCR product was tailed with Taq enzyme at 72 degrees for 10 minutes, it was directly connected to the pGEM-T vector (Promega company product). The connection reaction was carried out according to the kit instructions, and carried out at 4 overnigh...

Embodiment 3

[0044] Preparation of anti-NSPc1 specific antibody:

[0045] Antigen preparation: the full-length fragment was amplified by PCR from the cDNA template of NSPc1, and the primer was N-43.1-up / dn (see Example 1 for the sequence). Both ends of the PCR fragment contain restriction sites BamH1 and XhoI, so these two enzymes were used to digest the PCR product and vector plasmid pET43.1. Then, the digested PCR fragment and the vector were ligated with T4 ligase to form a recombinant plasmid (see FIG. 1 ).

[0046] PCR conditions: the annealing temperature is 63 degrees, the extension time is 1 minute, and other conditions are the same as ordinary PCR reactions. PCR reaction product 50ul (about 5ug) and vector plasmid (pET43.1)6 - 10ug were digested with BamH1 and XhoI respectively, and the DNA fragments were recovered by agarose electrophoresis, and ligated in a 10ul system, which contained 1u1 10x ligation buffer, 1ul T4 ligase, 100ng digested PCR fragments, 200ng digested vector ...

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Abstract

It is found out in the invention that the poly-comb protein NSPc1 of the neural system can highly express in the tumor cell lines from a variety source of neural system using the Western blot technique, it is also found out that the expression level of NSPc1 in human gliocytoma sample is far higher than that in the normal tissue around the cancer and it goes up as the grading increase of the neurogliocytoma pathology using the Real-time PCR technique. NSPc1 is found to have transcription inhibition capacity using the fluorescence detection of reporting gene technology, and p21Waf1 / Cip1 is found to be the target controlled gene of NSPc1. Promoter sequence mutation experiments and chromatin co-immunoprecipitation experiments of P21Waf1 / Cip1 showed that the inhibition effect of NSPc1 on the transcription of p21Waf1 / Cip1 depends on retinoic acid response elements (RARE). Overexpressions of retinoic acid receptor RXRalphacan eliminate the inhibition activity of NSPc1 on p21Waf1 / Cip1 promoter. These results indicate that NSPc1 highly expressing in nerve tumor inhibits the transcription of p21Waf1 / Cip1 through interfering the controlling access of the retinoic acid thus affects the differentiation of the nervous system tumor. Therefore, not only can NSPc1 be used as candidate markers for grouping diagnostic of nervous system malignant tumor type, but also can serve as a target of gene therapy. Meanwhile, in the invention, building the plasmid for prokaryotic expressing of full-length protein of NSPc1, transforming Escherichia coli to express the corresponding protein; After purifying the protein it can be used for the preparation of high-quality antibodies, as well as applied to the nervous system tumor diagnosis.

Description

technical field [0001] The invention belongs to the field of gene function, antibody preparation and clinical diagnosis. Specifically, the present invention relates to a full-length recombinant genetic engineering and protein purification process of a neural tumor cell differentiation-related gene NSPc1, involves the use of purified protein to prepare highly specific NSPc1 antibodies, and involves the use of Western Blot technology and Real-time PCR technology to detect The expression level of NSPc1 in tumor cell lines derived from nervous system and human glioblastoma specimens, involving the confirmation of NSPc1 downstream target gene p21Waf1 / Cip and the discovery of NSPc1 regulatory sequence retinoic acid receptor response element (RARE) and related regulation Confirmation of the mechanism, involving recombinant plasmids containing the NSPc1 gene and recombinant strains expressing the protein and their application in the diagnosis of glioblastoma, involving the NSPc1 prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/74C12N15/70C07K16/06C12Q1/68A61K48/00A61P35/00
Inventor 龚燕华岳继平吴旭东袁建刚彭小忠强伯勤
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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