New use of high-expressed human portein in androgen dependent/non-dependent prostatic cancer for prostatic tumour diagnosis and therapeutic prognosis
A technology for prostate tumors and prostate cancer, applied in gene therapy, antineoplastic drugs, material inspection products, etc., can solve the problem of lack of high-throughput functional genomes
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Embodiment 1
[0080] Example 1: Study on expression profile of androgen-independent prostate tumor tissue using cDNA chip
[0081] Gene chip or gene microarray (DNA Microarray) is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, Then use fluorescence detection and computer software to compare and analyze the data, so as to achieve the purpose of rapid, efficient and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as target DNA in gene chip technology for high-throughput research on new gene functions; finding and screening tissue-specific new genes, especially new genes related to diseases such as tumors; disease diagnosis, such as genetic diseases . Its specific method steps have multiple reports in the literature, as can refer to the literature DeRi...
Embodiment 2
[0109] Example 2: Northern blot analysis of the expression of highly expressed human protein genes in androgen-independent prostate tumor tissue
[0110]Total RNA was extracted by a one-step method [Anal. Biochem 1987, 162, 156-159]. The method includes acid guanidine thiocyanate phenol-chloroform extraction. That is, use 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0) to homogenate the tissue, add 1 times the volume of phenol and 1 / 5 volume of chloroform-isoamyl alcohol (49:1 ), mixed and centrifuged. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. 20 µg of RNA was electrophoresed on a 1.2% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transfer to nitrocellulose membrane. with α- 32 P dATP prepared by ran...
Embodiment 3
[0112] Example 3: Production of anti-human protein antibodies highly expressed in androgen-independent prostate tumor tissue
[0113] Use a peptide synthesizer (product of PE company) to synthesize human protein-specific peptides that are highly expressed in androgen-independent prostate tumor tissue:
[0114] These polypeptides are respectively coupled with hemocyanin and bovine serum albumin to form a complex. For the method, see: Avrameas, et al. Immunochemistry, 1969; 6:43. Rabbits were immunized with 4 mg of the above-mentioned hemocyanin polypeptide complex plus complete Freund's adjuvant, and then boosted with the hemocyanin polypeptide complex plus incomplete Freund's adjuvant once 15 days later. The titer of antibody in rabbit serum was determined by ELISA using a titer plate coated with 15 μg / ml bovine serum albumin-polypeptide complex. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The peptide was bound to a Sepharose4B column...
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