Mitochondrial membrane potential decrease related polynucleotide as well as coded polypeptide and uses thereof
A technology of mitochondrial membrane potential and polynucleotide, applied in gene expression regulation, biological field
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Embodiment 1
[0072] Embodiment 1, two-step method flux quantification RT-PCR technique amplifies target gene
[0073](1) The refseq database of NCBI was used to retrieve the predicted genes with unknown human functions to obtain the sequence of human unknown functional genes, and use the Human_est database to perform sequence correction through the BLASTn method, and the final sequence was set as the following sequence: SEQID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7. Design specific primers for genes MMPRG1, 2, 3, 4 according to such sequences:
[0074]
[0075] (2) Using the above primers, select a template from the existing cDNA library and tumor library according to the expression profile of the target gene, and perform initial amplification. The existing library includes 12 kinds of human normal tissues (heart, pancreas, testis, ovary, prostate, colon, small intestine, skeletal muscle, thymus, lymph node, tonsil, white blood cells); 6 kinds of human tumor tissues (lung cancer, ...
Embodiment 2
[0087] Embodiment 2, the construction of target gene eukaryotic expression vector
[0088] The purified product of secondary amplification and the eukaryotic expression vector pcDNA3.1 / V5-His-TOPO (Invitrogen, abbreviated as pcDT) were ligated according to the conditions suggested by the kit manufacturer. The ligation product was transformed into Escherichia coli DH5α by electric shock method, and the transformant was grown on a solid LB plate medium containing ampicillin, and the grown single clone colony was selected, the plasmid was extracted, digested with EcoRI, and the digested product was identified by agarose gel electrophoresis. Positive clones with inserts were selected, and the correct positive insert clones were selected by sequencing (ABI PRISM 3700 DNA analyzer), which were named MMPRG1, 2, 3, and 4 respectively.
[0089] At the same time, the culture solution was collected, and the precipitated protein was analyzed by SDS-PAGE to obtain MMPRG1, 2, 3, and 4 polyp...
Embodiment 3
[0091] Example 3. The use of JC-1 dye to determine the effect of the target gene on the reduction of mitochondrial membrane potential
[0092] After transfecting Hela cells with the target gene, stain the cells with JC-1 dye, operate as described in the product manual, and observe the changes in cell morphology under a fluorescent microscope. The specific operation steps are as follows:
[0093] In this experiment, a 96-well cell culture plate was used. Three parallel wells were set up for each gene to be tested, and pcDT empty plasmid and Bax plasmid were used as empty vector and positive control respectively. The amount used in the following steps is the amount used for a single hole.
[0094] (1) Cell culture: 1.2×10 4 One Hela cell (ATCC Number: CRL-11268) was plated on a 96-well cell culture plate (Costar, 3599) with DMEM (Dulbecco's modified Eagle's medium) medium (Hyclone, SH0022.02) containing 10% fetal bovine serum (100 μl medium / well) at 37°C, 5% CO 2 Cells in th...
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