Cloning And Preparation Of Monoclonal, Polyclonal Antibodies On Nuclear Respireatory Factor

Compared with normal cells, cancer cells display high rates of glucose uptake and glycolysis, but down-regulation of oxidative phosphorylation, that what we called cancer respiratory deficiency. A great number of cancer-related mitochondrial defects and mutations have been identified and described in the literature.That will be result in cancer energy metabolism disorder. The mechanisms responsible for the initiation and evolution of mtDNA defects and mutations, and their roles in the development of cancer, drug resistance, and disease progression are still unknown. The most well-known function of mitochondria is the production of adenosine triphosphate (ATP) through oxidative phosphorylation. This process is accomplished by a series of protein complexes, collectively known as the respiratory chain, encoded by both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). NRFs (nuclear respiratory factors, including NRF-1 and NRF-2) are key transcription factors involved in the regulation of mitochondrial biogenesis by activating directely the transcription of subunit genes of cytochrome c oxidase, ATP synthase and other nuclear-encode respiratory enzymes. NRF-1 is also a transcriptional activator of the nuclear genes encoding mitochondrial transcription factor mtTFA, it is necessary for mtDNA transcription and the MRP endonuclease involved in mtDNA replication. But, whether exists transcriptional regulative dysfunction of genes in OXPHOS is unknown. In our research, we cloned NRFs and mtTFA gene, constructed prokaryotic and eukaryotic vector of NRFs and produced NRF-1 monoclonal, polyclonal antibodies.1. NRF-1, NRF-2α, NRF-2βand mtTFA gene were cloned from the total RNA of SMMC7721 cells by RT-PCR, and their sequences were identical to the report in GenBank. The full length prokaryotic expression vector of NRF-1 were constructed and expressed in E.coli. The NRF-1 protein could be expressed well and then purified. The mice and rabbits were immunized with the purified protein respectively, and the monoclonal, polyclonal antibodies were obtained. The ELISA and Western blot results showed the antibodies could bind with the NRF-1 protein specifically.2. The eukaryotic expression vector pEGFP-C3-NRF-1 was constructed. The recombinant vector was transfected into Hela cells. NRF-1 was located in nucleus. To establish a NRF-1 knockdown cell line to investigate the function of NRF-1 in vitro, the double strand siRNA of NRF-1 corresponding to nucleotides 926 to 946 was synthesized and inserted into pSilencerTM3.1 H1 neo, hereafter referred to as pSilencer-siNRF-1. It was transfected into Hela cells to test its knock-down efficiency. The RT-PCR and Western blot results showed that pSilencer-siNRF-1 can effectively knockdown NRF-1 expression in Hela cells.In this study, the NRFs and mtTFA gene were cloned and expressed, and the NRF-1 monoclonal, polyclonal antibodies were produced. The eukaryotic expression vector pEGFP-C3-NRF-1 and RNA interference vector of NRF-1 were constructed. These results provid useful tools for the future study of NRFs.