Cloning Of Rat Oligomycin Sensitivity-conferring Protein And Construction Of Its Prokaryotic Expression Vector

Objective:Myocardial infarction(MI)has been one of the major cardiovascular diseases to harm the human healthy severely.In earlier period of MI,myocardial ischemic preconditioning(IPC)has profective effect on myocardium by improve the myocardial celluar energy metabolism.ATP synthase is the most important synthase in,and oligomycin sensitivity-conferring protein(OSCP)is the regulatory site of oligomycin in ATP synthase,and participates in the regulation of the energy metabolism directly.Accordingly,it can offer a new direction for study of the mechanism of preconditioning to protect the myocardium in the aspect of energy metabolism by study of the genetic transcription and expression regulation of the sensitive genetic locus of OSCP.Thus,in the experiments,the rat whole length of cDNA of OSCP gene was cloned and the prokaryotic expression vector was constructed in order to establish the basis for the OSCP expression,activity identification,antibody preparation.Methods:Amplication and clone of the OSCP gene were researched in the experiments,as follows:1.Extraction of total RNA from the tissues of rat brain:Total RNA from the tissues of fresh rat brain was obtained by one-step method.Its photodensity of the wavelength of 260 nm and 280 nm by ultraviolet spectrophotometer was measured, and the purity of total RNA by calculating A₂₆₀/A₂₈₀was determined;The concentration of the total RNA by A260 was calculated;The integrity of the total RNA by agarose gel electrophoresis of formaldehyde degeneration was identified.2.RT-PCR:According to the rat gene order of OSCP in GeneBank,the upstream primer and the downstream primer were designed by Primer 5.0 and Oligo 4.01 which are the current international software of molecular biology as follows:The upstream primer:5'-AGGATCCATGGCCGCACCAGCAAC-3'The downstream primer:5'-AGTCGACTCAGAGCAGATCCCGCATG-3'The recognition site GGATCC of restriction endonuclease BamH I in the upstream primer,and the recognition site GTCGAC of restriction endonuclease Sal I in the the dowanstream primer were introduced.The primers were synthetized by ShangHai Biology Engineering Company,and the primers were used in RT-PCR.The Oligo(dT)₁₅as the primers of the reverse transcription and the mRNA as the template were used,and the complementary single strand DNA was synthetized from mRNA by the reversed transcriptive enzyme.The OSCP was amplified by PCR with Pfu DNA polymerase to increase the fidelity of the OSCP.The amplification product was identified by 1.2%agarose gel electrophoresis.3.Construction and identification of the recombinant clone vector:The PCR product was recovered and purified,and the PCR product was ligated to the cloning vector pTA2.E.coli DH5αwas transformed with the ligated product,and the bacteria were amplified.The plasmid was extracted by alkaline lysis,and the positive recombinant plasmid was screened and identified by restriction endonuclease analyses of EcoR I and by PCR.Determination of DNA sequence and analysis of the result were performed by the software of Gene Runner and BLAST.4.Construction and identification of the prokaryotic expression vector:The recombinant plasmid pTA2 was cut by BamH I and Sal I.The purposed fragment OSCP was recovered and purified.The OSCP and the fragment of prokaryotic expression vector pQE30-Xa were ligated with T₄ DNA ligase.E.coli DH5αwas transformed with the ligation product,and the bacteria were amplified.The plasmid was extracted by alkaline lysis,and the positive recombinant plasmid was screened and identified by restriction endonuclease analyses of BamH I and Sal I and by PCR. Determination of DNA sequence and analysis of the result were performed by the software of Gene Runner and BLAST.Results:The total RNA,the RT-PCR product,the recombinant clone vector and the recombinant prokaryotic expression vector were all measured and identified,as follows:1.Measurement of the purity,the concentration and the integrity of total RNA:OD_{260 nm}/OD_{280 nm}of toal RNA was 1.87,purity was better;The concentration of total RNA was 2 989.8μg/ml;The result of agarose gel electrophoresis of formaldehyde degeneration showed that the integrity of total RNA was better.2.Amplification of the OSCP by RT-PCR:The RT-PCR product showed a specific limpid strap at 700 bp by 1.2%agarose gel electrophoresis,and the size of RT-PCR product was identical to the gene order of the published OSCP.3.Identification of the recombinant clone vector:The recombinant plasmid pTA2-OSCP was identified by restriction endonuclease analyses of EcoR I,and about 3 000 bp DNA linear fragment and about 700 bp DNA insert fragment were obtained. The result was consistent with the theoretical prediction.The recombinant plasmid pTA2-OSCP was identified by PCR,and about 700 bp PCR product was obtained. The result was consistent with the theoretical prediction.The positive recombinant plasmid was chosed to conduct DNA sequence and the result was analyzed by the software of Gene Runner and BLAST.Thus,the sequence analysis revealed that the homology was 100%to the published data in GeneBank.4.Identification of the recombinant prokaryotic expression vector:The recombinant plasmid pQE30-Xa-OSCP was identified by restriction endonuclease analyses of BamH I and Sal I,and about 3 500 bp DNA linear fragment and about 700 bp DNA insert fragment were obtained.The result was consistent with the theoretical prediction.The recombinant plasmid pQE30-Xa-OSCP was identified by PCR,and about 700 bp PCR product was obtained.The result was consistent with the theoretical prediction.The positive recombinant plasmid was chosed to conduct DNA sequence and the result was analyzed by the software of Gene Runner and BLAST. Thus,the sequence analysis revealed that the homology was 100%to the published data in GeneBank.Conclusions:The rat oligomycin sensitivity-conferring protein cDNA is cloned, and the recombinant cloning vector pTA2-OSCP and the recombinant prokaryotic expression vector pQE30-Xa-OSCP are constructed.