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

Objective:Because of its high morbidity and mortality,cardiovascular disease has become the number one killer of endangering human health.It is currently a hot research to search for effective treatment of energy dysbolism of drugs.As an important subunit in ATP synthase,oligomycin sensitivity-conferring protein(OSCP)plays an important role in the cell energy metabolism.The protective effect of myocardial ischemic preconditioning is closely related to the increase of ATP synthase activity. The study uses the genetic engineering method to clone the gene OSCP in ATP synthase and to construct its eukaryotic expression vector,it lays the foundation for the gene in eukaryotic cells over-expression,the protein activity detection and the research on the role of OSCP in the cellular energy metabolism,myocardial ischemic preconditioning.Methods:1.The amplification of the rat OSCP gene:First about 50 mg brain tissue was taken from SD rat.According to the manual of the kit,the whole RNA was extracted. Then a couple primers for PCR was designed according to the known sequence of OSCP gene.The whole OSCP gene by reverse transcription PCR(RT-PCR)was amplified,finally the PCR product was isolated and purified.2.The construction of recombinant cloning vector pTA2-OSCP:The PCR product was ligated to the cloning vector pTA2,then the ligated product was transformed into E.coli JM109 competent bacteria.After amplifying the bacteria,the plasmid DNA was extracted and identified with EcoRâ… .The positive recombinant plasmid was screened,and the DNA sequencing was conducted,finally the result was analyzed by the software of Gene Runner.3.The construction of recombinant expression vector pEGFP-N₁-OSCP: Recombinant cloning vector pTA2-OSCP and the eukaryotic expression vector pEGFP-N₁ were both cut by Bglâ…¡/EcoRâ… and linked together by T₄ ligase.The linking product was transferred into E.coli DH5αcompetent bacteria.After amplifying bacteria,the plasmid DNA was extracted.The transformants were identified by restriction enzyme Bglâ…¡/EcoRâ… ,the positive recombinant plasmid was screened,and DNA sequencing was conducted,finally the result was analysed by the software of Gene Runner. Results:1.Amplification the OSCP by RT-PCR:OD_{260 nm}/OD_{280 nm}of toal RNA is 1.87, purity meets the requirements;the result of agarose gel electrophoresis of formaldehyde degeneration shows that the integrity of total RNA is good.The length of PCR product on the agarose gel is about 700 bp,and it is the same as the expected results.2.Identification the recombinant cloning vector pTA2-OSCP:The recombinant cloning vector pTA2-OSCP is identified by restriction enzyme EcoRâ… ,about 3 000 bp DNA linear fragment and about 700 bp DNA insert fragment are obtained,and the result is consistent with the theoretical prediction;DNA sequencing shows the DNA sequence of cloned is the same as the published rat OSCP gene sequence.The result proves that recombinant vector pTA2-OSCP is successfully constructed.3.Identification the recombinant eukaryotic expression vector pEGFP-N₁-OSCP: PEGFP-N₁-OSCP is cut by Bglâ…¡/EcoRâ… ,about 47000 bp DNA linear fragment and about 700 bp DNA insert fragment are obtained,and the result is consistent with the theoretical prediction.The positive recombinant expression vector is chosen to conduct DNA sequencing,and the sequence analysis reveals the homology is 100% to the published rat OSCP sequence.The result proves that pEGFP-N₁-OSCP vector is successfully constructed.Conclusion:1.The rat OSCP gene is amplified by RT-PCR.2.The recombinant cloning vector pTA2-OSCP is successfully constructed.3.The recombinant eukaryotic expression vector pEGFP-N1-OSCP is successfully constructed.