Selenium Cysteine Decoding Related Factors Rpl30, Sbp2 And Efsec Role In The Expression Of Selenoprotein P Efficient Study

Objective: By cloning three trans-acting factor RPL30、SBP2and EFsec genes,constructing their mammalian expression vectors, and transfecting them into HEK293cellswith plasmid pcDNA3.1-iGFP-Sepp1, to study the transcription of Sepp1, lay foundation forover expression of sepp1in mammalian cells.Methods: The coding sequence of RPL30gene was amplified by RT-PCR from humanMCF-7cells, then cloned into pcDNA3.1(+) vector by Double Digests; The missingfragment of EFsec gene was synthesized and connected to the5’ end of EFsec by PartialSynthesis-Restrict Enzyme Ligation Method, then amplified by PCR and cloned intopcDNA3.1(-) vector; Eukaryotic expression plasmid pCMV-XL4-SBP2was bought fromOriGene biology company; The expression plasmids were transfected into HEK293cellswith pcDNA3.1-iGFP-Sepp1in different groups by LipofectamineTM2000, then detected thelevel of their mRNA.Results: Successfully cloned the coding sequence of RPL30gene and constructedpcDNA3.1-RPL30； Filled the missing sequence of EFsec gene next to5’ end accuratelyand effectively, built expression plasmid pcDNA3.1-EFsec; RT-PCR and analysis of meanoptical density of electrophoresis band showed that: comparing with the normal group andcontrol group, the mRNA level of transfecting recombinant plasmid group increasedobviously, indicating that the recombinant plasmids were transfected into HEK293cellssuccessfully, and expressed effectively.Conclusion: Construct human cell models HEK293/RPL30(SBP2or EFsec) whichcan expess RPL30(SBP2or EFsec) efficiently.