Myeloid Cell Leukemia-1 Mutatant Carriers Construction Research

Objective: This experiment adopts immune coprecipitation confirmed Mcl-1 proteins interacte with HSP90 proteins,but the specific binding sites is unknown.So the purpose of this research is to build the different domains lacking of Mcl-1 mutant carriers,so that providing the platform for subsequent researching which the specific binding sites.Methods: 1.By cell culture,protein extraction,immune coprecipitation and western blot to verify whether the Mcl – 1 protein interacte with Hsp90 protein.2.Search the Mcl-1's gene sequence in the NCBI gene pool,and according to the Mcl – 1's protein structure(including the CHT,BH1,BH2,BH3,PEST,five structure domains),using the Mcl – 1's CDs sequence and protein sequence and amino acid sequence which download from the NCBI gene pool,using Premier-Blast the design the different primers relating to the domains lacking of Mcl – 1's mutantions,then use the Standard Nucleotide Blast synthesis the primers and.And using the whole series Mcl-1 pc DNA3.1 Mcl-1 as a template to do inverse PCR which steps including restriction enzyme digestion,purposes DNA cyclization,PCR amplification to gained five structure domains lacking of Mcl-1 mutant carriers.3.Through those steps including agarose gel preparation,preparation of rubber sheet,add the sample,electrophoresis,to identify the PCR products which producted by reverse PCR.4.Using DNA agarose gel recovery kit to recycle reverse PCR amplification of the wild type Mcl-1 sequence,Mcl-1-? BH1 sequence,Mcl-1-? BH2 sequence,Mcl-1-? BH3 sequence,Mcl-1-? PEST sequence.Then do enzyme digestion to the purpose fragment DNA which recover by kit,and do enzyme digestion to the carriers and connect the purpose fragment DNA and carriers,and transformation,such as those steps to build different structure domains missing pc DNA3.1 /HA/Mcl-1 mutant carriers;5.Through steps such as plasmid extraction,double enzyme identification and sequencing analysising to whether the different domain lacking of Mcl-1 mutant carriers we build are successful.Results: 1.By the immune coprecipitation experiment we can find that Mcl-1 antibodies can precipitate Hsp90,Mcl-1 with Hsp90 antibodies can also precipitate?It means that Mcl-1 and Hsp90 exist interaction.2.According to the above methods and steps to design and synthesis the primers,we acquire the final primers sequence;3.By agarose gel electrophoresis for Mcl-1 the lack of structure domain mutant reverse PCR amplification results,we gain 6 line clear electrophoresis bands about 5 kb which consistent with expected DNA fragments size;4.Use of double enzyme appraisal examinate to the structure domain lacking of Mcl-1 mutant with HA eukaryotic expression plasmid,so that confirm that the lack of restructuring mutant carrier to be builded correctly;And do sequencing analysing to carriers for the construction of the lack of mutant,then compare the sequencing results to the results of NCBI,so that confirm five missing mutant genes encoding are correctly inserted into pc DNA3.1 / HA expression vector,and it means the different structure domain Mcl-1 missing mutant carrier building are successful.Conclusion: 1.Mcl-1 can interacte with Hsp90.2.Use the pc DNA3.1-Mcl-1 as the template,through reverse PCR amplification method to buiild five Mcl – 1 structural domains lacking mutants.2.3.Through the ecamination such as double enzyme appraisal examinate and sequence analysing to carriers for the construction domains lacking mutants,the result confirm five missing mutant genes encoding are correctly inserted into pc DNA3.1 / HA expression vector,and it means the different structure domain Mcl-1 missing mutant carrier are builded successfully.4.Through building five structure domain lacking mutants Mcl-1 HA eukaryotic expression vector,provide a platform for further experiment.