Construction And Expression Of The R127W Mutant Type HSPB1 Eukaryotic Expression Vector And Studying Of Interaction Between R127W Mutant Type HSPB1 And NFL

Backgroud:Charcot-Marie-Toothdisease(CMT), also named hereditary motor and sensory neuropathy(HMSN), is the most common hereditary peripheral neuropathy. The incidence of CMT is 40/10,0000. CMT is characterized by progressive and symmetric distal muscle atrophy, hyporeflexia and hypoesthesia in distal limbs. According to the electropsiological and histopathological criteria, CMT can be divided into two forms:the demyelinating form(CMT1) and the axonal form(CMT2). There is much research of molecular inheritance about CMT. In 2004, Evgrafov OV found S135F and R127W mutation in HSPBl gene respectively in a large Russian and a large Beigian CMT2F family, and confirm HSPBl as the disease causative gene of CMT2F. In 2005, Tang et al found R127W mutation in HSPBl gene in HSPB1 in four large Chinese CMT2F families. HSPB1 belong to sHSPs family. Recent studies revealed that HSPB1 is a molecular chaperone which can stabilize eytoskeletons and assist their correct assembly. NFL, as a important structure that can maintain cytoskeleton, is a main intermediate flament. Recent studies showed that SHSP and NFL form aggregates by itself after mutanting of SHSP and NFL. Evgrafov OV found there was interaction between the wild type NFL and S135F mutant HSPB1, But there were no reports concerning with the formation and distribution of polymer with R127W mutant HSPB1, nor with the interaction between NFL and R127W mutant HSPB1.Objective:Studing the formation and distribution of aggregates with wild type HSPB1 and R127W mutant HSPB1 and the interaction between NFL and R127W mutant HSPB1. This study should set up a foundation for researches on pathogenesis of CMT2F at last.Methods:In the studies, firstly we amplificated the cDNA of HSPB1 from the library of Hela cell by recombinant DNA technology and constructed 379C-T (R127W)mutation of HSPB1 by Overlap method. Secondly we constructed wild type HSPB1 with pEGFPN1 and R127W mutant type HSPB1 with pEGFPN1 eukaryotic expression vectors. At last we transfected wild type HSPB1/R127W mutant HSPB1 eukaryotic expression vectors into Hela cell. Furthermore, we co-transfected R127W mutant HSPB1 and wild type NFL eukaryotic expression vector into Hela cell. By laser scanning confocal microscope, we observe that the formation and distribution of wild type HSPB1 with pEGFPN1 and R127W mutant HSPB1 with pEGFPN1, and study whether there is interaction between R127W mutant HSPB1 with pEGFPN1 and wild type NFL by using indirect immunofluorescence technique. Meanwhile we observe R127W mutant HSPB1 co-locate with wild type NFL by using irnmunofluorescence technique, which showed that there is interaction between R127W mutant HSPB1 and wild type NFL indirectly.Results:By laser scanning confocal microscope, we found that wild type HSPB1 with pEGFPN1 distributed diffusively in the cytosol and nucleus in Hela cells; R127W mutant HSPB1 with pEGFPN1 form plaque aggregates which located around the nucleus predominantly.Conclusion:The molecular chaperone function could be affected for R127W mutant HSPB1 which result to protein floding and aggregating. R127W mutant HSPB1 co-locate with wild type NFL by using immunofluorescence technique, which showed the interaction between R127W mutant HSPB1 and wild type NFL indirectly. So we speculated that R127W mutant HSPB1 interact with construction and stability of neural network and the neuron.