Screening And Identificating Interaction Proteins Of CX30

Objective:Deafness is a frequent disability disease.The proportion of congenital deafness is about 0.1%in newborn infants,in addition,about half of men appear handicap of hearing at the age of 65 years. Approximately,50%of cases of deafness are inherited,more than 70% have been described for non-syndromic deafness.Gap junction protein 30 is one of gap junction protein gene families.CX30 involved in non-syndromic dominant heredity deafness(DFNA3)and non-syndrornic autosomal recessive deafness(DFNB1).CX30 mutation resulted in dysplasia linguofacialis;hidrotic ectodermal dysplasia,keratitis-ichthyosis-deafness syndrome and atrichia congenital et al too. Cloning deafness genes and detecting genetic defect is the first step to know hereditary deafness.We must recognit the relationships of acoustic physiology and mechanism of pathopoiesis and deafness genes through function studies of deafness genes in order to carry out intervention and therapeutics of deafness.The protein interaction network had been described since 2000.The studies of interaction protein became the hot spot of milestone of life science.The protein interaction network provided a significant framework for proteome functions.Screening and studying interaction proteins is a good idea to research genic function and mechanism of pathopoiesis.Assembly,intracellular transport,plaque assembly and stability and channel conductivity of gap junction protein are regulated and mediated by interaction proteins.we can further recognit CX30 by studying interaction proteins of CX30.Screening and identificating interaction proteins of CX30 by GST-pull down is to study assembly,intracellular transport,plaque assembly and stability and channel conductivity of CX30.Methods:(1)Constructing CX30-GST prokaryotic expression vector Designing primer and inletting enzyme cutting site by primer3.The C-terminal functional domain(211-261)of CX30 gene was amplified from normal human genomic DNA by PCR.CX30(211₂61)was introduced into pGEX-4T-2 vector,then converted to JM109. Plasmid was extracted.CX30(211₂61)was proved to has no mutation by double enzyme cut and Sequencing.there was no reading frame displacement in the pGEX-4T-2.Thus CX30 (211-261)-GST was succeeded to construct.(2)Affinity purified protein CX30(211-261)-GST and pGEX-4T-2 were converted to BL21 which can express a high protein.CX30 (211-261)-GST fusion protein and GST were induced to small dose express by IPTG,then a great quantity were expressed.CX30 (211-261)-GST fusion protein and GST were Affinity purified by GS4B beads which can specially conjunct GST.Purification proteins were segregated and detected by SDS-PAGE(3)Pull down interaction proteins The foetus brain tissue and hela cell were disrupted,then to extract proteins.Extracted proteins were incubated with GST and GS4B bead to stick in advance.then Extracted proteins were incubated with CX30(211-261)-GST fusion protein and GST to pull down possible interaction proteins.The pull down compound proteins were segregated and detected by SDS-PAGE.Differeal straps was not found by comparing CX30 (211-261)-GST fusion protein pulling down compound proteins with GST pulling down compound proteins and CX30(211-261) -GST fusion protein.Differeal straps were cut to enzymolysis to prepare for Micro Q-TOF mass chromatographic analysis,then to index and screen interaction proteins in NCBInr database.(4)Immunolocalization identificated interaction proteins of CX30 The CDS coding region of CX30 gene was amplified from normal human genomic DNA by PCR.CX30-pCDNA3.1-Myc-His(-)B expression vector was constructed.CX30 was proved to has no mutation by double enzyme cut and Sequencing.there was no reading frame displacement in the pCDNA3.1-Myc-His(-)B.CX30-pCDNA3.1 -Myc-His(-)B was transient transfected hela cell.The transfected hela cells were double immunofluorescence stained.The expression and subcellular localization of proteins were observed by the fluorescence microscope.Immunolocalization was observed,Scannied and photographed by laser confocal microscopyResults:(1)CX30(211-261)-GST eukaryotic expression vector and CX30-pCDNA3.1-Myc-His(-)B prokaryotic expression vector were constructed.(2)A great quantity CX30(211-261)-GST fusion protein and GST were purified to fit the requirement of pull down.(3)4 interaction proteins of CX30 were screened in the foetus brain tissue,as follow,Keratin 16,Camk2b,Tubulin beta-3,alpha-tubulin。We did not find interaction proteins in hela cell.(4)CX30 was proved to coexist with Keratin 16 and Tubulin beta-3 by Immunolocalization.CX30 was initially identificated to interact with Keratin 16 and Tubulin beta-3.Conclusion:(1)GST-pull down is an effective methods to screen interaction proteins.Keratin 16,Camk2b,Tubulin beta-3 and alpha-tubulin were screened the interaction proteins of CX30 by GST-pull down. Keratin 16 and Tubulin beta-3 was initially identificated to interact withCX30.(2)keratin 16,Camk2b,Tubulin beta-3 chain and alpha-tubulin had an effect on CX30 in intracellular transport and mediating channel conductivity.(3)keratin 16 and Camk2b are two new interaction proteins of Gap junction protein...