1.Construction And Identification Of Two Spider Peptide Toxin Bait Vectors 2.Construction Chimera Of Voltage-gated Sodium Channel And Detection Biological Activity

Yeast two-hybrid system is a direct method detecting protein interactions in eukaryotic cells with high sensitivity and specificity. It has been continuously refined and improved,also has been widely used in cell biology, oncology, proteomics in many oher fields, since 1989 which has been established by Fields and so on. Based on this, we intended to use the GAL4 yeast two-hybrid system 3 to build pGBKT7-HWTX-Ⅳ, pGBKT7-JZTX-Ⅲin order to find interact protein cDNA library from human cardiac cells in the fish can with sobtained gene. We constructed spider toxin bait vector pGBKT7-HWTX-Ⅳ, pGBKT7-JZTX-Ⅲ.Therefore, we constructed bait protein expression vector pGBKT7-HWTX-IV and pGBKT7-JZTX-Ⅲtransformed into AH 109 yeast.We find:the sequence is correct and fusion protein can not activate reporter gene transcription, without toxin effect on yeast cell growth. So bait vectors can be used to screen cDNA library, can be further used in yeast two-hybrid system to detect interaction protein of two spider toxins,for more applications and functions and interaction mechanism。 This family of voltage-gated sodium channel includes at least 9 different channels and by the emergence and spread around the control of the action potential. Nine different channels both include a large a subunit and auxiliaryβsubunits. A subunit is very large and contains four homologus domains (Ⅰ-Ⅳ), each domain has six transmembrane (S1-S6),with highly homology and similar structure. In this study,we try to recombinant a new sodium channel which four domains of a-subunit are from two different sodium channel subtypes.In oder to explore changes of the electrophysiological characteristics of the new sodium channel,we express recombinant sodium channel protein in HEK 293,which is a more direct method in vitro. Patch clamp technology detect analysis the difference with.he normal voltage-gated sodium channel in electrophysiological characteristics. Futher details about structure and function of the ion channel can be found.This process is divided into two steps:1 correctly construct recombinant Na+channel vector, which can express recombinant sodium channel protein.2 the whole cell electrophysiological characteristics of abnormal cell function in vitro expression (transient expression or stable expression),which is the more reasonable, and mammalian cell expression system,closer to human physiological conditions, which is currently functional research method.After successfully constructing expression vector, the expression of transfected cells and whole cell patch clamp analysis the sodium channel protein. We used human embryonic kidney epithelial 293 cells as transfected cell lines, transfection using liposomes as a medium, were transiently transfected wild-type and mutant two plasmids,3-7 days after transfection, whole cell patch-clamp technique, transient expression of the sodium channel protein was detected and analysis.The electrophysiological characteristics of the normal wild-type is detected but recombinant voltage-gated Na+channels has no electric current signal,We speculat that their action potential and possible mechanism has been changed.