摘要
第一部分两种蜘蛛多肽毒素酵母双杂交诱饵载体的构建及初步鉴定
酵母双杂交系统是一种直接在真核细胞内检测蛋白相互作用的方法,具有灵敏度高和特异性好的优点。自1989年由Fields等提出并初步建立以来,得到了不断的完善和改进,在细胞生物学,肿瘤学,蛋白质组学等诸多领域有着广泛的应用。基于此,我们拟用酵母双杂交GAL4系统,研究与虎纹捕鸟蛛多肽毒素-4(huwen toxinⅣ,HWTX-Ⅳ)和敬钊蜘蛛毒素-3(Jingzhao toxinⅢ, JZTX-Ⅲ)相互作用的蛋白质。故构建pGBKT7-HWTX-Ⅳ,pGBKT7-JZTX-Ⅲ融合蛋白即诱饵蛋白,以期从人心肌细胞cDNA文库中钓取可与之相互作用的蛋白的基因。我们从质粒PMD18-T-HWTX-Ⅳ, PMD18-T-JZTX-Ⅲ通过PCR获得目的基因,利用引物中的酶切位点,将目的片段酶切后连接于同一组酶切的载体pGBKT7上。从而得到蜘蛛毒素的酵母双杂交诱饵表达载体pGBKT7-HWTX-Ⅳ和pGBKT7-JZTX-Ⅲ,经测序鉴定全部正确。将所构载体与空载体pADKT7一起转入AH109酵母感受态细胞。经平板观察菌落以及X-α-gal滤纸法显色结果说明诱饵融合蛋白不能激活报告基因的转录,对酵母细胞的生长无毒性作用。同时针对实验中遇到的问题,总结了几点酵母双杂交实验上的经验,包括：1)酵母菌株,诱饵载体,滤纸显色法检测报告基因LacZ表达,2)酵母菌株的培养,转化效率问题,感受态制备,快速转化酵母等。正确构建的pGBKT7-HWTX-Ⅳ与pGBKT7-JZTX-Ⅲ可以用于筛选cDNA文库,可以进一步采用酵母双杂交筛选与这两种蜘蛛毒素的相互作用蛋白,为毒素的应用和功能及作用机制奠定了基础。
第二部分电压门控钠离子通道嵌合体的构建以及生理活性的检测
哺乳动物中发现的9种电压门控型钠离子通道,起主要功能作用的α亚基都是有4个结构域组成,而且每个结构域都有六个跨膜片段。在具有高度的结构同源性的同时,与河豚毒素作用表现出截然不同的生理活性,因此是研究离子通道蛋白结构与功能关系的理想模型。
在研究中,我们首次尝试将四种电压门控型钠离子通道的不同结构域通过基因工程的方法进行改造、构建异源嵌合体。新的离子通道嵌合体的四个结构域分别由自来源两个不同的离子通道互换后组成。为探索钠通道嵌合体电生理特征以及改变,采用一种较为直接的方法就是在体外细胞中,使重组钠通道蛋白表达,并应用膜片钳技术进行检测,分析钠通道嵌合体的电生理特性与正常钠通道的差异,以研究离子通道结构与功能的关系。
这一过程分为两步：1,要正确构建携带嵌合体钠通道cDNA的重组表达载体。2,质粒转染细胞并表达出钠通道蛋白及全细胞膜片钳分析。成功构建了15个离子通道嵌合体基因表达载体测全序列,序列完全正确。我们开始进一步进行功能表达研究。我们采用人胚肾上皮293细胞(HEK293)作为质粒转染的细胞系,使用脂质体作为转染媒介,分别瞬时转染含有野生型离子通道和所构嵌合体两种质粒,转染后的3-7日内,应用全细胞膜片钳技术,对瞬时表达的钠通道蛋白进行检测、分析,以期比较正常野生型与嵌合体的电压门控Na+通道在电生理特征上有何差异。嵌合体的已转染的HEK239都没有检测到电流信号,而作为对照组的野生型的nav1.5的Ⅰ-Ⅴ曲线和电流稳态失活曲线都正常。
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.
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