【作者】 孟尔；

【导师】 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 虎纹捕鸟蛛(Haplopelma schmidti)、敬钊缨毛蛛(Chilobrachys guangxiensis)、海南捕鸟蛛(Haplopelma hainanum)都分布于我国南方地区,属于体型较大、毒性较强且稀有的捕鸟蛛。在本实验室以前的工作中,分别从它们的粗毒中分离到了虎纹毒素-Ⅰ(HWTX-Ⅰ)、虎纹毒素-Ⅳ(HWTX-Ⅳ)、海南毒素-Ⅳ(HNTX-Ⅳ)、敬钊毒素-Ⅲ(JZTX-Ⅲ),并通过电生理实验了解这四种毒素对于电压敏感钠通道(VGSCs)具有不同的选择性及作用强度,但是具体相互作用机制仍然不清楚。本文试图利用酵母双杂交技术来部分解决这个问题。将编码毒素成熟肽的基因序列连入pGBKT7(酵母双杂交载体),将编码电压敏感钠通道膜外区的基因序列连入另一载体pGADT7。通过酵母双杂交的手段,分别检测到了以上四种毒素对hNav1.7、hNav1.5通道膜外区潜在的相互作用位点。HWTX-IV和hNav1.7的潜在的作用位点仅为通道结构域Ⅲ的S3-S4胞外连接片段(DⅢ-S3-S4)。HWTX-Ⅰ与hNav1.7的潜在的作用位点为DⅠ-S3-S4、DⅢ-S1-S2、DⅢ-S5-S6、DIV-S5-S6。HNTX-Ⅳ和hNav1.7的潜在的作用位点是DⅠ-S3-S4、DⅡ-S 1-S2、DⅢ-S 1-S2、DⅢ-S5-S6。JZTX-Ⅲ和hNav1.7的潜在的作用位点是DⅠ-S3-S4、DⅠ-S5-S6、DⅡ-S1-S2、DⅢ-S5-S6、DⅣ-S1-S2、DIV-S5-S6。进一步分析,发现JZTX-Ⅲ与hNav1.5的潜在的作用位点是DⅠ-S 1-S2、DⅡ-S3-S4、DⅢ-S3-S4。在本实验室以前的工作中,已经在膜片钳上检测了天然HWTX-Ⅳ对hNav1.7通道电流的抑制作用,其半数抑制量(IC50)为26 nM; JZTX-Ⅲ对hNav1.5有抑制作用,其IC50大约在300 nM附近(未发表数据)。本文中我们又在膜片钳上检测了天然的HWTX-Ⅰ, HNTX-Ⅳ, JZTX-Ⅲ对于hNav1.7通道电流的抑制能力。其中毒素HWTX-Ⅰ, HNTX-Ⅳ对于hNav1.7通道电流的IC50分别为640 nM和22nM。JZTX-Ⅲ在1μM浓度时,对hNav1.7通道电流没有明显抑制能力。这四个毒素对hNav1.7通道电流的抑制能力依次是：HNTX-Ⅳ-HWTX-Ⅳ>HWTX-Ⅰ>>JZTX-Ⅲ。通过综合分析酵母显色和电生理的结果我们可以得出：1)HNTX-Ⅳ和HWTX-Ⅳ对hNav1.7通道的S5-S6这些膜外区中的大部分并没有相互作用；2)虽然电生理的结果,HNTX-Ⅳ和HWTX-Ⅳ对hNav1.7电流的抑制能力十分接近,但是通过分析酵母相互作用的结果,两者的作用位点却差异明显。其中HWTX-Ⅳ能够检测到的作用位点仅有一个,而HNTX-Ⅳ有4处；3)通过分析酵母显色结果,我们发现通道hNav1.5与hNav1.7之间,很可能是由于DⅡ-S3-S4和DⅢ-S3-S4这两部分膜外区的差别,导致对JZTX-Ⅲ敏感性的不一样。虽然VGSCs在S1-S2、S3-S4膜外区的氨基酸在10个附近,但是在我们参看相关文献后,预测其核心序列在6个氨基酸左右,我们直接在酵母双杂交载体pGADT7的多克隆位点上插入编码随机的六个氨基酸的序列,从而组成一个随机六肽文库。我们通过2种方式构建了能够用于酵母双杂交的随机六肽文库。第一种方法是常规的,需要依赖酶切和连接的方式；而第二种方法是直接通过PCR来完成文库质粒构建的全新方法。两种方法均能得到库容量在10万左右的高质量随机六肽文库。随后对两种方法所得的文库,随机挑选20个克隆进行测序。测序结果表明：两个文库都有70%的序列是符合设计初衷的编码随机六肽的序列,并且没有一个重复序列,代表两个文库里面的复杂度良好。本文的另一个工作是建立了一种表达小分子生物活性多肽的方法。我们分别将编码虎纹毒素-Ⅰ(HWTX-Ⅰ),海南毒素-Ⅳ(HNTX-Ⅳ),敬钊毒素-Ⅴ(JZTX-Ⅴ)的序列通过聚合酶链式反应(PCR)手段获得,然后分别连接入表达载体pET-40b,接着转入表达菌株BL21(DE3),最后以乳糖为诱导物,来实现目的蛋白在大肠杆菌周质空间的融合表达。经过SDS-PAGE检测,均能检测到三种毒素以融合蛋白的形式表达。进一步将部分可溶的虎纹毒素-Ⅰ融合蛋白(带组氨酸标签)用镍柱纯化。纯化后的产物通过透析、除盐后,再加入肠激酶消化来释放重组的虎纹毒素-Ⅰ(rHWTX-Ⅰ),接着通过反相高效液相色谱法(RP-HPLC)来纯化rHWTX-Ⅰ。经质谱检测,rHWTX-Ⅰ的分子量和天然HWTX-Ⅰ一样,都为3750.69,表明rHWTX-Ⅰ已经形成了3对二硫键。随后通过全细胞膜片钳技术来检测rHWTX-Ⅰ的生理活性。rHWTX-Ⅰ抑制hNav1.7电流的IC50为640 nM,几乎和天然的一致(天然的HWTX-Ⅰ的IC50为630 nM)。更多还原

【Abstract】 The spiders(Haplopelma schmidti, Chilobrachys guangxiensis, and Haplopelma hainanum) distributing in the south of China are the relatively big, venomous, and rare bird spiders. In our previous studies, huwentoxin-I (HWTX-I), huwentoxin-IV (HWTX-IV), hainantoxin-IV (HNTX-IV), and jingzhaotoxin-III (JZTX-III) have been demonstrated that they have different selectivity and inhibiting ability on voltage-gated sodium channels (VGSCs) using whole-cell patch clamp assays. However, the interaction mechanism between spider toxins and VGSCs is still unclear. Here, we tried to solve it partially using yeast two hybird assay. The coding sequences of mature peptides of toxins were inserted to the yeast two hybird vector pGBKT7, and the coding sequences of outer-membrane segments of VGSCs were ligated to another vector pGADT7. Some potential interaction sites between these toxins and VGSCs (hNav1.5 and hNav1.7) were detected using yeast two hybird assays. The potential interaction site between HWTX-IV and hNav1.7 is only at the extracellular S3-S4 linker of domain III (DIII-S3-S4). The potential interaction sites between HWTX-I and hNav1.7 are at DI-S3-S4, DIII-S1-S2, DIII-S5-S6, and DIV-S5-S6. The potential interaction sites between HNTX-IV and hNav1.7 are at DI-S3-S4, DII-S1-S2, DIII-S1-S2, and DIII-S5-S6. The potential interaction sites between JZTX-III and hNav1.7 are at DI-S3-S4, DI-S5-S6, DII-S1-S2, DIII-S5-S6, DIV-S1-S2, and DIV-S5-S6. The potential interaction sites between JZTX-III and hNav1.5 are at DⅠ-S1-S2, DⅡ-S3-S4, and DⅢ-S3-S4.Previous studies in our laboratory have demonstrated that IC50 value of HWTX-IV is 26 nM for wild type (WT) hNav1.7. The IC50 value of JZTX-III is approximately 300 nM for WT hNav1.5 (unpublished datas). The inhibiting abilities of HWTX-I, HNTX-IV, and JZTX-III on the WT hNav1.7 were detected using electrophsiolgy assay. It was demonstrated that the IC50 value of HWTX-I and HNTX-IV were 640 nM and 22 nM for WT hNav1.7 channel, respectively. The hNav1.7 currents almost could not be inhibited after exposure to 1μM JZTX-III. The order of inhibition ability of toxins to hNav1.7 channel is HNTX-Ⅳ≈HWTX-Ⅳ>HWTX-Ⅰ>>JZTX-Ⅲ.According to the results of the electrophysiology and yeast two hybrid assays, key points can be concluded as follows:1) There is almost no interaction site between the outer-membrane S5-S6 segments of hNav1.7 and HNTX-IV or HWTX-Ⅳ, respectively.2) It is indicated that the inhibition ability of HNTX-Ⅳare identical to that of HWTX-IV for the hNav1.7 channel using electrophsiolgy assay, but the yeast two hybird results demonstrate that there are significant differences between them. The potential interaction site between HWTX-IV and hNav1.7 is only one, but HWTX-IV has four potential interaction sites to hNav1.7.3) The differences of segments of DII-S3-S4 and DIII-S3-S4 between hNav1.5 and hNav1.7 might result in the different sensitivity to JZTX-III. Outer-membrane S1-S2 and S3-S4 segments of VGSCs are almost no more than ten amino acids. We predicted that the core motif of these short out-membrane segments was about six amino acids by the analysis of related papers. The random-6-peptide library were constructed by inserting the coding sequences of the random-6-peptide into the expression vector-pGADT7, so the library could be used for the yeast two hybrid experiments. There were two different ways to build the random-6-peptide library. The first one was the regular method using the restriction endonuclease and T4 DNA ligase as some paper mentioned. But the second way was a novel method to build the library using the site-directed insertion mutagenesis. Two random-6-peptide libraries were constructed by two different ways mentioned above. Independent clones of each random-6-peptide library was over 100,000. Twenty clones were sequenced randomly from the two libraries mentioned above, respectively. The results of sequencing demonstrated that seventy percent sequences could be used for library screening, and there was no repetitive sequence, respectively. It was indicated that the libraries were of high quality.An efficiency method to express the small molecular bioactive peptides was developed. The coding sequences of HWTX-I, HNTX-Ⅳ, and JZTX-V were amplified by PCR. The cloned fragment was inserted into the expression vector pET-40b, and then the constructor was transformed into E.coli strain BL21(DE3), respectively. The expression of the soluble fusion protein was auto-induced into the periplasm of E. coli by lactose in the absence of IPTG. It was demonstrated that the three kinds of fusion proteins were expressed by the analysis of SDS-PAGE assay. After a partial purification using a Ni-NTA column and dialysis, the expressed fusion protein was digested using the enterokinase to release heteroexpressed HWTX-Ⅰ(rHWTX-Ⅰ) and further purified using RP-HPLC. The molecular weight of the rHWTX-I was 3750.69 using TOF/TOF MS spectrometer assays, which is identical to that of the natural toxin isolated from the spider venom. It was indicated there was three disulfide bonds formation of the rHWTX-I. The physiological properties of the rHWTX-I was further analyzed using the whole-cell patch clamp assay. The rHWTX-I was able to block the currents generated by human Nav1.7 at an IC50 of 640 nM, similar to that of the natural huwentoxin-I, which is 630 nM.更多还原