眼镜王蛇三指毒素新基因的克隆、表达及重组长链神经毒素的功能研究

【作者】 李晶；

【导师】 徐康森； 刘兢；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 三指毒素是一类由有着极其相似三级结构的低分子量（＜10kDa）毒素组成的家族。本文根据NCBI的检索结果，依据眼镜王蛇神经毒素高度保守的氨基酸序列设计简并引物进行RT-PCR，得到了眼镜王蛇神经毒素的部分基因序列，进而采用3’RACE法首次得到了眼镜王蛇6种三指毒素共19条新基因的全长cDNA序列。这6种三指毒素分别是长链神经毒素、短链神经毒素、弱神经毒素、心脏毒素、毒蕈碱毒素以及含有一个游离巯基的长链神经毒素。通过对cDNA编码区与非编码区同义及非同义突变频率的分析，论述了王蛇中三指毒素基因的进化方式为压力下的达尔文正选择。根据所构建的进化树在一种毒蛇内对五种三指毒素的进化关系进行了分析，并提出了眼镜王蛇的长链神经毒素与海蛇科的短链神经毒素有更近的进化关系。 通过序列分析，我们选取了眼镜王蛇三指毒素19种cDNA中的14种，利用pGEX-4T-1质粒在E.coli中对其进行了表达初筛，结果表明所有的重组毒素均能够大量表达，除心脏毒素外大多数毒素有部分可溶性表达。我们选取了两个最具代表性的长链毒素（rLNTX1与rLNTX3）进行更深入的研究，摸索表达与酶切条件，建立了使用GST柱-苯甲脒柱串连纯化酶切液中重组神经毒素的纯化工艺。经过Tris／tricine SDS-PAGE、体积排阻HPLC和RP-HPLC测定重组毒素的纯度以及飞行时间质谱测定分子量、N端氨基酸测序、圆二色散光谱等鉴定步骤，对其进一步定量定性，为下一步的功能研究打下了良好的基础。 通过急性毒性实验，我们测定了rLNTX1与rLNTX3的半数致死量，进而利用全细胞膜片钳技术对其抑制烟碱诱发电流的能力进行了检测，两个重组毒素均显示明显的突触后神经毒活性。但是，在LD₅₀测定中观察到小鼠呈现的一些异常表现结合克隆表达阶段中的现象，我们又通过MTT法对这两个毒素可能的细胞毒活性进行了检测，它们展示出与心脏毒素相似的细胞毒活性。我们从一级序列结构以及疏水性角度出发，对这两个重组毒素在神经毒与细胞毒活性上的差异进行了合理的解释。更多还原

【Abstract】 Three-finger toxins are a family of low molecular weight toxins （<10kDa） having a very similar tertiary structure. We designed the degenerative primers based on the highly conversed residue sequences of α-neurotoxins and obtained partial DNA sequences of neurotoxins from Ophiophagus Hannah. Followed by 3’RACE and RT-PCR, 19 novel cDNAs of three-finger toxins containing the sequence of signal peptide and 3’-UTR region were cloned from the venom glands of O.Hannah. Putative peptides encoded by the cDNAs were classified as six kinds of three-finger toxins: long chain neurotoxins, short chain neurotoxins, weak neurotoxins, cardiotoxins, muscarinic toxins and a toxin with a free SH group. It indicated that the accelated envolution and positive Darwinian selection had taken place in those genes according to the mathematical analysis based on the synonymous and non-synonymous substitutions in the coding and non-coding regions. Phylogenetic trees were constructed to describe the evolutionary relationship of these toxins in a specific snake. It also suggested that long chain neurotoxins of O.Hannah had closer evolutionary relations with short chain neurotoxins of HYDROPHIIDAE.According to the sequence alignment, we chose 14 cDNAs from the 19 genes and expressed them in E.coli. It shows that all the toxins could be expressed in a high level and most of them had the soluable forms except the cardiotoxins. We optimized the conditions of expression and digestion of two long chain neurotoxins and established the process of purifying recombinant toxins from the cleavage buffer by the GST column and the Benzamidine column connected in series. Recombinant peptides were characterized by MALDI-TOF mass, N-terminal residue sequencing, Tris/tricine SDS-PAGE and CD spectrometry. The purity of them was tested by RP-HPLC and SEC-HPLC.The results of LD₅₀ test and whole-cell patch clamp technology showed that recombinant toxins had potent neurotoxic acitivity and could be classified into postsynaptic neurotoxins. Whereas, some interesting phenomena observed in cloning and expressing stages and in LD₅₀ test indicated the existence of cardiotoxin-like cytolytic activity in these two recombinant peptides. Results of the MTT assay on HUV-EC and L929 cells confirmed this conjecture. By comparing the functional residues, we offered some possible explanations for the difference of their neurotoxic functions. Moreover, plausible elucidation on the extra cytolytic activity was carried out through the analysis of a hydropathy profile.更多还原