【作者】 唐建洲；

【导师】 谢锦云； 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， 2005， 硕士

【摘要】 虎纹捕鸟蛛(Ornithoctonus huwena Wang)是分布在我国广西省和云南省山区的一种稀有蜘蛛新种,在本实验室以前的工作中,从虎纹捕鸟蛛的粗毒中分离出了具有不同结构和功能的多种成分,并且已经克隆了其中9种毒素的cDNA,将其分为三个超家族。本文采用RACE方法克隆了HWTX-Ⅴ的cDNA,并将其归类于超家族中。根据毒素的氨基酸序列设计RACE引物,通过叠合3’RACE和5’RACE扩增的序列就得到了毒素蛋白的cDNA全序列。HWTX-Ⅴ的全长cDNA共有459个碱基。由5’UTR、可读框和3’UTR组成,将其与其它虎纹捕鸟蛛的毒素(HWTX-Ⅰ,HWTX-Ⅱ,HWTX-Ⅲ,HWTX-Ⅳ,HWTX-Ⅶ)cDNA进行同源比较,同时将Pre和Pro区与其他已知毒素的Pre和Pro区比较,结果表明HWTX-Ⅴ的核苷酸序列在Pre和Pro区与HWTX-Ⅰ,HWTX-Ⅲ,HWTX-Ⅳ高度同源,而与HWTX-Ⅱ,HWTX-Ⅶ的同源性较低。由此可知HWTX-Ⅴ,HWTX-Ⅰ,HWTX-Ⅳ属同一超家族,从而可以预测HWTX-Ⅴ的三级结构同HWTX-Ⅰ一样,六个半胱氨酸残基都通过C1-C4,C2-C5,C3-C6的配对方式形成抑制剂半胱氨酸结模体(inhibitor Cystine knot,ICK)。HWTX-Ⅴ的cDNA可读框编码86个氨基酸残基的毒素前体,包括N端的50个氨基酸残基组成的信号肽和间隔区,中间35个氨基酸残基组成的成熟毒素,以及C端的额外碱性氨基酸Lys。尾端的Lys在毒素前体的成熟过程中被切除。 敬钊缨毛蛛(Chilobrachis jingzhao)属于捕鸟蛛科,是分布于我国海南省山区的一种蜘蛛新种。本实验室以前的工作中,已经测定了约20种毒素的一级结构。本文采用RACE方法克隆了5种敬钊缨毛蛛的全长cDNA序列,它们都由5’UTR、可读框和3’UTR组成,其开放阅读框编码一个20-23个氨基酸残基组成的信号肽区域,一个30多个氨基酸残基组成的成熟蛋白区域和一个介于它们之间的pro区域构成的毒素前体。这几种毒素的pro区变化较大,从几个氨基酸残基(JZTX-Ⅰ,Ⅲ,Ⅶ)到30多个(JZTX-Ⅸ)氨基酸残基不等,且它们的同源性较低。 蜘蛛毒素蛋白由于其序列短而富含二硫键,所以采用基因工程的方法表达困难,本文采用原核分泌表达载体pMAL-p2X成功表达了HWTX-Ⅳ,更多还原

【Abstract】 The spider Ornithoctonus huwena (Wang) [=Selenocosmia huwena Wang] is distributed in the hilly areas of Guangxi and Yunnan in the south of China. In previous work, many components with different structures and functions were purified and characterized. The nine cDNA sequences of the huwena toxin were cloned and sequenced, It was classified into three different superfamilies according to the prepro region of cDNA sequences. In this study, the cDNA encoding HWTX-Vwas cloned and sequenced by RACE, classified into the three superfamilies. On the basis of their amino acid sequences, we designed and synthesized 3’ RACE and 5’ RACE primers. By overlapping the two partial cDNA sequences obtained by 3’ and 5’ RACE, the full-length sequence was obtained. The full length cDNA of HWTX-V contain 459 bases composed of 5’ UTR, ORF, 3’ UTR. Compared with the other cDNAs and the prepro regions of the huwena(HWTX- I, II, III, IV, VII), it was demonstrated high similarity to the HWTX- I, III, IV, but was not similarity to the HWTX-II, VII Thus, the HWTX-V shares the same superfamily. The pairing model of three disulfide bonds is Cys(I-TV), Cys(II-V), Cys(III-VI), the key structure composes of a triple-stranded antiparallel P-sheet and a disulfide knot, which is the characteristic property of ICK motif. The open reading frame encoded a precursor protein of 86 residues, including a signal peptide and prepro region of 50 residues and a mature peptide of 35 residues, an additional residue Lys was found at the C terminus of the mature peptide before the stop codon.The spider Chilobrachis jingzhao is distributed in the hilly areas of Hainan in China. In previous work, about 20 toxins was sequence and characterized. Five cDNAs encode JZTX-I, JZTX-III, JZTX-VI, JZTX-VII, JZTX-IX were cloned and sequenced. All the five cDNAs composed of 5’UTR, open reading frame (OFR) and 3’UTR. The OFR encode a precursor including a potential signal peptide of 20 to 23 residues, a mature toxin of about 30 residues and an intervening pro region. The length of the five toxins prepro regions varied from less than 10 to over 30 residues, further more, there have low similarity to these更多还原