【摘要】 目的:从金环蛇蛇毒中分离纯化名为bungaruskunin 1的一种新型胰蛋白酶抑制剂,并从其毒腺的cDNA文库中克隆出该胰蛋白酶抑制剂的cDNA全序列。方法:通过Sephadex G-50,CM-Sephadex C-25,HPLC,RP-HPLC(C4 col-umn)方法分离纯化bungaruskunin 1。样品的丝氨酸蛋白酶抑制剂活性则是在室温条件下50mmol·L-1Tris-HCl,pH7.8的缓冲液中通过对显色底物的水解抑制作用来检测的。金环蛇毒腺RNA用TRIZOL提取,并用SMARTM PCR cDNA syn-thesis kit(Clontech)建成cDNA文库。根据其信号肽的保守区域合成引物从该文库中扩增出bungaruskunin 1的cDNA全序列,进行胶回收,酶连到pMD18-T载体中转化测序。结果:bungaruskunin1的前体由83个氨基酸组成,其中信号肽含有24个氨基酸,成熟肽即:bungaruskunin 1含有59个氨基酸。bungaruskunin 1的cDNA序列与从红腹伊澳蛇Pseudechis porphy-riacus中分离纯化得到的丝氨酸蛋白酶抑制剂blackelin的cDNA序列的相似性高达64%。bungaruskunin 1是一种含有保守Kunitz端的Kuntiz蛋白酶抑制剂家族的一员,从而能够抑制蛋白酶和弹性酶的活性。在cDNA文库中,我们同时还筛选到了2种新的β-bungarotoxin B链的序列。结论:这些发现很好地证明了蛇中Kunitz/BPTI胰蛋白酶抑制剂和毒性神经的家族可能起源于共同的祖先。更多还原

【Abstract】 AIM:To purify and characterize a novel trypsin inhibitor named bungaruskunin 1 from the snake venom of Bungarus fasciatus,and to clone its cDNA from the cDNA library of B. fasciatus venomous glands. METHODS: Sephadex G-50,cation-exchange CM-Sephadex C-25,HPLC,and RP-HPLC(C4 column) methods were used to isolate bungaruskunin 1. The inhibition effects of the sample on the hydrolysis of synthetic chromogenic substrates by serine proteases were assayed in 50 mM Tris-HCl,pH 7.8 at room temperature.The total RNA of B.fasciatus venomous glands were extracted using TRIzol method and the cDNA library were constructed by a SMARTM PCR cDNA synthesis kit(Clontech).Two primers (positive primer,based on the conserved cDNA fragment encoding signal peptides of beta-bungarotoxin B chains and Kunitz inhibitors from Bungarus snakes,and reverse primer based on the adopter sequence in the cDNA library) were used to screen Kunitz inhibitor and beta-bungarotoxin B chain encoding sequences. The cDNA products with about 700 bp in length were reclaimed from 1% agarose gel,and purified before ligation into the pMD18-T vector. RESULTS: The predicted precursor was composed of 83 amino acid (aa) residues including a 24 aa signal peptide and a 59 aa mature bungaruskunin 1. Bungaruskunin 1 showed maximal similarity (64%) with the predicted serine protease inibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin 1 is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin and elastase. By screening the cDNA library,two new B chains of beta-bungarotoxin were also identified. CONCLUSION: The overall structures of bungaruskunin 1 and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.更多还原