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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。
OBJECTIVE: To isolate and purify a new type of trypsin inhibitor named bungaruskunin 1 from the snake venom of Rattus norvegicus and clone the complete cDNA sequence of the trypsin inhibitor from the cDNA library of its venom gland. Methods: bungaruskunin 1 was isolated and purified by Sephadex G-50, CM-Sephadex C-25, HPLC and RP-HPLC (C4 col- umn) The serine protease inhibitor activity of the sample was determined by the hydrolysis inhibition of the chromogenic substrate in 50 mmol·L-1 Tris-HCl, pH 7.8 buffer at room temperature. The Gall bladder snake venom gland RNA was extracted with TRIZOL and a cDNA library was constructed using SMARTM PCR cDNA syn-thesis (Clontech). Based on the conserved region of its signal peptide, the full-length cDNA sequence of bungaruskunin 1 was amplified from this library. The entire sequence of bungaruskunin 1 was recovered by gel filtration and ligated into pMD18-T vector for sequencing. Results: The precursor of bungaruskunin1 consists of 83 amino acids, of which the signal peptide contains 24 amino acids. The mature peptide, bungaruskunin 1, contains 59 amino acids. The cDNA sequence of bungaruskunin 1 is 64% similar to the cDNA sequence of serine protease inhibitor blackelin isolated and purified from Pseudechis porphyriacus. bungaruskunin 1 is a member of a family of Kuntiz protease inhibitors that contain the conserved Kunitz side, thereby inhibiting protease and elastase activity. In the cDNA library, we also screened two new β-bungarotoxin B chain sequences. CONCLUSIONS: These findings demonstrate well that the family of Kunitz / BPTI trypsin inhibitors and toxic nerves in the snake may originate from a common ancestor.