Bmeystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus
Bmeystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus
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DOI：
10.1016/j.bbrc.2006.06.018
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发表时间：
2006-08-18
影响因子：
3.1
通讯作者：
Tanaka, Aparecida S.
中科院分区：
文献类型：
--
作者：
Lima, Cassia A.;Sasaki, Sergio D.;Tanaka, Aparecida S.
牛微小扇头蜱（Rhipicephalus（Boophilus）microplus）是一种吸血动物，可传播Bababelus spp和边缘无形体（Anaplasma marginale）。从一个B。microplus脂肪体cDNA文库中，随机选择465个克隆进行测序，得到60个Contigs。开放阅读框（ORF）包含98个氨基酸，由于与来自肩突硬蜱的经典I型半胱氨酸蛋白酶抑制剂（GenBank登录号DQ 066227）的70%氨基酸同一性，被命名为Bmcystatin。Bmcystatin氨基酸序列分析显示两个半胱氨酸残基，理论pI为5.92，M-r为11 kDa。将Bmcystatin基因克隆到pET 26 b载体中，并在大肠杆菌BL 21 SI中表达。经Ni-NTA-aga-rose柱亲和层析和HiTrap Q柱离子交换层析纯化的重组Bmcystatin（rBmcystatin）分子量约为11 kDa，经SDS-PAGE和N端氨基酸序列测定，其N端含有未加工的pelB信号序列。纯化的rBmcystatin显示为C1半胱氨酸肽酶抑制剂，对人组织蛋白酶L和VTDCE（卵黄蛋白降解半胱氨酸内肽酶）的Ki值分别为0.1和0.6nM。通过半定量RT-PCR分析的rBmcystatin表达证实了脂肪体和卵巢cDNA制备物中特异性DNA序列（294 bp）的扩增。另一方面，蛋白质印迹法检测到的蛋白质带中的脂肪体，卵巢，和唾液腺提取物使用抗Bmcystatin抗体。目前的结果表明，Bmcystatin在卵巢中可能发挥作用，即使该基因是从脂肪体克隆的，这可能是该蛋白质合成的另一个位点。(c)2006年爱思唯尔公司All rights reserved.
The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type I cystatin from Ixodes scapularis tick (GenBank Accession No. DQ066227). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M-r of I I kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-aga-rose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of I I kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C I cysteine peptidase inhibitor with K-i value of 0.1 and 0.6 nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294 bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis. (c) 2006 Elsevier Inc. All rights reserved.