摘要
采用RT-PCR技术从美洲大蠊中扩增出变应原Cr-pII基因,并将其克隆至pMD18-T载体中。经序列测定和分析证实,所克隆的片段含有长度为1185bp的完整开放阅读框(ORF),与台湾株来源的Cr-pII(Per a1·0103)有95·9%的同源性。用NdeⅠ和NotⅠ将目的片段从T载体中切下,定向亚克隆入表达载体pET24a(+),然后成功转化大肠杆菌菌株BL21Star。经IPTG诱导得到45kDa的重组蛋白,Western-blot分析表明其具有良好的IgE结合活性。并通过生物信息学方法获得了目的蛋白的抗原表位和3D结构模型。
The Cr-pⅡ gone was amplified from Periplaneta Americana by RT-PCR and subsequently was cloned into the pMD18-T vector. Sequence analysis showed that this fragment contained a complete ORF of I 185 bp and had 95.9% homology with Per a 1.010 3 that was cloned from Taiwan strain. The fragment was subcloned into expression plasmid pgT24a （ ＋ ）, then the recombinant plasmid was transformed into E. coli BIll Star for expression. After induced by IPTG, 45kDa recombinant protein was obtained. Western-blot indicated it has good IgE binding potency. The antigen epitope and 3D structure model of Cr-pⅡ were predicted by biological information methods.
出处
《寄生虫与医学昆虫学报》
CAS
2006年第1期28-33,共6页
Acta Parasitologica et Medica Entomologica Sinica
基金
国家自然科学基金(No.39860071
No.30571625)
广东省科技计划重点项目(No.2003A3080502)
深圳市科技计划项目
关键词
美洲大蠊
变应原
Cr-PⅡ
克隆
表达
Periplaneta americana
Allergen
Cr-pⅡ
Cloning
Expression
