微小牛蜱唾液腺cDNA文库的构建

中国寄生虫学与寄生虫病杂志 ›› 2008, Vol. 26 ›› Issue (5): 16-391.

微小牛蜱唾液腺cDNA文库的构建

田占成,刘光远^*,谢俊仁,龚真莉

中国农业科学院兰州兽医研究所，家畜疫病病原生物学国家重点实验室、甘肃省动物寄生虫病重点实验室，兰州 730046

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2008-10-31 发布日期:2008-10-31
通讯作者: 刘光远

Construction of cDNA Expression Library of Salivary Gland from Boophilus microplus

TIAN Zhan-cheng,LIU Guang-yuan^*,XIE Jun-ren,GONG Zhen-li

Key Laboratory of Veterinary Parasitology of Gansu Province，State Key Laboratory of Veterinary Etiological Biology，Lanzhou Veterinary Research Institute，CAAS，Lanzhou 730046，China

Received:1900-01-01 Revised:1900-01-01 Online:2008-10-31 Published:2008-10-31
Contact: LIU Guang-yuan

摘要/Abstract

摘要：

从微小牛蜱唾液腺中提取RNA，纯化mRNA，合成双链cDNA，定向克隆到λSCREEN载体的两臂之间。用噬菌体蛋白包装产物对以上连接产物进行体外包装以形成完整的噬菌体，转染大肠埃希菌ER1647，构建成微小牛蜱唾液腺的cDNA表达文库。经测定，文库的库容量约为1.38×10⁶ PFU，重组率为100%，扩增后文库的滴度为2×10⁹ PFU/ml。用兔抗微小牛蜱唾液腺蛋白阳性血清，对文库进行初步免疫学筛选，获得一个细胞色素氧化酶第2亚基cDNA序列。将该基因序列与GenBank中的序列进行同源性比对，发现与已知的其他物种细胞色素氧化酶第2亚基的氨基酸序列同源性为60%～77%。微小牛蜱唾液腺cDNA表达文库构建成功。

关键词: 微小牛蜱, 唾液腺, cDNA文库构建

Abstract: Total RNA were isolated from salivary gland dissected from partially engorged Boophilus microplus. The mRNA was purified. A library of oligo(dT)-primed cDNA with added directional EcoR I/HindⅢ linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I/HindⅢarms of the λSCREEN vector. The recombinant phage DNA was packaged by phage-marker packaging extracts，resulting in a primary cDNA library with a size of 1.38×10⁶ PFU. Data showed 100% of the library were recombinant and the titer of the amplified library was 2×10⁹ PFU/ml. A partial cDNA encoding cytochrome oxidase C subunit II of B. microplus was screened from the expression library with rabbit serum against B. microplus salivary gland proteins. The results is suggested that the cDNA expression library has been constructed.

Key words: Boophilus microplus, cDNA expression library, Construction

田占成;刘光远;谢俊仁;龚真莉. 微小牛蜱唾液腺cDNA文库的构建[J]. 中国寄生虫学与寄生虫病杂志, 2008, 26(5): 16-391.

TIANZhan-cheng;LIUGuang-yuan*;XIEJun-ren;GONGZhen-li. Construction of cDNA Expression Library of Salivary Gland from Boophilus microplus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2008, 26(5): 16-391.

[1]	梁怡琳，高攀，柯泽楷，罗新萍，刘志刚. 微小牛蜱磷酸丙糖异构酶基因片段的克隆与原核表达[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(4): 20-331-332.
[2]	马米玲;关贵全;李有全;刘爱红;任巧云;牛庆丽;殷宏;罗建勋. 微小牛蜱Bm86基因的原核表达与表达条件的优化[J]. 中国寄生虫学与寄生虫病杂志, 2009, 27(6): 18-533.
[3]	程道新,王述姮,叶鑫生,卡胜宜,游本荣. 双抗体夹心法检测按蚊体内间日疟子孢子的研究[J]. 中国寄生虫学与寄生虫病杂志, 1988, 6(S1): 14-14.
[4]	李文盛,许派波,袁金富. 蝇类幼虫脑和唾液腺的初步观察[J]. 中国寄生虫学与寄生虫病杂志, 1988, 6(S1): 152-152.