摘要
从我国半饱血微小牛蜱雌性成蜱肠道和唾液腺中提取总RNA;参照GenBank中已发表的微小牛蜱Bm91基因的核苷酸序列,设计了1对特异性引物。采用RT-PCR技术扩增Bm91基因,测序正确后将其亚克隆到巴斯德毕赤酵母分泌型表达载体pPIC9K的EcoRⅠ酶切位点,构建重组表达载体pPIC9K-Bm91。再次测序正确后将其用SacⅠ内切酶线性化,电转化毕赤酵母GS115并经G418抗性筛选高拷贝重组菌株后用甲醇诱导表达。表达产物经SDS-PAGE和Western-blot分析,结果显示,Bm91基因获得了成功表达,诱导表达的培养上清液中表达出具有反应活性的83ku的重组Bm91蛋白。
Total RNAs were extracted from the salivary glands and the intestine of semiengorged adult female Boophilus microplus, respectively, According to the nucleotide sequences of Bm91 gene of B. microplus available in GenBank,a pair of primers were designed and the Bm91 gene was amplified by RT-PCR from the total RNAs. Then the verified gene was sub-cloned into EcoR Ⅰ site of the secretory expression vector pPIC9K,and the verified recombinant expression vector pPIC9k-Bm91 was linearized with Sac I and electrotransformed into yeast cell GSl15. The multicopy recombinant Pichia pastoris strains were screened by G418 and induced by methanol. The expressed product was analyzed by SDS-PAGE and Western-blotting. The results showed that Bm91 gene was expressed successfully and the expressed Bm91 protein of 83 ku in molecular mass in supernatant had good reactogenicity.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第7期569-575,共7页
Chinese Veterinary Science
基金
国家"十一五"高技术研究发展计划(863)项目(2006AA10A207)
国家自然科技资源共享平台项目(2005DKA21100)
国家"十一五"科技支撑计划项目(2007BAD40B06)
科技部欧盟项目
中国农业科学院杰出人才基金项目
欧盟EPIZONE
ICTTD3(INCO510561)项目
SSA-INCOME项目
关键词
微小牛蜱
Bm91基因
分泌表达
Boophilus microplus
Bm91 gene
secretory expression
