Chai Hui-ping, Liu Guang-yuan, Zhang Lin, Gong Zhen-li, Xie Jun-ren, Tian Zhan-cheng, Wang Lu, Jia Ning
Key Laboratory of Veterinary Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2009 Feb 28;27(1):6-10.
To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes.
Total RNA was isolated from unfed female ticks, mRNA was purified and a library of oligo (dT) -primed cDNA with added directional EcoR I /Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I /Hind III arms of the lambda SCREEN vector. Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8. Recombinant plasmids that were subcloned to E. coli BM25.8 were isolated and transformed into E. coli JM109. Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing.
The recombinant phage DNA was packaged by using phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.8 x 10(6) pfu. Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4 x 10(9) pfu/ml. Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library. Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H. longicornis tropomyosin mRNA, Rhipicephalus annulatus unknown larval protein mRNA, chromosome 2R of Drosophila melanogaster, mitochondrial DNA of H. flava, clones HqL09 unkown mRNA and Hq05 mRNA of H. qinghaiensis, and myosin alkali light chain protein mRNA.
The cDNA expression library from unfed female H. longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.
构建未吸血长角血蜱雌蜱的cDNA表达文库，用于筛选和克隆潜在的抗原基因。
从未吸血雌蜱中提取总RNA，纯化mRNA，并从纯化的mRNA构建添加了定向EcoR I/Hind III接头的寡聚（dT）引物cDNA文库。将构建的cDNA连接到λSCREEN载体的EcoR I/Hind III臂上。通过噬菌斑纯化收获纯噬菌体原种，并通过将噬菌体接种到宿主菌株BM25.8上转化为质粒亚克隆。将亚克隆到大肠杆菌BM25.8的重组质粒分离并转化到大肠杆菌JM109中。通过PCR和测序鉴定从JM109提取的重组质粒。
使用噬菌体标记包装提取物包装重组噬菌体DNA，得到一个大小为1.8×10⁶ pfu的初级cDNA文库。数据显示文库的重组率为100%，扩增文库的滴度为2.4×10⁹ pfu/ml。从cDNA文库中获得了42个编码免疫显性抗原的克隆。序列分析揭示了12个独特的cDNA序列，编码的推定蛋白与长角血蜱原肌球蛋白mRNA、环形扇头蜱未知幼虫蛋白mRNA、黑腹果蝇2R染色体、淡黄血蜱线粒体DNA、青海血蜱克隆HqL09未知mRNA和Hq05 mRNA以及肌球蛋白碱性轻链蛋白mRNA具有相似性。
成功构建了未吸血长角血蜱雌蜱的cDNA表达文库，保护性基因的筛选可为蜱的候选抗原提供依据。
