摘要
目的构建牛带绦虫(Taeniasaginata)成虫全长cDNA质粒文库,为寻找更多有效的牛带绦虫病疫苗候选抗原分子及对三种主要人体带绦虫的功能基因对比研究奠定基础。方法提取牛带绦虫成虫mRNA;进行反转录合成双链cD-NA。PCR产物经蛋白酶K消化、纯化后,进行SfiI酶切;用CHROMA SPIN-400柱将酶切产物进行分级分离,经1.1%琼脂糖凝胶电泳鉴定,并与改造载体pBluescript Ⅱ SK连接,将连接产物涂板,测定文库的滴度;用载体克隆位点两端的引物进行PCR扩增,以检测所构建文库质量。随机挑选质粒文库转化的阳性重组克隆,进行较大规模的5′端测序,归并UniGene。结果测定文库的滴度为1016个菌落/μL,共测1023个克隆,对这些序列进行UniGene归并,得到419条unigene。结论已成功获得高质量的牛带绦虫成虫全长cDNA表达文库。
The objective of the present study was to construct the full-length cDNA plasmid library for adult worms of Taenia saginata and then to form a gene expression pattern in order to establish a foundation for effective vaccine candidate antigen research and for comparative study of three main kinds of human Taenia. The mRNA was extracted from the T. saginata adult worms and the cDNA was synthesized. Products were digested by proteinase K and SfiⅠ, and then fractionated by CHROMA SPIN -400 column. The fractions were characterized by 1.1% agarose/EB gel electrophoresis, and the full-length cDNA plasmid library pBluescript II SK was constructed. The titer of the amplified library was evaluated and the quality of the constructed library was tested by PCR amplification using amphi-primers of the vector cloning sites.Furthermore,the positive recombinant clones were carried out with the 5’ terminus sequencing in large scale and then merged to unigenes. The results indicated that the titer of amplified library was 1016 pfu/μL. Of 1023 clone sequences were obtained and in with 419cDNA were merged. It was evident that the high-quality full-length cDNA plasmid library of adult T. saginata has been constructed successfully.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2010年第4期364-366,共3页
Chinese Journal of Zoonoses
基金
国家自然科学基金资助项目(No.30760227)
贵州省科技攻关项目〔黔科合NY字(2008)3060]
贵州农业攻关计划项目联合资助
