摘要
为构建毒害艾美球虫孢子化卵囊cDNA表达文库，用Trizol试剂提取毒害艾美球虫孢子化卵囊总RNA，采用SMART技术，在逆转录酶的作用下将总RNA反转录成第一链cDNA，经LD-PCR扩增合成双链cDNA（ds cDNA）。经蛋白酶K消化、SfiⅠ酶切，过CHROMA SPIN-400^TM柱去除小于400bp的片段，与λTriplEx2^TM噬菌体载体连接，用GigapackⅢGold^TM载体蛋白包装，构建了cDNA噬菌体表达文库。经测定，原始文库容量为4．72×10^6pfu／mL，扩增后的文库容量为2．62×10^10pfu／mL，重组率为97．5％，插入片段为500～1100bp。证实，该文库质量良好，为毒害艾美球虫新基因的筛选、克隆及功能研究奠定了基础。
To construct a cDNA expression library for Eimeria necatrix sporulated oocysts, the total RNA of the sporulated oocysts of E. necatrix was extracted using Trizol reagent and then was reverse-transcripted with reverse-transcriptase by SMART （switching mechanism at 5＇ end of RNA transcript） technique. The double-strand cDNA（ds cDNA） was synthesized with the first strand cDNA as template by amplification using long-distance PCR（LD-PCR）. cDNA fragments less than 400 bp fragments were removed by CHROMA SPIN-400^TM columns after the ds cDNA was digested with proteinase K and Sfi I ,and the purified fragments were ligated into λTriplEx2^TM vector and packaged with the Gigapack I]I GoldTM. The results showed that the capacity of the un-amplified library was 4.72× 10^6pfu/mL,the capacity of the amplified library was 2. 62 ×10^10 pfu/mL, the library recombination rate was 97. 5%, and the inserted cDNA fragments were 500-1 100 bp in length. These results revealed that the constructed cDNA expression library for E. necatrix sporulated oocysts had good quality which provided a basis for screening,cloning and further studies of new genes of E. necatrix.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第3期196-199,共4页
Chinese Veterinary Science
基金
国家“十五”科技基础条件平台建设项目(2005DKA21205-4)
国家“十一五”高技术研究发展计划(863)项目(2006AA10A207-1)
