摘要
应用定向克隆方法，将日本血吸虫虫卵ｃＤＮＡ片段重组入噬菌体载体λｇｔ１１Ｓｆｉ－Ｎｏｔ的ＥｃｏＲⅠ和ＮｏｔⅠ双酶切位点之间。所构建的基因表达文库的容量为１．０７×１０７重组子。经含有ＩＰＴＧ及Ｘ－Ｇａｌ的颜色选择平皿测定，初步提示重组效率达１００％。
To supply the basement for developing effective vaccine against schistosomiasis japonica, we constructed a cDNA library of Schistosoma japonicum(Sj) eggs using directional cloning technique. Schistosoma japonicum eggs were recovered from infected rabbits. cDNA fragments were obtained by in vitro transcription of the mRNA isolated from Chinese mainland S. japonicum eggs using reverse transcriptase and 5′ NotⅠ Oligo(dT) 18 Primer. After EcoRⅠ adaptor ligation, phosphorylation and NotⅠ restriction enzyme digestion, the cDNA with 5 EcoRⅠ end and 3′ NotⅠ end was directoinally ligated with the EcoRⅠ NotⅠ arms of the Lambda gt11 Sfi Not vector followed by Lamda DNA packing and transinfecting host bacterial strain Y1090. According to phage plaquesclear/blue selection in the presence of IPTG and X Gal,the proportion of recombinant phages in the S.japonicum egg libraries was 100%, while the cloning capacity was 1.07×10 7. We used primers on the Lambda gt11 arm located at several bases outer the insert position to amplify the inserted cDNA fragments by standard PCR. Of the 25 randomly selected clear phages, 21 had positive products with the amplified DNA fragments which were various from 564 bp to 1 500 bp, indicating the constructed library with big inserted cDNA fragments.
出处
《中国寄生虫病防治杂志》
CSCD
1998年第4期302-304,共3页
Chinese Journal of Parasitic Disease Control
关键词
CDNA文库
定向克隆
日本血吸虫
虫卵
cDNA library, directional cloning, Schistosoma japonicum , egg
