摘要
采用Ｑｕｉｃｋ Ｐｒｅｐ ｍ ＲＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ 试剂盒提取猪囊尾蚴ｍ ＲＮＡ；Ｔｉｍ ｅ－ Ｓａｖｅｒ ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ试剂盒反转录合成双链ｃＤＮＡ，并在末端加上ＥｃｏＲＩ／ＮｏｔＩａｄａｐｔｏｒ，修饰后的ｃＤＮＡ 与λｇｔｌｌＥｃｏＲＩ臂连接，体外包装为λ噬菌体颗粒；感染Ｅ．ｃｏｌｉＹ１０９０ 构建成功库容量为２×１０６、重组率为８０％ 的ｃＤＮＡ表达文库。利用兔抗猪囊尾蚴虫体抗原和囊液抗原血清分别对一部分文库进行免疫印迹筛选，分别获得８ 个阳性克隆。提取阳性克隆ＤＮＡ，利用ＰＣＲ法从重组噬菌体ＤＮＡ中扩增出ｃＤＮＡ片段，长度自４００—９００ｂｐ。本文工作为研制猪囊尾蚴核酸疫苗打下了基础。
mRNA of Cysticercus cellulosae was extracted with Quick Prep mRNA Purification kit,then double-strand cDNA was synthesized and EcoRI/NotI adaptor was added to the ends of ds cDNA using Time Saver cDNA Synthesis kit.The modified cDNA was ligated to the lambda gtll EcoRI arms and the ligated DNA was in vitro packaged into lambda phage.The phage than infected E.coli Y1090 culture and a cDNA expression library was constructed.The cDNA library was immunoscreened using rabit antibodies against the worm and cyst fluid antigens respectively,and 8 colonies were isolated respectively.The DNA of recombinant phage were extracted,and the insertion fragments were amplified using PCR.The lengthes of these fragmants are among 400-900bp.
出处
《中国人兽共患病杂志》
CAS
CSCD
北大核心
1999年第6期57-59,共3页
Chinese Journal of Zoonoses
关键词
猪囊尾蚴
CDNA表达文库
抗原基因
筛选
Cysticercus cellulasae\ cDNA expression library\ Antigenic genes
