猪囊尾蚴cDNA文库的构建

CONSTRUCTION OF cDNA LIBRARY FROM CYSTICERCUS CELLULOSAE

从猪囊尾蚴内提取 m RNA经反转录合成 c DNA,应用定向克隆的方法 ,将合成的 c DNA片段重组到噬菌体载体 Uni- ZAP XR的 Eco R 和 Xho 双酶切位点之间。5 μg poly(A) + RNA所构建的表达文库容量为 0 .98×10 6重组子。经含有 IPTG和 X- gal的颜色选择平皿测定 ,提示重组率达 86 %。随机取 6个噬菌斑做 PCR,检查插入片断的长度在 10 0～ 2 0 2 0

The mRNA was extracted from Cysticercus cellulosae and cDNA was synthetized using 5′ Xho Ⅰ oligo(dT) 18 primer and reverse transcriptase. After EcoR Ⅰ adaptor ligation, phosphorylation and Xho Ⅰ restriction enzyme digestion, the cDNA fragments with 5′ EcoR Ⅰ and 3′ Xho Ⅰ ends were directionally ligated with the EcoR Ⅰ Xho Ⅰ Arms of Uni Zap XR vector. The cDNA library was constructed after the recombinant DNA was packed into phage by Gigapack Ⅲ Gold Packaging Extract. It was demonstrated that the library contained 0.98×10 6 recombinant phages, and about 86 % phages contained inserts of cDNA fragment after the recombinant phages were transfected into XL 1 Blue MRF( strain and differentiated by clear/blue selection in the plate with IPTG and X gal. PCR was performed using 6 phageblots which were random selected from plate to detect the length of inserts of the library. The result showed that the length of the inserts was between 100 bp to 2 020 bp.