日本血吸虫cDNA基因库的构建

CONSTRUCTION OF A cDNA LIBRARY OF SCHISTOSOMA JAPONICVM

采用一种简单的互补脱氧核糖核酸(cDNA)克隆技术制备日本血吸虫的cDNA基因库.方法的主要过程如下:以血吸虫信使核糖核酸(mRNA)为模板,在禽成髓细胞白血病病毒(AMV)反向转录酶的作用下,合成第一股单链cDNA;用核糖核酸酶H和核糖核酸酶除去mRNA,并用AMV反向转录酶和T_4-脱氧核糖核酸(T_4-DNA)聚合酶催化合成第二股双链cDNA;通过耐克斯(NACS)小柱除去1千碱基对(kb)以下的cDNA片断:质粒pUC18作为载体,DNA末端转移酶催化加接寡聚鸟嘌呤核苷酸;血吸虫的cDNA加接寡聚胞嘧啶核苷酸。然后退火连接并转化大肠杆菌菌株MC1061,得到日本血吸虫的cDNA基因库。克隆效率为10~4转化子/μg mRNA,有cDNA插入片段的转化子占30％。

The template mRNA was extracted from Schistosoma japonicum. The first strand of cDNA was synthesized by AMV-reverse transcriptase. The second strand cDNA was first digested by RNase H to remove mRNA and was then synthesized by AMV-reverse tran-scriptase, T4-DNA polymerase- Sizing of cDNA was applied on a NACS column to remove small fragments of less than 1 kb. Homopolymeric tailing of vector (PUC18) was done with dGTP and DNA terminal transferase and tailing of the cDNA with dCTP was carried out under the same conditions. After annealing, the plasmids with cDNA were transformed into E. coli MC1061. The efficiency of cloning was about 104/μg mRNA with 30% of the transformants having the inserts of cDNA (Figs. 1-2).