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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 cDNA library of Schistosoma japonicum was prepared by a simple and complementary DNA cloning technique.The main process of the method is as follows: Using schistosome messenger RNA (mRNA) as a template, Under the action of transcriptase, the first strand of single-stranded cDNA was synthesized; mRNA was removed with RNase H and RNase, and second and thirdly synthesized with AMV reverse transcriptase and T_4-DNA (T_4-DNA) polymerase Strand double-stranded cDNA; cDNA fragments of less than 1 kilobase (kb) were removed on a Nixis (Cartridge): Plasmid pUC18 as a vector, DNA terminal transferase catalyzed addition of oligoguanine nucleotide; cDNA of Schistosoma japonicum Addition of oligomeric cytosine nucleotides. Then annealed and transformed into Escherichia coli strain MC1061 to obtain a cDNA gene library of Schistosoma japonicum. The cloning efficiency was 10-4 transformants per microgram of mRNA, with 30% of the transformants with cDNA inserts.