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目的 构建恶性疟原虫cDNA表达文库。方法 用Trizol试剂盒提取总RNA ,以经修饰的SMART寡聚核苷酸引物 CDSⅢ引物直接用总RNA选择性地反转录成单链cDNA ,再用长距PCR(LD)方法选择性地扩增出双链cDNA ,经蛋白酶K消化和酶切后 ,用玻璃奶试剂盒回收 2 0 0bp以上DNA片断 ,经与载体λTriplEX2连接和蛋白抽提物体外包装后 ,构建成cDNA文库并对文库进行了初步的鉴定。结果 构建了高滴度 ,高重组率的恶性疟原虫cDNA表达文库。结论 所构建的疟原虫cDNA文库适合进一步筛选抗疟原虫药物结合蛋白基因。
Objective To construct a cDNA library of Plasmodium falciparum. Methods Trizol kit was used to extract total RNA. The modified SMART oligonucleotide primer CDSⅢ was directly reverse transcribed into single-stranded cDNA with total RNA, and then amplified by long-range PCR (LD) The double-stranded cDNA was amplified and digested with Proteinase K. After digestion with digested proteinase K, a DNA fragment of 200 kbp or more was recovered by glass-milk kit. After being ligated with the vector λTriplEX2 and packaged with protein extracts, the cDNA library was constructed and subjected to library The initial identification. Results A high titer and high recombination rate of Plasmodium falciparum cDNA expression library was constructed. Conclusion The constructed plasmodium cDNA library is suitable for further screening anti-malaria drug-binding protein gene.