摘要
提取周期型马来丝虫总RNA。跟据已知的马来丝虫副肌球蛋白(BmPmy)基因序列,设计合成引物,并引入HindⅢ和BamHⅠ酶切位点,应用RT-PCR技术,扩增BmPmy基因片段,克隆至载体pGEM-T中,经PCR和双酶切鉴定后,亚克隆至真核表达质粒pcDNA3.1(+),成功构建了真核表达载体pcDNA3.1(+)-BmPmy,并转染COS-7细胞后进行RT-PCR分析。转染的COS-7细胞高水平表达周期型马来丝虫副肌球蛋白mRNA,结果与预期相符。
Total RNA was extracted from periodic Brugia malayL Specific primers were designed on the basis of known sequences of paramyosin gene from B.malayi （BmPmy）. The desired gene was amplified by PCR technique from cDNA. The PCR products were purified and cloned into plasmid pGEM-T by T-A cloning method, transformed into Escherichia coli （E.coli） strain DH5α. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The right gene fragments encoding BmPmy in positive clones for prokaryotic and eukaryotic expression plasmids were digested with restrictive endonuclease, and were subcloned into pcDNA3.1（＋）. The recombinant eukaryotic plasmid （pcDNA3.1-BmPmy） was then transfected into COS-7 cells. The transient expression of BmPmy was examined with RT-PCR. BmPmy mRNA was highly expressed in transfected COS-7 cells.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2007年第3期250-252,共3页
Chinese Journal of Parasitology and Parasitic Diseases
基金
江苏省教育厅自然科学研究项目(No.02KJD310002)~~
