[Construction of eukaryotic expression plasmids with paramyosin gene of periodic Brugia malayi].
Total RNA was extracted from periodic Brugia malayi 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 DH5alpha. 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.
Department of Parasitology, School of Basic Medical Sciences, Nantong University, Nantong 226001, China.
Yang
Y
Chen
Zheng
Z
Fang
Wei-qun
WQ
Huang
Dong-fang
DF
Xie
Sheng-yang
SY
Jiang
Jian-jun
JJ
Wu
D000818:Animals; D017178:Brugia malayi / Q000235:genetics*; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015870:Gene Expression; D015801:Helminth Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014335:Tropomyosin / Q000235:genetics*
D015801:Helminth Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014335:Tropomyosin