[Cloning of avastin gene of leishmania donovani isolates from Sichuan Province and its expression in eucaryotic system].
OBJECTIVE
To clone and express the avastin gene of two Leishmania donovani isolates from Sichuan Province of China.


METHODS
Avastin gene was amplified from nuclear DNA of two L.donovani isolates, cloned into pcDNA3.1(+), and sequenced by the dideoxy chain termination. NIH3T3 cell was transfected by recombinant plasmid. Transient expression of avastin gene was detected by immunofluoresence and stable expression was detected by RT-PCR and Western blot.


RESULTS
Avastin gene of both isolates was 552 bp. Sequence analysis showed that the similarity was 86% between the two isolates. A high green fluorescence was found on the cell membrane and inside the cell. The NIH3T3 cell was transfected by the recombinant plasmid successfully. Avastin gene was obtained by RT-PCR from the transfected NIH3T3 cells. Western blot analysis showed that there was a protein about Mr 20 000 in lysate of the transfected NIH3T3 cells, indicating that the avastin gene was expressed in the cells.


CONCLUSIONS
Avastin gene of the two L.donovani isolates has been cloned and the gene can be expressed stably in the NIH3T3 cell.
Li
Jin-Fu
JF
Department of Parasitology, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Chen
Jian-Ping
JP
Tian
Yu
Y
Yang
Zhi-Wei
ZW
Ma
Ying
Y
Hu
Xiao-Su
XS
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002681:China; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004285:Dogs; D015870:Gene Expression; D006801:Humans; D007893:Leishmania donovani / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D016054:DNA, Protozoan; D015800:Protozoan Proteins