[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. CONCLUSION: Avastin gene of the two L.donovani isolates has been cloned and the gene can be expressed stably in the NIH3T3 cell.