[四川省杜氏利什曼原虫分离株阿瓦斯汀基因的克隆及其在真核系统中的表达]

[Cloning of avastin gene of leishmania donovani isolates from Sichuan Province and its expression in eucaryotic system].

作者信息

Li Jin-Fu, Chen Jian-Ping, Tian Yu, Yang Zhi-Wei, Ma Ying, Hu Xiao-Su

机构信息

Department of Parasitology, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.

出版信息

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2007 Apr 30;25(2):124-8.

PMID:17633823

Abstract

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.

摘要

目的

克隆并表达来自中国四川省的两株杜氏利什曼原虫分离株的阿瓦斯汀基因。

方法

从两株杜氏利什曼原虫分离株的核DNA中扩增阿瓦斯汀基因，克隆至pcDNA3.1(+)中，采用双脱氧链终止法进行测序。用重组质粒转染NIH3T3细胞。通过免疫荧光检测阿瓦斯汀基因的瞬时表达，通过RT-PCR和蛋白质印迹检测稳定表达。

结果

两株分离株的阿瓦斯汀基因均为552 bp。序列分析表明两株分离株之间的相似性为86%。在细胞膜上和细胞内发现了高绿色荧光。重组质粒成功转染了NIH3T3细胞。通过RT-PCR从转染的NIH3T3细胞中获得了阿瓦斯汀基因。蛋白质印迹分析表明，转染的NIH3T3细胞裂解物中有一条约20 000 Mr的蛋白条带，表明阿瓦斯汀基因在细胞中表达。