Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites
Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites
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DOI：
10.1016/j.ijpara.2008.08.006
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发表时间：
2009-02-01
影响因子：
4
通讯作者：
McElwain, Terry F.
中科院分区：
文献类型：
--
作者：
Suarez, Carlos E.;McElwain, Terry F.
转染已成为一种有价值的技术，用于阐明基因在许多病原体中的功能。而瞬时转染巴氏杆菌属（Babalypspp.）在这项研究中，设计了一种质粒，将gfp-bsd基因靶向整合到牛巴氏杆菌ef-1 α基因座中。通过用环状或线性质粒电穿孔转染生物克隆的Mo 7菌株的牛巴氏杆菌感染的红细胞，并在电穿孔后24小时在具有不同量的杀稻瘟菌素的培养物中选择。几种抗稻瘟菌素的B.牛转染的细胞系在选择开始后5 - 26天以不同的速率出现。基于在致死浓度的杀稻瘟菌素存在下的快速生长速率和明亮的GFP荧光，选择一个转染的寄生虫系（1-2-124）用于进一步分析。通过逆转录-PCR、Western印迹分析和荧光显微镜观察证实了gfp-bsd融合基因在电穿孔后持续表达超过9个月。没有质粒或附加体DNA可以检测到在这个线，质粒回收大肠杆菌是不成功的。Southern印迹结果和推定插入位点侧翼的PCR扩增子的测序与至少一个gfp-bsd盒整合到转染的寄生虫系中的靶向ef-1 α基因座中一致。总的来说，我们认为这是第一次，外源基因在B中的染色体整合和稳定表达。牛有了B。牛的基因组，靶向稳定转染将提供一种手段，以确定特定基因在生物学，临床疾病和免疫B的作用。牛蜱是限制全球畜牧业生产的三大蜱传寄生虫之一。由Elsevier Ltd.代表Australian Society for Parasitology Inc.出版。
Transfection has been a valuable technique for elucidating gene function in many pathogens. While transient transfection of Babesia spp. has been reported previously, stable integration of exogenous genes in Babesia has proven difficult, In this study, a plasmid was designed to target integration of a gfp-bsd gene into the Babesia bovis ef-1 alpha locus. Babesia bovis-infected erythrocytes of the biologically cloned Mo7 strain were transfected by electroporation with either circular or linear plasmids and selected in cultures with varying amounts of blasticidin 24 h after electroporation. Several blasticidin-resistant B. bovis transfected cell lines emerged at different rates, ranging from 5 to 26 days after the start of selection. One transfected parasite line (1-2-124) was selected for further analysis based on a rapid growth rate and bright GFP fluorescence in the presence of a lethal concentration of blasticidin. Continued expression of the gfp-bsd fusion gene was confirmed by reverse transcriptase-PCR, Western blot analysis and fluorescence microscopy for longer than 9 months after electroporation. No plasmid or episomal DNA could be detected in this line, and plasmid recovery in Escherichia coli was unsuccessful. Southern blot results and sequencing of PCR amplicons flanking the putative insertion site are consistent with integration of at least one gfp-bsd cassette into the targeted ef-1 alpha locus in the transfected parasite line. Overall the results demonstrate, we believe for the first time, chromosomal integration and stable expression of a foreign gene in B. bovis. With the availability of the B. bovis genome, targeted stable transfection will provide a means to determine the role of specific genes in the biology, clinical disease and immunity of B. bovis, one of the three major tick-borne parasites that limit global livestock production. Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.