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重组大西洋三文鱼血清 C 型凝集素在黑腹果蝇 Schneider 2 细胞中的表达。

Expression of recombinant Atlantic salmon serum C-type lectin in Drosophila melanogaster Schneider 2 cells.

机构信息

Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, B3H 1X5, Canada.

出版信息

Cytotechnology. 2013 Aug;65(4):513-21. doi: 10.1007/s10616-012-9505-7. Epub 2012 Oct 18.

Abstract

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a soluble C-type lectin that binds bacteria, including salmon pathogens. This lectin is a cysteine-rich oligomeric protein. Consequently, a Drosophila melanogaster expression system was evaluated for use in expressing SSL. A cDNA encoding SSL was cloned into a vector designed to express it as a fusion protein with a hexahistidine tag, under the control of the Drosophila methallothionein promoter. The resulting construct was stably transfected into Drosophila S2 cells. After CdCl2 induction, transfected S2 cells secreted recombinant SSL into the cell culture medium. A cell line derived from stably transformed polyclonal cell populations expressing SSL was used for large-scale expression of SSL. Recombinant SSL was purified from the culture medium using a two-step purification scheme involving affinity binding to yeast cells and metal-affinity chromatography. Although yields of SSL were very low, correct folding and functionality of the recombinant SSL purified in this manner was demonstrated by its ability to bind to Aeromonas salmonicida. Therefore, Drosophila S2 cells may be an ideal system for the production of SSL if yields can be increased.

摘要

大西洋鲑鱼(Salmo salar)血清凝集素(SSL)是一种可溶性 C 型凝集素,可结合细菌,包括鲑鱼病原体。这种凝集素是一种富含半胱氨酸的寡聚蛋白。因此,评估了 Drosophila melanogaster 表达系统在表达 SSL 中的用途。将编码 SSL 的 cDNA 克隆到设计为与六组氨酸标签融合表达的载体中,受果蝇金属硫蛋白启动子的控制。所得构建体稳定转染入 Drosophila S2 细胞。用 CdCl2 诱导后,转染的 S2 细胞将重组 SSL 分泌到细胞培养基中。从稳定转化的多克隆细胞群体中衍生的细胞系用于 SSL 的大规模表达。使用两步纯化方案从培养基中纯化重组 SSL,该方案涉及与酵母细胞的亲和结合和金属亲和层析。尽管 SSL 的产量非常低,但通过其与鲑鱼气单胞菌结合的能力证明了以这种方式纯化的重组 SSL 的正确折叠和功能。因此,如果可以提高产量,Drosophila S2 细胞可能是生产 SSL 的理想系统。

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