Hsieh Ming-Shou, He Jie-Long, Wu Tzong-Yuan, Juang Rong-Huay
Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan.
Department of Post-Baccalaureate Veterinary Medicine, Asia University, Taichung 413, Taiwan.
J Immunol Methods. 2018 Aug;459:81-89. doi: 10.1016/j.jim.2018.06.001. Epub 2018 Jun 9.
A bi-cistronic baculovirus expression vector was constructed to facilitate the expression, detection, and isolation of the hemagglutinin (HA) fragment HA1 of H6N1 avian influenza virus (AIV) in an insect and a culture of its cells. In this construct, the GP67sp signal peptide promoted the secretion of the recombinant protein into the culture medium, and improved protein expression and purification. Enhanced green fluorescent protein, co-expressed through an internal ribosome entry site, served as a visible reporter for protein expression detection. The hemolymph of Spodoptera litura larvae infected with the bi-cistronic baculovirus was collected for the purification of the recombinant HA1, which was found to be glycosylated, and monomeric and trimeric forms of the recombinant HA1 were identified. Proteins expressed in both the cell culture and larvae served as effective subunit vaccines for the production of antiserum against HA. The antiserum recognized the H6 subtype of AIV but not the H5 subtype.
构建了一种双顺反子杆状病毒表达载体，以促进H6N1禽流感病毒（AIV）血凝素（HA）片段HA1在昆虫及其细胞培养物中的表达、检测和分离。在该构建体中，GP67sp信号肽促进重组蛋白分泌到培养基中，并改善了蛋白表达和纯化。通过内部核糖体进入位点共表达的增强型绿色荧光蛋白用作蛋白表达检测的可见报告基因。收集感染双顺反子杆状病毒的斜纹夜蛾幼虫的血淋巴用于重组HA1的纯化，发现其被糖基化，并鉴定出重组HA1的单体和三聚体形式。在细胞培养物和幼虫中表达的蛋白用作有效的亚单位疫苗，用于生产抗HA血清。该抗血清识别AIV的H6亚型，但不识别H5亚型。
