用杆状病毒表达载体在家蚕中表达犬γ干扰素及产物的抗病毒活性

Expression and Antiviral Activity of Canine Interferon-γ Expressed in Silkworm Baculovirus Expression System

利用杆状病毒表达载体在家蚕5龄幼虫体内高效表达犬γ干扰素(CaIFNγ)。将优化、合成的犬γ干扰素基因克隆到杆状病毒转移载体pVL1393的多角体蛋白基因启动子下游,与orf1629基因缺损的亲本病毒Bm-Bacmid DNA共转染BmN细胞进行同源重组,将获得的重组病毒接种家蚕5龄幼虫。Western blot检测感病幼虫的血淋巴中有分子质量约19 kD的目的蛋白质条带。用微量细胞病变抑制法在MDCK/VSV-GFP系统测定重组犬γ干扰素具有抗病毒活性,效价>5×106U/mL。研究结果为进一步研究犬γ干扰素的抗病毒活性和利用家蚕生物反应器生产犬γ干扰素奠定了一定的基础。

Canine interferon-gamma （CalFNy） was expressed efficiently in the 5th instar silkworm （Bombyx mori） larvae by baculovirus expression vector. Optimized canine interferon γ gene was artificially synthesized and cloned into transfer vector pVL1393 to construct the recombinant plasmid pVL-CalFNγ under control of polyhedron gene＇s promoter, pVL- CalFNγ and orfl629-defective Bm-Bacmid DNA were co-transfected into BmN cells for homologous recombination. The re- combinant virus was harvested and used to inoculate silkworm larvae of the 5th instar. Western blot assay showed that the target protein was detected in hemolymph of the infected silkworm larvae and its molecular weight was about 19 kD. The re- combinant CalFNγ expressed in silkworm hemolymph was further verified to have antiviral activity by mini-cytopathic effect inhibition assay using the MDCK/VSV-GFP system, with a titer as high as 5x10e U/mL. This result provides a reliable basis for further studying CalFNγ antiviral activity and producing CalFNγ using Bombyx mori as bioreactor.