利用家蚕杆状病毒表达系统表达人γ干扰素及产物的抗病毒活性检测

Expression of Human Interferon-γ by Bombyx mori Baculovirus Expression System and Detection of Antiviral Activity of the Product

干扰素-γ(IFN-γ)是免疫反应中重要的免疫调节因子,对多种免疫细胞具有激活作用。利用家蚕杆状病毒表达系统在家蚕中高效表达具有活性的人γ干扰素(hIFN-γ),首先根据家蚕密码子偏好性对hIFN-γ的编码基因进行优化合成,将优化合成后的序列克隆到杆状病毒转移载体pBR中,然后与orf1629基因缺失的失活拯救型杆状病毒BmBacmid共转染BmN细胞,获得重组病毒reBmBac-hIFN-γ,最后利用获得的重组病毒感染家蚕并收获表达产物。采用微量细胞病变抑制法,在Vero/VSV-GFP系统中检测到重组hIFN-γ的效价达到(3.69±2.02)×106 IU/mL。此外,重组hIFN-γ可以抑制猪蓝耳病病毒(PRRSV)在Marc-145细胞中的增殖。上述结果为利用家蚕生物反应器高效生产具有活性的人γ干扰素奠定了试验基础。

Interferon-γ(IFN-γ)is an important immunomodulatory factor in immune response,which can activate a variety of immune cells.In this study,silkworm baculovirus expression system was used to efficiently express active human interferon-γ(hIFN-γ)in silkworm.Firstly,the coding gene of hIFN-γwas optimized according to the codon preference of silkworm.The optimized sequence was cloned into the baculovirus transfer vector pBR.Then,the recombinant virus re-BmBac-hIFN-γwas obtained by co-transfection to BmN cell with the inactivated rescue baculovirus BmBacmid with the deletion of orf1629 gene.Finally,the recombinant virus reBmBac-hIFN-γwas used to infect the silkworm and the expression products were harvested.The titer of recombinant hIFN-γin Vero/VSV-GFP system was detected to be(3.69±2.02)×106 IU/mL by microcytopathic effect inhibition method.In addition,the recombinant hIFN-γcould inhibit the proliferation of porcine reproductive and respiratory syndrome virus(PRRSV)in Marc-145 cell.The above results laid an experimental foundation for the efficient production of active human interferon-γby silkworm bioreactor.