重组鸡γ-干扰素在家蚕中的表达及抗病毒活性  

Expression of Recombinant Chicken Interferon-γ in Bombyx mori with Baculovirus Expression System and Determination of Its Antiviral Activity

作　　者：赵泽 赵璐璐 刘兴健 王朋 李轶女[2] 张志芳[2] 易咏竹[1] Zhao Ze;Zhao Lulu;Liu Xingjian;Wang Peng;Li Yi'nyu;Zhang Zhifang;Yi Yongzhu(College of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212018,China;Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]江苏科技大学生物技术学院,江苏镇江212018 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《蚕业科学》2018年第6期898-904,共7页ACTA SERICOLOGICA SINICA

基　　金：国家重点研发计划项目(No.2017YFD0500706);国家重点基础研究发展计划项目(No.2016YFD0500108);国家自然科学基金项目(No.31670156)

摘　　要：鸡γ-干扰素(ChIFN-γ)是一类具有免疫调节及抗病毒活性的细胞因子。利用家蚕杆状病毒表达系统表达具有高生物活性的鸡γ-干扰素。首先,对ChIFN-γ基因序列进行优化与合成,再将其克隆到杆状病毒转移载体pVL1393多克隆位点中,与缺少orf1629基因的家蚕杆状病毒reBmBac共转染BmN细胞获得重组病毒reBm-ChIFN-γ。将reBm-ChIFN-γ注射感染家蚕5龄幼虫,检测感染后96~120 h之间发病蚕体的血淋巴。PCR扩增结果显示成功构建了重组杆状病毒reBm-ChIFN-γ,Western blot检测重组杆状病毒在家蚕体内表达了目的干扰素,微量细胞病变抑制法检测表达产物具有明显的抗病毒活性,效价约为(3.24±0.32)×10^6 U/mL。试验结果表明,利用家蚕杆状病毒表达系统成功地表达出具有较高抗病毒活性的鸡γ-干扰素,为鸡γ-干扰素相关生物制剂的研发奠定了基础。Chicken gamma interferon( Ch IFN-γ) is a kind of cytokine which plays a role in immune regulation and antiviral activity. Here,we expressed Ch IFN-γ with high biological activity by silkworm baculovirus expression system.First of all,the codons of Ch IFN-γ were optimized and the gene was synthesized and cloned into multiple cloning site of baculovirus transfer vector pVL1393. Then, we co-transfected Bm N cells with both the recombinant plasmid and re Bm Bac bacmid without orf1629 gene,obtained and purified the recombinant virus re Bm-ChIFN-γ. Subsequently,we injected 5 th instar larvae of Bombyx mori with re Bm-ChIFN-γ,collected the hemolymph of silkworm 96-120 h after injection and preserved for further detection. PCR results suggested that we constructed the recombinant baculovirus re BmCh IFN-γ successfully and target protein expression was confirmed by Western blot. In addition,biological potency of the protein was approximately( 3. 24± 0. 32) × 10^6 U/mL as determined by cytopathic effect inhibition assay. We conclude that,the Ch IFN-γ with high biological activity was expressed successfully in silkworm bioreactor through silkworm baculovirus expression system,which sets the good stage for subsequent biological agents related to Ch IFN-γ.

关 键 词：鸡γ-干扰素 杆状病毒表达系统 家蚕 抗病毒活性 

分 类 号：Q786[生物学—分子生物学] Q511