重组病毒/家蚕细胞系统稳定高效表达HBeAg基因的研究

Study for High-Expression of HBeAg Gene with Recombinant Baculovirus/BmN Cell System

以 HBe Ag(乙型肝炎病毒 e抗原 )基因置换家蚕核多角体病毒 (Bm NPV)中多角体蛋白基因编码序列 ,获得高效表达 HBe Ag的重组病毒 r Bm HBe。在重组病毒复制过程中 ,家蚕 Bm N细胞的病理变化与野生型病毒的感染相同 ,但不能形成多角体。重组病毒的复制为典型的一步生长曲线 ,感染后 2 4 h～ 36h,病毒效价递增并达到最高。感染后 2 4 h,培养液中可检测到表达产物HBe Ag,72 h达到高峰。病毒感染复数的改变对 HBe Ag表达的影响是时限性的。低感染复数时HBe Ag产量较高。细胞接种密度及病毒接种时间对 HBe Ag的表达有较大的影响 ,最适的细胞接种密度为 3.2～ 6.4× 1 0 5 细胞 /瓶 ,在细胞指数生长前期 (48h～ 60 h)接种重组病毒 ,对于获得较高的重组病毒复制效价和 HBe Ag的产量都是有利的。在采用最佳技术参数组合的条件下 ,ELISA法检测细胞培养液中 HBe Ag滴度可达 1∶ 32 0 0 0。以上研究结果有助于在生产中高效稳定地制备HBe Ag,以满足供应配套乙肝 HBe Ag/抗

A recombinant baculovirus rBmHBe was constructed with replacement of polyhedrin gene code sequence in Bombyx mori nuclear polyhedrosis virus (BmNPV) by HBeAg gene. It is demonstrated that excepting no polyhedra formed, the cytopathology of BmN cell infected with rBmHBe is similar to that of Wt BmNPV. During the period of 24～36 h post infection (pi), the propagation of rBmHBe is a typical one step growth curve and the titer of virus increases with incubation time up to the highest. The synthesis of HBeAg in the cell medium begins at 24 pi and reaches to the top at 72 h pi. The titer of HBeAg is 1∶32000 by analysis of ELISA. Affection of multiplicity of infection (MOI) on expression of HBeAg is association with incubation time. The result shows lower MOI (<0.4 PFU/Cell) is beneficial to HBeAg production. The cell density and infection time have also great affection on HBeAg synthesis. The optimal cell density is 3.2～6.4×10 5 cell/flask. It is available to propagation of recombinant baculovirus and expression of HBeAg when infection of virus is at early exponential phase (48～60h).