杆状病毒GP64表面展示元件与L-天冬酰胺酶Ⅱ融合表达的初步试验

A Primary Experiment on the Fusion Expression of L-asparaginaseⅡ Using Baculovirus GP64 Surface Display System

GP64是Ⅰ型杆状病毒的主要囊膜糖蛋白,利用基因重组技术在gp64中插入外源蛋白基因,二者在病毒表面融合表达活性肽或蛋白质,可以免去纯化表达产物的繁琐。L-天冬酰胺酶Ⅱ(ASP)是一种临床上广泛使用的蛋白类抗肿瘤药物,利用家蚕杆状病毒(BmNPV)GP64展示系统表面展示ASP,探讨以表面展示有ASP蛋白的出芽型病毒粒子(BV)生产抗肿瘤药物的可行性。构建重组转移载体pFast Bacdual-gp64sp-asp-gp64ctd,并转化E.coli DH10BacTM,通过蓝白斑筛选获得重组杆粒DNA,将重组杆粒DNA通过脂质体转染至Bm N细胞,获得了重组病毒。重组病毒大量感染Bm N细胞后72 h,收集感染病毒的细胞培养液超速离心纯化BV。采用奈氏试剂法检测接种重组病毒的Bm N细胞内表达融合ASP的酶活力为1 788.4U/mg;Western blot检测纯化后的BV中含融合ASP蛋白,然而BV中的ASP检测不出酶活性。试验结果提示,构建的杆状病毒GP64展示系统可以将ASP转到BV上,但产物的活性保持有待进一步研究。

GP64 is a major capsule membrane glycoprotein of group Ⅰ baculovirus. By gene recombination technique,the foreign protein-encoding genes were inserted into gp64 gene,and the fused active peptides and proteins were expressed on the surface of virus,which makes the expression products be purified easily. L-asparaginase Ⅱ（ ASP） is widely used as an antitumor drug in clinic. In this study,ASP protein was displayed by Bombyx mori baculovirus GP64 surface display system to explore the feasibility of using the budded virus（ BV） which displayed ASP protein on the surface as antitumor drugs. Firstly,the recombinant transfer vector p Fast Bacdual-gp64sp-asp-gp64 ctd was constructed and transformed into E. coli DH10 Bac^TM. Then,the recombinant bacmid DNA was screened by blue-white spot screening and transfected into silkworm Bm N cells mediated by lipofectin. The obtained recombinant viruses were harvested and used to infect Bm N cells. At 72 h post-transfection,BVs of the recombinant viruses were harvested and purified by ultracentrifugation. By using the Nessler reagent,the activity of fusion protein ASP expressed in Bm N cells after infection with the recombinant viruses was determined to be 1 788. 4 U / mg. The fusion protein ASP was detected in purified BV by Western blotting,but hadalmost no enzymatic activity. The results show that the ASP fusion protein can be transferred into BV by the constructed baculovirus GP64 surface display system,but how to keep the enzyme activity needs further research.