Optimization of Expression Conditions for Production of Anti-colorectal Cancer Monoclonal Antibody CO17-1A in Baculovirus-insect Cell System
Optimization of Expression Conditions for Production of Anti-colorectal Cancer Monoclonal Antibody CO17-1A in Baculovirus-insect Cell System
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DOI：
10.1089/hyb.2011.0049
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发表时间：
2011-10-01
期刊：
影响因子：
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通讯作者：
Ko, Kisung
中科院分区：
文献类型：
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作者：
Park, Da-Young;Lee, Jung-Hwan;Ko, Kisung
杆状病毒-昆虫细胞系统由于其高容量、灵活性和糖基化能力等优点，被认为是一种可行的重组糖蛋白表达系统。然而，为了确保重组杆状病毒在昆虫细胞中的高表达，需要使用商业和昂贵的免疫测定滴度试剂盒对重组杆状病毒进行准确的滴度测定，其中加利福尼亚多核多角体病毒（AcMNPV）型杆状病毒的包膜糖蛋白通过抗包膜糖蛋白抗体和辣根过氧化物酶（HRP）偶联的二抗进行检测。在本研究中，优化了CO17-1A免疫治疗单克隆抗体（MAb）在杆状病毒系统中对结直肠癌细胞的表达条件，而不使用商业滴度试剂盒。为了优化抗体在杆状病毒-昆虫细胞系统中的表达，研究了几个变量，包括杆状病毒传代，感染杆状病毒接种量（100、200、400和800 μ L），以及病毒感染后昆虫细胞或细胞上清液的收获时间（24、48和72 h）。构建了两种不同的pFastBac载体，分别携带CO17-1A单抗基因，有或没有KDEL内质网（ER）保留基元（Lys-Asp-Glu-Leu）与HC（分别为MAb CO17-1A K和MAb CO17-1A）融合，并用于生成杆状病毒。免疫印迹分析证实了MAb CO17-1A K和MAb CO17-1A在杆状病毒感染的昆虫细胞中的表达。采用密度分析法定量分析不同条件下蛋白条带的相对表达量。感染400 mL传代3杆状病毒（P-3 BV）后72h，携带编码CO17-1A MAb的基因的裂解细胞中无KDEL表达量最高。这些结果表明，在杆状病毒-昆虫细胞系统中，无需使用杆状病毒elisa滴度试剂盒，即可修改感染条件、病毒传代数、杆状病毒接种量和收获时间，以优化单抗的表达。
The baculovirus-insect cell system is considered a feasible expression system for recombinant glycoprotein production due to its several advantages, including high capacity, flexibility, and glycosylation capability. However, accurate titering of the recombinant baculovirus is required to ensure high expression in insect cells using a commercial and expensive immunoassay titer kit in which the envelope glycoprotein of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-type baculovirus is detected by anti-envelope glycoprotein antibody and a secondary antibody conjugated to horseradish peroxidase (HRP). In this study, conditions for the expression of the CO17-1A immunotherapeutic monoclonal antibody (MAb) against colorectal cancer cells in a baculovirus system were optimized without using a commercial titering kit. Several variables were investigated to optimize antibody expression in a baculovirus-insect cell system, including baculovirus passage, volume of the infecting baculovirus inoculum (100, 200, 400, and 800 mu L), and the harvest time of insect cells or cell supernatants after virus infection (24, 48, and 72 h). Two different pFastBac vectors carrying the CO17-1A MAb genes with or without the KDEL endoplasmic reticulum (ER) retention motif (Lys-Asp-Glu-Leu) fused to the HC (MAb CO17-1A K and MAb CO17-1A, respectively) were constructed and used to generate baculoviruses. Immunoblot analysis was conducted to confirm expression of MAb CO17-1A K and MAb CO17-1A in baculovirus-infected insect cells. Densitometry analysis of the protein bands was used to quantify the relative expression under different conditions. The highest expression was observed in lysed cells infected with 400 mL of passage 3 baculovirus (P-3 BV) carrying the gene encoding the CO17-1A MAb without KDEL at 72 h after virus infection. These results suggest that the infection conditions, the number of virus passages, baculovirus inoculum volume, and the harvest time can be modified to optimize MAb expression without using a BaculoELISA titer kit in a baculovirus-insect cell system.