Hüssy P, Faust H, Wagner J C, Schmid G, Mous J, Jacobsen H
PRP/Gene Technology, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
J Hepatol. 1997 Jun;26(6):1179-86. doi: 10.1016/s0168-8278(97)80450-3.
BACKGROUND/METHODS: The two envelope proteins of hepatitis C virus, E1 and E2, were expressed in E. coli and, as secretory proteins, in Sf9 insect cells using recombinant baculoviruses. Co-infection of insect cells with E1 and E2-recombinant baculoviruses was performed, which has been shown to result in formation of E1-E2 dimers. All envelope proteins were purified by Ni2+-NTA chromatography and used for screening of serum samples in a HCV EIA assay. Serum samples of normal blood donors, chronically HCV-infected patients, a mixed titer panel and several seroconversion panels were screened and compared to test results with Cobas Core Anti-HCV EIA.
Screening of the sera of chronically HCV-infected patients (100% positive in Cobas Core Anti-HCV EIA) revealed 10-40% anti-E1 positive sera using different Sf9-expressed, glycosylated proteins and 93% using E. coli-expressed, non-glycosylated E1 protein. When the same sera were tested with different E2 proteins expressed in Sf9 cells and in E. coli, about 70-73% showed anti-E2 reactivity. When the proteins from Sf9 cells co-infected with E1- and E2-recombinant baculoviruses were tested, 70-80% of the same sera showed anti-envelope reactivity.
Testing of these patient antisera, and those from the well-characterized mixed titer panel BBI-PHV203, showed that recombinant E1 expressed in E. coli and co-expressed E1 and E2 proteins from Sf9 cells could be used as additional tools for anti-HCV antibody screening.
背景/方法：丙型肝炎病毒的两种包膜蛋白E1和E2在大肠杆菌中表达，并利用重组杆状病毒作为分泌蛋白在Sf9昆虫细胞中表达。用E1和E2重组杆状病毒共感染昆虫细胞，已证明这会导致E1-E2二聚体的形成。所有包膜蛋白均通过Ni2+-NTA色谱法纯化，并用于丙型肝炎病毒酶免疫分析中血清样本的筛选。对正常献血者、慢性丙型肝炎病毒感染患者的血清样本以及一个混合滴度样本组和几个血清转化样本组进行筛选，并与Cobas Core抗-HCV酶免疫分析的检测结果进行比较。
对慢性丙型肝炎病毒感染患者的血清（Cobas Core抗-HCV酶免疫分析中100%呈阳性）进行筛选，结果显示，使用不同的Sf9表达的糖基化蛋白时，10%-40%的血清抗-E1呈阳性，而使用大肠杆菌表达的非糖基化E1蛋白时，93%的血清抗-E1呈阳性。当用在Sf9细胞和大肠杆菌中表达的不同E2蛋白检测相同血清时，约70%-73%的血清显示出抗-E2反应性。当检测用E1和E2重组杆状病毒共感染的Sf9细胞中的蛋白时，相同血清中有70%-80%显示出抗包膜反应性。
对这些患者抗血清以及来自特征明确的混合滴度样本组BBI-PHV203的抗血清进行检测，结果表明，在大肠杆菌中表达的重组E1以及在Sf9细胞中共表达的E1和E2蛋白可作为抗-HCV抗体筛选的额外工具。
