利用家蚕-HyNPV表达系统表达鼠抗人CD28分子单链抗体

Expression of Mouse Anti-human CD28 Single Chain Antibody Using Bombyx mori HyNPV Expression System

人CD28分子是T细胞表面的最重要的协同刺激分子。将抗人CD28的鼠源单链抗体基因(mouse anti-human CD28-ScFv)亚克隆至杂交病毒HyNPV Bac-to-Bac系统供体质粒pFastBacHTa中,获得重组转移载体pFast-BacHTa CD28-ScFv,并转化大肠杆菌(E.coli)DH10Bac获得重组杆粒rBacmid CD28-ScFv,脂质体介导转染Sf9昆虫细胞后获得重组杆状病毒rBV CD28-ScFv。该重组杆状病毒注射接种于家蚕5龄起蚕,经SDS-PAGE和Westernblotting分析表明,CD28-ScFv在家蚕体内得到了表达,即:人CD28单链抗体通过HyNPV这一宿主范围扩大的新型杂交杆状病毒表达载体在家蚕中获得了表达。

Human CD28 molecule is one of the most important antigen in T cell surface. In present study, the mouse anti-human CD28 single chain antibody （ScFv） gene was subcloned into the Bac-to-Bac donor plasmid pFastBacHTa of the hybrid virus HyNPV, and the recombinant plasmid pFastBacHTa CD28-ScFv was transformed into E. coil DH10Bac competent cells. The Bacmid rBacmid CD28-ScFv was obtained by screening of white plaque assay and identification by PCR. The Sf9 cells were transfected with the rBacmid CD28-ScFv DNA and the recombinant baculovirus rBV CD28-ScFv was harvested. Newly molted Bombyx mori larvae of the fifth star were inoculated with the recombinant baculovirus. Both SDS-PAGE and Western blotting analysis showed specific bands, implying that human CD28-ScFv gene had been successfully expressed in silkworm bodies through using the host-range expanded new hybrid baculovirus expression vector HyNPV.