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目的 :利用 Bac- to- Bac HT杆状病毒系统在 Sf9昆虫细胞中表达庚型肝炎病毒 (HGV) NS3蛋白 ,并对表达产物的免疫原性进行研究。 方法 :将 HGV NS3基因片段定向克隆至转座载体 p Fast Bac HTa,转化 DH10 Bac感受态细胞 ,37℃振荡培养 4h使发生转座 ,用抗生素平皿筛选重组 bacmid。脂质体介导转染 Sf9昆虫细胞 ,待细胞形态明显改变后收获细胞和培养上清液 ,将上清液再次感染 Sf9细胞以大量表达 HGV NS3重组蛋白。利用 SDS- PAGE和 Western- blotting方法分析重组蛋白。结果 :SDS- PAGE分析发现表达产物在 Mr43810处有一条明显的蛋白带 ,占细胞总蛋白量的 30 %以上 ;应用 Ni- NTA亲和层析柱获得了纯化的重组蛋白 ;Western- blotting显示该抗原可与 HGV RNA阳性血清发生特异反应。 结论 :获得了昆虫细胞内表达的 HGV NS3重组蛋白 ,并证明该蛋白有可能用于 HGV感染的抗体检测。
OBJECTIVE: To express the hepatomegalovirus (HGV) NS3 protein in Sf9 insect cells by Bac-to-Bac HT baculovirus system and to study the immunogenicity of the expressed product. Methods: The HGV NS3 gene fragment was cloned into the transposable vector p Fast Bac HTa and transformed into competent cells of DH10 Bac. Transfection was performed by shaking at 37 ° C for 4h. The recombinant bacmid was screened by antibiotic plate. Lipofectamine was transfected into Sf9 insect cells. After the cell morphology was changed obviously, the cells and culture supernatant were harvested. The supernatant was re-infected with Sf9 cells to express large amount of HGV NS3 recombinant protein. The recombinant proteins were analyzed by SDS-PAGE and Western-blotting. Results: SDS-PAGE analysis showed that Mr43810 expressed a significant protein band, accounting for more than 30% of the total cellular protein; purified recombinant protein was obtained by Ni-NTA affinity chromatography; Western-blotting showed that Antigen can react specifically with HGV RNA positive sera. Conclusion: The HGV NS3 recombinant protein expressed in insect cells was obtained and proved that the protein may be used for the detection of HGV infection.