HBsAg/GM-CSF融合蛋白的克隆及其在毕赤酵母中的表达

Cloning and Expression of HBsAg/GM-CSF Fusion Gene in Pichia pastoris

目的在毕赤酵母中表达HBsAg/GM-CSF融合蛋白。方法利用PCR扩增HBsAg和GM-CSF基因,通过15个氨基酸的连接肽将两个片段连接,获得融合基因S-GM,克隆入酵母穿梭质粒pPIC9K中。将重组质粒9K-S-GM电转化毕赤酵母后,G418筛选,甲醇诱导,HBsAg/GM-CSF融合蛋白表达。经SDS-PAGE检测表达水平,Western blot检测表达产物特异性。结果PCR扩增的片段与预期大小一致,HBsAg/GM-CSF融合蛋白在毕赤酵母中获得了表达。Western blot检测,该融合蛋白同时具有HBsAg和GM-CSF的特异性。结论该融合蛋白的获得为提高乙肝疫苗的免疫原性奠定了科学基础。

Objective To express HBsAg/GM-CSF fusion protein in Pichia pastoris. Methods Amplify HBsAg and GM-CSF genes by PER and link the two gene fragments by a linker consisting of 15 amino acids. Clone the obtained fusion gene fragment S-GM into yeast shuttie plasmid pPIC9K. Transform the recombinant plasmid 9K-S-GM to Pichia pastoris by electrotransformation, screen positive dorms with G418 and express HBsAg/GM-CSF fusion protein under induction of methanol. Determine the expression level of fusion protein by SDS-PAGE and the specificity by Western blot. Results The gene fragments with the expected lengths were amplified, and HBsAg/GM-CSF fusion protein was successfully expressed in Pichia pastoris. The expressed fusion protein showed specificities of both HBsAg and GM-CSF as proved by Western blot, Conclusion The expression of HBsAg/GM-CSF laid a foundation of improvement of immunogenicity of hepatitis B vaccine.