Li Jun-Wu, Liu Yan, Li Ke, Song Dong, Ye Qiu-Ping
Microbiology and Immunology Department, Medical College, Jinan University, Guangzhou, China.
Hepatogastroenterology. 2010 May-Jun;57(99-100):578-82.
BACKGROUND/AIMS: To construct the yeast expression vector containing cDNA sequence coding L protein of Hepatitis B Virus (HBV) and human granulocyte-macrophage colony stimulating factor (hGM-CSF), and to explore the method of secretory expression in Pichia Pastoris GS115 strain.
We used pVAX1-L-hGM as template to amplify L-hGM gene by PCR, and then the PCR products were cloned into the yeast expression vector pPICZaC with DNA recombination method. After linearized by Sac I, the recombinant plasmid pPICZa C-L-hGM was transformed into Pichia Pastoris GS115 by electrophoresis. The expressing protein was induced by methanol. SDS-PAGE and Western blot were used to analyze the expression of protein.
DNA sequence analysis revealed that the constructed genes sequences were totally consistent with the GeneBank reported. The results of SDS-PAGE and Western blot showed that the recombinant protein was induced by methanol and stably expressed in Pichia Pastoris.
The successful construction of a recombinant yeast expression vector containing gene coding L protein of Hepatitis B virus and hGM-CSF gene, and expressed in Pichia Pastoris, of which lays a foundation for the further researches on a better protective HB vaccine.
背景/目的：构建含乙型肝炎病毒（HBV）L蛋白编码cDNA序列与人粒细胞巨噬细胞集落刺激因子（hGM-CSF）的酵母表达载体，并探索在毕赤酵母GS115菌株中进行分泌表达的方法。
以pVAX1-L-hGM为模板，通过PCR扩增L-hGM基因，然后采用DNA重组方法将PCR产物克隆至酵母表达载体pPICZaC。经Sac I线性化后，将重组质粒pPICZa C-L-hGM通过电泳转化至毕赤酵母GS115中。用甲醇诱导表达蛋白。采用SDS-PAGE和Western blot分析蛋白表达情况。
DNA序列分析显示构建的基因序列与GenBank报道的完全一致。SDS-PAGE和Western blot结果表明重组蛋白经甲醇诱导后在毕赤酵母中稳定表达。
成功构建了含乙型肝炎病毒L蛋白编码基因和hGM-CSF基因的重组酵母表达载体，并在毕赤酵母中表达，为进一步研究更好的乙肝保护性疫苗奠定了基础。
