重组HBsAg在汉逊酵母中的分泌表达

目的构建重组乙型肝炎表面抗原(HBsAg)分泌型汉逊酵母工程菌株,并进行诱导表达。方法在HBsAg基因前加酿酒酵母α前导肽(MFα)基因序列作为信号肽,将整个序列优化为汉逊酵母优选密码子,采用搭桥PCR方法分别合成后,通过1对引物PCR法融合在一起。将融合基因插入汉逊酵母穿梭质粒pDGXHP2.0的甲醇氧化酶基因(MOX)启动子下游,构建成多拷贝分泌型重组表达载体。将其电转化多型汉逊酵母尿嘧啶缺陷型宿主菌ATCC34438(Ura3-),筛选分泌表达HBsAg的汉逊酵母工程菌株,并比较在YPD、BMMY、MM3种培养基中分泌表达HBsAg的水平,ELISA检测培养上清液中HBsAg的表达量,透射电镜观察类病毒颗粒(VLPs)。结果构建了HBsAg汉逊酵母多拷贝表达质粒pDGXHP2.0-2MFα-HBsAg和pDGXHP2.0-4MFα-HBsAg,筛选获得了分泌表达HBsAg的汉逊酵母HP/2MFα-HBsAg和HP/4MFα-HBsAg。经诱导,工程菌株在YPD培养基中分泌表达的HBsAg量高于在BMMY和MM中的表达量;ELISA定量检测表达量最高可达10μg/ml,培养上清液经透射电镜观察,形成了VLPs。结论HBsAg在汉逊酵母中能够分泌表达,并能形成VLPs,但表达量低于胞内表达量。

Secretory Expression of Recombinant HBsAg in Hansenula polymorpha

Objective To construct a recombinant Hansenula polymorpha strain for secretory expression of HBsAg.Methods The α-factor prepro peptide(MFα)from Saccharomyces cerevisiae was used as a signal peptide to secret HBsAg in Hansenula polymorpha.Both MFα and HBsAg genes were designed based on Hansenula polymorpha-preferred condons and synthesized by overlap PCR respectively,then fused together by PCR using a pair of primers.The fusion gene was inserted downstream to the MOX promoter of Hansenula polymorpha shuttle plasmid pDGXHP2.0,and the constructed multicopy secretory recombinant plasmid was transformed to Hansenula polymorpha ATCC34438(Ura3-)by electroporation.The recombinant Hansenula polymorpha strains for secretory expression of HBsAg were screened and cultured in YPD,BMMY and MM media respectively.The expression levels of HBsAg were determined by ELISA,and the virus-like particles(VLPs)were observed by transmission electron microscopy.Results Multicopy recombinant plasmids pDGXHP2.0-2MFα-HBsAg and pDGXHP2.0-4MFα-HBsAg were constructed.Three recombinant Hansenula polymorpha strains for secretory expression of HBsAg were screened.After induction,the expression level of HBsAg in the recombinant strains cultured in YPD medium was significantly higher than those in BMMY and MM media.The expression level of HBsAg reached 10 μg / ml at most,and VLPs were observed in culture supernatant of recombinant strain.Conclusion HBsAg was expressed in a secretory form in Hansenula polymorpha,and VLPs were formed.However,the HBsAg level was lower than that of intracellular expression.