摘要
目的构建可稳定表达脊髓灰质炎病毒(poliovirus,PV)类病毒颗粒(virus-like particles,VLPs)的整合型重组毕赤酵母,鉴定PV VLPs在毕赤酵母细胞中的表达及组装情况。方法根据毕赤酵母密码子偏好性优化salk株II型P1和3CD基因并连接到p Pic ZA载体,构建p Pic ZA-P1-3CD表达载体;用Bgl II线性化p Pic ZA-P1-3CD载体,电转至毕赤酵母GS115中。通过Zeocin抗性筛选获得整合型重组毕赤酵母,随后用高浓度Zeocin抗性筛选得到高表达菌株。甲醇诱导后,用Western Blot检测目的蛋白表达;蔗糖密度梯度离心纯化PV VLPs并进行透射电镜观察。结果成功构建p Pic ZA-P1-3CD表达载体,获得PV VLPs重组毕赤酵母。Western Blot在重组毕赤酵母裂解上清中检测到目的蛋白的表达;蔗糖密度梯度离心纯化后,在透射电镜中观察到直径为30 nm左右的VLPs,其形态与天然的PV颗粒相似。结论成功构建PV-2型VLPs的整合型重组酵母系统,并在毕赤酵母中组装形成了VLPs,为酵母表达系统中PV VLPs疫苗的研制奠定了基础。
Objective To construct an integrated recombinant Pichia Pastoris（ P. pastoris） with a stable expression of poliovirus virus-like particles（ PV VLPs）,and identification of the expression and assembly for VLPs. Methods According to P. pastoris codon preferences,the optimazation of salk II type P1 and 3 CD genes were carried out and synthesized,then cloned into p Pic ZA expression vector to construct p Pic ZA-P1-3 CD expression vector. After linearized by Bgl II endonucleases,the recombinant plasmid p Pic ZA-P1-3 CD was transformed into P. pastoris GS115 strain by using electroporation. Integrated recombinant P. pastoris colonies were screened with a low concentration of Zeocin,while high expression P. pastoris colonies were obtained by a high-resistance screening. After methanol induction,the expression of target protein was detected by western blot. Purification of PV was performed by sucrose density gradient centrifugation,and observation VLPs by transmission electron microscopy. Results The p Pic ZA-P1-3 CD recombinant plasmid was successfully constructed and expressed PV VLPs in recombinant P.pastoris were obtained. The expression of the target protein in the recombinant P. pastoris supernatant was detected by western blot. After purification by the sucrose density gradient centrifugation,VLPs with a diameter of about 30 nm were observed under the transmission electron microscope,which are similar in morphology to natural PV virus particles. Conclusion PV VLPs expression in recombinant P. pastoris system was successfully constructed and VLPs were assembled in recombinant P. pastoris,on which a foundation was set up in development of PV VLPs vaccines in yeast expression systems.
作者
赵辉
温晔
彭少丹
晏巧玲
马金
朱涛
ZHAO Hui;WEN Ye;PENG Shao-dan;YAN Qiao-ling;MA Jin;ZHU Tao(College of Biotechnology Tianjin University of Science ＆ Technology,Tianjin 300457,China)
出处
《微生物学免疫学进展》
2018年第4期7-13,共7页
Progress In Microbiology and Immunology
基金
国家"重大新药创制"科技重大专项资助项目(2015ZX09102009)
关键词
脊髓灰质炎病毒
毕赤酵母
蔗糖密度梯度离心
病毒样颗粒
Poliovirus
Pichia Pastoris
Sucrose density gradient centrifugation
Virus-like particles （VLPs）
