人促甲状腺激素受体A亚单位蛋白在昆虫细胞体系中的分泌性表达

Secretory expression of human thyroid stimulating hormone receptor A subunit in sf9 insect cell system

目的拟构建昆虫细胞分泌表达体系,在草地夜蛾卵巢细胞分泌性促甲状腺激素受体(thyroid stimulating hormone receptor,TSHR)A亚单位蛋白。方法将TSHR A亚单位蛋白(22-289)和绿色荧光蛋白(green fluorescence protein,GFP)基因连接到质粒,并通过转化、蓝白斑筛选获得重组杆粒,将其转染入sf9昆虫细胞收获重组杆状病毒,扩增并测定滴度。最终通过蛋白印迹实验鉴定重组蛋白的表达并优化表达条件。结果在蛋白表达体系的构建过程中,PCR鉴定和测序均证实了重组质粒和重组杆粒序列的正确性。将重组杆粒转染入细胞后观察到病毒出芽迹象,空斑实验鉴定P1代病毒的滴度为2×10^(7) pfu/m。蛋白印迹显示重组蛋白分子质量为55 ku,主要在培养基中。蛋白表达的最佳感染复数(multiplicity of infection,MOI)为1,最佳感染时程为72 h。结论本研究构建了TSHR A亚单位的昆虫细胞杆状病毒表达体系,该体系能够外泌性表达分子质量为55 ku左右的蛋白TSHR 22-289,且蛋白能够成功的糖基化修饰。该系统为其工业化平台的建设及生产提供前期基础,也针对日后TSHR蛋白的研究和Graves病的病因预防提供了有用的工具。

Objective To construct the secretory expression system of insect cells to express the secretory TSHR A subunit protein in the ovarian cells of Spotoma oryzae(sf9).Methods A recombinant plasmid containing the target protein was constructed,and then the positive bacmid was screened out by the blue and white spots experiment.The verified bacmid was transfected into SF9 insect cells to obtain recombinant baculovirus.The virus was amplified,and the titer level was detected by virus plaque assay.Finally,Western blotting was used to identify the expression of the recombinant protein and optimize the expression conditions.Results During the construction of the protein expression system,PCR identification and sequencing results confirmed the correctness of the sequences of the recombinant plasmid and the recombinant bacmid.After the transfection of the bacmid,the signs of virus budding were observed in sf9 cells.The virus was collected and amplified.The titer of P1 generation virus was 2×10^(7)pfu/m according to the plaque assay.The recombinant protein was identified by Western blotting and confirmed to be exogenous into the culture medium.The optimal condition for virus infection and protein expression was 72 h after the infection when the multiplicity of infection(MOI)was 1.Conclusion We constructed an insect cell expression system secreting TSHR 22-289(55 ku),and the protein could be successfully glycolyzed.This system provides a preliminary basis for the construction and production of its industrial platform and also provides a useful tool for studies on TSHR protein and prevention of GO in the future.