Secretory expression of human thyroid stimulating hormone receptor A subunit in sf9 insect cell system
- VernacularTitle: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
- Author:
Meng ZHANG
1
;
Kaining ZHANG
2
;
Ziyi CHEN
1
;
Ling WANG
1
;
Liping WU
1
;
Yue WANG
1
;
Bing LIU
2
;
Bingyin SHI
1
Author Information
- Publication Type:Journal Article
- Keywords: human thyroid stimulating hormone receptor A subunit; insect cell system; secretory expression; sf9 cell; Graves’ disease
- From: Journal of Xi'an Jiaotong University(Medical Sciences) 2023;44(3):409-414
- CountryChina
- Language:Chinese
- Abstract: 【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 2×107 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.
