Optimization of SARS-CoV-2 spike protein receptor binding domain expression in Pichia pastoris and evaluation of its immunogenicity
10.3760/cma.j.cn112309-20220224-00058
- VernacularTitle:新型冠状病毒刺突蛋白受体结合域在毕赤酵母中的分泌表达优化及其免疫原性研究
- Author:
Dongdong HU
1
;
Jiaduo SUN
;
Ziyan WANG
;
Haitao LIU
;
Yiran SUN
;
Dawei QIAN
;
Dong LI
;
Rongjun CHEN
;
Jiao AN
;
Chenliang ZHOU
;
Ge LIU
;
Jiang FAN
;
Yuanxiang JIANG
Author Information
1. 上海泽润生物科技有限公司研发部，上海 201203
- Keywords:
SARS-CoV-2;
Spike protein;
Receptor binding domain;
Pichia pastoris;
Recombinant protein vaccine;
Alum+ CpG adjuvant
- From:
Chinese Journal of Microbiology and Immunology
2022;42(7):520-526
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.
