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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.
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Objective:To summarize experience and effect of the stylized operation step in the neck endoscopic thyroidectomy for papillary thyroid microcarcinoma (PTMC).Methods:The clinical data of 51 patients who underwent transthoracic breast approach scarless neck endoscopic thyroidectomy (SET) from January 2018 to September 2019 in Tungwah Hospital Affiliated to Sun Yat-sen University were retrospectively analyzed. The operative procedure used stylized operation steps: establishment of operating space, three-step method thyroidectomy, central lymph node dissection (CLND), close operating space.Results:Among 51 patients, 25 cases underwent unilateral thyroidectomy plus isthmic resection, 26 cases underwent total/near-total thyroidectomy, and 16 cases underwent preventive CLND. The recurrent laryngeal nerve (RLN) was exposed in all 51 cases, the external branches of the superior laryngeal nerve (EBSLN) was exposed in 35 cases, no procedure was converted into open neck incision thyroidectomy. Forty-two cases were followed up, 2 cases had postoperative hoarseness and unilateral recurrent laryngeal nerve paralysis, which were recovered 55 and 75 d after operation respectively. There was no bucking, voice blunt or permanent low calcium. One patient had abnormal sensation of neck skin after operation, and recovered gradually 3 months after operation.Conclusions:The stylized operation step in the SET can increase safety of surgery.