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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 make comparison of duration of subarachnoid block with intrathecal ropivacaine between gravidas with diabetes mellitus and non-diabetic pregnancy, to evaluate the sensitivity of parturient with diabetes to ropivacaine. Methods 75 parturients who were presenting for elective cesarean section were randomly divided into pregestational diabetes mellitus group (group P, n=15) , gestational diabetes mellitus group (group G, n=30) and non-diabetic parturients group (group N, n=30). After entering the operating room, parturients were given spinal anesthesia spinal at the L3～4 interspace with 0.5％ hyperbaric ropivacaine 3 ml with left lateral decubitus position. To determine the level of sensory block by 10 g monofilament and evaluate the motor block with modified Bromage score. To record the time T6 sensory level was obtained, the onset time of sensory block, motor block, the duration of the motor block and sensory block. Results The time T6 sensory level was obtained of Group P were significantly shortened (P < 0.001). Compared with Group N and Group G, the duration of sensory (P < 0.001) and motor (P < 0.001) block were significantly prolonged. Conclusion Parturients with pregestational diabetes mellitus are more sensitive to 0.5％ hyperbaric ropivacaine compared to non-diabetic parturients. Compared with non-diabetic parturients, there are no difference in the sensitivity of parturient with gestational diabetes mellitus to 0.5％hyperbaric ropivacaine.