Objective To investigate the influence of streptozocin (STZ)'s dose on the inductive effect of diabetes in C57BL/6J mice, and investigate the dose-effect relationship and the optimal dose range. Methods 145 C57BL/6J mice were randomly divided into 9 diabetic groups (group A to group 1, n = 15 in each group) and I control group (n = I0) to receive intraperitoneal injection of STZ with the dosages of 30, 60, 80, 100, 120, 150, 180, 210, 240 mg/kg and same amount of buffer solution,respectively. Changes of blood glucose, body weight, survival rate at 45 day and serum insulin level were monitored, and the relationship with STZ doses was analyzed. Pancreas and kidneys of the mice were removed for morphological examination, and immunohistochemistry was used for determination of insulin in pancreas and CD<,68> in kidneys. Results Compared with control group, blood glucose in group C ～G increased significantly; body weigh, insulin level decreased significantly (P < 0.05), and the STZ dose was positively correlated with mean blood glucose (r = -0.984, P < 0.05) and was negatively correlated with mean serum insulin levels (r = 0.994, P <0.05). The diabetes modeling rates in group C ～ G (86.7% ～ 100%) were higher than those of group A and B (0 and 40%, P<0.05). At the 45th day, the survival rates of group C ～G (46.7% ～ 73.3%) were higher than those of group H and 1 (13.3% and 0, P <0.05). There was no obvious injury of pancreas and kidneys in group B, whereas, in group C and G, pancreatic island atrophy and decreased insulin secretion were observed; deposits of extracellular matrix and macrophage increased in the mesangium were also present. Conclusions 80 ～ 180 mg/kg of STZ dose was ideal for establishing diabetes model in C57BL/6J mice. Within this range, the modeling rate and survival rate was higher, and target organs injury was typical. The STZ dose was positively correlated with blood glucose and negatively correlated with serum insulin levels.

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.