Using immunohistochemical technique, the distribution and development of substance P (SP)-, L-enkephalin (L-Enk)- and serotonin (5-HT)-like immunoreactivity in Gudden's dorsal tegmental nucleus (GDTN) of human foetus (fetal age 11.5 to 35 weeks)and neonate (two days) were demonstrated. Gudden's dorsal tegmental nucleus in the human might be divided into pars centralis and pars peripheralis; and the time of initial appearance for SP-, L-Enk- and 5-HT-like immunoreactivity in GDTN was different. SP-like immunoreactive fibers and terminals appeared first and were distributed mainly in the pars centralis and their density was very high. L-Enk-like imunoreactive cells, fibers and terminals were distributed in the pars peripheralis. 5-HT-like immunoreactive cells, fibers and terminals were distributed in the medial part of the GDTN; As the foetus developed further, SP-, L-Enk- and 5-HT-like immunoreactivity in the GDTN showed their own special ontogenetic changes respectively

Nissl stain and immunocytochemical methods were used to observe the structure of the subnuclei of interpeduncular nucleus in human neonatal brain and their localization of substance P-, leu enkephalin-, and serotonin (5-HT)-like immunoreactive elements. It was detected that the human neonatal interpeduncular nucleus could be divided into five subnuclei, including: the dorsal, dorsolateral, lateral, central, and intermediate subnuclei. The immunocytochemical results showed that the substance P-containing cell bodies and fibers or terminals were distributed chiefly in the dorsolateral, lateral subnuclei and the ventral part of the intermediate subnucleus; the enkephalin-containing cell bodies and fibers or terminals were concentrated in the central subnucleus; and meanwhile the serotonin-containing cell bodies and fibers or terminals were found mainly in the dorsal subnucleus.

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.