Prediction and evolution of B cell epitopes of surface protein in SARS-CoV-2.

BACKGROUND: In order to obtain antibodies that recognize natural proteins, it is possible to predict the antigenic determinants of natural proteins, which are eventually embodied as polypeptides. The polypeptides can be coupled with corresponding vectors to stimulate the immune system to produce corresponding antibodies, which is also a simple and effective vaccine development method. The discovery of epitopes is helpful to the development of SARS-CoV-2 vaccine. METHODS: The analyses were related to epitopes on 3 proteins, including spike (S), envelope (E) and membrane (M) proteins, which are located on the lipid envelope of the SARS-CoV-2. Based on the NCBI Reference Sequence: NC_045512.2, the conformational and linear B cell epitopes of the surface protein were predicted separately by various prediction methods. Furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed, the sequences of the whole genome of SARS-CoV-2 were obtained from the GISAID. RESULTS: 7 epitopes were predicted, including 6 linear epitopes and 1 conformational epitope. One of the linear and one of the conformational consist of identical sequence, but represent different forms of epitopes. It is worth mentioning that all 6 identified epitopes were conserved in nearly 3500 SARS-CoV-2 genomes, showing that it is helpful to obtain stable and long-acting epitopes under the condition of high frequency of amino acid mutation, which deserved further study at the experiment level. CONCLUSION: The findings would facilitate the vaccine development, had the potential to be directly applied on the prevention in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus.

Characterization of a pathogenic variant in GBA for Parkinson's disease with mild cognitive impairment patients.

Parkinson's disease (PD) is the second most common neurodegenerative disease, and mild cognitive impairment (MCI) is a well-established risk factor for the development of dementia in PD. A growing body of evidence suggests that low expression of glucocerebrosidase (GBA) promotes the transmission of α-synuclein (α-Syn) interpolymers and the progression of PD. However, how GBA mutations affect the pathogenesis of PD via abnormal aggregation of α-Syn is unclear, and no clinically valid PD-MCI genetic markers have been identified. Here, we first located a GBA eQTL, rs12411216, by analysing DHS, eQTL SNP, and transcription factor binding site data using the UCSC database. Subsequently, we found that rs12411216 was significantly associated with PD-MCI (P < 0.05) in 306 PD patients by genotyping. In exploring the relationship between rs12411216 and GBA expression, the SNP was found to be associated with GBA expression in 50 PD patients through qPCR verification. In a further CRISPR/Cas9-mediated genome editing module, the SNP was identified to cause a decrease in GBA expression, weaken enzymatic activity and enhance the abnormal aggregation of α-Syn in SH-SY5Y cells. Additionally, using an electrophoretic mobility shift assay, we confirmed that the binding efficiency of transcription factor E2F4 was affected by the rs12411216 SNP. In conclusion, our results showed that rs12411216 regulated GBA expression, supporting its potential role as a PD-MCI genetic biomarker and highlighting novel mechanisms underlying Parkinson's disease.

[Preparation and identification of monoclonal antibody against human spermine oxidase].

AIM: To prepare mouse anti-human spermine oxidase (anti-hSMO) monoclonal antibody (mAb) and testify its application in the biological techniques including Western blotting and immunohistochemistry. METHODS: Plasmid pET-15b/SMO was first transferred into BL21 (DE3), and then SMO recombinant protein with 6×His tag was induced to express by IPTG and purified by Ni-NTA resin. The purified recombinant SMO was used to immunize BALB/c mouse. The spleen cells from the immunized mouse were harvested and hybridized with Sp2/0 myeloma cells to obtain a hybridoma cell line that could efficiently synthesize and secret anti-SMO mAb. The titer and antigen specificity of this antibody were identified by ELISA, Western blotting and immunohistochemistry. RESULTS: We successfully obtained the hybridoma cell line which could stably secret anti-SMO mAb. The mAb was of a high titer and antigen specificity and could be used in ELISA, Western blotting, and immunohistochemistry for SMO. CONCLUSION: The mouse anti-hSMO mAB with a high antigen specificity has been prepared successfully and used for a variety of bio-analytical techniques.