[Preparation and immungenicity of recombinant protein containing intramolecular adjuvant in SARS-CoV-2 RBD domain].

A fusion protein containing a tetanus toxin peptide, a tuftsin peptide and a SARS-CoV-2S protein receptor-binding domain (RBD) was prepared to investigate the effect of intramolecular adjuvant on humoral and cellular immunity of RBD protein. The tetanus toxin peptide, tuftsin peptide and S protein RBD region were connected by a flexible polypeptide, and a recombinant vector was constructed after codon optimization. The recombinant S-TT-tuftsin protein was prepared by prokaryotic expression and purification. BALB/c mice were immunized after mixed with aluminum adjuvant, and the humoral and cellular immune effects were evaluated. The recombinant S-TT-tuftsin protein was expressed as an inclusion body, and was purified by ion exchange chromatography and renaturated by gradient dialysis. The renaturated protein was identified by Dot blotting and reacted with serum of descendants immunized with SARS-CoV-2 subunit vaccine. The results showed that the antibody level reached a plateau after 35 days of immunization, and the serum antibody ELISA titer of mice immunized with recombinant protein containing intramolecular adjuvant was up to 1:66 240, which was significantly higher than that of mice immunized with S-RBD protein (P < 0.05). At the same time, the recombinant protein containing intramolecular adjuvant stimulated mice to produce a stronger lymphocyte proliferation ability. The stimulation index was 4.71±0.15, which was significantly different from that of the S-RBD protein (1.83±0.09) (P < 0.000 1). Intramolecular adjuvant tetanus toxin peptide and tuftsin peptide significantly enhanced the humoral and cellular immune effect of the SARS-CoV-2 S protein RBD domain, which provideda theoretical basis for the development of subunit vaccines for SARS-CoV-2 and other viruses.

Preparation of different fragments of SARS-co V-2 urine protein and its application in fluorescent layer system

Expression and purification of different fragments of the new coronavirus nucleocapsid (N) protein, establish a new coronavirus total antibody fluorescence immunochromatographic method and evaluate the influence of different protein fragments on the method. Using bioinformatics technology to analyze, synthesize, express and purify the N protein sequence, prepare different N protein fragments;use 1-ethyl-(3-dimethylaminopropyl) carbodiimide (1-( 3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) method of fluorescent microspheres coupled with antigen was established to establish a sandwich fluorescence chromatography antibody detection method, and the performance was evaluated respectively. In the prepared 4 N protein fragments, the full-length N protein (N419) is preferably coated, and N412 is labeled with 0.5mol/L NaCl as the optimal combination;the 91-120th amino acid (N412) of the N-terminus of the N antigen is deleted It can reduce 87.5% of non-specific interference;the linear range is 0.312-80U/L, the lowest detection limit is 0.165U/L, and the accuracy is above 95%. The fluorescence immunochromatographic detection method for total antibodies of the new coronavirus established by pairing the N protein fragments has a total coincidence rate of 98% compared with the Guangzhou Wanfu test strip. The improvement provides experimental basis and reference.

[Preparation of different fragments of SARS-CoV-2 N protein and its application in fluorescence chromatography].

Different fragments of SARS-CoV-2 nucleocapsid (N) protein were expressed and purified, and a fluorescence immunochromatography method for detection of SARS-CoV-2 total antibody was established. The effect of different protein fragments on the performance of the method was evaluated. The N protein sequence was analyzed by bioinformatics technology, expressed in prokaryotic cell and purified by metal ion affinity chromatography column. Different N protein fragments were prepared for comparison. EDC reaction was used to label fluorescence microsphere on the synthesized antigen to construct sandwich fluorescence chromatography antibody detection assay, and the performance was systemically evaluated. Among the 4 prepared N protein fragments, the full-length N protein (N419) was selected as the optimized coating antigen, N412 with 0.5 mol/L NaCl was used as the optimal combination; deleting 91-120 amino acids from the N-terminal of N412 reduced non-specific signal by 87.5%. the linear range of detection was 0.312-80 U/L, the limit of detection was 0.165 U/L, and the accuracy was more than 95%. A fluorescence immunochromatographic detection method for analysis of SARS-CoV-2 total antibody was established by pairing N protein fragments. The detection result achieved 98% concordance with the commercially available Guangzhou Wanfu test strip, which is expected to be used as a supplementary approach for detection of SARS-CoV-2. The assay could also provide experimental reference for improving the performance of COVID-19 antibody detection reagents.