N protein used for detection of novel coronavirus and preparation and application thereof
A protein and biological material technology, applied in the field of immunity, can solve the problems of high laboratory cleanliness and operator requirements, expensive RT-PCR instruments, high requirements for detection equipment or platforms, etc., and achieves broad application prospects and good specificity , the effect of high sensitivity
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Embodiment 1
[0041] Example 1: Construction of N protein expression strain E.coli BL21-N
[0042] 1. Design of N protein coding gene
[0043] In this study, the gene and protein homology analysis of the key gene N protein coding gene of the new coronavirus (NCBI number YP_009724397.2) and 6 other respiratory infection viruses including SARS and MERS, including SARS and MERS, and influenza virus were carried out. The N protein of the new coronavirus has 91.2% homology with SARS N protein, 47.6% homology with MERS N protein, and 8.8%-33.5% homology with other virus N proteins. We cut out the homologous sequence of the N protein gene of the new coronavirus and other viruses, especially those that do not cause disease, or only cause mild colds, and take the N protein coding gene of the new coronavirus spliced in multiple segments of specific sequences, and the optimized new coronavirus N Proteins increase the specificity of antibody responses.
[0044] The amino acid sequence of N protein ...
Embodiment 2
[0051] Embodiment 2: N protein expression and purification
[0052] 1. Activation and cultivation of strain E.coli BL21-N
[0053] Pick three single bacterium colonies of E.coli BL21-N obtained in Example 1, add respectively to 30mL shake flasks equipped with 5mL LB liquid medium, add kanamycin according to the concentration of 50mg / L, place in a shaker, Cultivate at 180 rpm at 37°C for 10 hours; then, add 1% inoculum into a 500mL Erlenmeyer flask containing 300mL LB liquid medium, and add kanamycin at a concentration of 50mg / L, and culture at 180rpm at 37°C in a shaker.
[0054] 2. Induced expression and identification of N protein
[0055] E.coli BL21-N cultured to OD 600 When it is 0.6-0.8, add 1mM IPTG to induce, after 6 hours of induction, collect the fermentation broth, detect whether the N protein is expressed; and ultrasonically disrupt the bacterial cells to determine the expression form of the N protein, the results are as follows figure 2 shown.
[0056] Depend...
Embodiment 3
[0062] Example 3: Purified N protein is used for antibody detection of novel coronavirus pneumonia
[0063] 1. Detect the serum of patients with new coronavirus and do sensitivity analysis
[0064] The truncated N protein collected in Example 2 was diluted, and 0.2 μg / mL truncated N protein and full-length N protein were used to coat the microtiter plate respectively, and the serum of the new crown patient was detected by indirect ELISA.
[0065] The specific operation steps are: 10-fold dilution of the serum sample with phosphate buffer solution containing 0.05% Tween 20, adding 100 microliters of the sample, reacting in the dark at 37°C for 30 minutes, and washing 5 times with phosphate buffer solution containing 0.05% Tween 20 , 30 seconds each time, add 100 μL of horseradish peroxidase-labeled mouse anti-human IgG diluted 5000 times with phosphate buffer containing 0.05% Tween 20, and react in the dark at 37°C for 30 minutes, then wash with 0.05% Tween 20 Wash 5 times wit...
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