Novel coronavirus N protein dominant epitope antigen and application thereof
A dominant epitope, protein technology, applied in the direction of antiviral immunoglobulin, virus, viral peptide, etc., can solve the problem of poor specificity of new coronavirus detection, and achieve the effect of improving specificity and avoiding false positives
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Embodiment 1
[0025] Example 1 Screening and preparation of novel coronavirus (SARS-CoV-2) N protein dominant epitope antigen
[0026] 1.1利用生物信息学手段,结合以往经验,筛选出来的SARS-CoV-2的N蛋白优势表位抗原的氨基酸序列为:ERSGARSKQR RPQGLPNNTASWFTALTQHG KEDLKFPRGQ GVPINTNSSPDDQIGYYRRA TRRIRGGDGK MKDLSPRWYF YYLGTGPEAG LPYGANKDGI IWVATEGALN TPKDHIGTRNPANNAAIVLQ LPQGTTLPKG FYAEGSRGGS QASSRSSSRS RNSSRNSTPG SSRGTSPARMAGNGGDAALALLLLDRLNQL ESKMSGKGQQ QQGQTVTKKS AAEASKKPRQ KRTATKAYNV (SEQ ID NO.1)
[0027] 1.2 Expression vector construction: Shanghai Sangon synthesized the nucleotide encoding the above amino acid sequence of SEQ ID NO.1 and ligated it between BamHI and XhoI of pET28a to obtain the PET28a-Nx plasmid
[0028] 1.3 Transform the constructed PET28a-Nx plasmid into BL21(DE)3 according to the routine test method of the second edition of "Molecular Cloning" for routine expression and purification. Specific steps: take the Escherichia coli BL21 (DE3) strain stored at -80°C, activate and culture it, and prepare competent ce...
Embodiment 2
[0052] Example 2 Using the N1-N8 proteins expressed and purified above as antigens to immunize rabbits (routine procedure)
[0053] Specific steps are as follows:
[0054] (1) Dissolve the antigenic protein in physiological saline (dissolved as 0.8mg / mL antigenic solution);
[0055] (2) Mix with an equal volume of Freund's complete adjuvant by ultrasonic method (at this time, the antigen concentration is about 0.4 mg / mL);
[0056] (3) Rabbits about 4 weeks old were immunized by multi-point subcutaneous injection in the abdomen (injection of about 0.3 mL);
[0057] (4) After 2 weeks, mix the same amount of antigen with incomplete Freund's adjuvant and then immunize;
[0058] (5) After another week, the above method (antigen mixed with incomplete adjuvant) was used to boost immunization once a week (antigen was halved), a total of 3 times;
[0059] (6) Separation and purification to obtain rabbit polyantisera R1-R8 (corresponding to N1-N8 proteins in sequence): immunize rabbi...
Embodiment 3
[0060] Embodiment 3 prepares test strip
[0061] Take the N1-N8 proteins as the T line and label the N1-N8 proteins with microspheres respectively to obtain the microsphere-labeled N1-N8 proteins, and make total antibody detection test strips K1-K8. The following takes the preparation of test strip K1 as an example, and the preparation of other test strips is the same as that of K1
[0062] Microsphere-protein complex preparation
[0063] (1) Take 40μL Eu 3+ Fluorescent microspheres were placed in a centrifuge tube (attached to the wall), and washed by adding 1 mL of deionized water;
[0064] (2) Centrifuge at 15000r / min for 15min at 4°C, discard the supernatant, and repeat washing twice;
[0065] (3) Add 1 mL of 0.05 mol / L borate buffer (PH=7.4) to resuspend the microspheres;
[0066] (4) Take 20 μL of 10 mg / mL EDC and NHS respectively in a centrifuge tube, mix thoroughly, and place on a shaker to protect from light for 20 minutes (temperature: 25-30°C);
[0067] (5) Cen...
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