A fully human monoclonal neutralizing antibody against sftsv and its application
A technology of monoclonal antibodies and antigens, applied in the fields of molecular biology and cellular immunology
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0085] Example 1 Screening of anti-SFTSV-Gn protein single chain antibody
[0086]1. Purification of JS-2010-014 virus particles
[0087] 1.1 Materials
[0088] JS-2010-014, obtained by the patent applicant in 2010 from the peripheral blood of a Jiangsu patient in the acute phase.
[0089] 1.2 Methods and results
[0090] After the virus was inoculated into Vero cells, at 37°C, 5% CO 2 Cultured for 5 days under certain conditions, the supernatant was aseptically collected and the 50% tissue culture infectious dose (50% tissue culture infective dose, TCID50) was measured. The virus suspension was inactivated by 1:4000β-propiolactone at 4°C for 24 hours, centrifuged at low speed to remove cell debris, suspended in PBS after ultracentrifugation for 2 hours, and further purified by molecular sieve chromatography. JS-2010-014 virus particles with high purity can be obtained through the above steps, and all virus operations are performed in a biosafety level 2 (BSL-2) laboratory...
Embodiment 2s
[0152] Example 2 Full molecular construction and eukaryotic expression of scFv antibody
[0153] 1. Construction of baculovirus recombinant plasmid
[0154] Use 4-6, 2F6, and 1B2 single-chain antibody plasmids as templates to amplify VH and VL genes by PCR respectively. Each amplification system contains the following contents: scfv plasmid 0.1μg, upstream primer 60pmol, downstream primer 60pmol, 10× PCR buffer 10 μL, dNTP 8 μL, MgCl 2 6 μL, Ex Taq 0.5 μL, add water to 100 μL. The reaction conditions are: 94°C for 5 min; 94°C for 15 sec, 56°C for 30 sec, 72°C for 1 min, 30 cycles; 72°C for 10 min. The gel extraction kit was used to recover the target band. The above PCR products were digested with XhoI / NheI (VH) and SacI / HindIII (Vk) respectively, and the eukaryotic baculovirus expression vector plasmid pAc-K-CH3 was first digested with XhoI / NheI, and the VH fragment was inserted Afterwards, carry out SacI / HindIII digestion and insert the VL fragment. The enzyme digestio...
Embodiment 3
[0157] Example 3 Binding Experiment of Whole Molecular Antibody and SFTSV
[0158] 1. Method
[0159] (1) Inoculate Vero cells in a 24-well plate, culture at 37° C., and inoculate each well with virus liquid when the fusion rate reaches 90%.
[0160] (2) Two days later, remove the virus liquid, add 400 μl of 4% paraformaldehyde to each well, and fix at room temperature for 30 minutes.
[0161] (3) Discard the paraformaldehyde, wash with PBS three times, add 400 μl 0.2% Triton X-100 to permeabilize the cell membrane, and keep at room temperature for 15 minutes.
[0162] (4) Discard the Triton X-100, wash with PBS 3 times, add 5 mg / ml BSA for blocking, room temperature for 30 min.
[0163] (5) Discard the blocking solution, add 300 μl of purified whole-molecular monoclonal antibody to each well, set up duplicate wells, and set up a blank control group without adding antibodies and 4-5 IgG1 monoclonal antibody (see patent literature: a human anti-SFTSV Source antibody, authori...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More