Single-domain antibody for neutralizing Xinjiang hemorrhagic fever virus
A technology of hemorrhagic fever virus and single domain antibody, applied in the direction of antibodies, antiviral agents, antibody mimics/scaffolds, etc., can solve the problems that have not yet been achieved, and achieve the effects of low production cost, short cycle and good tissue permeability
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Embodiment 1
[0039] [Example 1] Expression and purification of envelope protein of Xinjiang hemorrhagic fever virus
[0040] According to the gene sequence of Xinjiang hemorrhagic fever virus (GenBank No.gi|16271971), its amino acid sequence was analyzed, and the domain III (amino acid 406-521) of its envelope protein Gc was synthesized, and the gene was connected and transformed with the vector pET22b to construct the vector pET22b-Gc Domain III. 100ng of plasmid (pET22b-Gc DomainIII) was transformed into BL21(DE3) competent cells. Re-inoculate the bacteria in LB medium containing 100 μg / ml ampicillin (1L medium contains 10 g tryptone, 5 g yeast extract and 10 g NaCl), and wait until OD 600 When it reached 0.6-1.0, IPTG was added to a final concentration of 0.1 mmol / ml, and expression was induced at 30°C and 220 rpm for 16-20 hours. Collect the bacteria by centrifugation at 4°C, 6000rpm, 15min, discard the medium, resuspend the pellet in Buffer A (50mM Tris-HCL, 450mM NaCL, pH 8.0), a...
Embodiment 2
[0043] [Example 2] Construction and screening of phage display library
[0044] Using m0 as the backbone, a phage library was constructed according to existing literature (W Chen, et al., Methods Mol Biol, 2009:81-99), and screened with antigens expressed in Escherichia coli. After the purified antigen was incubated overnight at 4°C in a 96-well plate, it was panned with a phage library, and the specific phage was captured by the antigen, washed with PBS+0.05% Tween-20, and after four rounds of screening, the obtained One enriched clone was named XHF4RC8.
Embodiment 3
[0045] [Example 3] Expression and purification of XHF4RC8
[0046] XHF4RC8 was expressed and purified according to the existing literature (Gong R, et al., Methods Mol Biol., 2012). The prokaryotic XHF4RC8 expression vector was constructed and transformed into E.coli HB2151 competent cells. Re-inoculate the strains in SB medium containing 100 μg / ml ampicillin (1L medium contains 30g tryptone, 20g yeast extract and 10g MOPS, the pH value is adjusted to 7.0 with NaOH), and the OD 600 When it reaches 0.7-1.0, add IPTG to a final concentration of 200 μg / ml, and induce expression at 37° C. and 220 rpm for 14-16 hours. Collect the bacteria by centrifugation at 4°C, 6000rpm, 15min, discard the medium, resuspend the pellet in Buffer A (50mM Tris-HCL, 450mM NaCL, pH 8.0), and treat it with polymyxin B (polymyxin B) for 1 hour Collect the supernatant by centrifugation. Purified with Ni-NTA filler and verified its purity by SDS-PAGE. It was then concentrated by ultrafiltration using ...
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