Nanometer antibody specifically binding to hepatitis E virus capsid protein and application thereof
A technology of hepatitis E virus and nano-antibody, which is applied in the field of genetic engineering and can solve problems such as high R&D and production costs, large molecular weight, and complex antibody structure
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Embodiment 1
[0023] Screening of nanobodies against porcine HEV capsid protein:
[0024] The expressed and purified porcine HEV 239 protein was immunized with Bactrian camels, and after the antibody titer reached the standard, the peripheral blood was collected to separate lymphocytes and the genomic RNA was extracted, and the gene fragment encoding VHH was amplified by nested RT-PCR, and then ligated into pCANTAB5E to construct The recombinant phage vector was electrotransfected into TG1 competent cells to construct the variable region library of Bactrian camel heavy chain antibody; the specific nanobody against porcine HEV 239 protein was panned by phage display technology. Finally, two specific nanobodies against porcine HEV 239 protein were obtained, named porcine HEV-nb1 and porcine HEV-nb2. The nucleotide sequences are respectively shown in SEQ ID NO:1 and SEQ ID NO:2, and the amino acid sequences are respectively shown in SEQ ID NO:3 and SEQ ID NO:4.
Embodiment 2
[0026] Prokaryotic expression of nanobodies:
[0027] Using pET-21b as the prokaryotic expression vector, the constructed nanobody recombinant plasmid was transformed into BL21(DE3) expression competent cells, and the expression was induced at 37°C and 220 rpm for 6-8 h. Since nanobodies are mainly expressed in the form of inclusion bodies, first dissolve the precipitated bacteria with 8M urea and purify them through a nickel column, then gradient dialysis to 0 .01M PBS (PH7 .2) for renaturation, concentrate and filter with 0 .22 μm sterile Sterilize by membrane filtration, and finally store at -80°C until use. SDS and WB results such as figure 1 It was shown that a specific target band appeared around 15 kDa, indicating the expression of the fusion protein.
Embodiment 3
[0029] ELISA detection of Nanobody binding to human, porcine and rabbit HEV 239 proteins:
[0030] Because the homology between human, pig and rabbit HEV 239 proteins is as high as 93%. Pack human, pig and rabbit HEV 239 proteins at 400ng per well overnight, seal and wash the plates, add nanobodies, and then add mouse anti-his antibody and goat anti-mouse HRP-labeled secondary antibody for color development and identification. The result is as figure 2 It was shown that the nanobody reacted specifically with human, pig and rabbit HEV 239 proteins. It shows that these nanobodies have a broad spectrum.
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