Inhibition of viral infection using monovalent antigen-binding proteins
A technology that binds proteins and virus infection, and is applied in antiviral agents, antiviral immunoglobulins, antibodies, etc., and can solve the problems of not being economically attractive, not being obtained, not being able to provide, etc.
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Embodiment 1
[0062] Example 1 Induction of Humoral Immune Response in Llama
[0063]The phage P2 of Lactococcus lactis (1:9V / V, antigen in water: Specol (Bokhout et al. (1981), Immunology (Immunol.) Immunopathology (Immunopathology) .), 2, 491-500; Bokhout et al. (1986), Infectious Diseases (Infect.Dis.), 161-168).Each immunization site injects 0.75-1.5ml of water-in-oil emulsion containing 200μg phage protein (about 6*10 13 pfu). Immunizations were given on the following schedule: a second immunization three weeks after the first injection, and a third immunization two weeks after the second injection. The immune response was then detected by ELISA on maxi-sorbplates (coating solution 10 10 pfu / ml was titrated on serum samples of phages diluted with phosphate buffered saline). After incubation with the sera, polyclonal rabbit-anti-llama antisera (obtained by immunizing rabbits with llama immunoglobulins purified on ProtA and ProtG columns; ID-DLO) combined with horseradish peroxidized...
Embodiment 2
[0064] Example 2 Cloning, selection and screening of llama V capable of neutralizing Lactococcus lactis phage P2 H H fragment
[0065] 2.1 Isolation of V of Lactococcus phage P2 H H fragment
[0066] Blood samples of approximately 200 ml were taken from llamas that were positive for phage P2 in the ELISA assay and centrifuged through a Ficoll (Pharmacia) step gradient to obtain an enriched lymphocyte population. Total RNA was isolated from these cells by guanidium extraction (for example as described by Chomczynnski and Sacchi (1987), Analytical Biochem., 162, 156-159). After the first cDNA was synthesized using MMLV-RT (Gibco-BRL) and random oligonucleotide primers (Pharmacia), the following specific primers were used to amplify the coding V H DNA fragments of the H segment and part of the long or short hinge region:
[0067] PstIv H -2B 5'-AGGTSMARCTGCAGSAGTCWGG-3' (SEQ.ID NO.4) s=c and G, M=A and c, R=A and G, W=A and T,
[0068] HindIIILa...
Embodiment 3
[0097] Example 3.V H H Fragment Neutralizes Efficacy of Lactococcus lactis Phage P2
[0098] 3.1 Production of V in Saccharomyces cerevisiae H Re-cloning in Episomal Plasmid System of H Fragment
[0099] V of VHH#1, VHH#2 and VHH#3 clones H The H-encoded genes were respectively derived from E. coli phagemid vectors pUR3827, pUR3828 and pUR3829 with PstI (present in V H The 5' end of the H gene and was introduced by primers VH-2B (SEQ.ID.NO.1)) and BstEII (naturally present in most V H H gene 3' end) and BstEII were digested and cloned into the episomal Saccharomyces cerevisiae secretory plasmid pUR4547, thus obtaining pUR3834, pUR3835 and pUR3836, respectively. The replication plasmid pUR4547 (accession number CBS100012) containing an Ori for autonomous replication in Saccharomyces cerevisiae can be produced through an inducible Ga17 promoter; -123) fused to V H The amino terminus of the H product completes the secretion. Yield was determined by Coomassie blue stained p...
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