Modified hepatitis c virus proteins
A kind of technology of hepatitis C virus and composition, applied in the direction of virus, virus peptide, immunoglobulin, etc.
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Embodiment 1
[0283] Example 1: Single cysteine disulfide bond substitution mutation in the context of co-expression of full-length E2 glycoprotein and E1
[0284] As proposed by Krey et al., 2010 (ibid.), the assignment of disulfide bonds in the domain structure of E2, such as figure 1 And shown in Table 2. In the context of E1E2 derived from genotype 1a isolate H77c in HCVpp, the effect of replacing cysteine alone with alanine on E2 folding and function was evaluated. The substitution of Cys to Ala abolished the ability of HCVpp to infect Huh7 liver cancer cells, indicating that the 9 disulfide bonds of E2 are critical for cell entry ( figure 2 A). Western blot analysis of transfected cell lysates with MAb H52 (for E2) and MAb A4 (for E1) showed that E1 and E2 were expressed at wild-type levels ( figure 2 B). However, in the biosynthesis-labeled HCVpp immunoprecipitation, the use of conformation-dependent E2-specific MAb, H53 indicates that the mutation has caused defects in the glyco...
Embodiment 2
[0288] Example 2: Simultaneous mutagenesis of cysteine pairs involved in disulfide bond formation
[0289] Cys-to-Ala scanning of HIV envelope glycoprotein gp120 / gp41 complex shows that cysteine mutation alone is not conducive to the folding of functional protein, while Ala is substituted in 2 of the 10 disulfide bonds Both folding and function are saved (van Anken et al., Mol Biol Cell 19: 4298-309, 2008). In order to reduce the tendency of protein mismatch due to the presence of unpaired cysteine, Ala-substitution of each disulfide bond pair is performed at the same time. For the double Cys-to-Ala mutant, the intracellular expression and polyprotein processing of E1 and E2 were confirmed by Western blotting ( image 3 A), however, lacks HCVpp entry activity (data not shown). For C452A / C459A (domain II), C581A / C585A and C652A / C677A (domain III), the incorporation of H53-reactive E2 into HCVpp was observed, which did not form a heterodimer with E1 ( image 3 A). In these 3 ...
Embodiment 3
[0290] Example 3: The truncated E2 glycoprotein (E2 661 -his) single cysteine and pairwise disulfide bond substitution mutations
[0291] Next, in the receptor binding domain of E2 (residues 384-661, E2 661 -His) to evaluate the impact of the Cys-to-Ala mutation in the context of E2, which is independent of the E1 folding, retains the three domain structures described by Krey et al., 2010 (supra), and retains the CD81 and SRB1 binding function. All mutants were secreted from transfected 293T cells at the wild-type level, as shown by the immunoprecipitation of the metabolically labeled protein through the C-terminal 6-His tag and anti-His antibody ( Figure 4 A, upper block). E2 661 -His mutants except for one MAb H53-reactivity attribute mostly reflects the observed reactivity attributes of the corresponding E1E2 mutant ( Figure 4 A, blocks 2 and 3, see figure 2 B). Therefore, the Cys residues necessary for H53 reactivity include C494, C508 (DII), C552, C564 (DI), C607 and C6...
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