Murine monoclonal antibody to Zika virus envelope protein
A monoclonal antibody, envelope protein technology, applied in the direction of antiviral immunoglobulin, immunoglobulin, peptide, etc., to achieve the effect of good specificity, strong neutralization activity, and enhanced anti-aggregation
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Embodiment 1
[0015] Example 1: Obtaining of B8 antibody sequence
[0016] Based on the constructed immunized mouse Fab phage display library, (the acquisition method of the library comes from the fusion protein of the envelope protein of Flaviviridae virus disclosed in Chinese patent application 201811613477.1 and its preparation method and application, using the fusion protein as an immunogen After immunizing the mice, we obtained the cDNA and used it as the construction material of the phage display library), we carried out three rounds of screening, and through the monoclonal phage ELISA, the positive clones were sequenced, and the bases of the B0 candidate clones were obtained sequence, and through the amino acid translation software (https: / / web.expasy.org / translate / ), the amino acid sequence was obtained, the amino acid sequence of the light chain is shown in SEQ No.1, and the amino acid sequence of the heavy chain is shown in SEQ No.2 shown. .
Embodiment 2
[0017] Example 2: Using Tango software to optimize the obtained B0 amino acid sequence
[0018] Use Tango software (http: / / tango.crg.es / ) to predict the aggregation-prone region of the amino acid sequence of the B0 candidate clone, and find that there is a strong aggregation-prone region, such as Picture 1-1 , so point mutation is carried out in this region to obtain B8 antibody, its light chain amino acid sequence is shown in SEQ No.1, its heavy chain amino acid sequence is shown in SEQ No.3, the corresponding base sequence, and the light chain is shown in SEQ No. 4, the heavy chain is shown in SEQ No.5. The mutated amino acid sequence was re-predicted by Tango software, and it was found that the aggregation-prone region was significantly improved, as shown in Figure 1-2 , 1-3.
Embodiment 3
[0019] Example 3: 293F cells express B8 antibody and detect it by SDS-PAGE
[0020] The mutated base sequence was cloned into the dual promoter vector pVitro2-neo-mcs (InvivoGen), and the 293F cells were transfected with PEI to express the protein. After 5-7 days of expression, the culture supernatant of the 293F cells was treated with ProteinA (GE) was purified, and after the purified protein was concentrated and exchanged, the B8 antibody dissolved in PBS buffer was obtained. After SDS-PAGE electrophoresis detection, it was found that it presented two bands of light chain and heavy chain, such as figure 2 shown.
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