Monoclonal antibody targeting at Zika virus envelop protein conserved epitope and application thereof
A technology of antibody and recombinant protein, applied in the direction of antiviral immunoglobulin, application, antibody, etc., can solve the problem of no drug against Zika virus
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[0153] 3. Preparation and screening of hybridoma cells and subtype identification of secreted antibodies
[0154] With ZIKV E80 (10μg / mouse), (500μg / mouse) aluminum hydroxide ( Invivogen, USA) immunized 6-week-old female BALB / c mice intraperitoneally four times with an interval of 2 weeks each time. At the 8th week, blood was collected and the serum neutralizing titer was determined, and the highest one was selected for use as an antibody. Three days before the fusion, the tail vein of 50ug ZIKV E80 was boosted once. Splenocytes were then isolated and fused with SP2 / 0 cells using PEG1500. After culturing for 8 days, the supernatant of the hybridoma cells was collected, 50ul of which was tested for binding ability to ZIKV E80 by indirect ELISA, and 40ul was incubated with 16000pfu of ZIKV to detect its neutralization ability.
[0155] 4. Preparation and purification of monoclonal antibodies
[0156] The screened positive hybridoma cells were expanded and cultured, and abou...
Embodiment 1
[0177] Example 1 Screening and Identification of Monoclonal Antibodies
[0178] After three rounds of screening, the inventors screened three ZIKV-specific monoclonal antibodies (Table 1), among which 1C11 and 4C5 could only bind ZIKV E80, but could not neutralize Zika virus; while 5F8 could both bind ZIKV E80 and Can neutralize Zika virus. Monoclonal antibodies 4C5 and 5F8 belong to IgG1, and 1C11 belongs to IgG2a. Since only 5F8 has neutralization ability, follow-up experiments mainly analyze 5F8.
[0179] Table 1. Characteristics of Monoclonal Antibodies
[0180] monoclonal antibody hybridoma
light chain
Combined with ZKE80 capability a
Virus neutralization ability b
1C11
IgG2a
Kappa
++
-
4C5
IgG1
Kappa
++
-
5F8
IgG1
Kappa
++
+
[0181] a. OD450nm absorbance value of ELISA experiment; absorbance value greater than 1.0 is marked as "++".
[0182] b. "+" can i...
Embodiment 2
[0183] Example 2. Monoclonal antibody 5F8 can neutralize Zika virus
[0184] Firstly, the standard plaque reduction assay was used to analyze the neutralization ability of 5F8. Using D5 as a control, the 2-fold diluted monoclonal antibody was incubated with 100 PFU of Zika virus at 37°C for 1h, then added to the cells and incubated at 37°C for 1h, and sucked off. For samples, add overlay medium, keep at 4°C for 15mins, and culture at 37°C for about 80h. The result shows that the PRNT50 value of 5F8 is 5.709ug / ml, and D5 has no neutralizing effect ( figure 1 ).
[0185] In order to study the mechanism of action of 5F8, the adsorption analysis of virus and cells and the analysis of the neutralization ability of antibodies after virus adsorption to the cell surface were carried out respectively. The results showed that 5F8 can inhibit the adsorption of Zika virus to the surface of Vero cells, and can still play a neutral role after Zika virus is adsorbed on the cell surface ( ...
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