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

Active Publication Date: 2018-12-25
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Monoclonal antibody targeting at Zika virus envelop protein conserved epitope and application thereof
  • Monoclonal antibody targeting at Zika virus envelop protein conserved epitope and application thereof
  • Monoclonal antibody targeting at Zika virus envelop protein conserved epitope and application thereof

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preparation example Construction

[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

heavy chain

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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Abstract

The invention provides a monoclonal antibody targeting at Zika virus envelop protein conserved epitope and application thereof. specifically, a recombinant expressed Zika virus E-protein immune mouseis used herein to attain a murine monoclonal antibody specific to E-protein. Research results show that the antibody herein is an ideal Zika virus detection antibody and is applicable to the development antibody drugs against Zika virus. Identification of a conserved neutralizing epitope can also guide the development of broad-spectrum Zika virus vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a monoclonal antibody targeting a conserved epitope of Zika virus envelope protein and an application thereof. Background technique [0002] Zika virus belongs to the Flavivirus genus (Flavivirus) in the family Flaviviridae in biological classification, and the virus is a single positive-strand RNA virus. Zika virus was first isolated in Uganda Zika forest macaques in 1947, and is mainly transmitted through the bite of Aedes mosquitoes, and can be transmitted vertically to the fetus through the placenta. Zika virus infection is most harmful to pregnant women and fetuses. It is currently suspected to be the culprit of microcephaly in fetuses and newborns. Zika virus has been isolated from the amniotic fluid and brain tissue of fetuses with microcephaly. In addition, recent studies have found that Zika virus can damage the testes of adult male mice, and human ge...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/63A61K39/42A61P31/14
CPCC07K16/1081A61K2039/505C07K2317/92C07K2317/76C07K2317/34C07K2317/56C07K2317/565Y02A50/30
Inventor 黄忠屈攀科
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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