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Antibody aiming at yellow fever virus NS1 protein and application thereof

A yellow fever virus and antibody technology, applied in the field of biomedicine, can solve problems such as inability to be widely and conveniently applied, high cost, and complicated instruments, and achieve the effects of short detection time, low cost, and simple operation

Active Publication Date: 2021-11-23
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most reliable diagnostic method is to extract viral RNA and perform qRT-PCR. This method is time-consuming, expensive, and needs to be operated by professionals in a specific laboratory. The instruments used are also complicated and cannot be widely and conveniently applied.

Method used

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  • Antibody aiming at yellow fever virus NS1 protein and application thereof
  • Antibody aiming at yellow fever virus NS1 protein and application thereof
  • Antibody aiming at yellow fever virus NS1 protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1 Expression and purification of yellow fever virus nonstructural protein 1 (YFV-NS1)

[0063] The extracellular region gene fragment of YFV-NS1 was cloned into the pFastBac I vector through Nde I and Xho I, and transformed into Escherichia coli DH10 to obtain the recombinant plasmid Bacmid-NS1. Then, sf9 cells were transfected to obtain baculovirus containing NS1 gene. After baculovirus infects Hi5 cells, it can secrete and express NS1 protein into the culture supernatant. The protein was purified by affinity chromatography and molecular sieve chromatography, and the protein purity was identified by SDS-PAGE. The results showed that the NS1 protein existed in monomer and dimer forms, and the monomer size was 43kDa. The results were as follows figure 1 . Concentrate YFV-NS1 to 1 mg / mL and store it frozen at -80°C for future use.

[0064] For the construction, expression and purification of yellow fever virus YFV-NS1 C terminal and other flaviviruses WNV-N...

Embodiment 2

[0066] Example 2 Preparation of Human Monoclonal Antibody

[0067] 1. Single cell sorting and clone construction

[0068] With the informed consent of the recovered patients, peripheral blood samples were collected from two recovered patients infected with YFV for 6 months, peripheral blood mononuclear cells (PBMCs) were separated, and various blood cell-specific marker antibodies and specific YFV-NS1 antigen were added Incubated together. After sorting by FACSAria II (BD Biosciences), the YFV-NS1 positive memory B cells were collected into a 96-well plate, with 1 cell per well.

[0069] The inventors amplified antibody variable region gene sequences (VH and VL) from a single B cell by means of RT-PCR and nested PCR. First, the obtained B cells were reverse-transcribed using the SuperScript III reverse transcriptase kit. After obtaining the cDNA, nested PCR was performed using HotStar plus enzyme (QIAGEN) to amplify the antibody variable region sequence. The first round of...

Embodiment 3

[0074] Embodiment 3 YB36 and YD40 antibody functional identification

[0075] 1. ELISA detection of antibody specific binding to YFV-NS1

[0076] 1) Dilute antigen 2μg / mL (YFV-NS1, YFV-NS1 C terminal, WNV-NS1, ZIKV-NS1, DENV1-NS1, DENV2-NS1, DENV3-NS1, DENV4-NS1) with coating solution, add to enzyme In the target plate, 100 μL / well, overnight at 4°C.

[0077] 2) The next day, add 200 μL / well of PBST, wash the plate 5 times with a plate washer (BIO-TEK, 405_LS), add 5% skimmed milk powder blocking solution, 100 μL / well, and incubate in a 37°C incubator for 1 hour.

[0078] 3) Rinse 5 times with PBST, add antibody YB36 or YD40, 400ng / well, incubate at 37°C for 1 hour.

[0079] 4) Rinse 5 times with PBST, HPR-labeled goat anti-human IgG, 100 μL / well, and incubate at 37° C. for 45 minutes.

[0080] 5) Rinse with PBST for 5 times, add TMB chromogenic solution, 100 μL / well, keep in the dark at room temperature, react for 2 minutes, then add 2MH 2 SO 4 Stop the reaction, 100 μL / ...

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Abstract

The invention provides an antibody aiming at yellow fever virus NS1 protein and application thereof. The antibody comprises a YD40 antibody and / or a YB36 antibody; the invention provides a highly sensitive yellow fever virus ELISA double-antibody sandwich detection method. The method can be operated in a common laboratory, can be further developed into various kits, such as colloidal gold, ELISA kits and the like, and is convenient to use.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an antibody against NS1 protein of yellow fever virus and application thereof. Background technique [0002] Yellow fever virus (YFV) is an insect-borne virus transmitted by Aedes mosquitoes, and belongs to the Flavivirus genus of the Flaviviridae family, along with Zika virus (ZIKV), West Nile virus (WNV) and dengue virus (DENV). The vector of yellow fever virus is mosquitoes, so yellow fever outbreaks are high in mosquito-endemic areas and developing countries. Currently, it is mainly prevalent in tropical and subtropical regions of Africa and South America. Yellow fever virus can cause yellow fever, which seriously endangers human health, manifested as jaundice, hemorrhage, and even multi-system organ failure. Because the symptoms of yellow fever are similar to those caused by viruses of the same family, and yellow fever virus and other flaviviruses may also ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/569A61K39/42A61P31/14
CPCC07K16/1081G01N33/56983A61P31/14C07K2317/24C07K2317/52C07K2317/56C07K2317/565C07K2317/76C07K2317/92A61K2039/505G01N2333/185Y02A50/30
Inventor 高福严景华李燕仵丽丽王奇慧戴连攀马素芳
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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