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Heavy chain single-domain antibody of type-II dengue fever virus NS1 protein as well as preparation method and application of heavy chain single-domain antibody

A heavy chain single domain antibody and dengue fever virus technology, applied in the direction of anti-viral immunoglobulin, recombinant DNA technology, and resistance to vector-borne diseases

Active Publication Date: 2013-11-20
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no report on the heavy chain single domain antibody of NS1 protein of dengue virus type 2

Method used

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  • Heavy chain single-domain antibody of type-II dengue fever virus NS1 protein as well as preparation method and application of heavy chain single-domain antibody
  • Heavy chain single-domain antibody of type-II dengue fever virus NS1 protein as well as preparation method and application of heavy chain single-domain antibody
  • Heavy chain single-domain antibody of type-II dengue fever virus NS1 protein as well as preparation method and application of heavy chain single-domain antibody

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Experimental program
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Embodiment 1

[0047] A method for constructing a heavy chain single domain antibody against type II dengue virus NS1 protein, comprising the steps of:

[0048] Step 1, preparation of type II dengue virus NS1 protein:

[0049] (1) Design a pair of primers (upstream primer 1: 5′-TTA CATATG GATAGTGGTTGCGTTGTGAGC-3', see SEQ NO.24 and downstream primer 1: 5'-TTA GTC GAC GGCTGTGACCAAGGAGTTGA-3′, see SEQ NO.25), total RNA was extracted from the culture supernatant of C6 / 36 mosquito cells infected with type II dengue fever NGC strain, and RT- PCR amplification was performed to obtain a gene fragment with a full length of about 1.1 kb, which was then connected to the pET-30a prokaryotic expression vector (TaKaRa Company, Dalian, China) and sequenced to obtain the target gene with the correct gene sequence.

[0050] (2) Highly express NS1 recombinant protein with histidine tag in E. coli BL21 (DE3). The recombinant protein is expressed in the form of inclusion bodies, and after renaturation, it...

Embodiment 2

[0079] Application of Heavy Chain Single Domain Antibody in Detection of Dengue Virus Infection

[0080] (1) Use the heavy chain single domain antibody P2 and the monoclonal antibody against NS1 as the coating antibody, and assemble the colloidal gold test strip according to the conventional method (completed by Guangzhou Wondfo Biotechnology Co., Ltd.);

[0081] (2) Dilute the culture supernatant of cells infected with type 2 dengue fever virus at 1:10, 1:20, 1:100, and 1:150, and use the above two different colloidal gold test strips for detection, 1:150 is Minimum detection limit;

[0082] (3) Using the above two test strips to test 100 dengue-negative serum samples respectively, when using heavy chain single domain antibody P2 as the colloidal gold test strip assembled with the coated antibody, a false positive was detected, and a single When the cloned antibody was tested as an antibody-coated colloidal gold test strip, six false positives were detected, see Table 1.

...

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Abstract

The invention belongs to the technical field of biology and relates to a heavy chain single-domain antibody of a type-II dengue fever virus NS1 protein as well as a preparation method and application of the heavy chain single-domain antibody. The preparation method comprises the following steps of: biologically screening the recombination expressed dengue fever virus NS1 protein for many times by using an alpacas non-immunity heavy chain single-domain antibody library; picking by using an ELISA (Enzyme Linked Immunosorbent Assay) to obtain a specific bonded phage monoclone, and determining a DNA (Deoxyribose Nucleic Acid) sequence; and then, converting the DNA sequence into escherichia coli to express to obtain the heavy chain single-domain antibody. The heavy chain single-domain antibody obtained through screening can be efficiently expressed in the escherichia coli, can be used for preparing a colloid gold detection kit for detecting type-II dengue fever virus infection, and is higher in detection accuracy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a heavy chain single-domain antibody produced against the NS1 protein of type II dengue fever virus. The invention relates to an amino acid sequence encoding a heavy chain single domain antibody and a method for preparing the heavy chain single domain antibody by screening an alpaca non-immune library. The obtained heavy chain single domain antibody can be highly expressed in Escherichia coli, and can be used to prepare a colloidal gold detection kit to detect type 2 dengue fever virus infection. Background technique [0002] Dengue fever is a disease caused by dengue virus infection and transmitted by mosquitoes. In the past ten years, dengue fever infection has been increasing worldwide, and the number of infected people reaches 50 million to 100 million every year. The disease spreads rapidly and has a high incidence rate. It can be spread over long distances through modern means ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/70G01N33/577
CPCY02A50/30
Inventor 王菊芳王海鹰法蒂玛马毅王小宁
Owner SOUTH CHINA UNIV OF TECH
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