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Preparation of Zika virus multi-segment fusion protein and IgG/IgM antibody detection kit

A Zika virus and fusion protein technology, applied in the field of data biology, can solve the inevitable dengue virus cross-reaction and other problems

Inactive Publication Date: 2017-06-13
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Recently, some domestic and foreign manufacturers have released Zika virus diagnostic antigens, but the antigenic epitope corresponding to the upstream gene sequence of the antigen has not been modified in any way, resulting in inevitable cross-reaction with dengue virus

Method used

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  • Preparation of Zika virus multi-segment fusion protein and IgG/IgM antibody detection kit
  • Preparation of Zika virus multi-segment fusion protein and IgG/IgM antibody detection kit
  • Preparation of Zika virus multi-segment fusion protein and IgG/IgM antibody detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Expression and purification of fusion protein

[0040] 1. Preparation of linearized expression vector: (such as figure 1 ) select the prokaryotic expression vector pET28a, use restriction sites BamH I and Xhol I to double digest the prokaryotic expression vector, linearize it, recover and purify the linearized double digested vector for future use.

[0041] The double enzyme digestion system is as follows:

[0042]

[0043] 2. Preparation of multi-fragment PCR products (see attached figure 2 ): corresponding amplification primers were designed, and insert fragments 1, 2, and 3 were amplified by PCR respectively.

[0044] The PCR reaction system is as follows:

[0045]

[0046] PCR reaction conditions:

[0047]

[0048] PCR amplification products such as image 3 As shown, the desired target band can be seen to be amplified.

[0049] 3. Preparation of recombinant plasmids: Prepare a recombination reaction system with the linearized expression ve...

Embodiment 2

[0053] Example 2 Fusion antigen pairing screening experiment

[0054] 1. Label the purified fusion proteins ZIKV-Ag1, ZIKV-Ag2, and ZIKV-Ag3 with gold, respectively, and perform pairing experiments with the purchased anti-human IgG monoclonal antibody A371 and anti-human IgM monoclonal antibody A372, and titrate with checkerboard Cross-matching method, the paired antigen-antibody with the highest sensitivity and specificity. The results are shown in Table-4.

[0055] Table-4 Antigen-antibody pairing screening experiment results

[0056]

[0057] The results show that when ZIKV-Ag2 is used as the labeled antigen and A371 and A372 are used as the labeled antibody, the sensitivity and specificity are the highest (up to 100%), so ZIKV-Ag2, A371 and A372 are selected as the paired antigens of this detection reagent Antibody.

Embodiment 3

[0058] The composition of embodiment 3 kits

[0059] 1. Detection reagent card: ①sample pad, ②colloidal gold binding pad (glass fiber membrane): the membrane is adsorbed with dry gold-labeled antigen and gold-labeled streptavidin (flow band), ③chromatographic membrane (nitrocellulose membrane) ), the membrane is coated with antigen or antibody strips and quality control strips (detection strips) that can directly react with markers, ④ absorbent material (absorbent paper), ⑤ liner (PVC rubber sheet). The above components are connected end to end and attached to the PVC rubber sheet (such as Figure 5 shown).

[0060] 2. Sample treatment solution: 1 bottle (for 10 persons: 1.0mL; for 25 persons: 2.5mL; for 50 persons: 5.0mL), the main component is heterophile antibody blocking agent (concentration range: 1-50mg / mL). Preservatives: 0.02% ~ 0.06% NaN 3 .

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Abstract

The invention aims at providing a simple and quick Zika virus detection kit. The kit optimally selects fusion expression protein as diagnostic antigen; an anti-human IgG monoclonal antibody A374, an anti-human IgM monoclonal antibody A371 and a biotin-BSA conjugate are respectively coated on a nitrocellulose membrane as a detection line and a quality control line; colloidal gold labeled fusion expression protein and colloidal gold labeled streptavidin and other reagents are matched; and an immunochromatography capture method principle is used for qualitative detection of Zika virus specific IgM antibody and IgG antibody in human serum, thereby realizing quick and specific diagnosis of Zika virus infection.

Description

technical field [0001] In the field of data biotechnology, the present invention relates to the preparation and use of a Zika virus diagnostic antigen prepared by using genetic engineering multi-segment fusion rapid expression technology, and Zika virus IgG / IgM antibody rapid detection kit (colloidal gold method). Background technique [0002] In 2015, Zika virus spread rapidly around the world, with outbreaks reported in 24 countries and regions. The World Health Organization announced on February 1, 2016 that Zika virus and microcephaly were listed as global public health emergencies. [0003] Zika virus disease (Zika virus disease) belongs to the Flavivirus genus of the Flavivirus family, and is a self-limiting acute infectious disease caused by Zika virus. Zika virus is transmitted to humans through the bite of an infected Aedes mosquito, and may also be transmitted from a pregnant mother to the fetus or infant during pregnancy or childbirth, and may also be transmitted ...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/531G01N33/577G01N33/569
CPCG01N33/531G01N33/558G01N33/56983G01N33/577G01N2333/183G01N2333/46G01N2800/26Y02A50/30
Inventor 吴英松邓传欢董志宁李志雄
Owner GUANGZHOU DARUI BIOTECH
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