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Enterovirus 71 type specific recombinant protein antigen and application thereof

An enterovirus and recombinant protein technology, applied in the field of virus immunology detection of molecular biomedicine, can solve the problems of hidden dangers of biological safety, time-consuming and laborious, complicated process, etc., achieve good sensitivity and specificity, reduce cost, and easy to prepare Effect

Inactive Publication Date: 2012-07-11
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Solved technical problems: At present, in the diagnosis of enterovirus 71 infection, the methods of virus isolation and neutralizing antibody detection have the disadvantages of time-consuming and laborious and cannot be quickly diagnosed, and the use of molecular biology methods such as RT-PCR is cumbersome. It also requires expensive molecular biology reagents and corresponding instruments and equipment, and the use of whole virus particles as a diagnostic antigen al

Method used

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  • Enterovirus 71 type specific recombinant protein antigen and application thereof
  • Enterovirus 71 type specific recombinant protein antigen and application thereof
  • Enterovirus 71 type specific recombinant protein antigen and application thereof

Examples

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Embodiment 1

[0021] The preparation of embodiment 1 recombinant EV71 specific antigenic protein

[0022] 1.1 Sequence determination of VP1 gene of EV71 virus epidemic strain

[0023] The EV71 epidemic strain-MAS0901 virus liquid was inoculated onto a monolayer of African green monkey kidney cells (Vero), at 36 °C, 5% CO 2 Cultivate and observe the cytopathic effect (CPE) day by day. When the CPE reaches more than 80%, the virus is harvested, and 140 μL of the virus culture supernatant is taken, and the virus RNA is extracted with the QIAamp Viral RNA Mini Kit (QIAGEN), and the One-Step RT-PCR Kit ( QIAGEN) to amplify the VP1 gene, the upstream primer (F1-SEQ ID NO.3) is 5'-GCAGCCCAGAAGAACTTCAC-3', the downstream primer (R1-SEQ ID NO.4) is 5'-ACCACTCTAAAGTTGCCCAC-3', and the product size is 1015bp, the amplification system is as follows:

[0024] h 2 O(RNase-free) 13.5μL 5×One-Step RT-PCR Buffer 2.5μL dNTP Mix(10mM) 1.0 μL Primer F1 (10μM) 1.0 μL

[...

Embodiment 2

[0079] Example 2 Recombinant EV71-specific antigen multi-copy tandem expression and application

[0080] In order to further improve the immune reactivity of EV71-specific antigen, we added EV71-VP1 6-43 Recombinant protein coding gene was repeatedly cloned onto the expression plasmid pGEX-4T-2 to achieve EV71-VP1 6-43 Multiple copies of the recombinant protein are expressed in tandem.

[0081] 2.1EV71-VP1 6-43 Construction of multi-copy tandem expression plasmids for recombinant proteins

[0082] The recombinant plasmid pGEX-4T-2 / EV71-VP1 prepared in embodiment 1.3 6-43 Based on this, the target gene was obtained by double digestion with BamHI and EcoRI restriction enzymes, and the single-copy EV71-VP1 was obtained by double digestion with EcoRI and BglII restriction endonucleases. 6-43 The vector pGEX-4T-2 encoding the gene, the enzyme digestion system is as follows:

[0083] 10×buffer 6μL pGEX-4T-2 / EV71-VP1 6-43 ~1μg BamHI or BglII 2μL E...

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Abstract

The invention relates to enterovirus 71 type specific recombinant protein antigen and application thereof. The amino acid sequence is shown as SEQIDNO. 1; and the nucleotide sequence is shown as SEQIDNO. 2. The recombinant protein can be applied in preparation of monoclonal antibodies and polyclonal antibodies as well as detection of enterovirus 71 type specific antibodies. The invention provides a novel diagnostic antigen for serological detection of enterovirus 71 type infection. Experiments show that the enterovirus 71 type specific recombinant protein antigen has good sensitivity and specificity, can be identified by human serum after the enterovirus 71 type infection and inactivated full-virus immune serum of animals, can be applied in serological diagnosis of the enterovirus 71 type infection, and can also be applied in vaccine development and evaluation of immune effects in use. Compared with the full-virus particles as diagnostic antigens, the specific protein antigen can be prepared easily, and the cost of kit preparation is reduced.

Description

technical field [0001] The invention relates to the technical field of viral immunology detection in molecular biomedicine, in particular to a recombinant protein capable of specifically detecting enterovirus 71 antibodies and an application of the recombinant protein. Background technique [0002] Enterovirus 71 (Enterovirus 71, EV71) belongs to Picornaviridae, Enterovirus genus, and is one of the main pathogens of hand, foot and mouth disease. In addition to EV71, a variety of enteroviruses including coxsackievirus group A (types 4, 5, 9, 10, 16), coxsackievirus group B (type 2, 5) and echoviruses can also cause siblings. Mouth diseases, among which Coxsackie A16 (Coxsackie A16, CA16) is the most common. Usually, the clinical symptoms and signs of HFMD caused by EV71 infection are indistinguishable from those caused by other enteroviruses such as CA16, but some children may also develop aseptic meningitis, encephalitis, spinal A variety of nervous system complications su...

Claims

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Application Information

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IPC IPC(8): C07K14/085C07K16/10C07K19/00C12N15/41C12N15/63C12N1/21G01N33/569C12R1/19
Inventor 孟继鸿张建华董敏江炳福徐明杰程险峰董晨戴星
Owner SOUTHEAST UNIV
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