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Coxsackie virus CA16 VP1 recombinant antigens, and monoclonal antibodies and application thereof

A technology of CA16VP18 and Coxsackie virus, applied in the field of molecular biology, can solve the problem of outbreaks in children and achieve the effect of early screening

Inactive Publication Date: 2015-06-17
汪运山
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies believed that the symptoms of hand, foot and mouth disease caused by CA16 were generally mild, so the research on CV16 failed to attract enough attention, but it is easy to cause explosive epidemics in children

Method used

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  • Coxsackie virus CA16 VP1 recombinant antigens, and monoclonal antibodies and application thereof
  • Coxsackie virus CA16 VP1 recombinant antigens, and monoclonal antibodies and application thereof
  • Coxsackie virus CA16 VP1 recombinant antigens, and monoclonal antibodies and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Selection of Candidate Antigen Epitopes

[0028] By searching NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ), a total of 51 CA16 sequences of CA16 were analyzed, and according to the homology of amino acid sequences, they were sorted and screened sequentially. 8 linear epitope antigen sequences conserved in each genotype sequence. Their amino acid sequences are shown in Table 1, respectively.

[0029] Table 1 CA16VP1 linear epitope sequence of the present invention

[0030] serial number amino acid site amino acid sequence 1 1-15AA GDPIADMIDQTVNNQ 2 26-40AA LPTAANTEASSHRLG 3 47-61AA LQAAETGASSNASDK 4 94-108AA TMPTTGTQNTDGYVN 5 156-177AA VPPGAPKPTSRDSFAWQTATNP 6 183-199AA MTDPPAQVSVPFMSPAS 7 208-228AA YPTFGEHLQANDLDYGQCPNN 8 274-293AA KTNPNYKGNDIKCTSTSRDK

Embodiment 2

[0031] Example 2: Cloning and expression of CA16 VP1 recombinant antigen

[0032] 1. Construction of CA16 VP1 recombinant anti-expression plasmid

[0033] 1.1 Synthesis of CA16 VP1 recombinant antigen

[0034] According to the amino acid sequences of the above eight CA16 VP1 antigens, and using gene recombination methods, the above CA16 VP1 sequences were respectively added to a flexible short peptide (GGGGS or GGGGSGGGGSGGGGS or AAY) and linked together (the eight amino acid sequences were respectively combined with the flexible short peptide amino acid When connecting the sequences, each amino acid sequence was repeated 3 times), the amino acid sequence was translated into a nucleotide sequence using Escherichia coli preferred codons, and EcoRI and HindIII restriction sites were introduced at the 5' end and 3' end respectively (see the sequence listing), and commissioned Shanghai Jierui Bioengineering Co., Ltd. to synthesize the gene of the above-mentioned CA16VP1 antigen s...

Embodiment 3

[0052] Example 3: Preparation of monoclonal antibody by immunizing mice with recombinant CA16 VP1 antigen protein

[0053] 1. Preparation of immunization antigens: The preparation method of 8 kinds of CA16 VP1 recombinant antigens was carried out according to the method in Example 2.

[0054] 2. Mice immunization: BALB / c mice were immunized with recombinant CA16 VP1 protein. Immunization procedure: For the first immunization, take recombinant CA16VP1 protein mixed with Freund's complete adjuvant to emulsify, immunize BALB / c mice, 50ug / mouse, intraperitoneal injection; carry out the second immunization after an interval of 14 days: take recombinant VP1 protein and Freund's incomplete adjuvant The adjuvant was mixed and emulsified, and BALB / c mice were immunized with 50ug / mouse, and injected intraperitoneally; blood was collected 7 days later, and the CA16 antibody activity was measured by indirect ELISA. No adjuvant was added to the mouse with the highest antibody titer, and t...

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Abstract

The invention discloses a set of Coxsackie virus A16 (CA16) VP1 recombinant antigens which are prepared by respectively connecting 8 amino acids disclosed as SEQ ID NO.1-SEQ ID NO.8 with a flexible short peptide amino acid sequence. The invention also discloses monoclonal antibodies capable of being specifically combined with the CA16 VP1 recombinant antigens and application of the recombinant antigens in preparing an A16 Coxsackie virus antigen or antibody detection kit. The 8 recombinant antigens and 4 monoclonal antibodies have the characteristics of high sensitivity, high specificity and the like, and can be used as key raw materials for developing Coxsackie virus CA16 detection reagents.

Description

technical field [0001] The invention relates to a group of Coxsackievirus A16 (CA16) VP1 recombinant antigens and monoclonal antibodies and applications thereof, belonging to the technical field of molecular biology. Background technique [0002] Hand foot mouth disease (HFMD) is a global common acute infectious disease caused by enterovirus. The disease mostly occurs in children under 5 years old, mainly manifested as mouth pain, anorexia, low fever, small herpes or small ulcers in hands, feet, mouth and other parts, and a small number of children can cause complications such as myocarditis. Individual critically ill children develop rapidly and lead to death. Hand, foot and mouth disease is an infectious disease caused by enteroviruses. There are more than 20 types (types) of enteroviruses that cause hand, foot and mouth disease. The most common HFMD outbreaks in China are Coxsackievirus A16 (CA16) and Enterovirus 71 (EV71). Previous studies believed that the symptoms o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K16/10G01N33/68G01N33/569
Inventor 汪运山
Owner 汪运山
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