[Expression and activity determination of recombinant capsid protein VP2 gene of enterovirus type 71].

Abstract

OBJECTIVE:

To clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen.

METHODS:

VP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected.

RESULTS:

VP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay.

CONCLUSIONS:

VP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

• Authors: Xueyong Huang, Guohua Liu, Xiaoning Hu, Yanhua Du, Xingle Li, Yuling Xu, Haomin Chen, Bianli Xu
• Journal: Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine] (Zhonghua Yu Fang Yi Xue Za Zhi) Vol. 48 Issue 4 Pg. 324-7 (Apr 2014) ISSN: 0253-9624 [Print] China
• PMID: 24969459 (Publication Type: English Abstract, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Neutralizing
• Antibodies, Viral
• Capsid Proteins
• Immunoglobulin M
• Recombinant Proteins

Topics

• Antibodies, Neutralizing (blood)
• Antibodies, Viral (blood)
• Capsid Proteins (genetics, immunology)
• Enterovirus A, Human (genetics, immunology, isolation & purification)
• Enzyme-Linked Immunosorbent Assay
• Genetic Vectors
• Hand, Foot and Mouth Disease (immunology)
• Humans
• Immunoglobulin M (blood)
• Recombinant Proteins (genetics, immunology)