Expression of recombinant protein VP2 of human bocavirus and detection by indirect ELISA
10.3969/j.issn.1006-5725.2016.17.008
- VernacularTitle:人博卡病毒重组蛋白表达及间接酶联免疫法检测的建立
- Author:
Yunqi ZHANG
;
Taojun HE
;
Xuedong LU
;
Yinhui ZHANG
- Publication Type:Journal Article
- Keywords:
Human bocavirus;
Prokaryotic express;
Indirect ELISA
- From:
The Journal of Practical Medicine
2016;32(17):2803-2806
- CountryChina
- Language:Chinese
-
Abstract:
Objective By prokaryotic expression and purifying the human bocavirus recombinant protein VP2, to establish the indirect enzyme-linked immunoassay for detection of virus. Methods We amplified the human bocavirus recombinant protein VP2 gene fragments from WHL-1 template by PCR , and cloned into the expression vector pET28a, then conversed into the BL21 (DE3) and expressed the fusion protein detected by Western Blot detection , the obtained the antibody and detected the human bocavirus in serum in Guanghzhou area in healthy people. Results The Recombinant prokaryotic expression identified correct by double enzyme, and it could occur specific reaction with the virus positive serum. The best optimal antigen coating concentration were serum multiples and blocking BSA was 2 mg/mL , 1 ∶ 200 and 1%. The best working dilution of enzyme-labeled secondary antibody was 1 ∶ 4 000. The best working hours was 1h. This detection method had good specificity and reproducibility. The cut-off of the indirect ELISA method was 0.1 and the sensitivity and specificity of the developed ELISA method were 92% and 98% respectively. The coincidence rate of determination results by the developed kit and control kit was 97%. Conclusion The competitive ELISA established by prokaryotic expressing and purifying the human bocavirus protein VP2 protein , provides a basis in detecting the human bocavirus serum antibody.
