Preparation and characterization of an egg yolk antibody (IgY) against the recombinant human A rotavirus VP8
10.3760/cma.j.issn.0254-5101.2014.04.011
- VernacularTitle:人A组轮状病毒结构蛋白VP8的卵黄抗体制备与活性检测
- Author:
Zhi ZHANG
;
Weidong HU
;
Xiaoyang ZOU
;
Tianyou LI
;
Bingchao LI
;
Zaixin LI
- Publication Type:Journal Article
- Keywords:
Diarrhea;
Human A rotavirus;
VP8;
IgY
- From:Chinese Journal of Microbiology and Immunology
2014;(4):310-314
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare an egg yolk antibody ( IgY) against the recombinant human A rotavirus VP8 and evaluate its biological characteristics .Methods The complete VP4 gene was used as the template to clone VP8 DNA fragment by PCR .The VP8 gene was then cloned into the vector pET 28a for ex-pression of VP8 protein in the prokaryotic system.The expression plasmid of pET28a-VP8,identified by DNA sequencing,was transformed into E.coli BL21 to induce the expression of protein by 0.5 mmol/L IPTG at 30℃.The purified and refolded recombinant VP 8 protein was used to immunize laying hens in combination with complete Freund′s adjuvant ( CFA ) .Anti-VP8 IgY antibodies were extracted from egg yolks by using water dilution-ultrafiltration assay and were further analyzed by SDS-PAGE,ELISA,Western blot and Dot blot assay,respectively.Results The recombinant VP8 protein was expressed in E.coli BL21 as an inclusion body and its molecular weight was about 35 ×10 3 .The VP8 protein was well refolded in a buffer containing Tris-HCl (pH8.0).The isolated anti-VP8 IgY antibodies showed a high titer of 1∶100 000 and a high stabil-ity under the condition of pH3-11 and temperature 25-65℃.Moreover,the anti-VP8 IgY antibodies specific-ally recognized the wild type human A rotavirus .Conclusion The prepared anti-VP8 IgY antibodies showed the advantages of high titer ,good specificity and stability .It might be used as the tool in further investigation for the prevention and treatment of diarrhea caused by human A rotavirus .
