Cloning and expression of rotavirus SA11 VP7 and preparation of IgY antibodies against recombinant VP7 / 中华预防医学杂志
Chinese Journal of Preventive Medicine
;
(12): 526-530, 2009.
Article
in Chinese
| WPRIM
| ID: wpr-316140
ABSTRACT
<p><b>OBJECTIVE</b>To prepare eukaryotic expression of rotavirus (RV) SA11 capsid protein VP7, and to generate and purify yolk immunoglobulin (IgY) antibodies against the recombinant VP7 from Roman hens.</p><p><b>METHODS</b>MA104 cells were infected with the standard SA11 strain and the culture fluid was collected. A DNA fragment of 978 bp encoding SA11 VP7 was obtained by RT-PCR amplification from genomic RNA of RV SA11. The PCR products were ligated to pMD18-T vector following the confirmation by DNA sequencing and sub-cloned into pPICZalphaB. The recombinant pPICZalphaB-SA11 VP7 was transformed into E coli Top10. The plasmids were linearized by digestion of BstXI and transformed into Pichia pastoris X-33 through electroporation by DNA sequencing. The transformants were induced with methanol for expression. The cultural supernatant was subjected to SDS-PAGE and Western blotting. Fusion expression was purified through the column of affinity chromatography. IgY was identified and purified by SDS-PAGE and Western blotting from eggs of Roman hens immunized with recombinant SA11 VP7.</p><p><b>RESULTS</b>The RNA extracted from the RV culture fluid consisted of 11 bands visualized by silver staining. The expression vector pPICZalphaB-SA11 VP7 was constructed and the fusion protein in Pichia pastoris X-33 was harvested and purified. The recombinant SA11 VP7 with molecular weight of 40 200 was identified by Western blotting. The IgY antibodies against the recombinant SA11 VP7 were produced with a purity of 95 percent and yield of 10.2 mg per egg.</p><p><b>CONCLUSION</b>The preparation of IgY antibodies to recombinant SA11 VP7 might lay a foundation for the development of vaccines and diagnostic techniques.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Recombinant Proteins
/
Immunoglobulins
/
Chickens
/
Cloning, Molecular
/
Capsid Proteins
/
Allergy and Immunology
/
Genetics
/
Metabolism
/
Antigens, Viral
Limits:
Animals
Language:
Chinese
Journal:
Chinese Journal of Preventive Medicine
Year:
2009
Type:
Article
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