[Cloning and expression of rotavirus SA11 VP7 and preparation of IgY antibodies against recombinant VP7].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
The preparation of IgY antibodies to recombinant SA11 VP7 might lay a foundation for the development of vaccines and diagnostic techniques.
Zhang
Shi-Hai
SH
Department of Clinical Laboratories, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China.
Luo
Qing-Li
QL
Zhou
Yin-Di
YD
Li
Jing
J
Xu
Yuan-Hong
YH
Shen
Ji-Long
JL
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D002645:Chickens; D003001:Cloning, Molecular; D007136:Immunoglobulins / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000956:Antigens, Viral; D036022:Capsid Proteins; C016512:IgY; D007136:Immunoglobulins; D011994:Recombinant Proteins; C050779:VP7 protein, Rotavirus