|
|
|
| Fusion Expression and Purification of β Type Vacuole Processing Enzyme Gene(VvβVPE) in Vitis vinifera and Its Antibody Preparation |
| 1, Wei Rong1, 1 |
|
|
|
|
Abstract Vacuole processing enzyme(VPE) gene family plays an important role in mediating and executing programmed cell death in plants. β type VPE is specifically expressed in seed, which is indispensable in seed development. The ovules were harvested from V. vinifera cv. Pinot Noir in 8 different development stages. RNA was isolated from these ovules. VvβVPE gene was obtained by RT-PCR, and the mixture of cDNA was used as template. The results showed VvβVPE gene contained a complete ORF(open reading frame) sequence (1 485 bp) encoding 494 amino acid(GenBank accession number: KC136352). The full-length sequence was cloned into the prokaryotic expression vector of pGEX-6P-1. An about 81.3 kD fusion protein of inclusion body (GST-VvβVPE) was expressed in the Escherichia coli BL21 through IPTG inducing. After optimizing the inducing condition, two different methods were adopted to purify the protein, containing electro dialysis and dialysis renaturation by GdmCl treating, and then immunized the rabbit to obtain the polyclonal antibody. Western blot displayed a specific immunity-antigen signal, suggesting the obtained antiserum can be used for function analysis and detecting transgenic plants.
|
|
Received: 15 November 2012
|
|
|
|
|
|
|
