The cloning and sequencing of readthrough protein gene from BYDV GAV virus and its expression in E. coli

Chang ShengJun; Wang XiFeng; Li Li; Ma ZhanHong; Zhou GuangHe

Scientia Agricultura Sinica 33(4): 38-45

2000


ISSN/ISBN: 0578-1752
Accession: 003964815

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Abstract
The BYDV GAV isolate was maintained at the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences. The cDNA of BYDV GAV Readthrough protein (RTP) gene was amplified from the extracted RNA of BYDV GAV by using the polymerase chain reaction (PCR), and cloned into pGEM-7zf(+). Its complete nucleotide sequence has been determined by means of Sanger's Dideoxy-mediated chain-termination method. The result shows that BYDV GAV RTP gene has 1377 nt. It shares 87.4% and 87.1% identity with RTP gene of BYDV MAV in terms of nucleotide sequence and amino acid respectively. The IPTG-inducible expression vector containing the BYDV GAV RTP gene was constructed and transferred into E. coli BL21 (DE3). High-level expression of the specific protein was achieved by IPTG induction. The results of SDS-PAGE and Western blotting show that the expression product is 66 ku.