| Barley yellow dwarf virus (BYDV) is one of the mosteconomically important cereal viruses. Up to now, the mechanism of BYDV movingin vivo isn't clear, although ORF4 gene of BYDV was proposed being related to virusmovement in hosts. In this study, ORF4 gene of BYDV-GPV was cloned andexpressed in Escherichia coli. The antiserum against protein expressed by BYDV-GPV ORF4 was raised and used in detection of the 17kDa protein of BYDV-GPV ininfected oats. Fragment of ORF4 was amplified through RT-PCR after the primers beendesigned and synthesized according to the published nucleotide sequence. Thefragment was inserted into pGEM-T Easy vector and the recombinant plasmid wasobtained and named of pGEM-T-O4. Sequence analysis showed that ORF4 contains456 nucleotides that can code a protein of 152 amino acids with Mr about 17kDa.pET-O4, the prokaryotic expression plasmid (containing ORF4), was obtained byinserting the fragment, when it was recovered from pGEM-T-O4 by digested withNde I and Bam HI, into pET-5a plasmid. After induced with IPTG, the proteins wereanalyzed via SDS-PAGE. 17kDa protein was expressed in BL21(DE3)pLysS whichcontaining pET-O4 plasmid, compared with that of not induced pET-O4 and inducedpET-5a. The expression results of pET-O4 induced with different concentration ofIPTG or in different time course showed that the concentration of IPTG had noobvious effect on the induced expression, but there was a stronger expression alongwith the time increase before 8 hours. The antiserum with titer of 1:2048 was obtained from rabbit after injections with 68purified 17kDa protein, which had a specific reaction with 17kDa protein, purified orinduced in pET-O4/BL21(DE3)plysS. The antiserum was also used to detect theexistence of 17kDa protein in oat after infected with BYDV-GPV. The result showedthat the antiserum had a positive reaction with all of the samples of infected for 1 day,2 days 3 days and 4 days, and the reaction was stronger when time prolonged afterinfection.
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