Cloning and expression of ORF146 gene of Spodoptera litura multicapsid nucleopolyhedrovirus II (SpltMNPVII)

Abstract:

Abstract: 【Objective】To study the structure and function of a newly found virus strain Spodoptera litura multicapsid nucleopolyhedrovirus II (SpltMNPVII) ORF146 gene. 【Methods】The primers were designed according to the sequence of SpltMNPVII genome. The promoter of ORF146 was amplified by PCR. The promoter activities and the time course of mRNA transcription were analyzed. The fragment of the ORF146 gene was then cloned into the vector of pET28a(+) and expressed. The polyclonal antibody was prepared by using the purified fusion protein. Titration determination of anti-ORF146 antibody was evaluated by ELISA.【Result】Nucleotide sequence analysis demonstrated that this gene has a 1383 bp ORF, encoding 460 amino acids with a predicted molecular weight of 50.4 kDa. Analysis of both promoter activities and the time course of mRNA transcription of the ORF146 gene showed that ORF146 was transcripted in early stage as well as in late stage. The transcription began at 2 h post infection (hpi) and reached two peaks at 8 and 18 hpi and then the transcription level was slightly decreased from 24 hpi. pET-28a-ORF146 fusion protein expressed in prokaryotic and purified polyclonal antibody was with good specificity, with the titer above 1:3200. 【Conclusion】SpltMNPV II ORF146 gene is a composition structure protein, which was expressed at both early and late stage. ORF146 might be involved in viral DNA replication. The polyclonal antibody can be used to further study the biological characteristics and functions of proteins.