Prokaryotic expression and antiserum preparation for functional domain of Banana streak virus movement protein gene

【Objective】To provide antibody of Banana streak virus Guangdong isolate (BSV-GD) and to provide references for the function research of proteins encoded by BSV ORF3 further. 【Method】The MP functional gene sequence was obtained through bioinformatics analysis. The gene was cloned and inserted into the plasmid pET-28b(+) to construct the prokaryotic expression recombinant plasmid. The recombinant vector was induced by IPTG to express the fusion protein 6His·MP. Soluble analysis of the fusion protein was carried out by ultrasonic lysis method. The highly purified protein was obtained by His-tag purification kit. The special polyclonal antibody was generated by immunizing healthy rabbit using the purified protein as antigen. The specificity of antiserum was detected by Western-blot. The antibody titer was determined by indirect enzyme immunoassay. 【Result and conclusion】The analysis showed that the MP functional gene was 61-311 aa of the ORF3 sequence and the nucleotide sequence length was 753 bp. The prokaryotic expression recombinant plasmid pET28b-MP was constructed, and the expressed fusion protein 6His·MP was about 30 800 in size. Soluble analysis of the fusion protein indicated that it was an inclusion body. The highly purified target protein was obtained. The special polyclonal antiserum was generated according to the purified protein. The assay suggested that the antiserum had very strong specificity, and the antibody titer was higher than 1∶204 800.