Detection Of Banana Streak Virus And Expression Of The CP Peptide

Banana streak disease (BSD) is one of major diseases in banana and the pathogen is Banana streak virus (BSV) belonging to genus Badnavirus. We designed three pairs of primers based on the BSV sequences reported and an improved system of PCR for detecting BSV in banana was established.This system was used to detect the banana samples from hainan province,and no BSV was found.In order to express the peptide of BSV coat protein (BSVCPP) correctly, a pair of primers were designed in accordance with the reported BSV complete sequences and characteristic of badnavirus CP. The gene of BSVCPP was amplified from the banana streak virus particle and inserted into pGEM-T-easy vector. Sequencing result showed that a 657 nucleotides BSVCPP gene fragment was achived. Recombinant plasmid pGEM-T-BSVCPP was digested by HindⅢand EcoRⅠand the target band was purified for being ligated into prokaryotic expression vector pET-28b(+). Positive clone was named as pET-28b-BSVCPP after confirmation by PCR, digestion and sequencing. pET-28b-BSVCPP has a proper ORF encoding 213 amino acids. The recombinant vector was transformed into Rosetta (DE3)cells. Transformants were grown and induced by the addition of isothipropy- lgalactoside(IPTG) to a concentration of 1mM with continued shaking at 37℃. Two hours later, the amount of the expression yield of fusion protein accounting for the total protein of the cells was 23.6%. After SDS-PAGE, one part of the target protein band was added with incomplete Freund Adjuvant for emulsification and the other part was purified with elutrap system, then rabbits were immunized with the two part protein products, respectively. Blood was harvested by carotid artery and then antiserum was made from it. ELISA assay proved that the antiserum by tow methods of injection is different.