Efficient Expression Of Coat Protein Gene From Cucumber Mosaic Virus And Preparation Of Antiserum

In order to express coat protein (CP) of CMV correctly, a pair of primers were designed in accordance with the reported CMV-CP gene sequences. CP gene of CMV was amplified from the recombinant plasmid PBIC carrying CP gene by polymerase chain reaction(PCR).It was cloned into the prokaryotic expression vector pET-22b(+). The sequence of the CP gene was 657 nucleotides long determined by Sanger dideoxy method. Compared with a reported CMV-CP gene sequence, the homology of nucleotide sequences were 100%. The sequencing result also demonstrated the recombinant vector pET-22b-CP has a proper ORF encoding 218 amino acids.The recombinant vector was transformed into BL21(DE3) cells.Transformants were grown and induced by the addition of isothiopropylgalactoside(IPTG) to a concentration of ImM with continued shaking at 37癈. 6 hours later,the amount of the expression yield of CP accounting for the total protein of the cells was 28.9%(about 2mg/mL CP).After SDS-PAGE, the recovery of CP added the same volume of incomplete Freund Adjuvant emulsified completely,injected rabbits by subcutaneous injection. Every rabbit was injected 2mL emulsified antigen(300ug CP) once a week. 10 days after the fourth injection,blood was harvested by carotid artery and then antiserum was made from it.ELISA proved that the titres of the antiserum was 1/10000, it also proved that the acquired antiserum was specific to CMV. The assays lay a firm- foundation on detecting CMV fast and effectively and accumulate the experiences of efficient expressing exogenous gene in E.coli and preparation of antiserum.