In Vitro Expression And Antibody Preparation Of Marek's Disease Virus (MDV) UL13 Protein Kinase

Bac-to-Bac? Baculovirus Expressing System is frequently applied to generate heterogonous proteins owing to its special characters of high levels of heterogonous gene expression and high bioactivity, and the purified protein are contributed to the analysis of protein bio-function. The bio-function of conserved herpes viral protein kinases (CHPKs) in herpesvirus are one of the hot topic of herpesvirus'research. The roles of different CHPKs play differently among different herpesviridae. In this study, Bac-to-Bac? Baculovirus Expressing System was applied to express the Marek's disease virus (MDV) UL13 protein kinase which is a member of CHPKs. Three recombinant baculoviruses including rBac-His-UL13, rBac-His-EGFP and rBac-His were constructed successfully. Optimal expression conditions and harvest time for MDV UL13 protein kinase and EGFP were confirmed by optimization assays. In this study, the purification platform of Bac-to-Bac? Baculovirus Expressing System was preliminarily established based on Bac-to-Bac? HT vectors. Besides, the essential materials for MDV UL13 protein kinase bio-function research were obtained. To observe the effects on the viral growth of MDV, assays were performed through MDV UL13 protein kinase uptake. The results showed that MDV UL13 protein kinase had no significant effects on the plaque size of a very virulent (vv) MDV-1 strain– RB-1B.Polyclonal antibody against the full UL13 ORF was prepared by gene immunization. Antibodies against MDV UL13 protein kinase generated via different methods were applied to detected MDV UL13 protein kinase. In the mean time, GFP-UL13 fusion protein expression was applied to make further confirmation for the intracellular location of MDV UL13 protein kinase. In this test, it was demonstrated that MDV UL13 protein kinase shared the same intracellular localization with other members of CHPKs– in the nuclei.