Preparation of polyclonal antibody against human parvovirus B19 VP1u and effect of N-terminal amino acids outside its conserved domain on the sPLA2 activity

Abstract:

[Objective] To prepare the polyclone antibody of human parvovirus B19-VP1u and study the impact of anti-VP1u and N-terminal amino acids outside the VP1u conserved domain on the sPLA2. [Methods] In the present study, target genes were firstly cloned to the prokaryotic expression vectors to express and purify the full length and N terminal truncated VP1u-MBP fusion proteins by using prokaryotic expression system. Purified MBP-VP1u was used to make the polyclonal antibody in New Zealand rabbit. The sPLA2 activities of the purified proteins were measured using a PLA2 assay kit. [Results] Western blot and immunofluorescence experiments showed that the polyclonal antibody against VP1u was specific. The purified VP1u-MBP fusion protein exhibits sPLA2 enzyme activity which was inhibited by anti-VP1u antibody. Meanwhile, when first 12 amino acids were truncated, the sPLA2 enzyme activity decreased 53% compared to the full length VP1u protein, and the activity was completely abolished when 67 amino acids were truncated. [Conclusion] Our research firstly determined that amino acids outside the conserved domain of VP1u, especially for the first N terminal 12 amino acids and the region from 22 to 67 amino acids play important roles in maintaining its sPLA2 enzyme activity, suggesting that the truncation of N terminal sequences may affect protein conformation. The prepared polyclonal antibody may provide a basis for the further studies on viral replication life cycle of parvovirus B19.