人细小病毒B19 VP1u多克隆抗体的制备及其保守区外N端氨基酸对sPLA2活性影响的初步研究

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

摘要:

【目的】制备人细小病毒B19-VP1u的多克隆抗体，探究VP1u多克隆抗体及其保守区外N端氨基酸对病毒磷脂酶A2活性的影响。【方法】首先通过分子克隆方法构建相应原核表达载体；利用原核表达系统纯化含MBP标签的VP1u全长及N端系列截短突变融合蛋白；接着免疫新西兰大白兔制备全长VP1u蛋白的多克隆抗体；最后利用磷脂酶A2活性检测试剂盒检测了纯化蛋白的磷脂酶A2活性。【结果】Western blot及免疫荧光实验证实制备的多克隆抗体具有较高的特异性；磷脂酶A2活性检测发现全长VP1u-MBP融合蛋白具有一定的活性，该活性可以被VP1u的抗体抑制；N端保守区外截短系列蛋白的酶活检测发现，N端截掉12个氨基酸时酶活降低53%，截掉67个氨基酸时酶活性几乎完全丧失。【结论】首次发现VP1u保守区外N端氨基酸，尤其是第12个氨基酸前的区域以及第22?67个氨基酸之间的区域，对sPLA2活性的保持具有重要意义，推测该区域可能对维持正常的蛋白构象起重要的作用；而其特异性多克隆抗体的制备也为进一步研究B19病毒VP1u在病毒复制周期的作用奠定 基础。

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.