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对HBoV2 VP1U蛋白进行原核表达并检测其sPLA_2活性,对其关键氨基酸进行突变,检测突变对sPLA_2活性的影响。构建重组质粒pMD18T-VP1U,选取VP1U蛋白4个关键氨基酸为突变点,分别进行原核表达,蛋白纯化后用sPLA_2试剂盒检测sPLA_2活性,对比突变前后VP1U蛋白sPLA_2活性有无变化。野生型HBoV2VP1U蛋白的sPLA_2活性为0.243μmol/min/mL,具有sPLA_2活性;突变体L19P、P21R、D42H及Y45N蛋白的sPLA_2活性分别为野生型蛋白的1.2%、1.2%、0.82%、0.41%。野生型HBoV2VP1U蛋白具有sPLA_2活性,突变体蛋白均几乎完全丧失了sPLA_2活性,提示19、21、42、45位4个氨基酸是维持sPLA_2活性的关键氨基酸。该研究为靶向抗病毒药物的研究提供了理论基础。
The prokaryotic expression of HBoV2 VP1U protein was detected and the sPLA2 activity was detected. The key amino acids were mutated to detect the effect of mutation on sPLA2 activity. The recombinant plasmid pMD18T-VP1U was constructed and the four key amino acids of VP1U protein were selected as the mutants. The prokaryotic expression was carried out. The sPLA2 activity was detected by sPLA_2 kit before and after mutagenesis. The sPLA2 activity of wild type HBoV2VP1U protein was 0.243μmol / min / mL, which showed sPLA_2 activity. The sPLA_2 activity of mutant L19P, P21R, D42H and Y45N protein was 1.2%, 1.2%, 0.82% and 0.41% of the wild type protein respectively. The wild-type HBoV2VP1U protein has sPLA2 activity, and the mutant protein almost completely lost the sPLA2 activity, suggesting that the four amino acids 19, 21, 42 and 45 are the key amino acids for maintaining sPLA2 activity. This study provides a theoretical basis for the research on antiviral drugs.