环形泰勒虫HSP90原核表达与中药单体筛选

Prokaryotic expression and screen of TCM monomer of HSP90 in Theileria annulata

旨在克隆、表达环形泰勒虫HSP90基因并筛选与其结合的中药单体。对新疆地方环形泰勒虫株HSP90基因进行PCR扩增、克隆测序,并构建系统进化树。对HSP90蛋白理化特性、高级结构与亚细胞定位进行预测分析。构建原核表达载体HSP90-pET32a,筛选诱导表达条件,经镍株纯化重组蛋白并用Western blot检测其免疫原性。使用DrugRep服务器对靶向环形泰勒虫HSP90的中药单体筛选。结果显示,HSP90基因编码的蛋白序列与小泰勒虫序列(登录号:XP 766455)同源性较高。HSP90蛋白编码223个氨基酸,理论pI为8.4,具有28个磷酸化位点。HSP90蛋白二级结构以α-螺旋(66.37%)和无规则卷曲(19.28%)为主,存在多个B细胞抗原表位,亚细胞定位主要分布于细胞质。HSP90-pET32a蛋白相对分子质量约50 ku,0.2 mmol/L IPTG诱导3 h蛋白表达较好。Western blot结果显示,该蛋白具有较好的免疫原性。经虚拟筛选,发现了5种与环形泰勒虫HSP90有较好结合力中药单体,其中圣草次甙结合力最强(affinity=-8.5 kJ/mol),作用方式以氢键和疏水作用为主。该数据为研发抗环形泰勒虫疫苗、诊断方法和初步筛选治疗环形泰勒虫病的中药提供理论依据。

To clone and express the HSP90 gene of Th eileria annulate(T.annulata)and screened the Chinese medicine monomers combined with HSP90.The HSP90 gene of T.annulata isolated from Xinjiang was amplified by PCR,then it was cloned and sequenced,and a phylogenetic tree was constructed.The physicochemical properties,advanced structure,and subcellular localization of the HSP90 protein were predicted and analyzed.Prokaryotic expression vector HSP90-pET32a was constructed,the recombinant protein was purified by Ni-NTA affinity chromatography and immunogenicity was detected by Western blot.DrugRep server was used to screen the Chinese medicine monomers targeting HSP90 of T.annulata.The results showed that the protein sequence encoded by HSP90 gene has high homology with the sequence of T.parva(accession number:XP 766455).The HSP90 protein encoded 223 amino acids,theory of pI was 8.4,had 28 phosphorylation sites.The secondary structure of HSP90 protein was mainly composed ofα-helix(66.37%)and irregular coil(19.28%),multiple B-cell epitopes were present,subcellular localization was mainly distributed in the cytoplasm.HSP90-pET32a protein molecular weight was about 50 ku,0.2 mmol/L IPTG induction for 3 h had better protein expression.Western blot indicated that the protein had good immunogenicity.Through virtual screening,five kinds of Chinese herbal monomers with good binding ability to T.annulata HSP90 were found,among them,eriocitrin had the strongest binding ability(affinity=-8.5 kJ/mol),the main modes of action were hydrogen bonding and hydrophobic interaction.The data provided a theoretical basis for the development of anti-T.annulata vaccine,diagnostic methods,and preliminary screening of traditional Chinese medicine for the treatment of T.annulata disease.