美洲钩虫抗血小板聚集多肽NaHPI1的分离与活性鉴定

Isolation and identification of hookworm platelet inhibitor NaHPI1 from Necator americanus

目的 从美洲钩虫分离并重组制备一种含有精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)序列的库尼茨(Kunitz)型多肽-美洲钩虫血小板抑制剂1(Na HPI1),鉴定重组Na HPI1(r Na HPI1)对血小板聚集的抑制作用。方法通过美洲钩虫成虫全长转录组测序获得Na HPI1完整cDNA序列,通过在线生物信息学网站检索氨基酸序列同源性蛋白并预测信号肽。根据Na HPI1氨基酸序列合成大肠埃希菌密码子偏爱的成熟肽编码基因序列,将该序列连接入表达质粒构建原核表达重组质粒,鉴定正确的重组质粒pET32a-sumo/Na HPI1,转入大肠埃希菌BL21(DE3)中,用IPTG诱导表达,收集菌体超声破碎后,用Ni-NTA亲和层析纯化重组融合蛋白,并在层析柱上进一步用小泛素蛋白样修饰物(SUMO)蛋白酶切割融合伴侣,收集透析液获得r Na HPI1。体外检测r Na HPI1对凝血酶、二磷酸腺苷(ADP)诱导的血小板聚集的抑制作用。结果 通过全长转录组测序获得了编码Na HPI1的全长mRNA,预测其完整编码框编码包括16个氨基酸组成的信号肽和81个氨基酸残基组成的成熟肽。Na HPI1为Kunitz型结构多肽,并在其最后1个半胱氨酸残基之后的羧基端具有RGD序列。利用大肠埃希菌表达系统成功表达并分离纯化获得了r Na HPI1蛋白。10、20和40μg/mL的r Na HPI1蛋白对ADP(20μmol/L)诱导的血小板聚集的抑制率分别为(17.35±2.77)%、(23.33±2.31)%、(30.94±2.03)%,对凝血酶(1 U/mL)诱导的血小板聚集的抑制率分别为(12.85±1.55)%、(20.28±1.17)%、(27.30±2.11)%。r Na HPI1蛋白对凝血酶及ADP诱导的血小板聚集均有明显抑制作用,差异有统计学意义(P<0.05),并呈一定的剂量依赖关系。结论 本研究分离并鉴定了美洲钩虫的血小板聚集抑制剂Na HPI1的抑制血小板聚集活性,其可能通过抑制血小板聚集而在钩虫吸血时发挥抗凝血作用。

Objective To isolate and recombinantly prepare Necator americanus hookworm platelet inhibitor(r Na HPI1),a Kunitz-type peptide containing an arginine-glycine-aspartic acid(Arg-Gly-Asp,RGD)sequence in Necator americanus,and detect the inhibitory activity against platelet aggregation by r Na HPI1.MethodsThe complete cDNA sequence of Na HPI1 was obtained by sequencing the full-length transcriptome of N. americanus adults,and the homology search and signal peptide prediction of the coding amino acid sequence were carried out through online bioinformatics website. The codon-optimized synthesized gene encoding mature Na HPI1 was ligated into the expression plasmid to construct a prokaryotic expression recombinant plasmid pET32a-sumo/Na HPI1. The correct recombinant plasmid was identified and transformed into Escherichia coli BL21(DE3),and expression was induced with IPTG. After the cells were collected and sonicated,the recombinant fusion protein was purified by Ni-NTA affinity chromatography,and the fusion partner was further cleaved with small ubiquitin-like modifier(SUMO)protease on the column,and permeate was collected to obtain r Na HPI1. The inhibitory activity of r Na HPI1 on thrombin and adenosine diphosphate(ADP)-induced platelet aggregation was detected in vitro.ResultsThe full-length mRNA encoding Na HPI1 was obtained by full-length transcriptome sequencing,and its open reading frame was predicted to encode a peptide consisting of a signal peptide with 16 amino acid residues and a mature peptide with 81 amino acid residues. Na HPI1 is a Kunitz-type peptide and has an RGD sequence at its carboxy terminus after its last cysteine residue. The r Na HPI1 was successfully purified after expression in E.coli. The inhibition rates of 10,20 and 40 μg/mL r Na HPI1 on ADP(20 μmol/L)-induced platelet aggregation were(17.35±2.77)%,(23.33±2.31)% and(30.94±2.03)%,respectively. The inhibition rates of platelet aggregation induced by thrombin(1 U/mL)were(12.85±1.55)%,(20.28±1.17)% and(27.30±2.11)%,respectively. rNaHPI1 significantly inhibited thrombin and ADP-induced platelet aggregation(P<0.05),and showed a dose-dependent manner.ConclusionThe platelet aggregation inhibitor NaHPI1 of N. americanus was isolated and identified;it may play a role for N. americanus to prevent haemostasis by inhibiting platelet aggregation.