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目的克隆、原核表达人鞭虫(Trichuris trichiura)乳清酸蛋白TtWAP-3,分析其生物学活性。方法分别以鞭虫成虫前端及尾端cDNA为模板,通过PCR扩增TtWAP-3编码基因;将TtWAP-3编码基因克隆至原核表达载体pET32a-sumo,构建重组表达质粒转化至大肠埃希菌BL21(DE3),IPTG诱导表达目的蛋白并进行Ni-NTA亲和纯化和SUMO酶酶切,用发色底物法检测rTtWAP-3对蛋白酶的抑制活性。结果在成虫前端及尾端均成功克隆获得TtWAP-3编码基因序列,构建的原核表达重组质粒pET32a-sumo/TtWAP-3在大肠埃希菌表达rTtWAP-3,该蛋白对人中性粒细胞弹性蛋白酶(NE)和人蛋白酶3(PR3)有抑制活性,其抑制人NE(20nmol/L)、人PR3(200nmol/L)50%活性的浓度分别约为2.25μmol/L和5.34μmol/L。结论成功获得原核表达的重组TtWAP-3,该蛋白对人NE及人PR3均有抑制作用,为研究TtWAP-3对宿主的免疫调节作用奠定了基础。
Objective To clone and express the Trichuris trichiura whey protein TtWAP-3 and analyze its biological activity. Methods The cDNA of TtWAP-3 was amplified by PCR using the front and tail cDNAs of whipworm as template. The gene encoding TtWAP-3 was cloned into the prokaryotic expression vector pET32a-sumo to construct the recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3). The target protein was induced by IPTG and purified by Ni-NTA affinity purification and SUMO enzyme digestion. The inhibitory activity of rTtWAP-3 on protease was detected by chromogenic substrate method. Results The sequence of TtWAP-3 gene was successfully cloned from both the front and the tail of adult worms. The prokaryotic expression plasmid pET32a-sumo / TtWAP-3 was expressed in Escherichia coli which expressed rTtWAP-3, Protease (NE) and human protease 3 (PR3) showed inhibitory activity against human NE (20nmol / L) and human PR3 (200nmol / L) at concentrations of about 2.25μmol / L and 5.34μmol / L, respectively. CONCLUSION: The recombinant TtWAP-3 prokaryotic expression vector is successfully obtained. This protein can inhibit both human NE and human PR3, which lays the foundation for the study on the immunomodulatory effects of TtWAP-3 on the host.