线虫抗凝血蛋白AcaNAP7基因克隆、表达及抗凝活性鉴定

Cloning,expression and anticoagulant activities analysis of recombinant AcaNAP7

目的在大肠杆菌中表达犬钩虫线虫抗凝血蛋白NAP7(AcaNAP7)成熟蛋白,并鉴定表达产物的抗凝活性。方法根据AcaNAP7的核苷酸序列设计引物,从克隆质粒pUCm-T/AcaNAP7中扩增编码AcaNAP7成熟蛋白的基因;扩增产物经双酶切定向克隆到原核表达载体PET-32a中;构建的重组表达质粒转化至大肠杆菌BL21(DE3),用IPTG诱导表达;表达产物经镍琼脂糖凝胶FF纯化后,用凝血酶原时间(PT)和活化的部分凝血活酶时间(aPTT)检测体外抗凝血活性。结果克隆了编码AcaNAP7成熟蛋白的基因并构建了重组表达质粒pET-32a/AcaNAP7;在大肠杆菌BL21(DE3)中高效地表达了融合有硫氧还蛋白(Trx)的目的蛋白,产物主要以可溶形式存在;镍琼脂糖凝胶亲和纯化获得了纯度高达92%的目的蛋白;纯化产物能明显延长PT及aPTT,其延长PT比延长aPTT更有效。结论在大肠杆菌中成功表达了AcaNAP7融合蛋白,表达产物具有抗凝活性。该实验为进一步研究AcaNAP7的功能及应用奠定了基础。

Aim To express Ancylostoma caninum nematode anticoagulant protein 7 （AcaNAP7） gene （GenBank accession numbers： DQ435781 ） in Escherichia coli and analyze the anticoagulant activity of the recombinant protein in vitro. Methods After PCR amplification from the constructed cloning plasmids pUCm-T/AcaNAP7, the encoding mature AcaNAP7 gene was ligated into pET32a vector digested by Bam H Ⅰ and Hind Ⅲ, Then the recombinant plasmid of pET-32a/AcaNAP7 was transferred into E. coli BL21 （DE3）, and expressed by IPTG induction. The expressed products was purified by His affinity chromatography and its anticoagulant activity was measured with activated partial thrombohlastin time （aPTT） and prothrombin time （PT） assay in vitro. Results The gene encoding mature peptide AcaNAP7 was amplified and the prokaryotic expression plasmid pET-32a/ AcaNAP7 was constructed successfully. The recombinant fusion protein was mostly expressed in soluble form, its molecular weight was about 29 ku, and remarkably prolonged the PT and aPTT of human plasma in vitro. Conclusions We have successfully constructed prokaryotic recombinant expression plasmid pET- 32a/ AcaNAP7 and induced with IPTG to express AcaNAP7. The fusion protein of AcaNAP7-Trx has remarkable anticoagulant activities. The result of our study laid a basis for further research the function and application of AcaNAP7.