重组人中性粒细胞弹性蛋白酶的制备及鉴定

Preparation and identification of recombinant human neutrophil elastase

目的:利用基因工程技术制备纯化的重组人中性粒细胞弹性蛋白酶(HNE),为进一步制备HNE的相应抗体和建立精液HNE的检测方法奠定基础。方法:利用HNE的特异引物从人外周血粒细胞中获得HNEmRNA,并将其cDNA克隆入质粒pGEX-2T中以获得重组质粒pGEX-2T/HNE。重组质粒经PCR、双酶切和基因测序鉴定后转入感受态大肠埃希菌DH5α中,并用异丙基β-D-硫代半乳糖苷(IPTG)诱导表达重组融合蛋白GST/HNE。重组融合蛋白经凝血酶裂解后获得重组HNE,并经谷胱甘肽琼脂糖珠纯化后获得纯化的重组HNE。结果:成功制备重组表达质粒pGEX-2T/HNE,并转化入大肠埃希菌DH5α中。经IPTG在18℃过夜诱导后成功获得重组融合蛋白GST/HNE的表达。经凝血酶裂解和谷胱甘肽琼脂糖珠纯化后成功获得纯化的重组蛋白HNE。结论:纯化的重组HNE的获得为进一步制备HNE的相应抗体和建立精液HNE的检测方法奠定了基础。

Objective ： To prepare a purified recombinant human neutrophil elastase （HNE） using genetic engineering technology, and pave the way for the preparation of the antibody to HNE and establishment of semen HNE detection methods. Methods： HNE mRNA was obtained from human peripheral blood granulocytes with specific HNE primers, and the cDNA of HNE was cloned into the plasmid pGEX-2T to derive a recombinant plasmid pGEX-2T/HNE. After PCR identification, double-enzyme digestion and gene sequencing, the recombinant plasmid was transferred into competent Escherichia coli DH5α and further induced to express the recombinant fusion protein GST/HNE by isopropyl β-D-thiosulfate galaetosidase （ IPTG ）. The recombinant fusion protein was cleaved by thrombin and further purified with glutathione agarose beads to obtain purified recombinant HNE. Results： The recombinant plasmid pGEX-2T/HNE was successfully prepared and transferred into E. coli DH5α; the expression of the recombinant fusion protein GST/ HNE was successfully induced by IPTG at 18 ℃ overnight; and the purified recombinant protein HNE was successfully obtained by thrombin cleavage and purification of glutathione agarose beads. Conclusion ： The acquirement of purified recombinant HNE has prepared the ground for the preparation of the antibody to HNE and establishment of semen HNE detection methods.