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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 purified recombinant human neutrophil elastase (HNE) by genetic engineering and to lay the foundation for the further preparation of the corresponding antibody of HNE and the establishment of the detection method of semen HNE. METHODS: HNE mRNA was obtained from human peripheral blood neutrophils by using specific primers of HNE and cloned into pGEX-2T to obtain recombinant plasmid pGEX-2T / HNE. The recombinant plasmid was identified by PCR, double enzyme digestion and gene sequencing. The recombinant plasmid was transformed into competent E. coli DH5α, and recombinant fusion protein GST / HNE was induced by isopropyl β-D-thiogalactoside (IPTG). The recombinant fusion protein was purified by thrombin to obtain recombinant HNE, which was purified by glutathione sepharose beads to obtain purified recombinant HNE. Results: The recombinant plasmid pGEX-2T / HNE was successfully prepared and transformed into Escherichia coli DH5α. The expression of recombinant fusion protein GST / HNE was successfully obtained after overnight induction with IPTG at 18 ° C. After purified by thrombin and glutathione agarose beads, the purified recombinant protein HNE was successfully obtained. Conclusion: The purified recombinant HNE obtained the foundation for the further preparation of the corresponding antibody of HNE and the establishment of the detection method of semen HNE.