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目的在原核体系中表达人颗粒酶B(granzyme B,Gzm B)融合蛋白,并制备Gzm B免疫血清多克隆抗体。方法构建Gzm B原核表达重组质粒p ET32a-Gzm B,转化大肠埃希菌BL21(DE3),IPTG诱导表达重组Gzm B(r Gzm B)蛋白,镍离子螯合亲和层析柱纯化表达产物。以纯化的r Gzm B蛋白为免疫原,皮下多点注射BALB/c小鼠,共免疫3次,分别于免疫后2、4、6周经小鼠尾静脉采血,分离血清,间接ELISA法检测免疫血清中Gzm B特异性Ig G抗体水平,并确定其抗体最大稀释度,免疫斑点试验检测免疫血清多克隆抗体对Gzm B的特异免疫结合特性。结果质粒p ET32aGzm B经双酶切及测序鉴定证明构建正确;表达的r Gzm B蛋白相对分子质量为48 000;纯化的r Gzm B蛋白免疫小鼠血清中Gzm B特异性Ig G抗体水平随免疫时间延长至第4周达到高峰,随后维持一定水平;抗体最大稀释度为1∶1 600;r Gzm B免疫血清能够特异地结合Gzm B,显示了其免疫结合特性。结论制备了r Gzm B蛋白和Gzm B小鼠免疫血清多克隆抗体,为后续开展Gzm B的靶向杀伤、靶向检测等应用研究奠定了基础。
Objective To express human granzyme B (Gzm B) fusion protein in prokaryotic system and prepare Gzm B immune serum polyclonal antibody. Methods The recombinant plasmid p ET32a-Gzm B was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant Gzm B (r Gzm B) protein was induced by IPTG and purified by nickel ion chelate affinity chromatography. Purified r Gzm B protein was used as an immunogen. BALB / c mice were injected subcutaneously three times in total. Blood was collected from the tail vein of mice at 2, 4, and 6 weeks after immunization. The serum was separated and detected by indirect ELISA The level of Gzm B-specific Ig G antibody in the serum was determined and the maximum dilution of the antibody was determined. The immunolabeling assay was used to detect the specific immune-binding properties of Gzm B to the immune serum polyclonal antibody. Results The plasmid p ET32aGzm B was confirmed by double enzyme digestion and sequencing. The relative molecular mass of the expressed r Gzm B protein was 48 000. The level of Gzm B-specific Ig G antibody in the serum of the purified r Gzm B protein immunized mice The time was prolonged until the fourth week and then reached a certain level. The maximum dilution of antibody was 1: 1 600. R Gzm B immune serum could specifically bind to Gzm B and showed its immunological binding properties. Conclusions The polyclonal antibody of r Gzm B protein and Gzm B mouse serum were prepared, which lays the foundation for the subsequent research on the targeted killing of Gzm B and targeted detection.