人颗粒酶B融合蛋白的原核表达及其免疫血清的制备  

Expression of human Granzym B fusion protein in prokaryotic system and preparation of its immune serum

作　　者：汪文寰 冯方方[1] 蔡一奇 张婵琼[1] 岑丹维 宋易玲 郭刚强 张丽芳[1] 李文姝[1] WANG Wen-huan FENG Fang-fang CAI Yi-qi ZHANG Chan-qiong CEN Dan-wei SONG Yi-ling GUO Gang-qiang ZHANG Li-fang LI Wen-shu(Department of Microbiology and Immunology, Wenzhou Medical University, Wenzhou 325035, Zhejiang Province, Chin)

机构地区：[1]温州医科大学微生物学与免疫学教研室,浙江温州325035

出　　处：《中国生物制品学杂志》2017年第3期234-238,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81372447)

摘　　要：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 Granzym B （GzmB） fusion protein in prokaryotic system and prepare its specific immune serum. Methods Recombinant prokaryotic expression vector pET32a-GzmB was constructed and transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed recombinant GzmB （rGzmB） protein was purified by Ni-NTA agarose affinity chromatography, with which BALB/c mice were immunized s. c. in several sites for 3 times. The serum samples of mice were collected 2, 4 and 6 weeks after the first immunization and deteimined for GzmB specific IgG by indirect ELISA, based on which the maximum dilution of antibody was determined. Immunodot test was used to further confirm the specific binding of serum polyclonal antibody to GzmB. Results Restriction analysis and sequencing proved that recombinant plasmid pET32a-GzmB was constructed correctly. The relative molecular mass of expressed recombinant GzmB was 48 000. The specific IgG level against GzmB in sera of mice immunized with purified rGZmB increased as time went on and reached the peak value at week 4 after the first immunization, and was maintained at a certain level afterwards. The maximum dilution of antibody was 1 ： 1 600. The prepared polyelonal antibody in immune serum showed specific binding to GzmB. Conclusion Recombinant GzmB protein and its specific polyclonal antibody were successfully prepared, which laid a foundation of further study on the application of GzmB to target killing or target detection.

关 键 词：颗粒酶B 原核表达 多克隆抗体 

分 类 号：Q786[生物学—分子生物学] R392-33[医药卫生—免疫学]