河虾过敏原原肌球蛋白的基因克隆与原核表达  

Gene Cloning and Prokaryotic Expression of the Allergen Tropomyosin from Macrobrachium nipponense

作　　者：骆叶晴 郑双艳[2] 孙耀斌 陈娇[1,2,3] 刘鑫 陈红兵[1,2] 谢彦海 LUO Yeqing;ZHENG Shuangyan;SUN Yaobin;CHEN Jiao;LIU Xin;CHEN Hongbing;XIE Yanhai(State Key Laboratory of Food Science and Resources,Nanchang University,Nanchang 330047,China;Sino German Joint Research Institute,Nanchang University,Nanchang 330047,China;College of Food Science&Technology,Nanchang University,Nanchang 330047,China)

机构地区：[1]南昌大学食品科学与资源挖掘全国重点实验室,江西南昌330047 [2]南昌大学中德联合研究院,江西南昌330047 [3]南昌大学食品学院,江西南昌330047

出　　处：《食品科学》2024年第13期89-95,共7页Food Science

基　　金：国家自然科学基金地区科学基金项目(32060584);江西省自然科学基金项目(20232BAB205084)。

摘　　要：为了探寻天然原肌球蛋白的可替代物作为诊断和治疗虾类过敏的基础材料,本研究从河虾中提取总RNA,利用cDNA末端快速扩增技术克隆河虾过敏原原肌球蛋白基因的全长序列。以此序列设计表达引物,构建表达载体,最后通过异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)诱导表达重组原肌球蛋白。结果显示,河虾的原肌球蛋白基因cDNA序列全长1658 bp,开放阅读框855 bp,编码284个氨基酸,预测蛋白等电点4.70,预测分子质量32.8 kDa。河虾原肌球蛋白cDNA序列已上传至GenBank数据库,登录号为OP974621。本研究成功构建河虾原肌球蛋白重组表达质粒pET-30a-TM,并用IPTG进行体外诱导表达,最佳诱导条件为IPTG浓度1 mmol/L、37℃诱导4 h。可溶性分析结果表明重组原肌球蛋白主要以可溶性形式存在于细胞破碎液的上清液中,分子质量约38 kDa。To explore substitutes for natural tropomyosin as basic materials for the diagnosis and treatment of shrimp allergy,total RNA was extracted from Macrobrachium nipponense,and the full-length sequence of the tropomyosin gene was cloned by rapid amplification of cDNA ends(RACE).Specific primers were designed based on the cDNA sequence of tropomyosin and the expression plasmid pET-30a-TM was constructed.Finally,recombinant tropomyosin was expressed under the induction of isopropyl-β-D-thiogalactopyranoside(IPTG).The results showed that the full-length cDNA sequence of tropomyosin was 1658 bp in length(GenBank accession number:OP974621).Its open reading frame(ORF)was 855 bp in length,encoding 284 amino acids,with a predicted protein isoelectric point of 4.70 and a predicted molecular mass of 32.8 kDa.The highest expression was obtained after 4 h of induction with a final IPTG concentration of 1 mmol/L at 37℃.The recombinant tropomyosin mainly existed as a soluble form in the supernatant of cell lysate with a molecular mass of approximately 38 kDa.

关 键 词：河虾 原肌球蛋白 基因克隆 原核表达 重组蛋白 

分 类 号：Q78[生物学—分子生物学]