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弓形虫GRA1基因的原核表达与鉴定 预览

PROKARYOTIC EXPRESSION AND CHARACTERIZATION OF GRA1 GENE OF TOXOPLASMA GONDII
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摘要 为深入研究弓形虫致密颗粒蛋白GRA1及其单克隆抗体对弓形虫感染诊断的意义,本研究采用逆转录PCR方法扩增弓形虫RH株截短型GRA1基因,将其克隆至原核表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达并优化表达条件,SDS-PAGE分析表达情况,并分别通过Western blot和间接免疫荧光检测(indirect immunofluorescence assay,IFA)鉴定蛋白和单抗的免疫原性。结果表明重组蛋白GRA1高效可溶性表达,分子量约为24 kDa,且在28℃条件下诱导表达效果更好。Western blot显示,重组蛋白GRA1可以被感染弓形虫的犬阳性血清识别,具有良好的反应原性。IFA显示单克隆抗体能够特异性地识别弓形虫的天然蛋白,GRA1主要分布于虫体的细胞质中。本研究结果可为利用GRA1蛋白和其单抗诊断弓形虫感染提供借鉴和依据。 In the present study, the dense granule protein GRA1 of Toxoplasma gondii was prepared in prokaryotic expression system and specific monoclonal antibodies were generated for development of a diagnostic tool. The truncated GRA1 gene of T. gondii RH strain was amplified by RT- PCR and cloned into pET-28a(+) expression vector. Then the plasmid pET-28a(+)-GRAl was transfomed into Escherichia coli BL21 ( DE3) for expression via induction with IPTG. The results showed that the truncated fragment of GRA1 was highly expressed and released into supernatant with the molecular weight of approximates to 24 kDa. The optimal induction temperature was determined to be 28 ^. Western blot demonstrated that the recombinant GRA1 reacted strongly with serum samples from dogs infected with T. gondii RH strain. In addition, GRA1 was located in the c^oplasms of T. gondii in indirect immunofluorescence assay using the monoclonal antibodies. The availability of the recombinant GRA1 and its monoclonal antibodies provided valuable reagents for development of diagnostic method for Toxoplasma infection.
作者 韩阳瑞 蒋蔚 陈芸 薛俊欣 熊挺 龙梦瑶 凌娇 王权 HAN Yang-rui;HANG Wei;CHEN Yun;XUE Jun-xin;XIONG Ting;LONG Meng-yao;LING Jiao;WANG Quan(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Shanghai Entry-Exit Inspection and Quarantine Bureau,Shanghai 200135,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)
出处 《中国动物传染病学报》 CAS 北大核心 2019年第1期70-77,共8页 Chinese Journal of Veterinary Parasitology
基金 上海市自然科学基金(17ZR1437300) 上海市科学技术委员会科研计划项目(17140900400).
关键词 弓形虫 GRA1 原核表达 单克隆抗体 诊断 Toxoplasma gondii GRA1 prokaryotic expression monoclonal antibody diagnosis
作者简介 韩阳瑞,女,硕士研究生,兽医学专业;通信作者:王权,E-mail: wangquan@shvri.ac.cn.
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