Zheng Bin, Zheng Huan-Qin, He Ai, Li Zhuo-Ya, Cao Ai-Lian, Zhang Rui-Lin, Zhan Xi-Mei
Department of Parasitology, Zhongshan Medical College,Sun Yat-sen University, Guangzhou 510080, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2005 Apr 30;23(2):100-5.
To analyze the difference of GRA7 gene of Toxoplasma gondii different isolated strains and express GRA7 in Escherichia coli.
The GRA7 gene was amplified from genomes of T. gondii isolates by PCR and was cloned into pGEX-4T-1. The recombinant plasmid was transformed into JM109 and sent to be sequenced. The sequence was analyzed with CLUSTALW (an internet tool). The recombinant plasmid was induced by IPTG to express the fusion protein,which was identified by SDS-PAGE and Western blot with positive sera. The protein was purified and used as a diagnostic antigen for ELISA to test serum samples.
There was no difference among the sequences of T. gondii GRA7 gene from different isolates. The recombinant plasmid pGEX-4T-1/GRA7 induced by IPTG was expressed in E. coli. It was a GST fusion protein and could react with human and rabbit positive sera analyzed by Western blot.
The GRA7 gene of T. gondii isolates is highly conservative. The GRA7 is expressed as a recombinant protein in Escherichia coli, which shows an immunoreactivity.
分析不同分离株弓形虫GRA7基因的差异，并在大肠杆菌中表达GRA7。
采用PCR技术从弓形虫分离株基因组中扩增GRA7基因，将其克隆到pGEX-4T-1载体中。重组质粒转化至JM109感受态细胞并送去测序。利用CLUSTALW（一种网络工具）对序列进行分析。用IPTG诱导重组质粒表达融合蛋白，通过SDS-PAGE和Western blot用阳性血清进行鉴定。对蛋白进行纯化并用作ELISA诊断抗原检测血清样本。
不同分离株弓形虫GRA7基因序列之间无差异。经IPTG诱导的重组质粒pGEX-4T-1/GRA7在大肠杆菌中表达。它是一种GST融合蛋白，经Western blot分析可与人及兔阳性血清发生反应。
弓形虫分离株的GRA7基因高度保守。GRA7在大肠杆菌中表达为重组蛋白，具有免疫反应性。
