Yin Li-Tian, Wang Fen, Meng Xiao-Li, Wang Hai-Long, Liu Hong-Li, Shen Jin-Yan, Yin Guo-Rong
Department of Physiology, Key Laboratory of Cellular Physiology Co-constructed by Province and Ministry of Education, Shanxi Medical University, Taiyuan 030001, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2012 Apr 30;30(2):86-9.
To clone and express the phosphoglycerate mutase 2 (PGAM2) gene of Toxoplasma gondii, and analyze the antigenicity of the recombinant protein.
Total RNA was extracted from T. gondii tachyzoites of RH strain and reversely transcribed into cDNA. TgPGAM2 gene was amplified by PCR and cloned into pET30a(+) vector. The constructed pET30a(+)-TgPGAM2 was transformed into E. coli DH5alpha first and selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The correct plasmid was transformed into E. coli BL21 for expression induced by IPTG and the recombinant protein was further analyzed through SDS-PAGE followed by Coomassie brilliant blue staining. Western blotting assay with rabbit anti-T. gondii serum was used to analyze its antigenicity.
The length of PCR product was about 750 bp and the recombinant plasmid pET30a(+)-TgPGAM2 was successfully constructed. The results of SDS-PAGE and Western blotting revealed that the relative molecular weight (Mr) of the soluble recombinant protein was approximately 30 000 and could be recognized by rabbit anti-T. gondii serum.
The soluble TgPGAM2 protein has been expressed in the prokaryotic expression system and maintains its antigenicity.
克隆并表达刚地弓形虫磷酸甘油酸变位酶2（PGAM2）基因，分析重组蛋白的抗原性。
从RH株刚地弓形虫速殖子中提取总RNA并逆转录为cDNA。通过PCR扩增TgPGAM2基因并克隆到pET30a(+)载体中。首先将构建好的pET30a(+)-TgPGAM2转化到大肠杆菌DH5α中，通过菌落PCR筛选并经双酶切和测序确认。将正确的质粒转化到大肠杆菌BL21中，用IPTG诱导表达，通过SDS-PAGE和考马斯亮蓝染色进一步分析重组蛋白。用兔抗刚地弓形虫血清进行Western blotting分析其抗原性。
PCR产物长度约为750 bp，成功构建了重组质粒pET30a(+)-TgPGAM2。SDS-PAGE和Western blotting结果显示，可溶性重组蛋白的相对分子质量（Mr）约为30 000，能被兔抗刚地弓形虫血清识别。
可溶性TgPGAM2蛋白已在原核表达系统中表达并保持其抗原性。
