Yang Chung-Dar, Chang Gan-Nan, Chao David
Department of Biological Science, National Sun Yat-sen University, Kaohsiung, Taiwan, ROC.
Parasitol Res. 2004 Jan;92(1):58-64. doi: 10.1007/s00436-003-0992-5. Epub 2003 Nov 6.
The aim of this study was to test the protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2. The PCR-amplified SAG2 fragment (558 bp) digested with the restriction enzyme XhoI was inserted into the XhoI site of plasmid pGEX-6p-1, termed pGexSAG2. The PCR-amplified SAG1 fragment (1,008 bp) digested with restriction enzymes EcoRI and XhoI was cloned into the EcoRI/ XhoI sites of a separate plasmid pGEX-6p-1, termed pGexSAG1. The SAG2 fragment (HindIII/HindIII) excised from pGexSAG2 was inserted into the HindIII site of pGexSAG1 and a chimeric vector constructed, pGexSAG1/2. The fusion proteins GST-SAG1/2, GST-SAG1 and GST-SAG2 were expressed in BL21 Star (DE3) Escherichia coli and purified by GSTrap FF columns. After removing the GST tag, the recombinant proteins rSAG1/2, rSAG1 and rSAG2 were independently collected and injected into different groups of mice to evaluate their protective capability. The highest proliferation of splenocytes stimulated with tachyzoite sonicate antigen (TsoAg) was observed in BALB/c mice which had received two intraperitoneal injections of rSAG1/2. Maximum production of IFN-gamma was also found in the culture supernatants of TsoAg-stimulated splenocytes from rSAG1/2-immunized mice. Finally, 73% (11/15) of mice immunized with rSAG1/2 survived at least 28 days after a lethal challenge of 1 x 10(3) live tachyzoites which killed all non-immunized mice within 10 days. Moderate survival rates were observed in mice immunized with either rSAG1 (60%) or rSAG2 (53%). These results show that the chimeric protein rSAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice.
本研究的目的是测试嵌合蛋白rSAG1/2在小鼠体内诱导的针对刚地弓形虫的保护性免疫。用限制性内切酶XhoI消化的PCR扩增的SAG2片段（558 bp）插入质粒pGEX-6p-1的XhoI位点，命名为pGexSAG2。用限制性内切酶EcoRI和XhoI消化的PCR扩增的SAG1片段（1,008 bp）克隆到另一个质粒pGEX-6p-1的EcoRI/XhoI位点，命名为pGexSAG1。从pGexSAG2切下的SAG2片段（HindIII/HindIII）插入pGexSAG1的HindIII位点，构建嵌合载体pGexSAG1/2。融合蛋白GST-SAG1/2、GST-SAG1和GST-SAG2在BL21 Star（DE3）大肠杆菌中表达，并用GSTrap FF柱纯化。去除GST标签后，分别收集重组蛋白rSAG1/2、rSAG1和rSAG2，并注射到不同组的小鼠中以评估它们的保护能力。在接受两次腹腔注射rSAG1/2的BALB/c小鼠中，观察到速殖子超声抗原（TsoAg）刺激的脾细胞增殖最高。在rSAG1/2免疫小鼠的TsoAg刺激的脾细胞培养上清液中也发现了最大量的IFN-γ产生。最后，用rSAG1/2免疫的小鼠中有73%（11/15）在接受1×10(3)个活速殖子的致死攻击后至少存活28天，而所有未免疫的小鼠在10天内死亡。用rSAG1（60%）或rSAG2（53%）免疫的小鼠观察到中等存活率。这些结果表明，嵌合蛋白rSAG1/2可在小鼠体内引发针对刚地弓形虫感染的Th1相关保护作用。
