日本血吸虫DNA疫苗的构建及其保护性免疫

Construction of a novel schistosoma japonicum DNA vaccine pBK-Sj14-3-3 and studies on its immunoprotection in mice

目的 制备日本血吸虫裸露DNA疫苗 (pBK Sj14 3 3) ,观察其在小鼠抗血吸虫感染中的免疫保护作用。方法 以RT PCR方法扩增Sj14 3 3基因 ,并将其亚克隆入真核表达载体pBK ;提取、纯化重组质粒pBK Sj14 3 3,肌肉注射法接种BALB/c小鼠 ;日本血吸虫尾蚴攻击感染后 ,剖杀小鼠 ,计算减虫率和减卵率 ,观察pBK Sj14 3 3对小鼠肝脏虫卵肉芽肿形成的影响。结果 1%琼脂糖凝胶电泳显示 ,RT PCR扩增产物与EcoRⅠ +XhoⅠ双酶切重组质粒得到的插入片段大小相同 ,约为76 5bp ,核苷酸序列测定证实插入片段为Sj14 3 3;免疫注射pBK Sj14 3 3后 ,14 3 3组平均每只小鼠获检成虫数为 2 2 9± 9 6 ,与对照组成虫数 (31 3± 7 3)比较 ,14 3 3组小鼠平均成虫负荷下降了 2 7%。经 5 %氢氧化钾消化后 ,14 3 3组平均每克肝组织虫卵数为 16 6 0 0± 5 90 4 ,每对成虫平均虫卵数为 476 9± 115 6 ,与对照组平均每克肝组织虫卵数 (77875± 4 32 6 )和每对成虫平均虫卵数 (972 3± 2 0 74 )比较 ,减卵率为 79% ,每对成虫减卵率为 5 1%。肝脏虫卵肉芽肿平均直径下降了 2 9%。结论 DNA疫苗pBK Sj14 3 3构建成功 ,且在小鼠抗血吸虫感染中有一定的免疫保护作用 。

Objective To prepare Sj14 3 3 DNA vaccine and observe its immunoprotection against Schistosoma japonicum in mice Methods The Sj14 3 3 gene was amplified by reverse transcription polymerase chain reaction (RT PCR) and subcloned into eukaryotic expression vector pBK The recombinant plasmid pBK Sj14 3 3 was extracted, purified and inoculated into BALB/c mice by intramuscular injection Mice were attacked by Schistosoma japonicum cercariae and then killed Adult worm and egg were counted, respectively Diameter of the egg granulomas in the liver of infected mice was measured Results Electrophoresis on 1% agarose gel showed that the product of RT PCR and the inserted fragment of recombinant plasmid digested with EcoR I and Xho I had the same size, about 765 bp, confirming the latter was the 14 3 3 encoding gene by nucleotide sequencing Adult worm load declined by 27%, average egg load of per gram (EPG) of the liver tissues by 79%, average egg production per couple of adult worm (EPWP) by 51%, and mean diameter of egg granulomas by 29% in vaccinated mice Conclusion The recombinant plasmid pBK Sj14 3 3 was successfully constructed, which had some immunoprotection against Schistosoma japonicum in infected mice, indicating its potential to be vaccine candidate molecule of Schistosoma japonicum