摘要
目的 构建日本血吸虫 14kDa脂肪酸结合蛋白 (Sj14FABP)和细胞因子IL 12共表达质粒 ,并观察其能否在小鼠体内获得表达。方法 TRIzol试剂提取日本血吸虫成虫总RNA ,采用RT PCR方法逆转录Sj14FABP编码基因序列 ,经过双酶切后定向克隆到载体pVIVO2 IL12中 ,通过PCR、酶切及测序进行鉴定。大量制备重组质粒 pVIVO2 IL12 Sj14FABP并免疫BALB/c小鼠 ,取注射部位肌肉组织进行间接免疫荧光试验。结果 RT PCR扩增出一条大小为 4 4 0bp的片段 ;经PCR、酶切及测序鉴定表明所构建的重组质粒中含有Sj14FABP基因 ;间接免疫荧光试验结果为阳性。 结论 本试验成功构建重组质粒 pVIVO2 IL12 Sj14FABP ,并在小鼠体内获得表达。
To construct the recombinant plasmid co expressing 14 kDa fatty acid binding protein from Schistosoma japonicum(Sj14FABP) and IL 12 and to express this plasmid in mice, the total RNA was extracted from adult worms of S.japonicum;the gene encoding Sj14FABP was amplified by RT PCR and digested with restriction endonucleases BamHI and EcoRI for a directional gene cloning to the vector pVIVO2 IL 12. The resulting construct was identified by PCR, restriction endonuclease digestion and sequence analysis. This recombinant plasmid was then used to immunize BALB/c mice and the local muscle tissue was analyzed by means of indirect fluorescence assay (IFA). It was found that a 440 bp fragment was amplified through RT PCR, and the constructed recombinant plasmid containing the sequence of Sj14FABP gene was obtained after PCR, enzyme digestion and sequence analysis. It was proved by IFA that the recombinant plasmid pVIVO2 IL 12 Sj14FABP was expressed in muscle cells.It concludes that the recombinant plasmid pVIVO2 IL 12 Sj14FABP has been successfully constructed and expressed in mice.
出处
《中国人兽共患病杂志》
CAS
CSCD
北大核心
2005年第1期14-17,共4页
Chinese Journal of Zoonoses
基金
国家高技术研究发展计划 (863计划 )资助项目 (No .2 0 0 4AA2Z3 2 12 )
