摘要
目的 克隆、表达日本血吸虫（大陆株）3-磷酸甘油醛脱氢酶（SjcGAPDH）编码基因并作结构预测。方法 将编码SjeGAPDH的pBlueseript质粒用限制性内切酶XhoⅠ和SacⅠ消化后，收集目的片段，与经同样酶切的原核表达载体pET28b连接，筛选重组克隆并测序，将正确的重组质粒转化入BL21（DE3）感受态细菌，异丙基-β-硫代半乳糖苷（IPTG）诱导表达重组蛋白。用GeneRunner软件对该蛋白的结构及抗原表位进行预测。结果 十二烷基磺酸钠一聚丙烯酰胺凝胶电泳（SDS—PAGE）观察到大小约42．7kDa的特异性蛋白条带，与预计一致。免疫印迹试验（Western blot）结果表明致弱尾蚴免疫兔血清可识别该重组蛋白。结构预测表明SjcGAPDH的第50～70、102～122、135～148、165～175、187-205、254-272、278-285位氨基酸区域可能为蛋白质的抗原优势区域。结论 SjcGAPDH编码基因克隆、表达获得成功，为开展该分子功能和免疫原性研究奠定了基础，对GAPDH的结构和性质预测为探索该蛋白的抗原优势表位提供了理论依据。
Objective To clone and express the gene encoding Schistosoma japonicurn （Chinese strain） Glyceraldehyde-3-phosphate dehydrogenase （SjcGAPDH） and predict the protein structure and antigen determinants. Methods After digestion of pBluescript-SjcGAPDH with XhoⅠand Sac Ⅰ, the target DNA fragment was purified and subcloned into the proper prokaryotic expression vector pET28b. After identification of sequencing, the recombinant plasmid pET28a-SjcGAPDH was transformed into competent E. coli BL21 and expressed in the presence of isopropyl-β-D-thiogalactopyranoside （IPTG）. Results The results of SDS-PAGE showed that the molecular weight of expressed protein was around 42.7 kDa. The recombinant protein was recognized by Western blot with the sera from rabbits immunized with attenuated cercariae of S. japonicum. According to the prediction, the N-terminal No. 50-70, 102-122,135-148,165-175,187-205,254-272,278-285 may be antigen determinants. Conclusion The gene encoding SjcGAPDH has been coloned and expressed successfully, which will be helpful for identification of its function and antigenicity.
出处
《中国血吸虫病防治杂志》
CAS
CSCD
2007年第3期170-174,共5页
Chinese Journal of Schistosomiasis Control
基金
国家自然科学基金(30371262)
国家高技术研究发展计划(863计划)(2006AA02Z444
2004AA215240
2004AA2Z3520)
上海市科委科技攻关重大计划(03DZ19231)
生物医药重点科技攻关项目(064319026)
关键词
日本血吸虫
3-磷酸甘油醛脱氢酶
克隆
表达
Schistosoma japonicum
Glyceraldehyde-3-phosphate dehydrogenase （GAPDH）
Cloning
Expression
