摘要
目的：克隆表达华支睾吸虫脂肪酸结合蛋白（ Clonorchis sinensisfatty acid binding protein，CsFABP），并对其免疫原性进行分析。方法根据编码CsFABP的已知基因序列设计合成一对引物，应用RT－PCR技术扩增编码CsFABP的基因片段。将扩增的CsFABP的基因片段克隆到原核质粒pET30a（＋）中，转化入大肠埃希菌（ E． coli） DH5α中，经含卡那霉素琼脂平板筛选，小量抽提质粒，经双酶切、PCR及测序鉴定阳性克隆。将阳性重组质粒转化入E．coli BL21中，经异丙基－β－D－硫代半乳糖苷（ IPTG）诱导表达，表达产物经十二烷基硫酸钠－聚丙烯酰胺凝胶电泳（ SDS－PAGE）、考马斯亮蓝染色、Western blot分析鉴定。结果经酶切、菌液PCR以及测序确认，成功构建重组质粒pET30a（＋）－CsFABP，并在E．coli BL21中高效表达。 Western blot试验证实表达的Cs-FABP能被特异的兔抗华支睾吸虫免疫血清识别。结论本研究成功构建重组质粒pET30a（＋）－CsFABP，获得的CsFABP表达蛋白质具有一定的反应原性。
Objective To investigate the clone, expression and immunogenicity of fatty acid-binding protein of Clonorchis sinensi （ CsFABP） .Methods A pair of primers was synthesized according to the sequence of fatty acid-binding protein of C.sinensis （CsFABP） available in GenBank, which was then applied for amplification by RT-PCR. The amplified fragment of FABP was cloned into the plasmid pET30a （ ＋） which was then inserted into E.coli DH5αsusceptible cells.After screened in agar plate containing kanamycin, the resultant vectors were digested before PCR and DNA sequencing for identification of positive colons.The positive recombinant vector was transformed into E.coli BL21 followed by IPTG induction.The products obtained were analyzed by Western blotting.Results The recombinant vector CsFABP was successfully established and highly expressed in E.coli BL21.The purified protein was specifically recog-nized by the immune serum against C.sinesis from rabbits, according to Western blotting analysis.Conclusion The re-combinant vector CsFABP is established, resulting into CsFABP with immunogenicity.
出处
《徐州医学院学报》
CAS
2015年第6期351-354,共4页
Acta Academiae Medicinae Xuzhou
基金
国家自然科学基金（81171590,31302077）
家畜疫病病原生物学国家重点实验室开放基金（ SKLVEB2013KFKT005）
江苏高校优势学科建设工程资助项目（2013）
关键词
华支睾吸虫
脂肪酸结合蛋白
原核表达
免疫原性
Clonorchis sinensis
fatty acid-binding protein
prokaryotic expression
immunogenicity
作者简介
通信作者，E—mail：zky02@163．com
