犬新孢子虫(Neospora caninum)肌动蛋白解聚因子的克隆表达及应用
首发时间:2015-05-12
摘要:目的 克隆和表达犬新孢子虫肌动蛋白解聚因子(NcADF),分析其特征并评价其用于血清学诊断的价值。方法 通过对cDNA文库的免疫筛选克隆出NcADF基因、测序、用PCR扩增其开放阅读框,将其插入pGEX-4T中并转化大肠杆菌,诱导表达与谷胱甘肽巯基转移酶融合的重组蛋白后,用谷胱甘肽琼脂糖凝胶4B和thrombin纯化;用SDS-PAGE 和Western blotting 鉴定重组NcADF(rNcADF)的浓度、纯度、抗原性及分子量等。以rNcADF为抗原建立酶联免疫吸附试验(ELISA)检测血清特异抗体。结果 NcADF编码区为357 bp,编码含118个氨基酸和典型ADF结构域的多肽链;该基因表达产物rNcADF免疫鼠血清能特异地识别N. caninum的内源性NcADF,其表观分子量与rNcADF相符;ELISA检测N. caninum感染(29)和未感染(12)鼠血清的敏感性和特异性均为100%。然而部分T. gondii感染鼠血清与rNcADF产生交叉反应,疑为N. caninum与T. gondii的ADF蛋白具有92%相似性之故;宠物狗的血清抗体阳性率为4.7%(3/64)。结论 本实验成功克隆表达了NcADF基因,对其分子特征及血清学诊断潜能有了初步了解,为深入进行N. caninum入侵宿主机制等研究提供了理论资料。
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Cloning and Expression of Actin Depolymerizing Factor from Neospora caninum and its Potential for Serodiagnosis
Abstract:Objective To clone and express an actin depolymerizing factor gene of Neospora caninum (NcADF) and evaluate the potential of the recombinant NcADF for serodiagnosis on N. caninum infection. Methods NcADF gene was cloned by immunoscreening cDNA library using sera from mice infected experimentally with N. caninum. After sequencing, the open reading frame (ORF) of NcADF was amplified by PCR and inserted into pGEX-4T. Bacteria transformed with the recombinant pGEX-4T/NcADF were induced to express the recombinant NcADF protein (rNcADF) as a fusion protein with GST. rNcADF purified using glutathione sepharose 4B combined with thrombin was analyzed by SDS-PAGE and Western blotting analysis to examine its concentration, purity and antigenicity. Enzyme-linked immunosorbent assay (ELISA) with rNcADF as an antigen was used to detect specific antibodies in mice infected respectively with N. caninum, Toxoplasma gondii and uninfected mice. Sera from field dogs were also detected by ELISA with rNcADF as an antigen. Results The ORF of NcADF was 357 bp, encoding a polypeptide of 118 aa which contained a typical conserved ADF domain. NcADF gene was expressed successfully in the bacteria. The antiserum against rNcADF had a strong positive reaction to native NcADF, the molecular mass of which was correspondent to rNcADF. ELISA with rNcADF showed that the positive rates were 100% (23/23) in sera infected experimentally with N. caninum, 0 (0/12) in sera from uninfected mice, 30.7% (12/39) in serum infected with T. gondii. The cross reaction with T. gondii might be caused by the similarity of 92% between NcADF and a ADF from T. gondii. The positive rate in sera from the filed dogs was 4.7% (3/64) . Conclusion Results suggested that rNcADF was successfully expressed in E. coli and characterized. ELISA with rNcADF as an antigen was sensitive and specific for distinguish N. caninum infected from uninfected murine sera, however, there was certain cross reaction with T. gondii infected murine sera. This study provides not only the data for NcADF application for serodiagnosis but also some valuble information for further study of molecular mechanism for N. caninum invasion to host cells.
Keywords: Neospora caninum actin depolymerizing factor expression immunodiagnosis
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