【摘要】 目的 诱导表达华支睾吸虫一氧化氮合酶相互作用蛋白（CsNOSIP）的重组质粒并进行组织定位研究，初步了解重组产物免疫小鼠后细胞因子的变化及诱发宿主免疫应答类型。方法 应用生物信息学分析软件，分析Cs NOSIP的序列特征；将重组质粒pET-30a（+）-Cs NOSIP诱导表达并用镍离子金属螯合剂亲和层析柱进行纯化，用纯化的Cs NOSIP蛋白免疫BABL/c小鼠获得免疫血清；应用免疫荧光方法观察Cs NOSIP在华支睾吸虫成虫的定位；应用ELISA检测实验组（重组蛋白皮下免疫小鼠）和对照组（磷酸盐缓冲液免疫小鼠）细胞因子水平的变化。应用qPCR检测内皮型一氧化氮合酶（eNOS）和诱导性一氧化氮合酶（iNOS）转录水平的变化。结果 CsNOSIP编码288个氨基酸，SecP分值为0.955 097，无信号肽；理论分子量为35 000；免疫组化结果显示，该蛋白定位于虫体的肠支和虫卵。与对照组相比，r Cs NOSIP免疫组用5μg/mL ConA刺激后白细胞介素4、白细胞介素6水平均升高，差异均有统计学意义（t=12.16、8.91,P均<0.05）；与对照组相比，eNOS转录水平降低，iNOS转录水平增高，差异均有统计学意义（P均<0.05）。结论 CsNOSIP可在原核表达系统中呈现高效的可溶性表达，是华支睾吸虫分泌排泄抗原之一，具有良好的免疫学活性。更多还原

【Abstract】 Objective To induce the expression of recombinant Clonorchis sinensis nitric oxide synthase interacting protein(Cs NOSIP) and study its tissue localization, and the changes of cytokines and the type of induced host immune response after mice were immunized with the recombinant product. Methods analyzed by bioinformatics analysis software; the recombinant Cs NOSIP was expressed and purified by nickel ion metal chelate affinity chromatography column, and the purified recombinant Cs NOSIP protein was used to immunize BABL/c mice to obtain immune serum; the localization of Cs NOSIP in adult Clonorchis sinensis was observed by immunofluorescence method; the changes of cytokine levels in the experimental group(mice immunized subcutaneously with recombinant protein) and the control group(mice immunized with PBS) were detected by ELISA. The changes of transcription levels of endothelial nitric oxide synthase(eNOS) and inducible nitric oxide synthase(iNOS) were detected by qPCR. Results Cs NOSIP encodes 288 amino acids, with a SecP score of 0.955 097 and no signal peptide; the theoretical molecular weight is 35 000; immunohistochemical results showed that the protein was localized in the intestinal tract and eggs of the worm body; compared with the control group, the levels of interleukin 4(IL-4) and IL-6 in the r Cs NOSIP immunization group were increased after stimulation with 5 μg/mL ConA, and the difference was statistically significant(t=12.16, 8.91;all P<0.05); compared with the control group, the transcription level of eNOS was decreased, and the transcription level of iNOS was increased, and the difference was statistically significant(all P<0.05). Conclusion r Cs NOSIP could be expressed efficiently in a soluble form in the prokaryotic expression system, and had good immunological activity.更多还原