日本血吸虫核糖核酸酶T2的表达及其功能分析

目的制备重组151本血吸虫核糖核酸酶T2蛋白（rSj RNase T2），并分析其生物学功能。方法根据编码RNase T2开放阅读的基因序列设计、合成一对5’端带有酶切位点的引物。采用PCR方法从质粒CP1412/PEu—GST中扩增编码RNase T2蛋白成熟肽的基因片段，亚克隆到表达载体pET28a（＋）中，构建重组表达质粒Sj RNase T2-pET28a。将重组表达质粒转化到E．coli BL21中，用异丙基-β-D-硫代吡喃半乳糖苷（IPTG）进行诱导表达，用镍螯合亲和层析胶在变性条件下纯化rSj RNase T2蛋白。纯化蛋白通过尿素浓度梯度透析的方法进行复性，制备可溶性rSj RNase T2。以酵母RNA为底物进行消化，采用琼脂糖凝胶电泳的方法检测rSj RNase T2酶活性。用纯化的rSj RNase T2免疫小鼠，采用酶联免疫法（ELISA）检测小鼠血清特异抗体水平，观察其抗原性；采用检测Western blot抗rSj RNase T2抗体IgG与成虫可溶性虫抗原、成虫外分泌抗原、虫卯可溶性抗原和虫卵外分泌抗原的反应性，并观察Sj RNase T2在虫体中的分布。以rSj RNase T2刺激巨噬细胞RAW264．7，通过流式分析观察RAW264．7细胞表面标志物CD16／32、CD206表达水平的变化，采用RTPCR检测RAW264．7细胞内诱导型一氧化氮合酶与精氨酸酶基因mRNA的表达水平，采用ELISA检测RAW264．7细胞培养上清液中IL-12及IL-10水平的变化，以观察Sj RNase T2的免疫凋节功能。结果重组表达质粒Sj RNase T2-pET28a构建成功，经IPTG诱导，能表达重组Sj RNase T2蛋白。该蛋白以包涵体形式存在，经过复性能获得部分可溶性蛋白。rSj RNaseT2具有酶活性，能够水解酵母RNA。ELISA检测rSj RNase T2免疫小鼠血清特异性抗体滴度为1：200000，该抗血清能识别来源于血吸虫虫卯及虫卯外分泌抗原中的天然RNase T2。rSj RNase T2可诱导巨噬细胞向M2型方向转化，表达高水平的IL-10、精氨酸酶及CD206表面分子。结论rSj RNase T2表达成功。该蛋白具有天然的酶学特性和抗原性，能调节巨噬细胞向M2型方向分化。

Expression and characterization of RNase-T2 of Schistosoma japonicum

Objectives To prepare a recombinant Schistosoma japonicum ribonuclease T2 protein （rSj RNase T2） and investigate its biological function. Methods A pair of primers with restriction sites was designed in accordance with the DNA sequence of the open reading frame encoding Sj RNase T2. The DNA fragment encoding the mature peptide of Sj RNase T2 was amplified from the plasmid DNA of CP1412/pEU-GST. This DNA fragment was subcloned into the expression plasmid pET28a （2-） to construct the recombinant expression plasmid Sj RNase T2-pET28a. Then, the recombinant expression plasmid Sj RNase T2 pET28a was transformed into E. coli BL21 and protein expression was induced with isopropyl beta-D-galactoside （IPTG）. The product of large scale expression of Sj RNase T2 were prepared and purified with Ni affinity chromatography gel under denaturing conditions. The purified product was dialyzed in gradient urea at decreasing gradient concentrations for renaturing and refolding and was finally dialyzed with PBS to obtain soluble recornbinant Sj RNase T2 protein. The RNase activity of recombinant Sj RNase T2 was detected by agarose gel electrophoresis using yeast RNA as a substrate. Mice were immunized with the recombinant Sj RNase T2 protein, and antibody levels in serum were measured with an enzyme linked immunosorbent assay （ELISA） to observe the immunogenicity of recombinant Sj RNase T2. The soluble adult worm antigen （AWA）, soluble extracts of adult worms, and soluble egg anti gen （SEA） and secretory products of S. japonicum were analyzed using immunoblotting with purified IgG antibodies a- gainst rSi RNase T2 to observe the distribution of the protein. The maerophagocyte RAW 264.7 was stimulated with pu- rified recombinant Sj RNase T2, changes in the levels of expression of the cell surface markers CD16/32 and CD206 of the stimulated RAW 264.7 were monitored using flow cytometry, and mRNA levels of inducible nitric oxide synthase （iNOS） and arginase （Arg） were measured using RT-PCR with gene specific primers. The concentration of IL-10 and II. 12 in the supernatant of RAW264.7 culture was examined using ELISA with IL-10 and IL-12 kits to determine immunoregulation by recombinant Sj RNase T2. Results The recombinant expression plasmid Sj RNase T2-pET28a was successfully con- structed. Transform_ants containing the recombinant plasmid Sj RNase T2-PET28a expressed the Sj RNase T2 protein in the form of inclusion bodies after induction with IPTG. Partially soluble Sj RNase T2 protein was obtained after renatur ation. The recombinant Sj RNase T2 protein has RNase activity in that it hydrolyzes yeast RNA. Mice immunized with the recombinant Sj RNase T2 protein had higher titer antibody responses, and their sera recognized the natural Sj RNase T2 protein derived from schistosome egg protein, including soluble egg antigen （SEA） and egg-secreted protein. These findings indicated that the recombinant Sj RNase T2 protein had immunogenicity like that of natural Sj RNase T2 protein. The recombinant Sj RNase T2 protein induced the M2 polarization of macrophages with expression of high levels of IL 10, arginine, and CD206. Conclusion Sj RNase T2 protein was successfully expressed. This protein has natural enzymatic characteristics and immunogenicity, and it can modulate macropbage polarization to the M2 phenotype.