论文部分内容阅读
目的制备重组日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、虫卵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RT-PCR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的免疫调节功能。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 RNase T2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︰200 000,该抗血清能识别来源于血吸虫虫卵及虫卵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。
Objective To prepare recombinant Schistosoma japonicum RNase T2 protein and analyze its biological function. Methods According to the gene sequence encoding RNase T2 open reading, a pair of primers with restriction sites at 5 ’end were synthesized. The gene fragment encoding the mature peptide of RNase T2 protein was amplified by PCR from the plasmid CP1412 / PEU-GST and subcloned into the expression vector pET28a (+) to construct the recombinant expression plasmid Sj RNase T2-pET28a. The recombinant plasmid was transformed into E.coli BL21 and induced with isopropyl-β-D-thiogalactopyranoside (IPTG). Nickel chelating affinity chromatography gel was used to purify rSj RNase T2 protein. Purified proteins were renaturated by gradient dialysis of urea to prepare soluble rSj RNase T2. Yeast RNA as a substrate for digestion, the method of agarose gel electrophoresis rSj RNase T2 enzyme activity. The mice were immunized with purified rSj RNase T2, and the level of serum specific antibody was detected by enzyme-linked immunosorbent assay (ELISA). The anti-rSj RNase T2 IgG was detected by Western blot with soluble antigens of adult worms and adult exosomes , Egg soluble antigen and exocrine antigen, and observed the distribution of Sj RNase T2 in the parasites. RAW264.7 macrophages were stimulated with rSj RNase T2, and the expression of CD16 / 32 and CD206 on RAW264.7 cells was observed by flow cytometry. The expressions of inducible nitric oxide synthase Enzyme and arginase mRNA were detected by enzyme-linked immunosorbent assay (ELISA). The levels of IL-12 and IL-10 in supernatant of RAW264.7 cells were detected by ELISA to observe the immunoregulatory function of Sj RNase T2. Results The recombinant plasmid Sj RNase T2-pET28a was successfully constructed and induced by IPTG to express recombinant Sj RNase T2 protein. The protein is in the form of inclusion bodies, and the partially soluble protein can be obtained through refolding. rSj RNase T2 is enzymatically active and capable of hydrolyzing yeast RNA. Serum specific antibody titer of rSj RNase T2 was 1: 200 000 by ELISA. The antiserum could recognize natural RNase T2 derived from Schistosoma japonicum eggs and extraembryonic secretion antigens. rSj RNase T2 induces macrophages to transform into M2 type and express high levels of IL-10, arginase and CD206 surface molecules. Conclusion rSj RNase T2 expression was successful. The protein has the natural enzymatic properties and antigenicity, can regulate macrophages to M2-type differentiation.