赤子爱胜蚓谷胱甘肽和丙二醛含量变化指示重金属污染土壤的生态毒性

为探讨赤子爱胜蚓谷胱甘肽(GSH)和丙二醛(MDA)含量变化对重金属污染土壤生态毒性的指示作用,在试验室模拟条件下,利用人工土壤和自然土壤,研究了重金属含量与蚯蚓GSH和MDA含量变化的剂量效应关系。结果显示,在人工土壤试验中,Cu可诱导蚯蚓GSH和MDA含量上升,暴露2 d、7 d、14 d后,Cu浓度与蚯蚓GSH含量的相关系数分别为0.556、0.807和0.775,与MDA含量的相关系数分别为0.760、0.672和0.544,均存在显著的剂量效应关系(p<0.05)。自然土壤试验结果显示,赤子爱胜蚓的GSH和MDA含量与土壤Cu全量的偏相关系数分别为-0.830(p<0.001)和-0.599(p<0.05),与Cd全量的偏相关系数分别为-0.697(p<0.05)和-0.690(p<0.05)。同时发现,赤子爱胜蚓GSH含量变化对土壤重金属响应的敏感性要高于MDA。     

日本血吸虫钙网织蛋白在Sf9昆虫细胞中的表达及其活化树突细胞的初步分析

为获得在Sf9昆虫细胞中表达的日本血吸虫钙网织蛋白(Schistosoma japonicum calreticulin protein,SjCRT)并分析其活化小鼠骨髓来源树突状细胞的功能,将构建的重组杆状病毒转移载体pFastBacHTA-SjCRT转入DH 10Bac细胞,得到重组穿梭质粒reBacmid-SjCRT,再转染到Sf9昆虫细胞,进行重组蛋白的表达.用Westem blot和间接免疫荧光对表达蛋白进行鉴定.His柱亲和层析法纯化表达的蛋白,Westen blot鉴定纯化后的蛋白.从BALB/c小鼠产生骨髓来源的树突细胞mDCs,用纯化的重组SjCRT蛋白与mDCs共培养,流式细胞术检测mDCs细胞的表面分子MHCⅡ、CD40和CD86的表达.结果显示,在Sf9昆虫细胞中成功表达了SjCRT蛋白;纯化后的重组SjCRT蛋白既能被感染日本血吸虫42 d的兔阳性血清识别,也能被原核表达的重组SJC RT蛋白免疫鼠的血清所识别.流式细胞术结果显示,与对照组的相比,SjCRT蛋白刺激组mDCs细胞表面分子MHCⅡ和CD86的表达量显著增强(P＜0.05).可见,在Sf9昆虫细胞中表达的SjCRT蛋白能刺激小鼠骨髓来源树突细胞表型的成熟.     

日本血吸虫保护性免疫机制的研究——活化巨噬细胞和超敏反应测定

本文用辐照致弱日本血吸虫童虫疫苗,冷冻辐照童虫苗和速冻致死童虫苗免疫雌性C57-BL/6小鼠。免疫后对小鼠腹腔巨噬细胞(MФ)的吞噬杀伤活性和超敏反应进行动态测定。结果表明2个活苗免疫组小鼠腹腔MФ体外表现出强烈杀伤童虫活性,杀伤力显著强于死苗免疫组和对照组(P<0.01);免疫后第6周杀伤力达峰值,第8周杀伤力下降;MФ杀伤力与保护力存在显著正相关(r=0.630,r>r0.05);活化MФ在免疫血清调理后杀伤活力有所增加,而免疫血清对于未活化MФ则无调理作用;活苗和死苗免疫鼠都出现强烈的速发型超敏反应,但与保护力之间缺乏相关性;迟发型超敏反应在本次实验中出现较弱。     

Application of recombinant Sjc26GST for serodiagnosis of Schistosoma japonicum infection in water buffalo (Bos buffelus)

Schistosomiasis japonica is currently the most serious parasitic disease in mainland China and it is estimated that several million people are infected. Furthermore, it is also responsible for the deaths of many domestic animals. In order to establish an effective diagnostic method, the gene encoding Sjc26GST was cloned and expressed in Escherichia coli as a fusion protein with His-tag. The purified reSjc26GST was used as an antigen for an enzyme-linked immunosorbent assay (ELISA) and for immunoblotting detection of Schistosoma japonicum antibodies in water buffaloes. Our results showed that mean OD values of specific serum IgG antibodies from egg-positive buffaloes were 3.37-fold higher than what was found in egg-negative buffaloes from non-endemic areas. The data also showed the OD value of the endemic egg-negative group reached as high as 1.69 times as that found in non-endemic areas. The positivity rate of egg-positive buffaloes was 100%, but was 30.3% in the endemic egg-negative group. Infected bovine antisera also recognized reSjc26GST, a 27 kDa protein as determined by Western blot. These results suggest that the recombinant GST expressed in E. coli should be an effective diagnostic reagent for detection of antibody against S. japonicum in buffaloes. Due to straightforward production, excellent sensitivity and high specificity, the reSjc26GST described in this study can be considered as a candidate protein for immunological diagnosis of bovine schistosomiasis. Developing reSjc26GST, with its potential diagnostic values, will be useful for diagnosis and surveillance of schistosomiasis in controlling the spread of this parasitic disease in domestic animals.     

酵母双杂交诱饵蛋白日本血吸虫抱雌沟蛋白重组质粒的构建及鉴定

为筛选与血吸虫抱雌沟蛋白相互作用的因子,本研究以抱雌沟蛋白（gynecophoral canal protein ofSchistosoma japonicum,SjGCP）为诱饵蛋白构建了用于酵母双杂交筛选系统的重组质粒。根据日本血吸虫抱雌沟蛋白的基因序列设计引物,经过PCR扩增、酶切回收,与经相应酶切的线性化载体pBGKT7-BD连接构建重组质粒。随后将重组质粒pGBKT7-BD-SjGCP转化酵母菌株,测定融合蛋白BD-SjGCP在酵母菌中的自激活活性、对酵母菌生长的影响及在酵母中的表达情况。结果显示构建的重组诱饵蛋白质粒pGBKT7-BD-SjGCP能够在酵母细胞内正确表达,没有独自激活报告基因表达的活性,且其表达对酵母细胞没有毒性,不影响酵母细胞的生长。可见,重组质粒pGBKT7-BD-SjGCP可用于筛选与血吸虫抱雌沟蛋白相互作用的物质。     

家蚕谷胱甘肽-S-转移酶基因(BmGSTd1)的诱导表达定量分析

谷胱甘肽-S-转移酶(GST)在机体的解毒代谢和抗氧化中起重要作用。为探究家蚕谷胱甘肽-S-转移酶基因(BmGSTd1)在家蚕体内的解毒和抗性功能,采用实时荧光定量PCR方法,对BmGSTd1基因在正常饲养及添食NaF家蚕5龄幼虫不同组织中的转录水平进行检测,并采用家蚕Actin3、GAPDH、28S rRNA 3种内参照基因对检测结果进行归一化处理。BmGSTd1基因在家蚕5龄幼虫各组织的转录水平存在差异,且采用不同内参照基因结果不一致:分别以家蚕Actin3、GAPDH、28S rRNA基因为内参照,该基因转录水平最高的组织分别为中肠、丝腺、脂肪体。5龄第2天幼虫添食NaF对体内各组织中BmGSTd1基因的转录水平具有诱导作用,且在不同组织中诱导表达的情况不同,采用以上3种内参照基因进行归一化处理的结果数据表明,中肠和脂肪体组织中的诱导转录水平最高,可能与这2种组织是家蚕主要的解毒器官有关。研究认为,检测基因在不同组织中的转录水平,采用合适的内参照基因对保证检测结果的可靠性非常重要。