Contribution of the tdh 1 gene of Kanagawa phenomenon-positive Vibrio parahaemolyticus to production of extracellular thermostable direct hemolysin

Kanagawa phenomenon-positive strains of Vibrio parahaemolyticus contain two copies of the tdh gene (tdh1 and tdh2) encoding thermostable direct hemolysin (TDH). Previous studies suggested that the tdh2 gene, but not the tdh1 gene, was responsible for production of extracellular TDH. In this study, a tdh2-deficient isogenic mutant of Kanagawa phenomenon-positive strain AQ3815 was constructed by a suicide vector-mediated in vivo recombination method. The intact tdh1 gene in the mutant contributed little to Kanagawa phenomenon on Wagatsuma agar but produced TDH in broth media, accounting for 0.5–9.4% of total extracellular TDH of AQ3815.     

橙皮苷对免疫功能低下小鼠免疫调节作用的实验研究

目的研究橙皮苷(hesperidin,HDN)对小鼠免疫调节的作用。方法腹腔注射环磷酰胺(Cy)复制免疫功能低下的动物模型,测定胸腺、脾脏重量计算脏器指数;碳廓清法测定网状内皮系统吞噬功能;分光光度法测定血清溶血素IgM、IgG(HCIgM、HCIgG);绵羊红细胞(SRBC)的定量溶血测定法(QHS)测定脾溶血空斑形成细胞(PFC);噻唑蓝(MTT)法测定脾淋巴细胞增殖反应;二硝基氟苯(DNFB)诱导迟发型变态反应模型,观察HDN对小鼠迟发型变态反应(DTH)和T淋巴细胞亚群的影响。结果HDN可提高免疫低下小鼠的脏器指数、碳廓清指数K和吞噬指数α;HDN可以改善免疫抑制小鼠低下的脾淋巴细胞增殖反应,恢复小鼠DTH和提高CD4+、CD8+细胞数;对特异性HCIgM、HCIgG和PFC无影响。结论HDN对小鼠的非特异性免疫和特异性细胞免疫反应有促进作用,对特异性体液免疫反应无影响。     

快速检测单核细胞增生李斯特菌LAMP方法的建立

目的:建立一种检测单核细胞增生李斯特菌核酸的快速、特异的环介导等温扩增(Loop-mediated isothermalamplification,LAMP)方法。方法:针对单核细胞增生李斯特菌的李斯特菌溶血素hlyA基因设计4条LAMP引物,在水浴箱内65℃保温约60 min,完成对单核细胞增生李斯特菌的扩增,扩增产物经肉眼、SYBR Green I染色和电泳鉴定。利用LAMP和PCR方法同时检测1株单核细胞增生李斯特菌和10株非单核细胞增生李斯特菌(对照组)来验证LAMP方法的特异性。用LAMP和PCR方法分别检测一系列稀释后的单核细胞增生李斯特菌菌液,比较两者的敏感性。结果:经肉眼、加染料和电泳均能观察到1株单核细胞增生李斯特菌LAMP扩增阳性反应,10株非单核细胞增生李斯特菌没有出现LAMP扩增。LAMP敏感度比PCR高,LAMP检测单核细胞增生李斯特菌的检测下限为3 cfu/ml,PCR检测下限>300 cfu/ml。LAMP检测速度比PCR更快,只需要60 min。结论:建立了一种检测单核细胞增生李斯特菌的快速、特异的LAMP方法。     

梅毒螺旋体溶血素生物信息学分析

目的本研究旨在对梅毒螺旋体(Tp)的5种溶血素进行进一步的生物信息学分析,为后续研究溶血素家族蛋白在Tp致病过程中的致病机制提供依据。 方法通过使用NCBI、BLAST、CDD、BioEdit、ExPASy、SignalP、TMHMM、MEGA、PSORTb 3.0、ABCpred等生物信息学分析方法分析梅毒螺旋体溶血素家族蛋白的一般性质、信号肽、糖基化、磷酸化位点、亚细胞位置、二级结构、功能结构域及抗原表位。 结果预测了Tp中5个溶血素家族蛋白一般性质,Tp1037功能结构域不同于其余4种,Tp0028含有1个信号肽,Tp0027含有1个糖基化位点,Tp0027和Tp1037有多个跨膜结构域,5个溶血素家族蛋白均含有多个磷酸化位点和B细胞、T细胞表位。 结论Tp含有5个溶血素家族蛋白基因,提示Tp入侵宿主时这些基因产物极有可能通过其膜成孔毒性损伤靶细胞,从而致病。     

白藜芦醇对小鼠免疫系统调节作用的研究

目的研究白藜芦醇对小鼠免疫功能的影响。方法通过碳粒廓清试验,测定白藜芦醇对小鼠腹腔巨噬细胞功能的影响;通过比色法测定鸡红细胞免疫后小鼠血清溶血素抗体水平,研究白藜芦醇对小鼠体液免疫的影响。结果白藜芦醇30 mg/kg和15 mg/kg剂量对环磷酰胺致小鼠细胞免疫抑制有对抗作用,能使免疫功能低下小鼠吞噬指数和吞噬系数得到恢复;白藜芦醇30 mg/kg剂量可对抗环磷酰胺致小鼠体液免疫抑制,提高血清溶血素抗体水平。结论白藜芦醇对小鼠免疫功能有较好的调节作用。     

壮尔颗粒对小鼠免疫功能的影响

目的观察壮尔颗粒对小鼠免疫功能的影响。方法采用氢化可的松造模法制作免疫抑制小鼠模型,进而观察壮尔颗粒对其腹腔巨噬细胞吞噬功能、溶血素形成、淋巴细胞转化的影响。结果壮尔颗粒能明显提高正常小鼠和免疫抑制小鼠巨噬细胞吞噬百分率、吞噬指数、血清溶血素含量和淋巴细胞转化率。结论壮尔颗粒对机体免疫功能有显著增强作用。     

马齿苋提取物对正常小鼠免疫功能的影响

目的：观察马齿苋提取物对正常小鼠免疫功能的影响.方法：观察马齿苋提取物对小鼠腹腔巨噬细胞吞噬功能、溶血素形成、溶血空斑形成及淋巴细胞转化功能的作用.结果：马齿苋提取物可显著提高小鼠腹腔巨噬细胞的吞噬百分率和吞噬指数;促进溶血素及溶血空斑的形成;促进淋巴细胞的转化.结论：马齿苋提取物有提高免疫功能的作用.     

马齿苋提取物对正常小鼠免疫功能的影响

目的:观察马齿苋提取物对正常小鼠免疫功能的影响。方法:观察马齿苋提取物对小鼠腹腔巨噬细胞吞噬功能、溶血素形成、溶血空斑形成及淋巴细胞转化功能的作用。结果:马齿苋提取物可显著提高小鼠腹腔巨噬细胞的吞噬百分率和吞噬指数;促进溶血素及溶血空斑的形成;促进淋巴细胞的转化。结论:马齿苋提取物有提高免疫功能的作用。     

兔脑磷脂的抗炎与免疫调节作用

兔脑磷脂(RBP)100～200 mg·kg~(-1)ip或sc,对4种急性渗出性炎症模型均有显著的抗炎作用,卵磷脂和脑磷脂为RBP抗急性炎症的有效成分.但RBP对慢性增生性炎症无抗炎作用.对正常和免疫受抑动物,RBP能增加脾重,提高碳粒廓清率和腹腔巨噬细胞的吞噬指数,提高淋巴细胞转化率和溶血素抗体生成及肝细胞.cGMP含量.在体外,小剂量RBP促进正常人淋巴细胞增殖反应,大剂量呈抑制作用.     

ShlB mutants of Serratia marcescens allow uncoupling of activation and secretion of the ShlA hemolysin

The ShlB protein in the outer membrane of Serratia marcescens secretes hemolytic ShlA protein into the culture medium. In the absence of ShlB, nonhemolytic ShlA remains in the periplasm. ShlB mutants were isolated in which secretion was uncoupled from activation. Mutants with a tetrapeptide insertion after residues 136 or 224 of mature ShlB and a mutant with an insertion after residue 154 and a deletion secreted inactive ShlA. In vitro, secreted nonhemolytic ShlA was converted into hemolytic ShlA by isolated wild-type ShlB and by complementation with an N-terminal ShlA fragment of 255 residues (ShlA-255). The isolation of secretion-competent, but activation-negative mutants indicates that secretion alone is not sufficient for activation of ShlA. Rather, ShlB is required for activation and secretion, and the mutants define sites in ShlB which are involved in activation. According to a predicted transmembrane model of ShlB, the mutations that retain secretion competence but abolish activation competence are located in the most prominent surface loop and the following transmembrane loop. In one tetrapeptide insertion mutant, ShlB-332, most of the ShlA remained cell-associated in an inactive form and low amounts (6%) were hemolytic. Secreted inactive ShlA(o) was completely degraded by trypsin, in contrast to hemolytic ShlA, which was cleaved into two fragments of 60 and 100 kDa. This result indicates that the conformational change from a highly trypsin-sensitive to a highly trypsin-resistant protein with only a single cleavage site in a polypeptide of 1,578 residues occurs upon activation of ShlA and not during secretion.