福氏志贺菌O-抗原磷酸乙醇胺修饰特异抗血清的制备

目的 制备针对福氏志贺菌O-抗原磷酸乙醇胺（PEtN）修饰的抗血清。方法 用血清型Yv菌株036_Yv（O-抗原PEtN修饰）免疫兔子制备抗血清,用036_Yv 的宿主菌036（O-抗原无PEtN修饰）吸附后,制备针对O-抗原PEtN修饰的特异抗血清,并用135株福氏志贺菌检测血清的特异性。结果 制备的血清能够特异地与O-抗原存在PEtN修饰的福氏志贺菌发生凝集,效价为1:32;除了O-抗原携带PEtN修饰的福氏志贺菌血清型Xv,Yv和4av外,制备的血清不能与其他血清型发生交叉凝集,具有很好的特异性。结论 成功制备了针对福氏志贺菌O-抗原PEtN修饰的特异抗血清,可应用于痢疾检测和监测。     

志贺菌属肠毒素ShET1/ShET2的基因型PCR分析

目的 对福氏2a志贺菌肠毒素ShET1和肠毒素ShET2进行基因分型，提高福氏2a志贺菌同源克隆鉴定系统的分析水平。方法 对93株分离自不同地区、不同时间的福氏2a志贺菌用PCR法检测志贺菌肠毒素ShET1／ShET2基因并进行基因分型。结果 93株福氏2a志贺菌按志贺菌肠毒素ShET1／ShET2基因分为4种基因型，即12株ShET1(-)／ShET2( )，14株ShET1( )／ShET2(-)，59株ShET1( )／ShET2( )，8株ShET1(-)／ShET2(-)。结论 福氏2a志贺菌肠毒素ShET1／ShET2基因PCR检测可用于福氏2a志贺菌的初步筛检。     

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T

AIM: To screen the immunogenic membrane proteins of Shigella flexneri 2a 2457T. METHODS: The routine two-dimensional polyacrylamide gel electrophoresis (2-DE) and Western blotting were combined to screen immunogenic proteins of S. flexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was performed to determine the molecular weight of peptides. Electrospray ionization (ESI-MS/MS) was performed to determine the sequences of the interesting peptides. RESULTS: A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein (YaeT) was detected, which might be a candidate target of vaccine. CONCLUSION: Membrane proteins of 5. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.     

59株痢疾杆菌药敏及耐药性R质粒检测结果分析

我科分离的59株痢疾杆菌中有弗氏菌34株(57.6％),其1型和2型占82.4％(28/34)、宋氏菌19株(32.2％),其R型占78.9％(15/19),鲍氏5株(8.5％),志贺氏2型1株(1.9％)。全部菌株对10种抗生素各有不同程度的耐药,对3种以上耐药者在弗氏菌株中为61.8％(21/34)、宋氏菌则为89.5％(17/19),后者有1株对10种抗生素均耐药。本组34株可传递耐药性菌株的R质粒分离率占59株的57.6％,其中有3种以上可传递的R~+质粒的菌株占85.3％(29/34)。从菌型分布、耐药率和耐药R质粒传递率表明,弗氏菌以1、2型为主,宋氏菌有明显增多的趋势。     

福氏和宋内氏痢疾双价杂交菌苗株FSM21—17人体试验观察

32人服用福氏2a和宋内氏痢疾菌双价杂交菌苗株,24人服安慰剂(奶粉PBS液)作为对照。每次服苗或服安慰剂后连续3d观察副反应。轻型腹泻:服苗组2人(6.3%),安慰剂组1人(4.2%);腹部不适:服苗组5人(15.6%),安慰剂组5人(20.8%);恶心:服苗组3人(9.4%),安慰剂组1人(4.2%)。上述反应均很轻微,为一过性的。用BA-ELISA法测定了20人的粪抗体应答,抗福氏2a菌的IgA和IgG阳转率分别为60.0%和55.0%;抗宋内氏菌的IgA和IgG阳转率均为75.0%。菌苗株在肠道内停留时间平均为2.2d。该菌苗株经人体肠道后未发现毒力回复突变。     

Killed oral Shigella vaccine made from Shigella flexneri 2a protects against challenge in the rabbit model of shigellosis

The protective efficacy of an orally administered heat-killed virulent strain of Shigella flexneri 2a (5 weekly oral doses) was evaluated in 25 rabbits (14 immunized and 11 non-immunized controls) against challenge with the same strain of Shigella using the rabbit model of shigellosis. All 11 non-immunized rabbits developed bloody diarrhoea following challenge and 6 (54%) died. None of the 14 immunized rabbits developed diarrhoea (all had pellet stools) but 3 (21%) died from causes not associated with diarrhoea. Protection from diarrhoea and dysentery following oral immunization with a killed Shigella species was 100% and highly significant. Death following challenge was 2.5-fold higher in the non-immunized group (p = 0.115) but was not significant. These promising results suggest that further studies should be undertaken to develop a killed oral vaccine against shigellosis.     

抗福氏痢疾杆菌单克隆抗体的制备及其特性研究

本文用福氏痢疾杆菌4a、4b煮沸致死菌体免疫BALB/c小鼠,取其脾淋巴细胞在PEG介导下与Sp2/0细胞融合,经筛选、克隆化获得3株抗福氏痢疾杆菌McAb的杂交瘤细胞系。经连续传代6个月、液氮冷存6个月后,复苏仍能稳定分泌高效价的McAb。经鉴定,3株代号为401、420、421的McAb的Ig类型为IgG2b、IgG3和IgG3,轻链为λ、κ、κ;染色体数目为101～105条。交叉试验表明,3株McAb与志贺氏、宋内氏、鲍氏菌及常见肠道菌如大肠杆菌,变形杆菌、枯草杆菌、伤寒杆菌、鼠伤寒沙门氏菌、克雷伯氏菌、小肠结肠耶氏菌等及革兰氏阳性杆菌等均不发生交叉反应。McAb的表位分析表明,McAb真正识别的表位在福氏菌脂多糖的特异性多糖即O-侧链上,从而进一步证实了McAb的特异性。     

痢疾多糖-蛋白结合疫苗小鼠母子抗体传递试验

目的　以小鼠为试验动物模型 ,用不同的痢疾多糖 蛋白结合疫苗免疫母鼠 ,观察IgG抗体的母婴传递情况。方法　用福氏 2a痢疾多糖 重组铜绿假单胞菌外毒素A(rEPA)结合疫苗 (F2a O SP rEPA)和宋内痢疾多糖 重组铜绿假单胞菌外毒素A结合疫苗 (S O SP rEPA)分别皮下免疫母鼠 ,待其怀孕、分娩 ,用ELISA分别检测母体、子体小鼠的IgG抗体滴度。结果　子体小鼠出生第 2周的IgG抗体水平较高 ,随着小鼠周龄的增加 ,抗体水平逐渐下降。结论　痢疾多糖 蛋白结合疫苗免疫母体后 ,诱导的特异性抗体可经母体传递给子体 ,抗体滴度随时间的推移而逐渐降低。     

痢疾菌体内诱导基因表达文库的构建

目的 构建筛选痢疾菌福氏2a体内诱导基因的融合基因表达文库。方法 以自杀载体pGP704为骨架，用氯霉素乙酰转移酶基因作为报告基因，构建了用于筛选体内诱导基因的载体pGP-cat。把痢疾菌福氏2a基因组DNA的随机酶切片段（0．6－1kb)亚克隆到pGPcat载体报告基因上游的BglⅡ位点，构建了融合基因文库。结果 与痢疾菌福氏2a2457T结合转移后，获得融合基因表达文库。以氯霉素为选择标记，经过体外初步筛选，得到3．2％的具有氯霉素抗性的菌株。结论 构建的融合基因表达文库适用于筛选福氏2a痢疾菌的体内诱导基因。     

HeLa细胞感染痢疾杆菌前后差异表达的新EST序列的电子延伸及验证

目的:研究痢疾杆菌福氏2a入侵前和入侵后3h的人上皮细胞差异表达的新基因.方法:采用增毒的痢疾杆菌福氏2a 2457T侵袭HeLa细胞,检查HeLa细胞中细菌的数量并用RT-PCR方法从HeLa细胞中提取总RNA,制备探针.采用含有约3000个代表新基因的芯片进行芯片杂交,分析杂交结果.将所获得的新的156条EST序列为种子序列用cDNA微阵列实验,以人类EST数据库为基础库,应用siClone软件进行EST拼接、校对并尽可能延长获得大片段或全长cDNA.选取基因组未注释的编码区序列作为新基因进行RT-PCR实验验证.结果:共鉴定到≥3倍或≤0.33的差异表达的代表新基因的EST序列45条,其中25个延伸序列鉴定为与重要的肠道功能相关的已知基因,并有3个在痢疾杆菌侵袭期间高表达的存在显著差异的新EST序列,他们分别名为:NPCCKH12、ADBCSB02和HTBAMG05,这3个基因是新的人基因或假基因.结论:鉴定出了在HeLa细胞感染痢疾杆菌前后差异表达的新EST序列,为进一步研究痢疾杆菌与寄主相互作用奠定了基础.