Ⅰ型大肠杆菌粘附素的表达受环境因素影响的初步探讨

本文探讨了葡萄糖、半乳糖、麦芽糖、ＰＨ、温度因子对尿路感染Ⅰ型大肠杆菌粘附素表达的影响。结果发现葡萄糖、半乳糖和低温可使粘附素的表达减少,而且葡萄糖和半乳糖与低温因子累加可使细菌几乎完全不表达粘附素。另外分析了与此有关的一些现象。     

抗大肠杆菌987P粘着素单克隆抗体及其初步应用

共研制出ll株抗大肠杆菌987P粘着素单克隆抗体,对其部分免疫学特性作了测定。这些单抗不仅效价很高,而且特异性强,对不具有987P抗原的大肠杆菌及所试的其他肠道杆菌均无交叉反应。以FlTc或Hrpo标记的987P单抗作实验室诊断,具有简易、快速、敏感和准确的优点。酶标EPN3株单抗检测的灵敏度可达2×l03个／ml 987P+菌,对人工发病仔猪的粪样和小肠内容物的阳性检出比分别为4／4和2／2。 EP22株荧光标记单抗对病猪小肠粘膜触片的阳性检出比为6／6。 11株单抗中7株能不同程度地阻断987P+菌对仔猪小肠上皮细胞的吸附作用。EL1sA和荧光阻抑试验表明,1株单抗是针对987p粘着素上三种不同的抗原决定簇。EPN2株单抗的免疫胶体金定位还表明,987P粘着素似呈螺旋状结构,且含有许多相同的抗体结合位点。这些单抗不仅可用于ETEC菌株的987P柔毛鉴定,而且可用于987P分子结构和生物学特性的研究。     

在大肠杆菌中表达人防御素-1

构建了一组HNP-1原核表达载体,其中有几个含SD或序列的载体,在表达产物中检测到了HNP-1的存在,说明宿主菌是否在任何情况下都降解外源小分子多肽是一个值得考虑的问题．另外从表达载体在大肠杆菌JM109和JM109（DE3）中蛋白表达量的差异也可推测HNP-1发生了低水平的表达,HNP-1对细菌的作用与HNP-1浓度和细菌本身的生理状况密切相关.     

大肠杆菌α-溶血素分泌系统基因表达调控研究进展

引起人泌尿道感染的大肠杆菌(UPEC)的α-溶血素分泌系统属于Ⅰ型分泌系统,能够分泌一种RTX毒素蛋白-α-溶血素。近年来α-溶血素分泌系统的应用日益广泛,特别是在减毒活载体疫苗中呈递外源抗原。本分析了大肠杆菌α-溶血素分泌系统的遗传特性,详细介绍了其基因的表达调控,为构建更加有效的分泌型载体提供了理论基础。     

血管生成抑制素基因工程大肠杆菌的高密度发酵研究

用5L发酵罐研究了E.coli TG1/pBVA2和E.coli TG1/pBVK13的高密度培养工艺,确定了诱导及补料策略,在不降低外源基因表达量的前提下,工程菌TG1/pBVA2高密度发酵菌体干重为16.8g/L,hAGN(K1-4)的表达量为菌体总蛋白的24.1%,相当于1.39 g/L;同样的方法, 工程菌TG1/pBVK13菌体干重可达16g/L, hAGN(K1-3)占菌体总蛋白25.8%,相当于1.45 g/L。     

纤维结合素分子中Asp1961～Glu1978序列对三结构域多肽在大肠杆菌中表达的影响

构建了人纤维结合素（ＦＮ）的三功能结构域重组多肽的两个表达质粒,分别编码两个重组多肽：ＣＨ６２（ＦＮ的Ｐｒｏ１２３９～Ｓｅｒ１５１５经Ｍｅｔ和Ａｌａ１６９０～Ｖａ１２０４９相连）和ＣＨ６３（从ＣＨ６２中删除了Ｉｌｅ１８５０～Ｇｌｕ１９７８）．ＣＨ６２在大肠杆菌中的表达效率很低,而ＣＨ６３的表达效率则很高,结果提示ＦＮ分子中的Ａｓｐ１９６１～Ｇｌｕ１９７８序列是影响三结构域多肽在大肠杆菌中表达的关键结构．ＣＨ６３经过溶解和复性后,可通过肝素－琼脂糖亲和层析得到纯品,所得纯品具有结合肝素和结合细胞的功能,且结合细胞的能力比双结构域ＦＮ多肽更强,表明两个结合细胞的功能结构域均有活性．ＣＨ６３的制备为进一步研究具有更强的抑制肿瘤转移作用的基因工程制品奠定了基础．     

大肠杆菌α-溶血素分泌途径研究进展

随着生物技术的不断发展,大肠杆菌胞外分泌分子机理日渐明晰,大肠杆菌表达系统成为实现规模化制备胞外重组蛋白的途径之一。本文综述了大肠杆菌α-溶血素分泌途径的转运机制及应用前景。     

动物源产肠毒素大肠杆菌(ETEC)黏附素研究进展

动物源产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)是引起动物(尤其是幼龄动物)腹泻的主要病原菌。已知黏附素和肠毒素是ETEC中两种重要的毒力因子,在致病性中两者缺一不可。其中黏附素结合到宿主易感肠上皮细胞是ETEC感染的第一步,也是最重要的关键步骤。动物源ETEC的菌毛黏附素主要包括K88、K99、987P、F18、F17和F41等。人们从20世纪60年代就开始了ETEC菌毛黏附素的相关研究,包括菌毛的基因、结构组成、生物合成、菌毛表达的调控机制以及黏附素和宿主受体相互作用等,这些研究基础有助于我们深入了解ETEC病原菌的感染机理;并且在疾病诊断和新疫苗的开发中具有重大意义。     

THE PHENOTYPIC AND FITNESS EFFECTS OF COLICIN RESISTANCE IN ESCHERICHIA COLI K-12

Previous studies indicate that most natural isolates of Escherichia coli are resistant to most or all colicins (antibiotics produced by E. coli) when assessed in the laboratory. Additionally, resistance to different colicin types appears to arise in a nonindependent manner. One possible mechanism to explain this nonindependence is pleiotropy: Multiple resistances are selected after exposure to a single colicin. This study, which was designed to address the role of pleiotropy in the generation of colicin resistance, revealed that 96% of colicin resistant mutants were resistant to two or more colicins. Mutational class was important because putative translocation mutants (Tol pathway mutants) resisted fewer colicins than putative receptor mutants. To determine whether colicin resistance is costly, the effects of colicin resistance mutations on maximal growth rate in a rich medium were also examined. Relative to the sensitive ancestor, translocation mutations lowered maximal growth rates by 17%, whereas putative receptor mutations did not significantly lower growth rates. Thus, when nutrients are abundant, the most advantageous forms of colicin resistance may not impose a cost. The ecological consequences of pleiotropic colicin resistance could involve population cycling between colicin sensitivity and resistance. Additionally, if the cost of resistance depends on the environment, ecological diversification could result.     

HIGH LEVELS OF COLICIN RESISTANCE IN ESCHERICHIA COLI

Colicins are plasmid-encoded antibiotics that are produced by and kill Escherichia coli and other related species. The frequency of colicinogeny is high, on average 30% of E. coli isolates produce colicins. Initial observations from one collection of 72 strains of E. coli (the ECOR collection) suggest that resistance to colicin killing is also ubiquitous, with over 70% of strains resistant to one or more colicins. To determine whether resistance is a common trait in E. coli, three additional strain collections were surveyed. In each of these collections levels of colicin production were high (from 15 to 50% of the strains produce colicins). Levels of colicin resistance were even higher, with most strains resistant to over 10 colicins. A survey of 137 non-E. coli isolates revealed even higher levels of resistance. We discuss a mechanism (pleiotropy) that could result in the co-occurrence of such high levels of colicin production and colicin resistance.