志贺菌对HeLa细胞侵袭力的研究

通过使用噬斑形成试验、透射电镜及检测志贺菌蛋白表达等方法,研究志贺菌对Hela细胞的侵袭能力。研究发现:37℃培养条件下,胞质内出现成堆的志贺菌;30℃培养条件下,细菌主要分布在细胞外。SDS-PAGE显示,与30℃培养条件相比,在37℃培养下,志贺菌表达蛋白质的种类和数量明显增加。噬斑形成发现,12株福氏志贺菌强毒株,有9株噬斑数>1000个／ml,而2株弱毒株噬斑数则在50个／ml以下。透射电镜证实了志贺菌对细胞的黏附、侵入和释放过程。结果表明:志贺菌对细胞的侵袭能力受温度的影响;不同志贺菌流行株对细胞的侵袭力存在着差异:实验也显示,应用HeLa细胞研究志贺菌侵袭力是一种简便易行且价廉的方法。     

In vitro selection of a DNA aptamer targeted against Shigella dysenteriae

To identify DNA aptamers demonstrating binding specificity for Shigella dysenteriae, a whole-bacterium Systemic Evolution of Ligands by Exponential enrichment (SELEX) method was applied to a combinatorial library of single-stranded DNA (ssDNA) molecules. After several rounds of selection using S. dysenteriae as the target, the highly enriched oligonucleotide pool was sequenced and then grouped into different families based on primary sequence homologies and similarities in the secondary structures. Aptamer S 1, which showed particularly high binding affinity in preliminary studies, was chosen for further characterisation. This aptamer displayed a dissociation constant (K_d value) of 23.47 ± 2.48 nM. Binding assays to assess the specificity of aptamer S 1 showed high binding affinity for S. dysenteriae and low apparent binding affinity for other bacteria. The ssDNA aptamers generated may serve as a new type of molecular probe for microbial pathogens, as it has the potential to overcome the tedious isolation and purification requirements for complex targets.     

Virulence phenotypes of Shigella flexneri 2a avirulent mutant 24570 can be complemented by the plasmid-coded positive regulator virF gene

Avirulent mutation of an opaque colony variant of Shigella flexneri 2a designated 24570 has been believed to be linked with the glpK locus of the chromosome. However, avirulent phenotypes of the 24570 strain could be complemented by the invasion plasmid-coded virF gene, a positive regulator for invasion genes. The 24570 strain had a DNA structural alteration upstream of the virF gene.     

肠毒素性大肠杆菌CS3纤毛抗原在痢疾菌中的表达及其免疫效果研究

首先通过体内外重组的方法,构建了福氏2a痢疾菌T32asd基因缺陷的突变体FaD,作为抗原载体菌;同时,构建包含asd基因的表达质粒pYX102,与FaD一起,构成宿主-载体平衡致死系统,用于在没有抗生素条件选择的情况下,稳定表达克隆在表达质粒上的外源抗原基因.将肠毒素性大肠杆菌的CS3菌毛抗原基因克隆至pYX102,构建成重组表达质粒pYX103,ELISA检测结果证实CS3在痢疾菌中可以很好地表达.免疫小鼠后可诱生相应的抗体,虽然口服免疫和注射免疫产生的CS3抗体效价有一定差别,但对痢疾菌的毒株攻击均可提供较好保护.该结果为细菌性腹泻疫苗的研制提供了候选株.     

Proteomic analysis and protein structure prediction of Shigella phage Sfk20 based on a comparative study using structure prediction approaches

Bacteriophages are the natural predators of bacteria and are available abundantly everywhere in nature. Lytic phages can specifically infect their bacterial host (through attachment to the receptor) and use their host replication machinery to replicate rapidly, a feature that enables them to kill a disease-causing bacteria. Hence, phage attachment to the host bacteria is the first important step of the infection process. It is reported in this study that the receptor could be an LPS which is responsible for the attachment of the Sfk20 phage to its host (Shigella flexneri 2a). Phage Sfk20 bacteriolytic activity was examined for preliminary optimization of phage titer. The phage Sfk20 viability at different saline conditions was conducted. The LC–MS/MS technique used here for detecting and identifying 40 Sfk20 phage proteins helped us to get an initial understanding of the structural landscape of phage Sfk20. From the identified proteins, six structurally significant proteins were selected for structure prediction using two neural network systems: AlphaFold2 and ESMFold, and one homology modeling software: Phyre2. Later the performance of these modeling systems was compared using various metrics. We conclude from the available and generated information that AlphaFold2 and Phyre2 perform better than ESMFold for predicting Sfk20 phage protein structures.     

Construction of prophylactic human papillomavirus type 16 L1 capsid protein vaccine delivered by live attenuated Shigella flexneri strain sh42

To express human papillomavirus （HPV） L 1 capsid protein in the recombinant strain of Shigella and study the potential of a live attenuated Shigella-based HPV prophylactic vaccine in preventing HPV infection, the icsA/virG fragment of Shigella-based prokaryotic expression plasmid pHS3199 was constructed. HPV type 16 L1 （HPV16L1） gene was inserted into plasmid pHS3199 to form the pHS3199-HPV16L1 construct, and pHS3199-HPV 16L1 was electroporated into a live attenuated Shigella strain sh42. Western blotting analysis showed that HPV 16L 1 could be expressed stably in the recombinant strain sh42-HPV 16L 1. Sereny test results were negative, which showed that the sh42-HPV16L1 lost virulence. However, the attenuated recombinant strain partially maintained the invasive property as indicated by the HeLa cell infection assay. Specific IgG, IgA antibody against HPV16L1 virus-like particles （VLPs） were detected in the sera, intestinal lavage and vaginal lavage from animals immunized by sh42-HPV 16L1. The number of antibodysecreting cells in the spleen and draining lymph nodes were increased significantly compared with the control group. Sera from immunized animals inhibited mufine hemagglutination induced by HPV 16L1 VLPs, which indicated that the candidate vaccine could stimulate an efficient immune response in guinea pig＇s mucosal sites. This may be an effective strategy for the development of an HPV prophylactic oral vaccine.     

弗氏2a志贺氏菌2457T株yciD基因缺失突变株的构建

目的:构建弗氏2a志贺氏菌2457T株yciD基因缺失突变体,以研究yciD基因的功能。方法:根据弗氏2a志贺氏菌2457T株基因组全序列,采用Red重组系统对yciD基因进行缺失,并经PCR和SDS-PAGE证实;对野生株和突变株的生长状态及生化反应进行比较研究。结果:构建了弗氏2a志贺氏菌2457T株的yciD基因缺失突变株2457TΔyciD,该突变株外膜蛋白样品中缺失了一条相对分子质量与从yciD基因推导的蛋白相当(约22000)的蛋白带。该突变株比野生株生长快,利用葡萄糖和甘露醇的能力也比野生株大为增强。结论:获得了弗氏2a志贺氏菌2457T株的yciD基因缺失突变株。