稳定、无抗药的痢疾福氏2a和宋内双价菌苗候选株的构建

通过体内外基因重组,将大肠杆菌粘附因子cs3基因定位整合到痢疾杆菌福氏2a疫苗株T32菌染色体的asd基因内,使asd基因灭活;将来内O抗原基因克隆至无抗药性表达载体pXL378,获得重组质粒pXL390,将其转化asd^-的T32受体菌,构建成福氏2a和宋内双价苗苗株FS01。实验表明:重组质粒pXL390在不带任何抗菌素基因的情况下,在asd-的T32受体菌内是稳定的。FS01株遗传稳定,能表达两种痢疾菌的PLS-O抗原,无明显毒性作用。动物试验表明,以FS01株皮下免疫的小鼠对福氏2a和宋内有毒株的腹腔攻击有100％的保护。     

利用宿主-载体平衡致死系统构建志贺氏菌3价疫苗候选株

选择福氏2a志贺氏菌疫苗株T32为受体菌,通过基因同源重组交换技术,对其染色体asd基因进行定位突变,使之不能在LB培基上生长;同时利用链球菌asd基因构建Asd⁺的无抗药性互补载体,两者组成1套T32宿主载体平衡致死系统.进一步应用该系统克隆和表达了具有重要免疫保护功能的宋内I相O抗原基因和志贺氏毒素B亚单位基因(stxB),构建成福氏2a-宋内-StxB3价菌苗候选株FSD0l.结果显示:该菌株遗传稳定,重组质粒不需用抗生素选择,能有效表达3价抗原和产生针对上述3种野生型毒株的免疫保护反应.     

福氏2a志贺氏菌△aroA突变减毒株的构建

志贺氏菌芳香族氨基酸合成酶基因缺陷能够使菌体明显减毒,并有可能成为新一代痢疾疫苗．用ＰＣＲ技术从野生型福氏２ａ志贺氏菌２４５７Ｔ中克隆出ａｒｏＡ基因,在体外进行精确的缺失突变,并通过体内同源重组,构建成△ａｒｏＡ突变体ＲＳ４２６．实验结果表明,这种突变体仍保持了侵袭能力和保护性Ｏ抗原的表达,但其毒力已明显降低,不能产生豚鼠角结膜炎,小鼠半数致死量明显提高．免疫保护试验显示,ＲＳ４２６可在小鼠中产生对福氏２ａ野生菌１００％的保护作用．     

痢疾基因工程三价菌苗候选株的构建

通过DNA体内外同源重组, 用霍乱毒素B亚单位基因(ctxB)完全取代了福氏志贺氏2a T32株染色体上的asd基因, 获得了稳定表达CtxB的DAP依赖株FWL01. 随后, 用T32株的asd基因标记志贺氏宋内S7株的Ⅰ相大质粒, 并将其诱动至FWL01, 构成三价菌苗候选株FSW01. 在该菌苗候选株中, 表达宋内Ⅰ相O抗原的大质粒与宿主菌是平衡致死的. 因此, 该候选株在没有任何抗生素存在情况下, 能稳定地表达福氏 2a, 宋内O抗原和CtxB. 豚鼠眼角膜试验和HeLa细胞侵袭试验证明FSW01无毒, 家兔免疫试验证实了其有很好的免疫原性. 小鼠和猴体免疫保护试验显示该候选株对相应的有毒株攻击具有很好的保护效果.     

口服福氏2a和宋内氏痢疾双价活菌苗FS人群粪抗体检测

本文测试了经三剂（共１×１０１１ｃｆｕ）ＦＳ双价活菌苗口服免疫的１６３名中学生免前及免后１、３和６个月时特异性ｓＩｇＡ粪抗体水平。结果发现,服苗后半年内,分别有７１％和７７．９％的免疫人群特异性抗福氏２ａ和抗宋内氏ｓＩｇＡ有四倍以上升高,且有８２．９％的人群粪便中,两种抗体的应答状况表现一致,说明该菌苗具有良好的免疫原性,并具有双价菌苗的免疫学特性。     

宋内氏福氏2a双价志贺氏菌芳香族氨基酸营养缺陷型减毒株的研究:Ⅰ.减毒株的构建

FS54是本室构建的宋内氏福氏2a双价志贺氏菌杂交株,有与志贺氏菌毒株相同的毒力。本研究通过遗传重组技术,给FS54株引入了aroD基因的转座子Tn10插入失活物(aroD∶∶Tn10),构建了FS54株的芳香族氨基酸营养缺陷型减毒株,并进一步去除了Tn10所携带的四环素抗性和宋内氏Ⅰ相大质粒(pSS120∶∶Tn5)上Tn5所携带的卡那霉素抗性,得到了宋内氏福氏2a双价志贺氏菌芳香族氨基酸营养缺陷型减毒株FS54—7b。     

痢疾福氏2a asd基因的克隆及其序列分析

本文根据大肠杆菌（Ｅ．ｃｏｌｉ）Ｋ１２ａｓｄ基因两侧序列设计了一对引物,用全菌ＰＣＲ扩增了福氏２ａ Ｔ３２株的ａｓｄ基因及其两侧序列。对ＰＣＲ产物的初步结果表明,在ａｓｄ基因两端存在ＢａｍＨ Ｉ位点。为了防止由ＰＣＲ扩增带来的差错,我们又从福氏２ａ Ｔ３２株染色体中克隆了全长的ａｓｄ基因。序列分析了结果表明,福氏２ａＴ３２株ａｓｄ基因的序列与Ｅ．ｃｏｌｉ Ｋ１２的完全一致,全长１６８０ｂｐ,其两侧     

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。     

Developing live Shigella vaccines using lambda Red recombineering

Live attenuated Shigella vaccines have shown promise in inducing protective immune responses in human clinical trials and as carriers of heterologous antigens from other mucosal pathogens. In the past, construction of Shigella vaccine strains relied on classical allelic exchange systems to genetically engineer the bacterial genome. These systems require extensive in vitro engineering of long homologous sequences to create recombinant replication-defective plasmids or phage. Alternatively, the lambda red recombination system from bacteriophage facilitates recombination with as little as 40 bp of homologous DNA. The process, referred to as recombineering, typically uses an inducible lambda red operon on a temperature-sensitive plasmid and optimal transformation conditions to integrate linear antibiotic resistance cassettes flanked by homologous sequences into a bacterial genome. Recent advances in recombineering have enabled modification of genomic DNA from bacterial pathogens including Salmonella, Yersinia, enteropathogenic Escherichia coli, or enterohemorrhagic E. coli and Shigella. These advances in recombineering have been used to systematically delete virulence-associated genes from Shigella, creating a number of isogenic strains from multiple Shigella serotypes. These strains have been characterized for attenuation using both in vivo and in vitro assays. Based on this data, prototypic Shigella vaccine strains containing multiple deletions in virulence-associated genes have been generated.     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S. sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey models, vaccinated animals were protected against the challenges of wild S. flexneri 2a strain 2457T and S. sonnei strain S9.     

基因工程抗体表达系统的研究进展

本文就基因工程抗体几种常用表达系统包括大肠杆菌、昆虫细胞、酵母、植物、哺乳类细胞等系统的研究近况作一综述,并分别介绍了各表达系统的优缺点。     

不同蛋白载体的痢疾多糖结合疫苗小鼠免疫原性对比试验

试验中以小鼠为动物模型,对不同蛋白载体的痢疾多糖结合疫苗进行免疫效果观察。3种福氏2a痢疾结合疫苗和3种宋内氏痢疾结合疫苗分别皮下免疫NIH小鼠,同时设置O-SP(O-特异性多糖)对照组,免疫3针,在不同免疫针次间采血,用ELISA测定抗体滴度。单独使用福氏2aO-SP和宋内氏O-SP免疫后,小鼠血清中几乎没有抗LPS IgG抗体产生,而用结合疫苗免疫后,小鼠血清中产生了抗LPS IgG抗体,且第二次、第三次免疫后,小鼠血清中抗LPS IgG抗体水平有显著升高,表明结合疫苗具有加强免疫应答效应。三种不同的痢疾结合疫苗相比较,F2a-O-SP-rEPA结合疫苗较F2a-O-SP-TT结合疫苗和F2a-0-SP—DT结合疫苗的小鼠抗LPS IgG抗体水平高,S-O-SP-rEPA结合疫苗较S-O-SP-TT结合疫苗和S-O-SP—CRM9,结合疫苗的小鼠抗LPS IgG抗体水平高。以rEPA作为载体的痢疾结合疫苗比DT,TT作为载体的痢疾结合疫苗的免疫原性要强。