伤寒─鼠伤寒重组株Vi4072在小鼠中定居及血清学效果观察试验

以伤寒─鼠伤寒双价重组株Ｖｉ４０７２的３×１０８ＣＦＵ一次口服感染ＢＡＬＢ／Ｃ小鼠,４天后即可从小鼠的集合淋巴结、肝、脾中分离到该菌,４９天后该菌始被小鼠机体彻底清除。血清和小肠匀浆液中Ｖｉ抗体检测结果证明Ｖｉ４０７２菌株有刺激特异性免疫应答的功能。血清、小肠匀浆液中Ｖｉ抗体明显升高。     

伤寒─鼠伤寒重组株Vi4072在小鼠中定居及血清学效果观察试验

以伤寒─鼠伤寒双价重组株Ｖｉ４０７２的３×１０８ＣＦＵ一次口服感染ＢＡＬＢ／Ｃ小鼠,４天后即可从小鼠的集合淋巴结、肝、脾中分离到该菌,４９天后该菌始被小鼠机体彻底清除。血清和小肠匀浆液中Ｖｉ抗体检测结果证明Ｖｉ４０７２菌株有刺激特异性免疫应答的功能。血清、小肠匀浆液中Ｖｉ抗体明显升高。     

伤寒─鼠伤寒重组株Vi4072的效力试验研究

伤寒─鼠伤寒重组株Ｖｉ４０７２所产生Ｖｉ抗原以伤寒Ｔｙ２株所产Ｖｉ抗原作对照,通过ＥＤ５０测定和小白鼠被动保护试验作了比较,结果表明Ｖｉ４０７２株Ｖｉ抗原的半数有效剂量为０．０１３６μｇ,Ｔｙ２株的半数有效剂量为０．０１８３μｇ,说明Ｖｉ４０７２─Ｖｉ对小白鼠的保护作用不低于Ｔｙ２─Ｖｉ。被动保护试验证明,在同等条件下,两种Ｖｉ抗原的免疫血清对小白鼠提供的保护作用相同。     

伤寒─鼠伤寒重组株Vi4072的效力试验研究

伤寒─鼠伤寒重组株Ｖｉ４０７２所产生Ｖｉ抗原以伤寒Ｔｙ２株所产Ｖｉ抗原作对照,通过ＥＤ５０测定和小白鼠被动保护试验作了比较,结果表明Ｖｉ４０７２株Ｖｉ抗原的半数有效剂量为０．０１３６μｇ,Ｔｙ２株的半数有效剂量为０．０１８３μｇ,说明Ｖｉ４０７２─Ｖｉ对小白鼠的保护作用不低于Ｔｙ２─Ｖｉ。被动保护试验证明,在同等条件下,两种Ｖｉ抗原的免疫血清对小白鼠提供的保护作用相同。     

国产与法国Merieux产伤寒Vi多糖菌苗人体接种反应及血清抗体应答比较

作者对国产伤寒Ｖｉ多糖菌苗和法国Ｍｅｒｉｅｕｘ产伤寒Ｖｉ多糖菌苗人体接种反应及血清抗体应答情况进行比较,接种后６～８小时法国菌苗的体温反应同国产菌苗相比有显著性差异（ｘ２＝５．３４７,０．２５＞Ｐ＞０．０１）,２４小时后反应消失,免后一月国产菌苗和法国菌苗Ｖｉ抗体四倍增长率分别为９１．８０％和８９．６％（ｘ２＝０．１６４,Ｐ＞０．０５）,ＧＭＴ分别为３６．９４和３５．４９（ｔ＝０．６５３,Ｐ＞０．０５）,二者均无显著性差异。     

PhoQ基因重组鼠伤寒沙门氏菌株的构建及毒力研究

应用PCR技术从鼠伤寒沙门氏菌基因组DNA中克隆phoQ基因片段,构建原核表达pUC18重组质粒,测定序列(GenBank登录号为DQ787014),并转入鼠伤寒沙门氏菌,经异丙基硫代半乳糖苷(IPTG)诱导,进行高效表达。对重组菌株、野生菌株进行毒力检测对比实验,通过口腔注入45日龄健康无菌KM小鼠,测定其半数致死量(LD50)。结果发现：重组菌株与野生菌株的毒力存在显著差异,其半致死量分别为3.981×107 cf u/ mL and 5.012×102 cf u/ mL,PhoQ基因重组菌株的毒力远远低于非重组菌株。说明phoQ基因是调节鼠伤寒沙门氏菌致病机制中一个重要的调节因子。     

鼠伤寒沙门氏菌诱导昆明小鼠肠道感染模型的建立

目的:建立鼠伤寒沙门氏菌诱导昆明小鼠肠道感染模型。方法:先用5 mg/mL链霉素预处理2 d,提高小鼠对鼠伤寒沙门氏菌的敏感性,然后正常饲养1 d,攻毒前禁水禁食4 h,再分别以不同剂量灌胃攻毒2次,间隔24 h。观察小鼠临床症状,并通过组织病理切片、透射电镜和免疫组织化学的方法,分别观察小鼠肠道组织病理变化、小肠上皮细胞超微结构变化及肠道淋巴细胞增殖状况。结果:攻毒后昆明小鼠会出现昏睡、食欲不振、寒颤,甚至死亡的现象,解剖后发现小鼠肠道充血膨胀。组织病理切片显示小鼠肠粘膜受损,小肠绒毛肿胀,排列杂乱,炎性细胞浸润;透射电镜观察超微结构显示小肠上皮细胞线粒体空泡化,嵴和膜发生融合消失,粗面内质网发生扩张;免疫组织化学的方法显示肠道感染后,淋巴结肿大,T淋巴细胞大量增殖。结论:该模型对探索鼠伤寒沙门氏菌引发肠炎的发病机制、病理生理、免疫等方面作用具有重要意义,并为特异性卵黄抗体被动免疫保护效果的后续评价奠定基础。     

鼠伤寒沙门氏菌外膜蛋白诱发BALB／C小鼠免疫保护的研究

本文采用超声破碎ＴｒｉｔｏｎＸ—１００和超速离心技术提取了鼠伤寒杆菌（Ｓａｌｍｏｎｅｌｌａｔｙｐｈｉｍｕｒｉｕｍ,ＳＴＭ）的外膜蛋白（Ｏｕｔｅｒｍｅｍｂｒａｎｅｐｒｏｔｅｉｎｓ,ＯＭＰｓ）,并发现ＯＭＰｓ中脂多糖ＬＰＳ的含量约为５％,ＯＭＰｓ经ＳＤＳ—ＰＡＧＥ显示１０余条蛋白带。对ＯＭＰｓ诱发ＢＡＬＢ／Ｃ小鼠产生典型迟发型变态反应ＤＴＨ和ＩＬ—２的水平进行了检测。经腹腔免疫的小鼠用５００ＬＤ５０鼠伤寒杆菌（５０１１５）攻击,１００％可得到保护;用５００ＬＤ５０伤寒杆菌（Ｅ６８６）攻击,３３．３％可得到交叉保护。免疫ＢＡＬＢ／Ｃ小鼠的Ｔ淋巴细胞,经尾静脉注射给非免疫小鼠,可使后者得到８５．７％的被动免疫保护,上述结果说明ＯＭＰｓ能诱发ＢＡＬＢ／Ｃ小鼠细胞免疫和保护性免疫,并提示成为分子疫苗的可能性。     

用体内激活的启动子调控HCV核心抗原表达可增强重组鼠伤寒沙门氏菌的免疫原性

为提高抗原表达质粒在重组伤寒沙门氏菌中的稳定性以增强重组伤寒沙门氏菌诱导的免疫应答 ,克隆鼠伤寒沙门氏菌pagC基因启动子 ,以其为转录调控元件构建HCV核心抗原表达质粒 ,转化到减毒鼠伤寒沙门氏菌中。体外培养时 ,Mg2 能够剂量依赖性抑制该重组菌表达HCV核心抗原。将该重组菌和组成性表达的重组菌分别口服接种BALB/c小鼠 ,观察质粒的稳定性和小鼠的免疫应答。结果表明 ,体内激活的pagC基因启动子能明显提高质粒在重组鼠伤寒沙门氏菌中的稳定性和增强重组菌诱导的体液和细胞免疫应答 ,这为发展高效免疫、成本低廉的口服丙肝疫苗提供了一个新思路     

伤寒Vi多糖菌苗多糖含量及分子大小测定试验

伤寒Ｖｉ多糖菌苗是我国新近研制成功的一种多糖菌苗。为了严格控制该制品的质量,经反复试验,建立了多糖含量和分子大小的测定方法。本文报导了（１）用火箭电泳法测定伤寒Ｖｉ多糖菌苗多糖含量。经对不同实验条件进行比较,选择出较为理想的条件。用该法测定８批样品。结果均符合规程要求。对其中５批样品进行６次重复试验表明,该法的重复性好,操作简单,是测定多糖含量较为理想的方法。（２）用琼脂糖柱层析法对２８批伤寒Ｖｉ多糖菌苗的分子大小进行测定。对用该法所得柱层析收集液分别用Ｈｅｓｔｒｉｎ法和２０６ｎｍ扫描法测定其多糖回收率,对测定结果进行比较。结果表明,两种方法的测定结果无显著性差异（Ｐ＞０．０１）,而且重复性均好。可根据实验室条件选择测定方法。     

Taenia solium: Immune response against oral or systemic immunization with purified recombinant calreticulin in mice

Recombinant functional Taenia solium calreticulin (rTsCRT) confers different degrees of protection in the experimental model of intestinal taeniosis in hamsters. The aim of this study was to evaluate the immune response induced after oral or systemic immunization with an electroeluted rTsCRT in BALB/c mice. Oral immunization elicited high fecal IgA and the production of IL-4 and IL-5 by mesenteric lymph node cells after in vitro stimulation with rTSCRT, indicating a Th2 response. Mice subcutaneously immunized produced high amounts of serum IgG, being IgG1 (Th2-related) the predominant isotype, while in vitro stimulated spleen cells synthesized IL-4, IL-5 and also IFN-γ, indicating a mixed Th1/Th2 cellular response after systemic immunization. Our data show that purified rTsCRT induces polarized Th2 responses after oral immunization of mice, a common characteristic of protective immunity against helminths and, consequently, a desirable hallmark in the search for a vaccine.     

Immune responses dependent on antigen location in recombinant attenuated Salmonella typhimurium vaccines following oral immunization

The subcellular location of a recombinant antigen in recombinant attenuated Salmonella vaccines may influence immunogenicity dependent on exposure of the recombinant antigen to cells involved in systemic immune responses. It has been shown that a recombinant attenuated Salmonella vaccine secreting the recombinant Streptococcus pneumoniae PspA (rPspA) antigen specified by pYA3494 induced protective anti-rPspA-specific immune responses (Kang et al. (2002) Infect. Immun. 70, 1739-1749). A recombinant plasmid pYA3496 specifying a His(6)-tagged rPspA (His(6)-rPspA) protein (no apparent signal sequence) caused the rPspA antigen to localize to the cytoplasm of Salmonella. Salmonella vaccines carrying pYA3494 or pYA3496 expressed similar amounts of rPspA. After a single oral immunization in BALB/c mice with 10(9) colony-forming units (CFU) of the recombinant Salmonella vaccines carrying pYA3494 or pYA3496, IgG antibody responses were stimulated to both rPspA and Salmonella lipopolysaccharide (LPS) antigens. The anti-rPspA IgG titer induced by Salmonella carrying pYA3494 (1.9 x 10(7)) was 10(4) times higher than induced by Salmonella carrying pYA3496 (<2.4 x 10(3)).     

Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains

 Human papillomaviruses (HPV) are present in approximately 95% of all cervical carcinomas and the HPV E6 and E7 genes are continuously expressed in these lesions. There is also circumstantial evidence that often natural immunity against HPV is generated and that this is of influence on HPV-induced lesions. Stimulation of the immune system by proper presentation of relevant HPV antigens might, therefore, lead to a prophylactic or therapeutic immunological intervention for HPV-induced lesions. For this purpose we have expressed the E6 and E7 protein of HPV 16 in an attenuated strain of Salmonella typhimurium (SL3261, aroA mutation), which has been used extensively as a live vector. Live recombinant Salmonella vaccines have the ability to elicit humoral, secretory and cell-mediated immune responses, including cytotoxic T cells, against the heterologous antigens they express. This report describes the construction of recombinant Salmonella strains expressing the HPV 16 E6 and E7 proteins, and the induction of an HPV-16-specific immune response in mice after immunization with these live vectors. Received: 25 June 1996 / Accepted: 6 August 1996     

IgA response of BALB/c mice to orally administered Salmonella typhimurium flagellin‐displaying T2 bacteriophages

Salmonella typhimurium antigens were displayed on the capsid of a T2 bacteriophage to explore the potential of phage display for an oral vaccine. Segments of the flagellin proteins FliC (H1 antigen) and FljB (H2) were fused to the N‐terminal of T2 phage SOC to give two recombinant phages, T2FliCm and T2FljBm. Over 14 days, 19 BALB/c mice were orally administered twice, either with purified recombinant FliCm and FljBm protein, or T2FliCm and T2FljBm with or without host Escherichia coli. Feces were sampled over 10 weeks and examined for phage by plaque assay and for the presence of mucosal IgA by ELISA. Relatively few phages were detected relative to the amount administered (up to 8.21 × 10³ PFU/g faeces) and none were detected five days after initial administration. The administration of a large number of phages appeared to cause no clinical symptoms. IgA concentration in feces peaked around four weeks after the second administration and subsided after eight weeks. The highest relative titers were observed in the protein group (0.37% for anti‐FliCm and 0.22% for anti‐FljBm) and the mouse group which received no E. coli (0.33% and 0.35%) despite the theoretical amount of protein contained in a phage dose being at least 80–465 times lower than the protein dose administered. The possibility that the immuno‐stimulatory properties of the phage create an adjuvant effect to enhance the immunogenic properties of the displayed proteins is discussed. We conclude that phage may be valuable as a vector for oral vaccines. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

重组质粒在减毒沙门氏菌中的稳定性及其对细菌侵袭力的影响

将含有外源基因的重组真核表达质粒pcDNA3-F和pCI-F转化减毒鼠伤寒门氏菌,探讨质粒类型和插入片段对重组质粒在细菌内的稳定性和细菌侵袭力的影响。结果表明,外源质粒可降低减毒沙门氏菌在体外的增殖能力和侵袭力,也影响细菌在鸡体内的存活力;就质粒类型而言,pCI的影响大于pcDNA3,而以携带外源基因的重组质粒影响较为显著;外源基因插入也影响质粒在宿主菌内的稳定性。提示利用减毒鼠伤寒沙门氏菌为载体传递DNA疫苗研究时,要考虑质粒类型与其在宿主菌内稳定性的关系、携带外源基因重组质粒对载体菌侵袭力和存活力的影响等问题。