CVB3-VP1基因免疫诱导特异性抗病毒免疫应答及保护作用

目的 :构建表达柯萨奇病毒B3(CVB3)主要包膜蛋白VP1的基因疫苗 ,并研究该疫苗诱导CVB3特异性免疫应答及免疫保护的作用。方法 :抽提CVB3RNA ,以RT PCR扩增VP1基因 ,克隆于真核表达载体 pcDNA3中 ,构建质粒pcDNA3 VP1。将该质粒转染Hela细胞 ,观察其表达情况 ;以 5 0 μgpcDNA3 VP1质粒DNA肌注免疫BALB/c小鼠 3次 ,检测CVB3特异性体液和细胞免疫应答。间隔 4wk以 5×LD50 的CVB3攻击小鼠 ,观察攻击后小鼠的存活情况。结果 :构建了重组质粒 pcDNA3 VP1,并在体外获得有效表达。以该质粒肌肉免疫BALB/c小鼠 ,可诱生高水平的IgM和IgG ,VP1多肽特异性淋巴细胞增殖反应及CTL活性均显著高于 pcDNA3免疫的对照组。病毒攻击试验表明 ,pcDNA3 VP1免疫组33.3%小鼠可长期存活 ,其心肌组织未见明显的病理学改变 ;而对照小鼠平均仅存活 6 .7d ,心肌显示大量的局灶性坏死和炎性细胞浸润。结论 :pcDNA3 VP1免疫可诱生CVB3特异性体液及细胞免疫应答 ,保护免疫小鼠抵抗CVB3的致死性攻击     

人IL-2信号肽基因增强柯萨奇病毒B3型VP1 DNA疫苗诱导的中和抗体应答

目的将人白细胞介素2(hIL-2)信号肽基因与柯萨奇病毒B组3型(CWB3)的VP1基因融合，构建分泌型VP1真核表达质粒pcDNA3／sVP1；免疫小鼠后，通过测定血清特异性中和抗体效价及对致死量CVB3攻击的保护作用，评价疫苗的免疫效果。方法采用重叠区基因扩增法，将hIL-2信号肽基因连同其下游11个氨基酸残基的基因与CVB3 VP1基因拼接，获得分泌型VP1(sVP1)的基因；将sVP1基因克隆至真核表达载体pcDNA3，构建分泌型VP1真核表达质粒pcDNA3／sVP1；肌注免疫小鼠，测定血清中CVB3特异性中和抗体的效价；第3次免疫后2周，腹腔内注射1000TCID50的CVB3，观察小鼠生存情况。结果成功构建了分泌型VP1真核表达质粒pcDNA3／sVP1，插入的sVP1基因中含有hIL-2信号肽及其下游11个氨基酸残基的基因以及VP1基因；pcDNA3／sVP1可比对照质粒pcDNA3／VP1诱导小鼠产生更高水平的中和抗体。结论hIL-2信号肽基因增强了柯萨奇病毒B3型VP1 DNA疫苗诱导的中和抗体应答，为进一步研制高效的CVB3 DNA疫苗提供了实验基础。     

Chitosan-DNA基因免疫诱导粘膜特异性免疫应答及其抗CVB3感染作用

目的：构建新型粘膜基因疫苗，诱生CVB3VP1特异性粘膜免疫应答，探讨粘膜免疫抗CVB3感染的作用，为病毒性心肌炎的特异性防治奠定基础。方法：抽提CVB3 RNA，以RT-PCR扩增得VP1基因，插入真核表达载体pcDNA3中，构建质粒pcDNA3-VP1，以Chitosan多糖包裹形成Chitosan-DNA基因疫苗。将该质粒转染Hela细胞，观察其体外表达情况。以50辉DNA剂量的Chitosan-DNA疫苗滴鼻免疫BALB／C小鼠3次，检测CVB3特异性体液和细胞免疫应答；隔4周以5LD50活CVB3致死攻击小鼠，观察攻击后存活情况。结果：制备了直径为80-100nm的Chitosan-DNA复合物颗粒，体外转染证实Chitosan-DNA复合物中VPl的表达高于pcDNA3-VP1质粒-Lipofectamine的表达水平。该疫苗滴鼻3次免疫后，不仅诱生了高水平的IgG，而且诱生了高水平的粘膜IgA抗体，第6周抗体P/N值分别达3．5和3．2。特异性细胞免疫应答研究发现，该疫苗诱生了较强的VP1特异性CTL杀伤作用，并显著高于pcDNA3-VP1和pcDNA3组。CVB3攻击后，可保护33．3％小鼠长期存活，而pcDNA3和pcDNA3-VP1质粒滴鼻免疫对照组的平均存活天数分别为8．5和10．8天。病理学研究显示：Chitosan-DNA免疫小鼠心肌组织基本正常，而对照小鼠死亡前心肌显示大量的灶性坏死和炎性浸润。结论：Chitosan-DNA基因疫苗滴鼻免疫可诱生CVB3特异性粘膜IgA应答及CTL反应，具有一定的抗CVB感染的免疫保护力。     

CVB3腺病毒载体疫苗Ad/VP1的构建及免疫效果研究

 目的 构建柯萨奇病毒B3(coxsackievirus B3,CVB3)VP1基因重组腺病毒疫苗Ad/VP1,并观察其对小鼠的免疫效果。方法 利用AdEasy-1系统构建、包装重组腺病毒Ad/VP1,并检测目的蛋白的表达。BALB/c小鼠随机分为Ad/VP1、Ad和PBS 3组,肌肉注射免疫,共免疫2次,每次间隔16d。用ELISA法和微量中和试验法分别检测血清CVB3 VP1 IgG和中和抗体滴度;CCK-8法检测特异性CTL杀伤活性;用致死量CVB3攻击小鼠后,检测血中病毒滴度并观察小鼠的存活率。结果 成功构建、包装了重组腺病毒Ad/VP1,并在293细胞中检测到VP1蛋白的表达。免疫小鼠后,Ad/VP1组血清CVB3 VP1 IgG滴度、中和抗体水平明显高于PBS和Ad对照组(P<0.01),CTL杀伤活性和对小鼠的保护率也高于对照组(P<0.05),血清病毒滴度低于两对照组(P<0.05)。结论 重组腺病毒疫苗Ad/VP1能显著提高小鼠的细胞和体液免疫应答及免疫保护作用。     

柯萨奇B3病毒结构和非结构蛋白DNA 疫苗诱导小鼠产生免疫应答的研究

目的 制备4种柯萨奇B3病毒（CVB3）结构和非结构蛋白重组质粒DNA疫苗,并探讨其诱导机体产生体液和细胞免疫应答的效果。方法 用基因重组技术构建4种CVB3结构和非结构蛋白重组质粒,将各重组质粒体外转染真核细胞,用Western blot检测表达产物;于BALB／c小鼠后腿胫骨前肌注射免疫,于0、4、8周共免疫3次,100μg/次。免疫后不同时间检测体液和细胞免疫应答指标。结果 4种重组质粒酶切出相应大小的片段,经测序证实为CVB3序列,Western blot证实能够在体外真核细胞中表达。pcDNA3/vp2、pcDNA3/VP1、pcDNA3/2A和pcDNA3/3D均可诱导小鼠产生相应的特异性抗体、细胞毒性T淋巴细胞（CTL）和淋巴细胞增殖反应、迟发型超敏反应（DTH）,并对致死量的CVB3m、CVB5和CVB2攻击具有保护作用,表现为病毒攻击后第3天血中病毒滴度降低,第10天心肌病理变化比对照组明显减轻,且小鼠生存率显著提高。其中以pcDNA/VP1和pcDNA3／3D组保护作用最明显。结论 CVB3结构蛋白VP1和非结构蛋白3D质粒DNA有可能用作CVB DNA疫苗的候选基因,值得进一步深入研究。     

Chitosan-pcDNA3-VP1 DNA疫苗预防病毒性心肌炎的实验研究

为研究新型chitosan DNA疫苗预防CVB3病毒性心肌炎的效果 ,以天然生物多糖chitosan包裹含CVB3主要结构蛋白VP1编码基因的质粒pcDNA3 VP1,制备新型chitosan pcDNA3 VP1疫苗。以含 5 0 μgDNA的该疫苗于 0、 7、 14、 2 1d滴鼻免疫小鼠 4次 ,末次免疫后 3周以 3×LD50 剂量CVB3经腹腔感染小鼠 ,称量小鼠体重、心脏重 ,并行心脏HE染色。结果 :chi tosan pcDNA3 VP1疫苗滴鼻免疫诱生了高水平的血清特异IgG和肠粘膜IgA ;同时诱生了较高强度的特异性CTL活性。以致病毒性心肌炎剂量CVB3感染小鼠 7d后发现 :pcDNA3组小鼠 10 0 %出现病毒性心肌炎 ,其心室壁呈现严重灶性坏死和炎性浸润 ;而chitosan pcDNA3 VP1组仅 16 7%小鼠产生心肌炎 ,坏死灶少且程度轻。其它病毒性心肌炎体征显示 :pcDNA3组体重降幅为 4 5 0 % ;而chitosan pcDNA3 VP1组类似正常小鼠 ,体重略增 1 75 % ;pcDNA3免疫组体重 /心脏重比为 171 75 ;chi tosan pcDNA3 VP1免疫组为 186 36。提示chitosan pcDNA3 VP1疫苗滴鼻可诱生全身及粘膜特异免疫 ,并可有效预防病毒性心肌炎的发生 ,可能成为CVB3及病毒性心肌炎预防性候选疫苗     

柯萨奇病毒B组3型亚单位疫苗与基因靶向疫苗免疫小鼠的效果比较

目的:观察柯萨奇病毒VP1基因靶向核酸疫苗pcDNA3/C3d3-sVP1、重组腺病毒rAd/C3d3-sVP1和亚单位疫苗VP1蛋白的免疫效果。方法:雄性BALB/c小鼠随机分为4组,每组18只,分别肌肉注射PBS、pcDNA3/C3d3-sVP1、rAd/C3d3-sVP1和VP1蛋白,除重组腺病毒rAd/C3d3-sVP1组免疫2次,间隔2周外,其余3组均免疫3次,间隔3周。质粒每次每只接种100μg/100μl,腺病毒每次每只接种1.2×107pfu/100μl,蛋白每次每只接种50μg。分别用ELISA法和微量中和试验法检测血清CVB3 VP1特异性IgG抗体和中和抗体;CCK-8法检测脾淋巴细胞特异性CTL杀伤活性;以致死量的CVB3病毒液感染已免疫小鼠,检测小鼠血中病毒滴度,观察存活情况以评价各种疫苗的免疫保护作用。结果:各实验组小鼠的特异性IgG抗体和中和抗体滴度随免疫次数增加而提高,末次免疫后,VP1蛋白组的特异性IgG抗体和中和抗体滴度均明显高于pcDNA3/C3d3-sVP1组和rAd/C3d3-sVP1组(P<0.05),但rAd/C3d3-sVP1组CTL杀伤活性高于pcDNA3/C3d3-sVP1组和VP1蛋白组;经致死量CVB3感染后,VP1蛋白组血中病毒滴度低于其他实验组,而生存率明显高于其他各组。结论:VP1蛋白疫苗诱导小鼠产生较强的特异性免疫应答,提高了小鼠的生存率,免疫效果优于靶向基因疫苗pcDNA3/C3d3-sVP1和rAd/C3d3-sVP1。     

rAd/MDC-VP1初免pcDNA3/MDC-VP1加强策略抗CVB3的免疫效果研究

目的:应用柯萨奇病毒B3(CVB3)腺病毒载体疫苗rAd/MDC-VP1初免,核酸疫苗pcDNA3/MDC-VP1加强免疫的策略免疫小鼠,观察其免疫效果。方法:BALB/c小鼠随机分为A～D 4组,分别肌肉注射PBS、rAd/MDC-VP1、pcD-NA3/MDC-VP1、rAd/MDC-VP1+pcDNA3/MDC-VP1,用ELISA和微量中和试验法分别检测CVB3 VP1特异性IgG和中和抗体滴度,CCK-8法检测脾淋巴细胞增殖活性和特异性CTL杀伤活性;用致死量的CVB3攻击小鼠后,检测小鼠血中病毒滴度并观察动物的存活情况。结果:D组CVB3 IgG、非特异性淋巴细胞增殖活性及特异性CTL杀伤活性明显高于其他各组(P<0.05);CVB3攻击后,D组小鼠血中病毒滴度较其他各组显著降低,生存率为41.67%,明显高于其他各组(P<0.05)。结论:rAd/MDC-VP1初免pcDNA3/MDC-VP1加强的免疫策略能显著提高小鼠细胞和体液免疫水平,提高致死量病毒攻击后的保护率。     

柯萨奇病毒B组3型VP1蛋白的原核表达及免疫效果研究

目的:用原核细胞表达柯萨奇病毒B组3型VP1蛋白并观察其免疫效果.方法:构建原核表达质粒pET-his/VP1,转化大肠杆菌BL21(DE3)pLysS,诱导VP1蛋白的表达并纯化.BALB/c小鼠随机分为实验组和对照组.实验组腹腔注射VP1蛋白,对照组注射PBS,每3周免疫1次,共免疫3次.每次免疫后2周取血清,用微量中和试验法检测血清CVB3特异性中和抗体滴度;末次免疫后3周,每组随机取3只小鼠,用细胞计数试剂盒检测淋巴细胞增殖活性和特异性CTL杀伤活性;每组另随机抽取3只腹腔注射3LD50CVB3,第7天检测血中病毒滴度;用致死量的CVB3攻击每组剩余12只小鼠,观察免疫保护作用.结果:实验组小鼠血清中和抗体滴度随免疫次数增加而提高(P＜0.05),第3次免疫后中和抗体滴度达70.79±1.31,明显高于PBS对照组(P＜0.05);非特异性淋巴细胞增殖活性亦明显高于PBS组(P＜0.05),但特异性淋巴细胞增殖活性和CTL杀伤活性两组间无统计学差别(P＞0.05);CVB3攻击后,实验组小鼠血中病毒滴度显著降低,生存率达33.33%,而PBS组小鼠无存活,差异有统计学意义(P＜0.05).结论:VP1蛋白可显著增强小鼠的体液免疫应答水平,提高致死量CVB3感染后小鼠的生存率.     

接头长度对MDC与CVB3VP1融合基因疫苗免疫效果的影响

目的构建表达不同接头（linker）长度的巨噬细胞源趋化因子（MDC）与CVB3VP1融合基因疫苗，观察接头长度对融合基因疫苗免疫效果的影响。方法构建表达接头长度分别为10、15和19个氨基酸的重组质粒pcDNA3／MDC-L-VP1；将6—8周龄雄性BALB/c小鼠随机分为A-E5组，分别肌肉注射pcDNA3、pcDNA3／VP1、pcDNA3／MDC-L10-VP1、pcDNA3／MDC-L15-VP1和pcDNA3／MDC-L19-VP1，每次接种100μg/只，4周注射1次，共3次，每次免疫后第14天眼眶静脉采血，用微量中和试验滴定血清中和抗体效价。第3次免疫后3周，每组取3只小鼠，制备脾细胞，用CCK-8法检测特异性CTL杀伤活性；每组取3只小鼠以3LD50 CVB3病毒攻击，第7天取血处死，检测血中病毒滴度。结果成功构建了3种不同接头长度的质粒；第3次免疫后，E组中和抗体滴度和小鼠脾淋巴细胞特异性CTL杀伤活性显著高于其他各组，血中病毒滴度显著低于其他各组（P〈0．01）。结论融合基因疫苗pcDNA3／MDC-L19-VP1能诱导小鼠对CVB3VP1产生较强的体液和细胞免疫，有效地抑制了病毒的增殖。     

Hybrid hepatitis B virus core-pre-S proteins synthesized in avirulent Salmonella typhimurium and Salmonella typhi for oral vaccination.

Avirulent salmonellae expressing foreign genes are attractive for use as oral vaccine carriers. To facilitate the stable expression of heterologous genes without conferring antibiotic resistance, a deletion of the asdA1 gene was introduced into Salmonella typhimurium and S. typhi delta cya delta crp mutant vaccine strains. An asd-complementing plasmid expressing hybrid hepatitis B virus nucleocapsid-pre-S (HBcAg-pre-S) particles was constructed. These hybrid HBcAg-pre-S particle genes were stably expressed in S. typhimurium and S. typhi delta cya delta crp mutant vaccine strains in this balanced, lethal host-vector combination. A single oral immunization of BALB/c mice with a recombinant S. typhimurium delta cya delta crp mutant synthesizing hybrid HBcAg-pre-S elicited potentially virus-neutralizing anti-pre-S serum immunoglobulin G antibodies. In addition, serum immunoglobulin G recognizing S. typhimurium lipopolysaccharide was induced. Distribution in tissue after oral immunization was analyzed in one plasmid-strain combination. The recombinant S. typhimurium colonized the gut-associated lymphoid tissue and the spleen and persisted for over 4 weeks, retaining the HBcAg-pre-S expression plasmid. An isogenic virulence plasmid-cured S. typhimurium delta cya delta crp strain expressing the same HBcAg-pre-S gene had reduced immunogenicity for the carried antigen after oral immunization.     

Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: persistence and induction of humoral responses in rats.

Recombinant Salmonella typhimurium has been used as an oral vaccine for various microbial pathogens. Here we report immune responses in Fischer rats orally immunized with a recombinant S. typhimurium strain encoding surface protein antigen A (SpaA) of Streptococcus sobrinus. The attenuated S. typhimurium chi 4072 delta cya delta crp delta asd mutant used in this study contains the Asd+ plasmid pYA2905 expressing a fragment of the SpaA protein. Salmonella cells were cleared from spleens by 7 days and from Peyer's patches by 14 days in rats receiving a single oral immunization of 10(9) CFU of chi 4072. In animals receiving multiple (i.e., days 0 and 7 or days 0, 7, and 21) immunizations, Salmonella cells were cleared from the Peyer's patches by 25 days following the initial immunization. Antigen-specific systemic and mucosal antibody responses were greater in rats receiving multiple immunizations than in those receiving a single immunization. Serum anti-Salmonella activity was potentiated following boosting on day 21. Mucosal immunoglobulin A antibody responses were also greater in rats receiving multiple immunizations than in rats receiving a single immunization. Anti-Salmonella and anti-Streptococcus immunoglobulin A activity persisted longer in rats boosted on day 21 than in rats immunized on days 0 and 7. These data indicate that oral immunization of rats with the recombinant S. typhimurium chi 4072(pYA2905) vaccine induces systemic as well as mucosal antibody responses specific to the Salmonella cells and to the cloned SpaA protein. This is the first report of the use of an attenuated mutant of the murine pathogen S. typhimurium as an oral vaccine in rats.     

Recombinant avirulent Salmonella vaccine strains with stable maintenance and high level expression of cloned genes in vivo

Salmonella typhimurium strains with deletion (delta) of the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) genes are avirulent for mice and induce a high level of protective immunity to oral challenge with up to 10,000 times what would be a lethal dose of wild-type virulent S. typhimurium cells. This immunity begins as early as seven days after immunization and lasts for at least four months. S. typhimurium delta cya delta crp mutants stably maintain plasmids and give high-level expression of cloned gene products; in this they appear superior to other avirulent S. typhimurium strains. S. typhimurium delta cya delta crp strains with a delta asd mutation (abolishing production of aspartate beta-semialdehyde dehydrogenase), have an obligate requirement for diaminopimelic acid (DAP). This strain can be used in conjunction with plasmid vectors lacking antibiotic resistance markers but having the wild-type asd+ gene from Streptococcus mutans to complement the delta asd chromosomal mutation. The Asd+ plasmid vector can be used to express a diversity of colonization and virulence antigens from other pathogens. In the delta cya delta crp delta asd S. typhimurium vaccine strain, the plasmid is completely stable in the absence of any exogenous selective pressure either in vitro or in vivo.     

Control of colonization by virulent Salmonella typhimurium by oral immunization of chickens with avirulent delta cya delta crp S. typhimurium

Oral immunization with a delta cya delta crp Salmonella typhimurium strain has been shown to preclude colonization by wild-type, virulent S. typhimurium and induces humoral and cellular immune response in chickens. Intestinal tract colonization by the virulent challenge strain was used to determine the level of protection conferred by immunization with the delta cya delta crp mutant. The associated humoral and cellular immune responses were measured by ELISA and delayed-type hypersensitivity (DTH) tests, respectively. The levels of colonization by both Salmonella strains were determined by enumeration of viable cells in the intestinal tract. A reduction in faecal excretion of the wild-type strain was observed with a single oral immunization with the delta cya delta crp mutant, but caecal colonization was not affected. However, double oral immunization with the delta cya delta crp mutant precludes caecal colonization by the virulent strain. IgM, IgA and IgG were detected against sonicated Salmonella whole-cell antigens. Outer membrane and flagella proteins induced DTH responses, whereas lipopolysaccharide failed to do so. The effectiveness of the delta cya delta crp strain in reducing caecal colonization by the highly virulent challenge strain in chickens demonstrates that oral vaccination with the delta cya delta crp S. typhimurium should aid in eliminating Salmonella carriers in chickens. The elimination of these carriers on the poultry farm should help to control Salmonella contamination of poultry products, therapy improving public health.     

以减毒沙门菌作为DNA疫苗载体的研究

目的 以真核表达质粒pCMVβ为报告基因，研究用芳香族氨基酸合成缺陷的沙门菌SL7207为载体以提高DNA疫苗免疫应答的可行性。方法 携带质粒pCMVβ的SL7207体外感染小鼠腹腔巨噬细胞后，用X-gal染色方法和RT-PCR方法检测巨噬细胞内β-gal的表达。小鼠口服免疫SL7207(pCMVβ)后，用RT-PCR方法检测β-gaⅠ基因在淋巴组织中的转录产物；用ELISA方法检测体液免疫；用^3H-TdR掺入法检测脾淋巴细胞增殖；用JAM试验检测杀伤性T淋巴细胞反应。结果 SL7207能有效地将质粒DNA传递到体外培养的巨噬细胞中并进行表达；小鼠口服携带有pCMVβ的SL7207后，能在脾脏、派伊尔结、肠系膜淋巴结中检测到目的基因的转录，并可诱导小鼠产生特异性体液免疫和细胞免疫。与肌内注射pCMVβ相比较，口服SL7207(pCMVβ)能更有效地诱导出细胞免疫应答。结论 减毒沙门菌SL7207作为DNA疫苗的载体，可经口服途径进行免疫，并可将质粒直接传递给抗原递呈细胞，诱导出以细胞免疫为主的免疫应答。     

优化密码对牛乳头瘤病毒L1基因粘膜免疫的增强作用

为探讨优化密码对牛乳头瘤病毒 1型主要晚期基因 (BPV1L1)免疫原性的影响 ,分别将含野生型和优化密码的人源化BPV1L1基因的真核表达质粒转入减毒沙门菌S BRD5 0 9后 ,通过滴鼻、阴道免疫、静脉注射、腹腔注射等途径免疫小鼠 ,经VLPELISA方法测定抗L1构象性抗体产生 ,MTT法观察诱导的特异性细胞免疫反应。抗体检测结果显示 ,经粘膜免疫途径 ,与含有野生型L1基因的表达质粒pcDNA3WBPVL1相比 ,含优化密码L1基因的pcDNA3HBPVL1诱导产生的SIgA、IgG明显升高 ,而细胞免疫反应无明显差异。表明优化密码能促进乳头瘤病毒晚期基因L1诱导的体液免疫反应 ,减毒沙门菌是PVL1DNA疫苗粘膜免疫的有效载体。     

Enhancement of mucosal antibody responses to Salmonella typhimurium and the microbial hapten phosphorylcholine in mice with X-linked immunodeficiency by B-cell precursors from the peritoneal cavity.

The observation that approximately half of the B cells in the murine intestinal lamina propria are derived from peritoneal CD5 B-cell precursors raises the question of their contribution to mucosal protection. Using mice with X-linked immunodeficiency which are deficient in CD5+ B cells, we showed that they mount little serum and virtually no intestinal immunoglobulin M (IgM), IgG, and IgA antibody responses following oral inoculation with live Salmonella typhimurium. Nonresponsive Xid mice were reconstituted with responsive CBA/Ca donor cell preparations which were constitutively enriched or depleted of CD5 B-cell precursors. Reconstitution of irradiated Xid mice with CD5 B-cell-deficient bone marrow from CBA/Ca donors marginally improved IgM responses in the intestinal mucosa but had no effect on IgG or IgA in response to oral immunization with live S. typhimurium. Whenever Xid mice were reconstituted with donor cells from the peritoneal cavity, which are enriched for CD5 B-cell precursors, strong IgA and in some cases IgG responses in the intestinal mucosa were stimulated in response to oral immunization. When mucosal and serum antibody responses were compared, the peritoneal donor cells again reinstated maximal serum antibody responses to S. typhimurium. Serum and mucosal responses to the bacterial hapten phosphorylcholine could be induced in Xid mice after immunization with S. typhimurium or hapten-carrier conjugates but only following reconstitution with donor cells containing CD5 B-cell precursors. These observations suggest that different lymphoid compartments are enriched for regulatory or effector cells which vary in their contributions to the mucosal antibody response against epitopes on S. typhimurium.     

以减毒沙门菌为载体布鲁氏菌DNA疫苗的构建及免疫原性分析

目的 构建携带布鲁氏菌BLS-L7/L12融合基因的重组减毒沙门菌并进行免疫原性分析,为口服布鲁氏菌DNA疫苗研究奠定基础.方法将BLS-7/L12融合基因克隆到真核表达载体asd -pVAX1,依次将重组质粒转化减毒沙门菌X3730、X4550得到重组沙门菌X4550(asd -pVAX1-BLS-L7/L12).以1×109CFU/只的剂量口服免疫Balb/C小鼠,3次免疫后进行免疫效果的评价.结果构建的重组减毒沙门菌质粒转染COS-7细胞经免疫组化和Western-blot试验证明BLS-L7/L12融合蛋白在细胞中得到了瞬时表达,ELISA检测到免疫小鼠血清和肠黏液中有特异性抗体IgG和sIgA产生.通过淋巴细胞增殖实验、细胞因子和CD分子测定表明DNA疫苗以诱发Th1型免疫为主.结论 所构建的以重组沙门菌为载体口服布鲁氏菌DNA疫苗具有诱导特异性细胞免疫和体液免疫应答的能力,且以细胞免疫应答为主.可作为潜在的布鲁氏菌新型疫苗.     

A recombinant Salmonella typhimurium vaccine induces local immunity by four different routes of immunization.

Immunization of mice with an attenuated Salmonella typhimurium strain (Phopc) carrying a plasmid encoding a hybrid form of the hepatitis B virus core antigen (HBc) induced specific antibody responses against the bacterial lipopolysaccharide (LPS) and HBc. Different mucosal routes of immunization, i.e., oral, nasal, rectal, and vaginal, were compared for their ability to induce a systemic as well as a mucosal response at sites proximal or distant to the site of immunization. Anti-LPS and anti-HBc immunoglobulin A (IgA) antibodies were measured in saliva, in feces, and in genital, bronchial, and intestinal secretions. Specific antibodies in serum and secretions were observed after immunization via all routes; however, the response to LPS was independent of that against HBc. In serum, saliva, and genital and bronchial secretions, high amounts of anti-HBc IgA were obtained by the nasal route of immunization. Vaginal immunization resulted in two different responses in mice: high and low. We observed a correlation between the level of specific immune response and the estrous status of these mice at the time of immunization. Rectal immunization induced high amounts of IgA against HBc and LPS in colonorectal secretions and feces but not at distant sites. These data suggest that S. typhimurium is able to invade different mucosal tissues and induce long-lasting local IgA responses against itself and a carried antigen after a single immunization.     

恶性疟原虫113肽抗原基因重组的减毒鼠伤寒沙门菌在家兔中免疫应答的研究

我们已经在减毒鼠伤寒沙门菌ＳＬ３２６１以融合蛋白的形式表达了人工合成的恶性疟原虫杂合１１３肽基因ＡＢ（ＧＺ－ＡＢ）。活菌以２×１０９ＣＦＵ经口服免疫新西兰家兔，用ＥＬＩＳＡ测定抗体水平，结果于首次免疫或加强免疫后都可检测到一定水平的特异性抗体。所免疫的家免可以诱发针对恶性疟原虫抗原及ＧＺ－ＡＢ的迟发性超敏反应（ＤＴＨ）。我们的研究表明，含有多个恶性疟原虫抗原表位的人工合成基因可以在减毒鼠伤寒沙门菌中表达，活菌可诱发家兔产生特异的体液免疫及细胞免疫，为恶性疟口服活菌苗的制备打下基础。