H9亚型禽流感病毒黏膜DNA疫苗与灭活油乳苗的联合免疫研究

目的构建减毒沙门氏菌介导的H9亚型禽流感病毒黏膜DNA疫苗,并探讨黏膜DNA疫苗与灭活油乳苗联合应用的免疫效力。方法以RT-PCR法扩增H9N2亚型禽流感病毒分离株(A/Chicken/China/N/2005)的血凝素(HA)基因,然后克隆入pmcDNA3.1+载体中,获得重组质粒pmcDNA3.1-HA。通过电穿孔法将重组质粒转入减毒鼠伤寒沙门氏菌SL7207,构建沙门氏菌介导的黏膜DNA疫苗SL7207(pmcDNA3.1-HA)。重组菌与灭活油乳苗联合免疫1日龄商品代伊莎褐蛋鸡,并设立重组菌SL7207(pmcDNA3.1-HA)单独免疫组、油苗免疫组、空载体免疫组及空白对照组。用HI或ELISA测定血清和小肠黏膜抗体效价,以H9N2亚型禽流感病毒攻击,计算免疫保护力。结果联合免疫组和重组菌SL7207(pmcD-NA3.1-HA)单独免疫组能激发机体产生黏膜免疫应答,且与其他试验组之间存在显著性差异(P<0.05)。联合免疫组和重组菌SL7207(pmcDNA3.1-HA)单独免疫组的免疫保护力均与空载体组、空白对照组有显著差异(P<0.05),且联合免疫组的免疫保护率最高,达100%。结论成功构建H9亚型禽流感病毒黏膜DNA疫苗,DNA疫苗与灭活油乳苗联合应用具有良好的免疫协同作用。     

抗沙门氏菌O抗原特异单克隆抗体的制备及其免疫生物学特性的鉴定

本研究应用淋巴细胞杂交瘤技术制备了41株分别针对沙门氏菌O_2、O_3、O_4、O_7、O_8、O_9、O_(11)、O_(19)等O抗原的特异单克隆抗体(下称单抗)。在多种血清学试验中,这组单抗只选择性地与所试52株沙门氏菌中具有相应O抗原的菌株反应,而不与其它沙门氏菌或其它肠道杆菌反应。应用这组单抗对864个现场分离菌株的鉴定和分解结果与常规O血清的试验结果一致,表明这组单抗完全可以取代常规O血清,具有广泛的应用前景。     

结核分枝杆菌CFP32蛋白的原核表达及其细胞免疫学检测价值评价

目的 克隆、表达与纯化结核分枝杆菌CFP32蛋白,并对其进行细胞免疫学特性评价。方法 PCR扩增cfp32基因片段,与pET-30a(+)构建重组表达质粒,在大肠杆菌中诱导表达,对融合蛋白进行纯化,ELISPOT试验测定其细胞免疫应答,通过牛结核外周血IFN-γ释放试验,评价其作为刺激原的能力。结果 构建了正确基因序列的重组质粒,并在BL21(DE3)中高效可溶性表达,融合蛋白大小为32 ku,纯度达91.8%。ELISPOT显示CFP32蛋白刺激机体分泌IFN-γ和IL-4的细胞数相当,呈现Th1/Th2的平衡。在牛结核外周血IFN-γ释放试验中,其阳性符合率和阴性符合率分别为40%和73.3%,与牛型PPD刺激结果的相关系数为0.684。结论 成功构建CFP32蛋白重组表达菌,该蛋白引起的免疫应答趋向于Th1/Th2的平衡,并且有作为刺激原用于牛结核病检测的潜力。     

屠宰生猪多重耐药沙门菌I类整合子与耐药基因的检测

目的了解屠宰生猪沙门菌分离株多重耐药与I类整合子及耐药基因的携带关系。方法采用K-B纸片法对屠宰生猪78株沙门菌分离菌株进行22种抗生素敏感试验;应用PCR技术对沙门菌分离菌株进行I类整合子及耐药基因检测。结果78株沙门菌分离株中有41株(52.56%)对2种以上抗生素耐药,属于多重耐药株,强力霉素-四环素-卡那霉素-氯霉素是主要多重耐药谱;41株多重耐药菌中有19株(46.34%)携带I类整合子,tetB、aph(3)-IIa和Flor基因分别检出最高。结论沙门菌多重耐药性与整合子的携带之间关系密切,耐药表型测定结果与耐药基因检测结果一致。     

表达绿色荧光蛋白的单核细胞增生李斯特菌重组菌株的构建及其生物学特性

目的为了对单核细胞增生李斯特菌运送外源抗原所诱导免疫应答特性进行评价,开展了以原核方式运送模式蛋白GFP的研究。方法利用SOEing PCR的方法把LLO的启动子与GFP融合在一起,通过同源重组的方式整合到yzuLM1-2actA和plcB基因片段之后,在LLO启动子的作用下实现GFP的表达。结果PCR扩增证实目的基因gfp融合到李斯特菌基基因组中,重组菌对小鼠的LD50为4.31×108,毒力比yzuLM1-2显著降低。以重组菌株免疫小鼠后获得的血清进行Western blot显示在27kD处出现特异印迹带;ELISA测定结果显示能诱导小鼠产生较高的抗GFP的抗体水平。结论研究结果表明减毒LM所运送的GFP能诱导小鼠产生较强的免疫应答的特性,这为减毒株yzuLM1-2运送外源保护性抗原的研究奠定了基础,也为开展减毒重组菌与抗原递呈细胞之间的相互作用及其诱导的免疫应答分析提供了必要条件。     

单克隆抗体测定沙门氏菌鞭毛蛋白属共同抗原表位的分布特性

本研究应用抗沙门氏菌鞭毛蛋白共同抗原的４株羊克隆抗体所建立的直接ＥＬＩＳＡ法，测定了该类抗原表位在沙门氏菌属内外的分布特征。对１８４株覆盖Ａ至Ｏ６７血清群沙门氏菌、９６株其他肠道杆菌和８株革兰氏阳性菌的测定结果显示，该共同抗原表位广泛分布于沙门氏菌全属内，而在属外出现的频率很低。这类表位既存在于Ⅰ相较毛，也存在于Ⅱ相鞭毛。它们的上述分布特性不同于分型抗原Ｏ、Ｈ的分布特征。由此可见，沙门氏菌鞭毛蛋白共同抗原表位具有高度的属特异性，可作为该菌属识别标志，在沙门氏菌快速检验和抗原结构研究方面具有重要应用价值。     

重组沙门菌表达结核分枝杆菌ESAT-6及其特异性免疫应答分析

Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.     

4株沙门氏菌鞭毛蛋白属共同抗原组分免疫原性及免疫保护性的研究

采用酸解法、单抗亲和层析提纯了鼠伤寒沙门氏菌（Ｓ．ｔｙｐｈｉｍｕｒｉｕｍ）鞭毛蛋白Ｈｉ。经电镜和ＳＤＳＰＡＧＥ鉴定，发现４２ｋＤ的蛋白带，即鞭毛蛋白（Ｆｌａｇｅｌｌｉｎ）。它能够刺激小鼠产生特异性抗体，酶联免疫吸附试验（ＥＬＩＳＡ）、凝集试验（ＤＡ）测定其Ｈｉ抗体效价分别为１：１０４、１：１０３。用其他非Ｈｉ抗原的沙门氏菌包板，ＥＬＩＳＡ效价可达１：１００～１：１０００，ＤＡ阴性。这表明沙门氏菌鞭毛蛋白上存在着属特异共同抗原表位，且免疫原性较好，该类表位免疫性比分型Ｈ抗原弱，提示沙门氏菌分型Ｈ抗原表位是鞭毛蛋白分子上的优势表位。用沙门氏菌鞭毛蛋白属特异共同抗原表位单抗ｄｅ７研究被动保护作用，结果显示该表位被动保护作用很小。本研究为沙门氏菌鞭毛蛋白抗原多样性及特性研究提供了新的认识，并为临床上诊断沙门氏菌感染提供了新的检测对象。     

重组沙门菌表达结核分枝杆菌ESAT-6及其特异性免疫应答分析

Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.