Ⅱ型B族链球菌表面免疫相关蛋白基因的克隆和原核表达

目的重组表达Ⅱ型B族链球菌表面免疫相关蛋白(Surfaceimmunogenicprotein,SIP)基因,为进一步免疫学研究提供目标蛋白。方法用PCR的方法从GBSⅡ型标准株的基因组DNA中扩增出SIP基因,用T/A克隆法将其插入pMD18T载体,构建原核表达载体pET32aSIP,用BL21(DE3)/pET系统表达TrixSIP融合蛋白,SDSPAGE和质谱分析鉴定表达产物,并对表达蛋白进行初步纯化。结果PCR扩增产物经测序,证实与GenBank中Ⅰa/c型GBS的SIP的基因序列同源性为99%。SDSPAGE显示,经IPTG诱导后BL21(DE3)/pET32aSIP总蛋白中出现一条相对分子质量为66000的新蛋白带。质谱分析和蛋白质库的比较证实其为B族链球菌表面免疫相关蛋白(SIP)的可能性分数为74。结论已成功表达并初步纯化SIP,为SIP在细菌致病中的作用研究以及相关疫苗的制备奠定了基础。     

改进的蒽酮法检测肺炎链球菌荚膜多糖结合物中多糖浓度

目的改进检测肺炎链球菌荚膜多糖结合物中多糖浓度的蒽酮法。方法分别对蒽酮法中蒽酮的浓度和处理温度进行优化,并对改进的方法进行验证和重复性检测。结果适宜的蒽酮浓度为0·3g/300ml,处理温度为40℃,在检测肺炎链球菌多糖结合物原液中的多糖浓度时,标准曲线回归系数高,方法稳定,重现性好。结论改进的蒽酮法可以有效、准确、稳定地检测肺炎链球菌结合物多糖原液中的多糖浓度。     

肺炎链球菌荚膜多糖-破伤风类毒素结合疫苗两种免疫途径对小鼠抗体应答的比较

目的比较肺炎链球菌荚膜多糖-破伤风类毒素结合疫苗两种免疫途径对小鼠的抗体应答。方法选NIH小鼠,分别经皮下和腹腔途径免疫3次同等剂量的结合疫苗,每次间隔2周,用ELISA方法检测两种途径免疫小鼠的血清抗体滴度。结果结合疫苗经腹腔免疫后的抗体滴度优于皮下免疫。小鼠经腹腔免疫3次后,血清抗体的几何平均滴度分别是第1次免疫的6·6倍和3·5倍。结论结合疫苗经腹腔免疫较皮下免疫获得的抗体应答水平高。     

Revealing the Changes in Saliva and Serum Proteins of Pigs with Meningitis Caused by Streptococcus Suis: A Proteomic Approach

Meningitis due to Streptococcus suis causes high mortality and morbidity on pig farms and has increasing zoonotic potential worldwide. Saliva proteome analysis would potentially be useful in elucidating pathophysiological changes and mining for new biomarkers to diagnose and monitor S. suis infection. The objective of this study was to investigate the changes in the salivary and serum proteome profile of piglets with meningitis. The LC-MS/MS TMT proteomic approach was used to analyze saliva and serum samples from 20 male piglets: 10 with meningitis and 10 healthy. In saliva, 11 proteins had higher and 10 had lower relative abundance in piglets with meningitis. The proteins with the highest relative abundance were metavinculin (VCL) and desmocollin-2 (DSC2). Adenosine deaminase (ADA) was selected for validation using a spectrophotometric assay and demonstrated excellent performance in the differentiation between healthy and pigs with meningitis due to S. suis. In serum, the most protruding changes occurred for one SERPIN and haptoglobin (HP). In saliva and serum, the highest number of proteins with altered abundance were linked, via the enrichment analysis, with platelet and neutrophil pathways. Overall, meningitis caused by S. suis resulted in specific proteome changes in saliva and serum, reflecting different pathophysiological mechanisms, and marking new potential biomarkers for this infection.     

传统乳制品来源乳酸菌的发酵特性及其在酸奶中的应用

对从传统乳制品中分离出的4株保加利亚乳杆菌和4株嗜热链球菌发酵特性及应用进行研究。利用脱脂乳进行培养,以发酵时间、风味物质及贮藏过程中的p H、滴定酸度、持水力、粘度变化为指标,研究单菌株的发酵性能。进一步对较优发酵性能菌株复配,考察不同接种比例时酸奶的酸度、粘度、风味、感官等指标,研究其在酸奶中的应用。结果表明,保加利亚乳杆菌Lb.YNF-5和嗜热链球菌St.GST-6具有良好的发酵性能。通过对此两株菌进一步复配,最终确定当保加利亚乳杆菌（Lb.YNF-5）:嗜热链球菌（St.GST-6）配比为1∶1时,酸奶产品品质最佳,发酵时间为5.8 h,粘度为7186.47 m Pa·s,感官评价得分为76分。本研究综合表明,菌株Lb.YNF-5和St.GST-6具有开发优良酸奶发酵剂的潜在价值。      

富硒乳酸菌的分离、筛选、驯化及富硒研究

从酸奶中分离纯化出保加利亚杆菌(LactobacillusBulgaria,LB)和嗜热链球菌(StreptocociusThermopiles,ST),通过耐硒筛选,确定嗜热链球菌为耐硒菌种,以浓度梯度法对其驯化,从而得到富硒菌株,并进行富硒实验。初步确定富硒条件为:采用蕃茄酵母培养基,接种量10%,培养温度39℃,pH7,第6小时时加硒,硒浓度为15μg/mL,培养11h,富硒率为72.8%。     

乳酸菌冷冻保护剂配方设计

乳酸菌浓缩发酵剂的制备过程中,冷冻对菌体的损伤比较明显。通过在保加利亚乳杆菌和嗜热链球菌混菌菌悬液中添加不同的保护剂并测定冷冻前后乳酸菌菌数,筛选出适用的保加利亚乳杆菌和嗜热链球菌混菌的保护剂。最终确定的保加利亚乳杆菌和嗜热链球菌混菌保护剂配方为:蔗糖浓度为10%,脱脂乳浓度为10%,谷氨酸钠浓度为5%。菌体经冷冻处理后,冷冻存活率可达到95.0%,比不添加保护剂的菌体存活率大大提高。此配方也简单易得,完全可以满足生产需要。     

食品生物防腐剂产生菌门间远缘细胞融合研究

乳酸链球菌素(Nisin)和ε-聚赖氨酸是由微生物产生的安全的天然防腐剂,分别由乳酸链球菌和白色链霉菌产生。这两种生物防腐剂的抑菌效果不同,存在互补性。为了获得具有更广谱抑菌作用的优良性状的菌株,探索远缘细胞融合的可行性,以自主分离的ε-聚赖氨酸产生菌白色链霉菌YB12和Nisin产生菌NZ9700作为亲本菌株,通过原生质体融合技术将两个菌株进行门间的细胞融合;通过抗药性筛选、形态学观察、SRAP-PCR分子鉴定,成功获得两株稳定遗传的融合菌株。同时对融合菌株的抑菌活性进行了检测。     

Molecular Epidemiology and Population Structure of Bovine Streptococcus uberis

The molecular epidemiology and population structure of 30 bovine subclinical mastitis field isolates of Streptococcus uberis, collected from 6 Portuguese herds (among 12 farms screened) during 2002 and 2003, were examined by using pulsed-field gel electrophoresis (PFGE) for clustering of the isolates and multilocus sequence typing (MLST) to assess the relationship between PFGE patterns and to identify genetic lineages. The 30 isolates were clustered into 18 PFGE types, using a similarity cutoff of 80%, and 3 PFGE types accounted for almost half of the isolates (46.6%). These major types were herd specific, suggesting either cow-to-cow transmission or infection with isolates from the same environmental reservoirs. The remaining unrelated PFGE types of isolates were from different herds strongly suggesting environmental sources of Strep. uberis infection. All 30 isolates were analyzed by MLST and clustered into 14 sequence types (ST). These ST were found to be novel, either with 10 new alleles of 6 housekeeping genes or with different combinations of previously assigned alleles. Five of these ST were clustered into 3 clonal complexes (lineages), ST-143, ST-86, and ST-5, known to include bovine isolates from several geographic locations (Australia, New Zealand, United Kingdom, Sweden, and Denmark) and 9 singletons. To our knowledge, this is the first report that documents molecular typing studies of bovine isolates of Strep. uberis from Portugal, which were shown to represent novel genomic backgrounds of this pathogen.     

Lactobacillus delbrueckii subsp. bulgaricus和Streptococcus thermophilus接种比例对Bifidobacterium lactis益生菌发酵乳品质的影响

将Lactobacillus delbrueckii subsp.bulgaricus ND02(LB-ND02)和Streptococcus thermophilus ND03(ST-ND03)按1∶1、1∶10、1∶100、1∶1000接种于脱脂乳中,同时接入益生菌Bifidobacterium lactis V9(B.lactis V9,接种量为2.0×107g-1),于42℃进行发酵。通过对发酵及贮藏过程中发酵乳指标的测定,评价LB-ND02和ST-ND03的接种比例对发酵乳品质的影响。结果表明,随着LB-ND02接种比例减小,凝乳时间显著延长,B.lactis V9活菌数显著提高。4℃贮藏28 d后,随LB-ND02接种比例减小,B.lactis V9存活率差异显著,后酸化也显著减弱。研究发现,LB-ND02和ST-ND03的接种比例,显著影响发酵乳的发酵时间、B.lactis V9活菌数、后酸化及黏度。