牙龈卟啉单胞菌菌毛相关外膜蛋白pgmA基因的克隆和表达

目的 :克隆牙龈卟啉单胞菌 (Porphyromonasgingivalis,Pg) pgm A基因 ,构建 pgm A基因表达载体 ,鉴定其表达产物的免疫性。方法 :采用高保真 PCR分别从牙龈卟啉菌 ATCC332 77和 4 7A- 1菌株中扩增 pgm A基因 ,T- A克隆后测定核苷酸序列 ,构建 p ET32 a的 pgm A表达载体 ,在 E.coli BL2 1DE3宿主菌中用不同浓度的 IPTG诱导表达 ,采用兔抗融合蛋白血清的 Western blot鉴定其免疫反应性和免疫原性 ,采用 ELISA检测 6 5株临床分离牙龈卟啉单胞菌与 Pgm A融合蛋白抗血清的反应情况。结果 :所克隆的牙龈卟啉单胞菌 ATCC332 77与 4 7A- 1菌株 pgm A基因的核苷酸序列同源性为 10 0 %,与报道的相应核苷酸序列同源性为 98.98%,氨基酸序列同源性高达99.18%。p ET32 a- pgm A- BL2 1DE3系统的 Pgm A融合蛋白表达量为细菌总蛋白的 5 0 %左右。Pgm A融合蛋白免疫家兔能获得相应抗体并与 Pgm A蛋白发生结合反应。 ELISA结果证实 92 .3%的牙龈卟啉菌临床菌株 (6 0 / 6 5 )能与Pgm A融合蛋白抗血清发生结合反应。结论 :本研究成功地构建了 Pg pgm A高效表达系统 ,所表达的 Pgm A融合蛋白有良好的免疫原性和免疫反应性 ,可作为 Pg检测试剂盒和 Pg疫苗的候选抗原。

海豚链球菌simA和pgmA真核表达质粒对尼罗罗非鱼免疫保护的研究

尼罗罗非鱼(Oreochromis niloticus)是世界水产养殖业中的重要经济鱼类,但在养殖生产中易受海豚链球菌(Streptococcus iniae)感染而致病致死,使用疫苗是一种相对理想的防感染措施。该研究采用海豚链球菌simA、pgmA基因构建的真核表达载体作为DNA疫苗,肌肉注射罗非鱼评估疫苗保护效果。免疫后在DNA和RNA水平上,在注射鱼体内检测到2个目的基因。首次免疫后第7至第28天,鳃、肝、肾脏、头肾中疫苗组的白介素1 (Interleukin,IL-1β)与肿瘤坏死因子(Tumor Necrosis Factor,TNF-α)表达量高于PBS对照组;疫苗组的抗体滴度、血清抗菌活性显著(P<0.05)高于PBS对照组。攻毒后,注射pcDNA3.1-pgmA、pcDNA3.1-simA、pcDNA3.1-pgmA与pcDNA3.1-simA等比例混合疫苗的相对保护率(Relative percent survival,RPS)分别为60.7%、49.9%和75.0%。结果表明所制备的疫苗具有免疫保护效果,可作为候选疫苗。

慢性牙周炎龈下菌斑中牙龈卟啉单胞菌外膜蛋白A的水平与牙周病变程度关系的研究

目的检测慢性牙周炎(chronic periodontitis,CP)患者龈下菌斑标本中牙龈卟啉单胞菌外膜蛋白A(porphyromonas gingivalis outer membrane protein A,PgmA),了解PgmA水平与牙周病变程度的关系。方法采用SDS-PAGE检测原核表达系统重组PgmA(rPgmA)表达和Ni-NTA亲和层析法提取的rPgmA纯度,rPgmA免疫家兔获得抗血清,建立ELISA检测56例CP患者209个龈下菌斑标本中的PgmA,并分析PgmA水平与牙周病变程度的关系。结果rPgmA表达产量约为细菌总蛋白的50%。提纯的rPgmA SDS-PAGE后仅见单一的蛋白条带。90.0%的龈下菌斑标本(188/209)PgmAELISA阳性。重度CP龈下菌斑标本中PgmA含量明显高于轻度CP标本(P<0.05),但轻度和中度、中度和重度CP标本之间PgmA含量差异无统计学意义(P>0.05)。结论慢性牙周炎病变程度与龈下PgmA水平有关,PgmA可能是Pg毒力因子之一。

Facile synthesis of wormlike quantum dots-encapsulated nanoparticles and their controlled surface functionalization for effective bioapplications

Semiconductor quantum dots (QDs) are considered as ideal fluorescent probes owing to their intrinsic optical properties. It has been demonstrated that the size and shape of nanoparticles significantly influence their behaviors in biological systems. In particular, one-dimensional (1D) nanoparticles with larger aspect ratios are desirable for cellular uptake. Here, we explore a facile and green method to prepare novel 1D wormlike QDs@SiO2nanoparticles with controlled aspect ratios, wherein multiple QDs are arranged in the centerline of the nanoparticles. Then, an excellent cationic gene carrier, ethanolamine-functionalized poly(glycidyl methacrylate) (denoted by BUCT-PGEA), was in-situ produced via atom transfer radical polymerization on the surface of the QDs@SiO2nanoparticles to achieve stable surfaces (QDs@SiO2-PGEA) for effective bioapplications. We found that the wormlike QDs@SiO2-PGEA nanoparticles demonstrated much higher gene transfection performance than ordinary spherical counterparts. In addition, the wormlike nanoparticles with larger aspect ratio performed better than those with smaller ratio. Furthermore, the gene delivery processes including cell entry and plasmid DNA (pDNA) escape and transport were also tracked in real time by the QDs@SiO2-PGEA/pDNA complexes. This work realized the integration of efficient gene delivery and real-time imaging within one controlled 1D nanostructure. These constructs will likely provide useful information regarding the interaction of nanoparticles with biological systems. [Figure not available: see fulltext.] © 2016, Tsinghua University Press and Springer-Verlag Berlin Heidelberg.