抗粘放菌5519Ⅰ型菌毛单克隆抗体的制备和鉴定

目的：制备抗粘放菌５５１９Ⅰ型菌毛单克隆抗体。方法：采用ＢＡＬＢ／Ｃ小鼠免疫后取脾细胞与ＳＰ２／０小鼠骨髓瘤细胞杂交融合,制备单克隆抗体,并以ＥＬＩＳＡ法和双向琼脂扩散法进行鉴定。结果：共得到６株杂交瘤细胞,抗体交价达１：１０－１００万,类型为ＩｇＧ,亚类为ＩｇＧ１。结论：经ＢＡＬＢ／Ｃ小鼠免疫可得到高纯度的抗粘放菌Ⅰ型菌毛单克隆抗体,而且效价高,细胞株稳定分泌抗体。     

粘放菌5519Ⅰ型菌毛兔抗血清的分离及鉴定

本实验在纯化粘性放线菌５５１９Ⅰ型菌毛的基础上，以粘放菌Ｉ型菌毛蛋白作为抗原，采用多途径免疫方法进行了兔抗Ｉ型菌毛抗血清的分离和提取，并以免疫扩散和琼脂凝胶免疫电泳进行了鉴定。实验结果显示，粘放菌５５１９Ｉ型菌毛蛋白的分子量大部分为５４ＫＤ，少量为３８ＫＤ，抗体效价可达１：３２，且与粘放菌５９５１Ⅱ型菌毛无交叉反应出现。     

粘放菌5519Ⅰ型菌毛氨基酸组成分析

为阐明粘放菌Ⅰ型菌毛的粘附作用,在对粘放菌５５１９Ⅰ型菌毛进行提取和鉴定的基础上,进行了菌毛的氨基酸组成分析,结果表明Ⅰ型菌毛含有大量的天冬氨酸,甘氨酸,丙氨酸和赖氨酸以及少部分其它氨基酸,其中非极性氨基酸为３４％,极性不带电荷氨基酸为２１．２２％。这些结果提示Ⅰ型菌毛亚单位具有高度的疏水片断和较强的粘附能力。     

Western-Blotting在粘放菌5519 I型菌毛鉴定中的应用

目的：检测粘放菌５５１９ Ｉ型菌毛的纯度及 Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ 方法的特异性。方法：采用 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 电泳和 Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ 免疫印迹法。结果： 粘放菌 ５５１９ Ｉ型菌 Ｍｒ 大部分为５４ ０００,少部分为３８ ０００。 Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ 检测结果与 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ结果相一致。结论： Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ 在粘放菌菌毛鉴定中具有高度特异性。     

抗粘放菌Ⅰ型菌毛单克隆抗体抗粘附作用的研究

目的 :为了解抗粘放菌 Ⅰ型菌毛单抗的抗粘附机理 ,在提取和鉴定抗粘放菌 Ⅰ型菌毛单克隆抗体和多克隆抗体的基础上 ,进行了抗粘附作用的研究。方法 :实验采用体外抗体阻断粘放菌 5 5 19和 5 95 1对牙釉质片的粘附 ,并结合扫描电镜和Ca+ + 测定方法进行。结果 :结果显示 ,单克隆和多克隆抗体能有效地阻断粘放菌 5 5 19对牙釉质片的粘附。扫描电镜发现未经抗体处理的牙釉质片表面粘附大量的细菌 ,而经抗体处理的牙釉质片表面细菌粘附明显减少 ,与牙釉质表面脱矿程度及钙离子浓度差异结果相吻合。结论 :这些结果提示 ,Ⅰ 型菌毛主要介导细菌对牙釉质片的粘附 ,抗 Ⅰ型菌毛的抗体可以有效地阻断细菌的粘附。     

粘放菌5519I型菌毛兔抗血清的分离及鉴定

本实验在纯化粘性放线菌５５１９Ｉ型菌毛的基础上，以粘放菌Ｉ型菌毛蛋白作为抗原，采用多途径免疫方法进行了兔抗Ｉ型菌毛抗血清的分离和提取，并以免疫扩散和琼脂凝胶免疫电泳进行了鉴定。实验结果显示，粘放菌５５１９Ｉ型菌毛蛋白的分子量大部分为５４ＫＤ，少量为３８ＫＤ，抗体效价可达１：３２，具有粘放菌５９５１Ⅱ型菌毛无交叉反应出现。     

粘性放线菌5519Ⅰ型菌毛的分离,鉴定

目的：对粘性放线菌５５１９Ⅰ型菌毛进行分离和部分特性研究。方法：采用超声粉碎方法从粘放菌５５１９细胞壁上分离Ⅰ型菌毛，然后经１６００００×ｇ２４ｈ高速离心，硫酸铵沉淀和葡聚糖凝胶Ｇ１００过柱后取得纯化菌毛，并经电镜和ＳＤＳ－ＰＡＧＥ电泳检查。结果：粘放菌５５１９Ⅰ型菌毛大部分分子量为５３５７３．４ｕ，少量为３７６９９．８ｕ。经电镜检查证实为Ⅰ型菌毛。结论：通过超声，结合高速离心，沉淀和葡聚糖凝胶Ｇ１００过程可得到纯化的粘放菌５５１９Ⅰ型菌毛     

粘放菌5951Ⅱ型菌毛多克隆抗体抗血清的制备

目的制备粘放菌５９５１Ⅱ型菌毛兔抗血清。方法采用物理,化学方法提纯Ⅱ型菌毛,多途径免疫制备兔抗血清,双向免疫扩散法鉴定兔抗血清。结果提纯的Ⅱ型菌毛分子质量主要为３４ｋｕ,多途径免疫后效价棕右１：６４。结论粘放菌５９５１Ⅱ型菌毛蛋白分子质量为３４ｋｕ,抗体效价为１：６４,与Ⅰ型菌毛间有轻度交叉反应。     

抗Ⅰ型菌毛单克隆抗体抗粘附作用机制的研究

目的探讨Ⅰ型菌毛粘附机理和抗Ⅰ型菌毛单克隆抗体抗粘附作用.方法应用提取的抗粘放菌Ⅰ型菌毛单克隆抗体,采用3H标记法,在体外测定细菌对SHA的粘附率及抗体的粘附抑制率.结果抗粘放菌Ⅰ型菌毛单抗抑制细菌对SHA的粘附达80%,与多克隆抗体无显著差异.结论Ⅰ型菌毛主要介导粘放菌对SHA的粘附,抗Ⅰ型菌毛抗体可以有效地阻抑细菌的粘附.     

粘性放线菌5951菌毛亚单位组成分析

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Fimbriae of Porphyromonas gingivalis are important for initial invasion of osteoblasts, but not for inhibition of their differentiation and mineralization

Background: Porphyromonas gingivalis is etiologically associated with chronic periodontitis. The major fimbriae of this periodontal pathogen mediate binding to host gingival epithelial cells and fibroblasts, a critical function in the initiation of periodontitis. However, the role of fimbriae in P. gingivalis–osteoblast interactions remains unknown. In the present study, the involvement of major fimbriae in the initial and long‐term interactions between P. gingivalis and osteoblasts is investigated. Methods: Primary mouse calvarial osteoblast cultures were established and inoculated with P. gingivalis ATCC 33277 or YPF1, a major fimbriae–deficient mutant of P. gingivalis. Confocal microscopy images were acquired to assess bacterial invasion. DNA content measurement, real‐time polymerase chain reaction, and alizarin red S staining and calcium content analysis were used to study the impact of bacteria on the proliferation, differentiation, and mineralization of osteoblasts, respectively. Results: Compared to the parent strain, YPF1 was significantly reduced in invasion of osteoblasts after 3 hours interaction. However, extended culture of infected osteoblasts did not reveal significant differences in persistence between the two strains. Proliferation of osteoblasts was not affected by either strain, and differentiation and mineralization of osteoblasts were inhibited by both strains to comparable levels. Conclusion: This study reveals that major fimbriae are involved in the initial invasion of osteoblasts by P. gingivalis, but are not essential for the subsequent inhibition of osteoblast differentiation and mineralization in long‐term culture.     

单克隆抗体对唾液凝集素介导的茸毛链球菌(S.sobrinus)聚集的影响

目的;用制备的抗茸毛链球菌主要表面蛋白的单克隆抗体作用于茸毛链球菌,体外观察单克隆抗体对唾液凝集素介导的细菌聚集的影响。方法;于紫外分光光度计上通过光密度值检测１ｈ内的细胞凝集情况。结果：单克隆抗体对唾液凝集素介导的茸毛链球菌之间的聚集有一定程度的影响,但作用不明显。     

猪发育期牙胚釉原蛋白的分离纯化及其多克隆抗体的制备

目的　制备猪釉质蛋白多克隆抗体,为釉原蛋白的检测提供条件。方法　选择1月龄乳猪,分离埋于上下颌骨内的牙胚,刮取牙胚表面干酪样尚未完全矿化的釉质基质,通过盐酸胍抽提及SephadexG-200柱层析分离纯化猪发育期牙胚釉原蛋白,联合免疫家兔,抗血清经DE-52纤维素纯化,并经ELISA测定抗体效价。结果　 SDS-PAGE电泳结果发现采用SephadexG-200葡聚糖凝胶过滤能达到较为理想的釉原蛋白分离纯化效果,用所提纯的猪釉原蛋白免疫家兔,成功地制得兔抗猪釉原蛋白抗血清,抗血清稀释达1∶32 000时,采用ELISA法仍有明显的抗原抗体反应。结论　本实验成功制备得到抗釉原蛋白多克隆抗体,为釉原蛋白的检测提供了条件。     

远缘链球菌GTF催化活性蛋白及其多克隆抗体的制备和鉴定

目的:在大肠杆菌中表达重组远缘链球菌葡糖基转移酶(glucosyltransferase,GTF)的催化活性区(catalytic region,CAT)蛋白并制备其多克隆抗体。方法:扩增远缘链球菌OMZ176基因组的CAT区的基因片段,克隆入pQE31载体诱导表达,鉴定表达产物。重组蛋白纯化后免疫小鼠制备多克隆抗体,ELISA测定抗体的效价,West-ern blotting检测抗体的特异性和亲和性。结果:重组质粒成功在大肠杆菌中表达,经抗His tag单克隆抗体免疫印迹法检测,有阳性条带出现,证实重组蛋白有抗原特异性。ELISA测定免疫小鼠所得抗CAT多克隆抗体效价可达到1∶5000。Western blotting检测证明该抗体有较好的针对CAT蛋白的专一性。结论:GTF区CAT基因原核表达质粒构建成功,表达的融合蛋白具有良好的抗原性。抗CAT多克隆抗体特异性和效价良好,能够满足针对CAT免疫印迹和细胞免疫组化检测等实验要求,为深入研究同时包含变形链球菌和远缘链球菌两种致龋菌的主要抗原的新型防龋DNA疫苗奠定了必要的物质基础。     

牙龈类杆菌内毒素与牙周炎关系的研究 Ⅲ.牙龈类杆菌单克隆抗体的研制

应用杂交瘤技术建立了三株分泌牙龈类杆菌单克隆抗体的细胞系CY—4、CY—5和CY—6。CY—4、CY—5和CY—6分泌的单克隆抗体均属IgM,能与牙龈类杆菌内毒素发生特异性结合,且经ELISA证实具有较高的种特异性。该3株杂交瘤细胞体外培养7个月余和冻存3个月后复苏培养仍能稳定地分泌单克隆抗体。     

Direct detection of cell surface interactive forces of sessile, fimbriated and non-fimbriated Actinomyces spp. using atomic force microscopy

Actinomyces species are predominant early colonizers of the oral cavity and prime mediators of inter-bacterial adhesion and coaggregation. Previous workers have evaluated the adhesion of Actinomyces spp. by quantitative assessment of sessile, as opposed to planktonic cells attached to substrates, but did not quantify the cell surface interactive forces. Therefore we used atomic force microscopy to directly detect the interactive force between an approaching silicon tip and sessile Actinomyces spp. adhering to a substrate, at nanonewton (nN) range force levels. A total of eight strains each belonging to fimbriated and non-fimbriated Actinomyces species were employed, namely A. bovis, A. gerencseriae, A. israelii, A. meyeri, A. naeslundii genospecies 1 and 2, A. odontolyticus and A. viscosus. The sterile mica discs, used as the adhesion substrate, were immersed in mono-species bacterial suspensions for five days to obtain a thin bacterial biofilm. Interactive forces were measured using a silicon nitride cantilever attached to a Nanoscope IIIA atomic force microscope. The interactive forces between the approaching silicon nitride tip and bacterial biofilm surfaces were randomly quantified at three different locations on each cell; namely, the cell surface proper, the periphery of the cell and the substrate and, the interface between two cells. When the interactive forces at these locations of the same species were compared, significantly higher force levels at the cell-cell interface than the other two locations were noted with A. gerencseriae (P < 0.001), A. viscosus (P < 0.01) and A. israelii (P < 0.05). When the interactive forces of different Actinomyces spp. at an identical location were compared, fimbriated A. naeslundii genospecies 2 showed the greatest interactive force at the cell surface proper (-32.6 +/- 8.7 nN, P < 0.01). A. naeslundii genospecies 1, 2 and A. viscosus demonstrated greater interactive force at the cell-mica periphery than the other five species (P < 0.05); A. viscosus (-34.6 +/- 10.5 nN) displayed greater interactive force at the cell-cell interface than the others (P < 0.01), except for A. gerencseriae (P > 0.05). These data indicate that fimbriated Actinomyces spp., including A. naeslundii genospecies 1, 2 and A. viscosus exert higher cell surface interactive forces than those devoid of fimbriae and, such variable force levels may modulate their adhesion and coaggregation during biofilm formation.     

A probable common disturbance in the early stage of odontoblast differentiation in Dentinogenesis imperfecta Type I and Type II

Deciduous teeth from 7 patients with dentinogenesis imperfecta (DI) Type I and Type II were examined by conventional microscopy. A defective layer was found which runs parallel with the dentinal surface in the outer portion of dentin in teeth of both types. Dentinal tubules were interrupted in the vicinity of this layer. When the ground sections were examined after being stained by the phosphophoryn staining method, the DI Type I dentin was found to contain phosphophoryn at the same low level as the DI Type II dentin, suggesting similar deficiency in phosphophoryn concentration. These results suggest that both types of DI have a common primary disturbance in the early stage of odontoblast differentiation.