唾液蛋白质组学唾液样本制备的方法学初探

目的：通过对唾液淀粉酶单克隆抗体的制备和鉴定,寻找蛋白质组学研究中样本制备关键环节的可行性方案,为日后唾液蛋白质组学研究奠定基础。方法：将腮腺液经超滤法浓缩蛋白后,进行SDS-PAGE分析,切取分子量为50～65kDa的高丰度蛋白条带,研磨后注射BALB/c小鼠,诱导产生免疫应答并制备人唾液淀粉酶单克隆抗体,应用酶联免疫吸附试验和蛋白免疫印记等方法鉴定。结果：成功制备和鉴定了人唾液淀粉酶单克隆抗体。结论：成功制备和鉴定了唾液淀粉酶单克隆抗体,为唾液蛋白质组学研究中样本制备提供解决方案。     

涎腺疾病

耳后区和口内联合进路切除下颌下腺的近期疗效观察；腺样囊性癌细胞凋亡抗原负载的树突状细胞诱导抗肿瘤效应的体外研究；切胶免疫制备腮腺液高丰度蛋白多克隆抗体；HER-2基因在涎腺恶性肿瘤中表达及临床意义的研究；db／db自发性糖尿病小鼠颌下腺PCNA的表达及细胞凋亡关系的研究.     

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。     

牙齿发育相关基因adam28多克隆抗体的制备及鉴定

目的制备及鉴定抗ADAM28的多克隆抗体。方法采用反转录-多聚酶链式反应(RT-PCR)扩增出adam28基因的蛋白编码区序列,克隆到中间载体pMD18-T Vector中,再经双酶切得到adam28片段定向克隆到pGEX-4T-1载体中,构建融合表达载体pGEX-4T-adam28,在大肠杆菌中用IPTG进行诱导表达,得到GST-ADAM28融合蛋白,经过初步纯化及SDS-PAGE电泳,在35300的新蛋白带处直接割胶,作为抗原免疫新西兰大白兔后获取抗体。结果成功构建融合表达载体pGEX-4T-adam28,得到初步纯化的GST-ADAM28融合蛋白,免疫兔子1个月后,获得免疫血清,盐析法纯化得到多克隆抗体。Western印迹结果显示所得到的抗体具有较高的特异性,ELISA分析证实其效价可达1∶16000。结论成功制备出高效价的抗ADAM28多克隆抗体,为进一步研究ADAM28在牙齿发育中的作用和表达分布奠定基础。     

牙龈卟啉单胞菌牙龈蛋白酶K催化结构域融合蛋白的纯化及其多克隆抗体的制备

目的纯化牙龈卟啉单胞菌牙龈蛋白酶K催化结构域（KGPcd）融合蛋白并制备其多克隆抗体，为下一步的实验提供条件。方法KGPcd蛋白与载体pET-16b中的His标签融合表达，用Ni—NTA亲和层析纯化融合蛋白，复性后用Westernblot检测。以重组、纯化的KGPcd蛋白为抗原，免疫新西兰大白兔，制备多克隆抗体，用间接ELASA法检测抗体效价，WesternBlot进行抗血清特异性试验。结果经亲和层析、复性得到纯化融合蛋白His．KGPcd，Westernblot进一步证实纯化的蛋白为His．KGPcd融合蛋白。抗血清稀释达1：3200时，ELASA法仍有明显的抗原抗体反应，Westernblot也进一步证实抗血清能够与KGPcd发生特异性反应，抗体仅特异识别目的蛋白。结论成功制备兔抗KGPcd多克隆抗体，为后续研究奠定基础。     

小鼠cbfa1多克隆抗体制备及在骨和牙胚中的表达

目的　制备cbfa1多克隆抗体并进行鉴定和效价分析 ,观察其在骨和牙胚中的表达情况。方法　用自制的融合蛋白免疫新西兰大白兔 ,制备多抗血清 ,提取小鼠牙胚、颅骨和肝脏组织的总蛋白进行westernblot ,同时采用免疫组化染色观察其组织表达特性。结果　cbfa1蛋白在小鼠颅骨和牙胚组织中均有表达 ,在人骨肉瘤细胞系中呈强阳性表达。在免疫组化和westernblot的有效作用滴度分别为 1:40 0和 1:10 0 0。结论　成功地制备了兔抗小鼠cbfa1多克隆抗体     

小鼠cbfal多克隆抗体制备及在骨和牙胚中的表达

目的 制备cbfal多克隆抗体并进行鉴定和效价分析，观察其在骨和牙胚中的表达情况。方法 用自制的融合蛋白免疫新西兰大白兔，制备多抗血清，提取小鼠牙胚、颅骨和肝脏组织的总蛋白进行western blot，同时采用免疫组化染色观察其组织表达特性。结果 cbfal蛋白在小鼠颅骨和牙胚组织中均有表达，在人骨肉瘤细胞系中呈强阳性表达。在免疫组化和western b1ot的有效作用滴度分别为1：400和1：1000。结论 成功地制备了兔抗小鼠cbfal多克隆抗体。     

牙龈卟啉单胞菌肽酰精氨酸脱亚氨酶的克隆与表达

目的:构建牙龈卟啉单胞菌外膜蛋白肽酰精氨酸脱亚氨酶(PAD)克隆表达重组子,转化于大肠杆菌BL21中,并在最适宜条件下诱导表达。方法:以牙龈卟啉单胞菌ATCC33277全基因组DNA为模板,利用PCR技术获得目的基因PAD,将扩增得到的PAD基因定向插入线性克隆载体PMD18-T Vector中,得到克隆重组子PMD18-T-PAD。经PCR和双酶切鉴定正确的克隆重组子PMD18-T-PAD与表达载体PET-28a经Xhol和Ncol双酶切后,在一定连接体系下,连接构建表达质粒PET-28a-PAD。鉴定正确的原核重组表达质粒PET-28a-PAD,转化大肠杆菌BL21感受态细胞,在不同浓度异丙基硫代-β-D-半乳糖苷(IPTG)及时间诱导下表达融合蛋白。以抗His Tag单克隆抗体为一抗,Western免疫印迹鉴定。结果:DNA测序结果表明,PAD与NCBI核酸数据库中收录的PAD序列同源性达100%;37℃,IPTG浓度为0.5mmol/L,250r/min振摇培养6h的诱导条件下,PAD可高效表达。结论:本实验成功构建了PAD的克隆表达重组子,并在大肠杆菌中表达了PAD蛋白,为进一步研究PAD的免疫学性能及相应的抗体制备奠定了基础。     

牙龈卟啉单胞菌和具核梭杆菌多克隆抗体的制备及应用效果评价

目的    制备高效价高纯度的牙龈卟啉单胞菌（P. gingivalis）和具核梭杆菌（F. nucleatum）多克隆抗体，并评价其应用效果。方法    将新鲜培养的P. gingivalis和F. nucleatum菌液灭活后与佐剂乳化混匀，作为抗原皮下多点注射免疫雄性新西兰大白兔，定期免疫并在抗体效价达到预期后采用颈动脉取血获得抗血清。采用间接ELISA法测定多克隆抗体交叉反应。应用硫酸铵沉淀法纯化多克隆抗体，并通过Western-blot方法鉴定抗体的纯度。荧光显微镜观察多克隆抗体应用于免疫荧光实验的效果。结果    免疫6周后，血清抗体效价达到1∶320 000 ~ 1∶640 000，相互之间未出现交叉反应，且纯度较高。免疫荧光实验中可观察到明显的荧光，并可见P. gingivalis的球杆状形态和F. nucleatum的短杆状形态。结论    成功制备了P. gingivalis和F. nucleatum多克隆抗体，其特异性良好，为后续的免疫学相关实验奠定了基础。     

Quantification of human myeloperoxidase in oral fluids

Peroxidase activity in human whole saliva is derived from salivary peroxidase and myeloperoxidase. Present spectrophotometric assays are relatively nonspecific and influenced by ions present in salivary secretions, resulting in an over and/or underestimation of peroxidase activities. Specific polydonal or monoclonal antibodies would greatly simplify the identification of salivary peroxidase and myeloperoxidase in human saliva and determine the relative contribution of each enzyme to the total peroxidase activity in human saliva. In the present study, a highly purified preparation of myeloperoxidase was used to raise polydonal antibodies against the antigen. The antibodies were purified and extensively characterized in terms of their ability to interact with the antigen, with other mammalian peroxidases, and with other proteins present in salivary fluids. The antibodies recognized only myeloperoxidase and did not cross-react with any of the substances tested, showing that these antibodies can be used to detect and differentiate myeloperoxidase from other peroxidases in saliva. We have also developed and tested a sandwich ELISA which can be used in a clinical setting to quantify myeloperoxidase in whole saliva and gingival crevicular fluid.     

Human salivary peroxidase and bovine lactoperoxidase are cross-reactive.

Peroxidases are abundant in nature, and the primary function of mammalian peroxidases is to catalyze the peroxidation of halides and pseudohalides. Previous studies have shown that antibodies raised against bovine lactoperoxidase moderately cross-react with human salivary peroxidase, a feature that has been used in the present study to examine epitopes common to the antigen and human salivary peroxidase. Polyclonal antibodies against a highly purified preparation of bovine lactoperoxidase were raised in rabbits, and their properties were examined. In double-immunodiffusion experiments, the two enzymes showed partial identity, and in competitive radioimmunoassay and enzyme-linked immunosorbent assay, lactoperoxidase replaced the labeled and coated antigen, while salivary peroxidase did not. However, salivary peroxidase from human and rat saliva samples and the purified enzyme in its non-reduced, reduced, and de-glycosylated forms were recognized by these antibodies, as analyzed by Western blot analysis and immunodetection. The major activity of these antibodies was directed against the protein core of the antigen. Immunodetection of the peptide fragments of bovine lactoperoxidase and human salivary peroxidase revealed structural differences in the two enzymes. These antibodies also precipitated an in vitro translation product from rat-parotid-gland cell lysate that, on SDS-PAGE, compared favorably with the expected molecular weight of a de-glycosylated peroxidase. The antibodies partly inhibited the enzyme activity of salivary peroxidase and the peroxidase in rat parotid gland lysate, but the enzyme activity of lactoperoxidase was not affected by addition of anti-lactoperoxidase IgG between 25 and 400 micrograms/mL. The enzyme activity remained unchanged in all samples when pre-immune IgG was used.     

Adsorbed salivary acidic proline-rich proteins contribute to the adhesion of Streptococcus mutans JBP to apatitic surfaces

Experimental pellicles formed on hydroxyapatite (HA) beads from parotid or submandibular saliva promoted the adhesion of Streptococcus mutans JBP cells to a greater extent than did pellicles prepared from buffer, human plasma, or serum. The nature of the salivary components responsible was studied by the preparation of pellicles from fractions of parotid saliva obtained by chromatography on Trisacryl GF 2000 columns. Two groups of fractions promoted attachment of the organism. Components migrating in the high-molecular-weight mucin fraction were most effective, but a later-eluting fraction also possessed adhesion-promoting activity. Subfractionation of the latter material indicated that the adhesion-promoting activity was associated with the acidic proline-rich proteins (PRPs). Pellicles prepared from 10-20-micrograms/mL solutions of pure PRP-1 were effective in promoting attachment of S. mutans JBP cells. PRP-3 was less effective, while human salivary statherin, fibrinogen, fibronectin, type 1 collagen, and the amino-terminal tryptic peptide derived from PRP-1 were ineffective. The quantities of 150-residue and 106-residue PRPs and of statherin, which became incorporated into experimental pellicles prepared from saliva, were estimated with use of radiolabeled protein tracers. The data obtained suggest that these proteins compete for similar binding sites on HA, and that their ratios in saliva would therefore influence the quantity of the larger PRPs that become incorporated into the pellicle. Such competition may contribute to the variability observed in the adhesion-promoting activities of different saliva samples.     

Multiple components contribute to ability of saliva to inhibit influenza viruses

Introduction:  Saliva is a potentially important barrier against respiratory viral infection but its mechanism of action is not well studied.
Methods:  We tested the antiviral activities of whole saliva, specific salivary gland secretions, and purified salivary proteins against strains of influenza A virus (IAV) in vitro .
Results:  Whole saliva or parotid or submandibular/sublingual secretions from healthy donors inhibited IAV based on hemagglutination inhibition and neutralization assays. This differs from human immunodeficiency virus (HIV), for which only submandibular/sublingual secretions are reported to be inhibitory. Among purified salivary proteins, MUC5B, scavenger receptor cysteine-rich glycoprotein 340 (salivary gp-340), histatins, and human neutrophil defensins (HNPs) inhibited IAV at the concentrations present in whole saliva. In contrast, some abundant salivary proteins (acidic proline-rich proteins and amylase) had no activity, nor did several other less abundant salivary proteins with known activity against HIV (e.g. thrombospondin or serum leukocyte protease inhibitor). Whole saliva and MUC5B did not inhibit neuraminidase activity of IAV and viral neutralizing and aggregating activity of MUC5B was potentiated by the neuraminidase inhibitor oseltamivir. Hence, MUC5B inhibits IAV by presenting a sialic acid ligand for the viral hemagglutinin. The mechanism of action of histatins requires further study.
Conclusions:  These findings indicate that saliva represents an important initial barrier to IAV infection and underline the complexity of host defense activity of oral secretions. Of interest, antiviral activity of saliva against IAV and HIV differs in terms of specific glandular secretions and proteins that are inhibitory.     

Immunological characterization of plasminogen activators in human parotid saliva

These were characterized in stimulated saliva from 21 healthy volunteers using antibodies specific for human tissue-type plasminogen activator (t-PA) or urokinase. After pre-incubation with immunoglobulins, with and without specific antibodies, the fibrinolytic activity was assayed on fibrin plates containing plasminogen. Fibrinolytic activity was demonstrated in all salivas but one; it was completely quenched by antibodies against t-PA, but antibodies against urokinase-like plasminogen activator had no effect. The fibrinolytic activity was expressed in international units (IU) using a standard curve obtained by serial dilution of the international tissue-plasminogen standard. The normal range was 0.05-0.35 IU/ml, and the maximal value 2 IU/ml. There is thus considerable individual variation in fibrinolytic activity in parotid saliva, where it is regulated by t-PA under physiological conditions.     

Fractionation of salivary micelle-like structures by gel chromatography

Globular structures have been demonstrated in human parotid saliva by transmission electron microscopy and photon correlation spectroscopy. The aim of this study was to fractionate these salivary globular structures for analytical and preparative purposes using a gel-filtration material capable of separating spherical particles up to 300-400 nm in diameter. Freshly obtained parotid saliva was applied to a Sephacryl S-1000 column. Peak fractions were collected and prepared for transmission electron microscopy (TEM) or for amino acid analysis. Bovine milk was included as the casein micelles by TEM appear to be similar to the salivary aggregates and their elution profiles are known. The salivary globular structures were eluted in one major peak. TEM of negatively stained samples from the peak fractions demonstrated globular protein aggregates consistent with the salivary structures in parotid saliva. Amino acid analysis showed characteristic amino acid profiles with unusual high levels of proline, 40-45%. The casein micelles were eluted in one major peak and separated from the whey proteins. This study indicates that the salivary globular structures can be isolated by gel chromatography. The amino acid analysis indicates that proline-rich proteins may be an important fraction of the salivary globular structures.     

Monoclonal antibody-mediated inhibition of adhesion of Candida albicans and Candida dubliniensis to human epithelial cells

The aim of this study was to assess the role of whole saliva, four saliva-derived preparations, and six monoclonal antibodies (mAbs), directed against components of the cell wall of Candida albicans , on the adhesion of C. albicans and Candida dubliniensis to human epithelial cells (HEC). C. albicans serotype A NCPF 3153 and C. albicans serotype B ATCC 90028 showed higher adhesion to HEC than C. dubliniensis NCPF 3949. Pooled whole saliva was more efficient than salivary secretory immunoglobulin A, partially purified by chromatography, at inhibiting the adhesion of C. albicans serotype A NCPF 3153 to HEC. Monoclonal antibodies C7, 14-8, and 26G7 were the most potent inhibitors of adhesion. Our results show that mAbs can mimic the inhibition of adhesion of C. albicans to HEC that is mediated by human saliva.     

The association of basic proline-rich peptides from human parotid gland secretions with caries experience

To address whether there are associations between the peptide composition of human parotid saliva and dental decay (caries) experience, we have characterized the peptides from parotid ductal saliva collected from nine adults who have remained free from dental caries (mean age = 59.2; Decayed Missing Filled Surfaces index [DMFS] = 0) and nine individuals who have experienced caries (mean age = 51.2; mean DMFS = 38.4). Ethanol-soluble peptides were size-fractionated on columns of Bio-Gel P-2; the salivary peptides derived from caries-susceptible subjects appeared larger than those found in the saliva of caries-free subjects. Peptides were then resolved into 19 species by cation exchange HPLC. Sequence analysis identified 18 peptides that appear to be proteolytic cleavage products of the basic proline-rich proteins IB-4, IB-5, IB-7, IB-8b, and P-B. The peptides that were more abundant in saliva obtained from the caries-free group differed from those isolated from the caries-susceptible group. The median peptide concentration of one possible precursor protein, IB-7, was found to be higher in saliva collected from caries-free individuals than in that from caries-susceptible individuals. Although differences were found in the phenotypes of proline-rich proteins expressed by these groups of caries-free and caries-susceptible subjects, no statistically significant associations were observed among proline-rich phenotypes and the level of any peptide. Collectively, our results indicate that proteolytic processing of parotid salivary proteins differs among individuals who have remained caries-free and those who have experienced dental decay.     

Specific IgA subclass responses in serum and saliva: a 12-month follow-up study after parenteral booster immunization with tetanus toxoid

Specific IgA subclass antibodies against tetanus toxoid in serum, parotid saliva, and whole saliva were quantified after booster immunization. Samples from 14 healthy individuals were collected before and 1, 6, and 12 months after subcutaneous injection with Duplex ® (0.25 ml tetanus toxoid 30 Lf/mL and diphtheria 7.5 Lf/mL). Samples of whole saliva were also collected after 2 weeks. Specific IgA1 and IgA2 subclass antibodies to tetanus toxoid were quantified by enzyme-linked immunosorbent assay (ELISA). In this quantitative method, chimeric IgA1 and IgA2 antibodies directed against NP (4-hydroxy-3-nitrophenacetyl) were used as standards. Total levels of IgA1 and IgA2 were measured using a nephalometer or ELISA. Immunization with tetanus toxoid resulted in raised mean values of specific IgA1 and IgA2 antibodies against tetanus toxoid in serum after 1 month. Compared with the baseline, the mean value of specific IgA1 antibodies showed a 2.6-fold increase (mean value 10.47 µg/mL) in serum, and that of specific IgA2 antibodies a 2.7-fold increase (mean value 0.93 µg/mL). Specific IgA subclass antibody levels in parotid and whole saliva were unchanged after 1 month. The ratio of specific IgA subclass antibodies to total IgA subclass antibodies was 3 to 10 times higher in parotid saliva compared with whole saliva. In conclusion, subcutaneous booster immunization with tetanus toxoid induced immune responses of both antigen-specific IgA1 and IgA2 subclass antibodies in serum with the same increase, whereas the levels of specific IgA subclass antibodies in secretory fluids were unchanged. The ratio of specific IgA subclass antibodies to immunoglobulins was higher in parotid saliva compared with whole saliva.