牙龈卟啉单胞菌血凝素2与氯化血红素结合位点分析

目的 通过竞争性ELISA方法明确牙龈卟啉单胞菌血凝素2(Porphyromonas gingivalis hemagglutinin-2,Pg HA-2)与氯化血红素结合的多肽位点,为牙周病保护性抗体的制备奠定基础.方法 人工合成疑为Pg HA-2氯化血红素结合位点的多肽片段,制备抗Pg HA-2单克隆抗体(MAb QB),通过间接竞争性ELISA,进一步分析血红素特异性结合位点.结果 Pg HA-2氯化血红素结合位点的氨基酸序列为DGFPGDHYAVMISK.MAb QB可以抑制Pg HA-2与氯化血红素结合.结论 明确了Pg HA-2氯化血红素结合位点的氨基酸序列,确定了Pg HA-2氯化血红素结合位点的位置.为今后Pg HA-2氯化血红素结合位点的鉴定、功能结构区分析和多肽疫苗的制备奠定基础.     

牙龈卟啉单胞菌血凝素2氯化血红素结合位点多肽对牙龈卟啉单胞菌生长抑制作用

目的探讨牙龈卟啉单胞菌血凝素2（Porphyromonas gingivalis hemagglutinin-2,PgHA-2）的氯化血红素结合位点多肽对牙龈卟啉单胞菌（Porphyromonasgingivalis,Pg）摄取氯化血红素生长的影响。方法合成多肽DHYAVMISK（肽1）,DEYAVMISK（肽2,肽1中第2位氨基酸突变为谷氨酸）,ALHPDHYLI（肽3,HA-2结合位点不相关多肽）。将肽l、肽2、肽3分别与氯化血红素琼脂糖珠预孵育,加入Pg重组血凝素2（Porphyromonas gingivalis recombinant HA-2,PgrHA-2）,收集与氯化血红素结合的PgrHA-2,SDS—PAGE电泳,分析多肽对PgrHA-2与氯化血红素结合的抑制作用。肽1、肽2、肽3与氯化血红素预孵育后,加入到CDC液体培养基中培养Pg,测定菌液A600值,分析多肽对Pg生长的抑制作用。结果肽1浓度依赖性抑制PgrHA-2与氯化血红素结合,而肽2和肽3对PgrHA-2与氯化血红素的结合无抑制作用。在24、48和72h时间点,肽1组的A600值较肽2、肽3和PBS组明显降低（P〈0．05）。结论本研究表明PgHA-2氯化血红素结合位点多肽DHYAVMISK与Pg竞争结合氯化血红素,抑制Pg的生长,为开发新的牙周病防治方法奠定基础。     

多肽内切酶鉴定牙龈卟啉单胞菌血凝素2结合位点

目的利用多肽内切酶分析牙龈卟啉单胞菌凝血素2(Porphyromonas gingivalishemagglutinin-2,PgHA-2)与氯化血红素结合位点的氨基酸序列。方法Endoproteinase Lys-C多肽内切酶水解获得与氯化血红素结合的功能性的多肽片段,质谱技术鉴定多肽片段的氨基酸序列。结果质谱鉴定与氯化血红素结合的多肽片段的氨基酸序列为YAVNDGFPGDHYAVMISK。结论进一步明确了HA-2与氯化血红素结合位点的氨基酸序列,为牙周病的预防和治疗方法的改进奠定基础。     

牙龈卟啉单胞菌分子生物学研究进展

牙龈卟啉单胞菌已公认为是牙周炎的致病菌,它的一些表面结构诸如细胞外囊泡,菌毛,外膜蛋白,凝集素介导该菌对牙周组织粘附、定植,或作为毒性因子破坏牙周组织,随着分子生物学技术的发展,已对这些结构进行了分子克隆,本文拟就牙龈卟啉单胞菌(Porphyromonasgingivalis)简称Pg)分子生物学进展作一简要综述。1　菌毛基因的分子克隆牙龈卟啉单胞菌菌毛作为该菌表面结构之一介导了该菌对牙周组织的粘附和定植。已纯化了41kda菌毛亚单位蛋白,并克隆了菌毛蛋白基因[1]。使用寡核苷酸M1和M2作为引物,采用PCR从9株Pg菌株中扩增了1.3kb的DNA片段,E…     

牙龈卟啉单胞菌HA2基因和白细胞介素IL-15基因重组质粒的构建

构建抗牙龈卟啉单胞菌的牙周炎基因疫苗p VAX1-HA2、pVAX1-HA2/IL-15,体外检测其在293T细胞的表达。以HA2基因(牙龈卟啉单胞菌牙龈素—血凝素基因编码区的核心功能区)为目的基因与IL-15基因为免疫佐剂构建真核表达质粒,用Lip2000介导瞬时转染293T细胞,RT-PCR检测目的基因mRNA水平及酶联免疫吸附试验检测IL-15蛋白表达水平。重组质粒p VAX1-HA2、pVAX1-HA2/IL-15经酶切及DNA测序鉴定构建正确,转染的293T细胞能够检测到目的基因的表达,也可以检测到IL-15蛋白的表达。说明我们成功构建了真核共表达质粒pVAX1-HA2和p VAX1-HA2/IL-15,为下一步研制抗牙龈卟啉单胞菌DNA疫苗奠定了基础。     

多肽免疫抑制牙龈卟啉单胞菌引起的小鼠感染及牙槽骨吸收作用的研究

目的探讨牙龈卟啉单胞菌(Porphyromonasgingivalis,P.g)血凝素2(hemagglutinin-2,HA-2)的氯化血红素结合位点多肽作为抗原免疫小鼠,对降低P.g感染后毒性和抑制牙周炎牙槽骨吸收的作用。方法将多肽与牛血清白蛋白(Bovine serum albumin,BSA)偶联作为抗原免疫BALB/c小鼠。P.g国际标准菌株ATCC33277厌氧培养,皮下注射菌液,监测小鼠脓肿的大小及转归,评价多肽对P.g皮下感染的保护作用;口腔接种细菌感染小鼠,检测小鼠牙周炎牙槽骨吸收量,评价其对于P.g引起的牙周炎牙槽骨吸收的保护作用。结果多肽DGFPGDHYAVMISK作为抗原免疫小鼠可减小小鼠皮下接种P.g处形成脓肿的大小,并加快皮下脓肿的愈合;可抑制口腔感染P.g后小鼠牙周炎模型牙槽骨的吸收。结论多肽DGFPGDHYAVMISK免疫小鼠对P.g感染小鼠具有一定的保护作用;可减少小鼠牙周炎模型牙槽骨的吸收。为开发新的牙周病防治方法奠定基础。     

牙龈卟啉单胞菌蛋白酶及疫苗的研究进展

牙龈卟啉单胞菌 (P g) ,革兰阴性厌氧菌 ,是人类牙周炎的主要致病菌[1] 。动物实验表明它在小鼠、大鼠和灵长类动物的龈下定植与牙周炎的发生和进展相关。P g可以调整真核细胞信号转导途径 ,为了满足新陈代谢的需要 ,P g基因表达的调节可以控制在转录水平。证据表明 ,P g的感染会导致严重的全身系统疾病如心血管疾病和分娩早产儿。P g含有大量毒性因子[2 ] ,如菌毛 ,血凝素[3 ] ,脂多糖等 ,其中Arg、Lys样半胱氨酸蛋白酶在牙周致病作用中占据了一个重要地位 ,他们通过激活宿主前体酶 ,例如 :血纤维蛋白酶原 ,或通过暴露细胞隐位及改变血…     

牙龈卟啉单胞菌相关致病因子的研究进展

牙龈卟啉单胞菌是一种生长于口腔内的革兰阴性厌氧茵.它能够利用脂多糖、荚膜多糖、菌毛、牙龈蛋白酶等一系列致病因子,侵袭局部牙周组织并逃避宿主的免疫防御机制,是诱发牙周炎的重要因素之一,引起了学者们的广泛关注.因此,探讨牙龈卟啉单胞菌的致病因子对于牙周炎的防治具有重要意义.     

牙龈卟啉杆菌W83单克隆抗体的研制与鉴定

牙龈类杆菌曾是重要的产黑色素类杆菌群菌株,最近重新命名为牙龈卟啉杆菌,该菌为牙周病龈下菌斑中关键性厌氧菌,与成人慢性牙周炎的发生、发展有密切关系。作者用牙龈卟啉杆菌侵袭型菌株W83,作为免疫原,通过免疫小鼠、细胞融合、筛选、克隆化,最后得到一株能够稳定分泌抗牙龈卟啉杆菌W83的单克隆抗体杂交瘤细胞系,经鉴定该单抗特异性良好,可用于临床该菌的检出和生态学研究。     

牙龈卟啉单胞菌酪氨酸激酶致病性的分子机制

目的 探讨牙龈卟啉单胞菌（Porphyromonas gingivalis,P. gingivalis）酪氨酸激酶（Ptk1)致病性的分子机制。方法 采用重组PCR技术构建P. gingivalis野生菌株ATCC 33277的Ptk1单基因缺失的突变菌株（ΔPtk1）,通过Real-time PCR技术检测并比较参与调控P. gingivalis（野生型P. gingivalis ATCC 33277与突变型ΔPtk1）细胞外多糖（extracellular polysaccharides,EPS）合成的转录因子SinR的表达情况,同时采用激光共聚焦显微镜观察Ptk1缺失的突变菌株与野生型菌株EPS的形成情况,最后通过ELISA试剂盒检测并比较Ptk1缺失的突变菌株与野生型菌株白细胞介素-1β(IL-1β)表达情况。结果 与野生菌株P. gingivalis ATCC 33277比较,Ptk1单基因缺失的突变菌株转录因子SinR的表达量没有显著变化（t=–1.572,P>0.05）;ELISA检测发现,Ptk1单基因缺失的突变菌株IL-1β的表达量较野生型菌株显著下降,差异有统...     

Production and characterisation of monoclonal antibodies to the principle sonicate antigens of Porphyromonas gingivalis w50

Abstract Protein antigens from whole cell sonicates of Porphyromonas gingivalis W50, previously shown to be discriminatory antigens for patients with adult periodontitis, were purified using SDS-PAGE. Electroeluted proteins were used to immunize mice for the production of monoclonal antibodies (mAbs). A combination of enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen hybridoma supernatants for mAbs. MAbs were successfully raised against M _r 115 000, M _r 55 000 and M _r 47 000 antigens together with a second M _r 55 000 polypeptide which was a contaminant of the M _r 55 000 antigen. No immunological cross-reactivity was found between these four proteins. The mAbs were used to examine the distribution of these antigens among fifteen P. gingivalis strains together with related oral bacteria using immunostaining of dot blots and Western blots. The antigens were confined to P. gingivalis with the M _r 115 000 and M _r 47 000 antigens being present in all strains tested . The distribution of the M _r 55 000 antigens were slightly more restricted: one M _r 55 000 (outer membrane location) was present in nine of the fifteen P. gingivalis strains tested, while the other M _r 55 000 (location unknown) was only absent from one strain. Whole cell ELISA demonstrated that the M _r 115 000 and the outer membrane M _r 55 000 antigen possess epitopes which are located on the surface of the bacterium.     

鼠抗衣原体属抗原单克隆抗体的研制

本文利用杂交瘤技术,成功地建立了３株稳定分泌小鼠抗衣原体属脂多糖抗原单克隆抗体的杂交瘤细胞。实验结果表明,３株单抗均为ＩｇＭ类,特异性强,识别相似抗原位点,为今后的应用打下了基础。     

Expression of single-chain antibody against RgpA protease of Porphyromonas gingivalis in Lactobacillus

AIMS: The monoclonal antibody 61BG1.3, recognizing the RgpA protease, has been reported to confer protection against recolonization by the periodontal pathogen Porphyromonas gingivalis in humans. The aim of this study was to express a functional scFv derived from the monoclonal antibody 61BG1.3 on the surface of Lactobacillus paracasei for potential use in the prevention or treatment of periodontal diseases. METHODS AND RESULTS: The scFv was fused to an E-tag and cloned in the Escherischia coli/Lactobacillus shuttle vector pLP501, which mediates surface expression of the scFv. FACS analysis using an anti-E-tag antibody revealed that the scFv was expressed on the surface of the transformed lactobacilli and binding of the scFv to RgpA was shown by ELISA. Lact. paracasei expressing the scFv against RgpA was able to agglutinate P. gingivalis whereas the Lact. paracasei expressing an irrelevant scFv fragment did not. Scanning electron microscopy demonstrated efficient binding of the lactobacilli expressing the scFv anti-RgpA to P. gingivalis. CONCLUSIONS: We have expressed a functional scFv antibody directed against the RgpA protease of P. gingivalis in Lactobacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest a potential of Lactobacillus expressing scFvs against P. gingivalis to be used to combat periodontal disease.     

牙龈卟啉单胞菌及其细胞外膜泡多克隆抗体的比较研究

分别制备牙龈卟啉单胞菌４７Ａ－ １ 及其膜泡的多克隆抗体,并用间接免疫荧光法对６７ 个临床标本进行检测。结果经统计学检验,两种抗体显著相关,Ｐ＜ ０．０１ ,符合率为８２．１ ％ ;膜泡抗体的检出率高于菌体抗体,Ｐ＜０．０５ ;膜泡抗体相对于菌体抗体的敏感性为９３．５ ％ 。膜泡抗体与牙龈卟啉单胞菌以外的其他口腔细菌无交叉反应,说明膜泡抗体的特异性较强。     

Cloning and characterization of heavy and light chain genes encoding the FimA-specific monoclonal antibodies that inhibit Porphyromonas gingivalis adhesion

FimA of Porphyromonas gingivalis, a major pathogen in periodontitis, is known to be closely related to the virulence of these bacteria and has been suggested as a candidate for development of a vaccine against periodontal disease. In order to develop a passive immunization method for inhibiting the establishment of periodontal disease, B hybridoma clones 123-123-10 and 256-265-9, which produce monoclonal antibodies (Mabs) specific to purified fimbriae, were established. Both mAbs reacted with the conformational epitopes displayed by partially dissociated oligomers of FimA, but not with the 43 kDa FimA monomer. Gene sequence analyses of full-length cDNAs encoding heavy and light chain immunoglobulins enabled classification of the genes of mAb 123-123-10 as members of the mVh II (A) and mVκ I subgroups, and those of mAb 256-265-9 as members of the mVh III (D) and mVκ I subgroups. More importantly, 50 ng/mL of antibodies purified from the culture supernatant of antibody gene-transfected CHO cells inhibited, by approximately 50%, binding of P. gingivalis to saliva-coated hydroxyapatite bead surfaces. It is expected that these mAbs could be used as a basis for passive immunization against P. gingivalis-mediated periodontitis.     

牛血清白蛋白单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清白蛋白(BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,培养上清经过双抗体夹心法检测初步筛选分泌鼠IgG的杂交瘤细胞,将此种杂交瘤细胞注射小鼠产生的腹水用间接ELISA法筛选,获得4株能稳定分泌抗BSA单克隆抗体的杂交瘤细胞株,分别命名为2A5、3A3、3G6、4A8。鉴定结果显示,2A5细胞分泌IgG2a/κ,其余3株细胞分泌IgG1/κ;纯化后4株腹水单抗的纯度达90%以上,对BSA的ELISA滴度均可达到1∶100000以上;4株单抗均不与人以及马、猪、羊、兔、豚鼠等血清发生交叉反应;W estern B lotting试验证明4株单抗均识别分子量为68000的BSA;用间接ELISA法测定4株单抗相对亲和力及相对敏感度大小依次为3A3>2A5>3G6>4A8;杂交瘤细胞株连续培养3个月以及冻存半年后复苏,细胞生长良好,杂交瘤细胞分泌的抗体效价稳定。     

大黄鱼病原哈维氏弧菌单克隆抗体的制备及其应用

用甲醛灭活哈维氏弧菌（Vibrio harveyi）GYC1108-1制备免疫原免疫8周龄雌性BALB/c小鼠,利用淋巴细胞杂交瘤技术,用间接酶联免疫吸附试验（ELISA）对杂交瘤进行筛选,阳性克隆经2－3次亚克隆后,共获得5株针对GYC1108-1的单克隆抗体。选取1B7制备小鼠腹水抗体,其细胞上清及腹水效价分别为1∶3200和1∶32000。同样用灭活的哈维氏弧菌免疫新西兰大白兔,5次免疫后颈动脉采血,离心取血清,并用饱和硫酸铵法进行纯化,制备了兔抗哈维氏弧菌多克隆抗体,其ELISA效价为1∶51200。利用1B7单克隆抗体和兔抗哈维氏弧菌多克隆抗体以及山羊抗小鼠HRP酶标二抗,建立了检测哈维氏弧菌的三抗体夹心酶联免疫吸附试验（TAS-ELISA）方法。该方法对哈维氏弧菌的最小检出浓度为1×104个/mL。用该TAS-ELISA方法检测大黄鱼（Pseudosciaena crocea）样品,54尾患病大黄鱼中有45尾检出哈维氏弧菌,而14尾健康大黄鱼都没有检出哈维氏弧菌。由此可见,本试验建立的TAS-ELISA方法,可以用于患病大黄鱼哈维氏弧菌的快速诊断。     

庆大霉素单克隆抗体的制备及试剂盒的配制

目的建立庆大霉素直接竞争酶联免疫吸附分析方法。方法应用戊二醛法制备庆大霉素完全抗原,通过杂交瘤技术筛选分泌特异性庆大霉素抗体的杂交瘤细胞株,并建立庆大霉素竞争酶联免疫吸附分析检测方法。结果获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,建立了庆大霉素竞争酶联免疫吸附分析检测方法,该方法操作简单具有良好的线性、特异性和精密度;庆大霉素质量浓度在1.5625～50.0000 ng/mL范围内,呈现良好的线性,r2=0.9913,50%抑制浓度为(IC50)为7.37 ng/mL,检测限(LOD)为1.54 ng/mL,该试剂盒与链霉素等8种药物无交叉反应。结论获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,研制的庆大霉素竞争ELISA检测试剂盒具有良好的线性、特异性和精密度。