牙龈卟啉菌牙龈蛋白酶K催化结构域基因克隆、表达、纯化及预防牙周炎的初步研究
|
摘要
牙龈卟啉菌(Porphyromonas gingivalis，P.gingivalis，简称Pg)是牙周炎的重要优势致病菌，牙龈蛋白酶是P.gingivalis的重要致病因子。牙龈蛋白酶K(gingipain K，简称KGP)基因全长由6.4Kbp组成，前22个氨基酸为信号肽，接下来23～229残基是N-端前序列，230～739个氨基酸为成熟肽蛋白酶结构域(KGPcd)，740～1723的氨基酸为β亚单位即凝集素结构域。KGPcd为KGP的生物活性部分，β亚单位其保守的位点几乎存在于所有P.gingivalis，并具有粘附、凝集功能。由于近年来分子生物学和免疫学的发展，牙龈蛋白酶基因已被克隆和测序，对其蛋白质分子中的各个功能区、抗原表位及相应的编码基因片段已有了更深入的了解，为研制疫苗提供了可靠的依据。
本课题为研究基因免疫在预防牙周炎中的作用，采用分子生物学技术，对P.gingivalis KGPcd进行了克隆、原核表达和纯化，并制备了抗KGPcd抗体；同时构建KGPcd真核表达质粒，并免疫SD大鼠，观察其在防治牙周炎中的作用，实验结果表明：KGPcd可作为基因候选疫苗，有减缓牙槽骨吸收的作用，这为今后进一步研究预防牙周炎的基因疫苗奠定了实验基础，为控制牙周炎的发生提供一种新型的预防方法。本研究分为以下二部分：
第四军医大学博十学位论文
lpgingivalisKGpcd的基因克隆、表达、纯化及抗体制备
1.Ik即cd基因克隆及其序列分析
采用细菌基因组DNA抽提试剂盒从尸gi眼ivalis ATCC 33277中
提取DNA组，然后利用PCR技术，从尸gtngivalis ATCC 33277 DNA
组中扩增人邵，cd的基因片段(1467bp)，将所得的基因片段与克隆载
体pGEM一T easy Vector连接，转化大肠杆菌E.coli XL一10并进行蓝白
筛选，随机挑选数个克隆，提取质粒DNA，通过限制性酶切和核普
酸序列分析鉴定。结果表明:其序列与GeneBank核酸数据库中收录
的尸gl’ngl’vall’s 381的切，cd的序列完全一致。因此通过PCR方法从
尸gl’ngtvalis ATCC 33277基因组中成功获得了及霎，cd基因片段。
1.2 KGPcd在大肠杆菌中的表达
将编码KGPed的基因片段克隆到原核表达载体pET-16b上，使
其受控于T7启动子，构建表达质粒pET- 1 6b/k即cd，以大肠杆菌E. coli
BL21(DE3)为宿主菌，用IPTG诱导其表达，优化IPTG诱导浓度与诱
导时间，并进行蛋白表达形式分析，然后进行大量诱导表达，表达产
物用镍金属鳌合柱纯化，采用稀释加透析方法进行复性处理。再以抗
His标签抗体通过认飞stem blot检测纯化的蛋白。结果表明:经IPTG
诱导后，工程菌在SDS一PAGE上出现一条新蛋白带，分子量与预期大
小一致(约56kDa)。在IPTG不同浓度，不同时间的诱导条件下，对
融合蛋白表达量影响不大，表达量差异不明显。表达形式分析证实表
达的融合蛋白主要以不溶形式存在于包涵体中。经裂菌、洗涤后，SM
尿素溶解，过Ni一SePhrose亲和层析并经透析复性。结果获得了高纯
度融合蛋白His一KGPed，其分子量与预期相符。Westem blot结果也进
一步证实纯化的蛋白为His一KGPed融合蛋白。
1.3抗KGped抗体的制备
以重组、纯化的KGPed蛋白为抗原，与等体积弗氏佐剂充分乳
化后，免疫新西兰大白兔，制备KGPcd的多克隆抗体，将获得的多
第4页
第四军医大学博士学位论文
克隆抗血清初步纯化，并用ELASA法检测抗体效价，用认乞Stem blot
鉴定抗血清的特异性。结果表明:抗体效价为1:6400，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。多克隆抗
KGPcd抗体的获得为后续研究奠定了基础。
2 KG子七d基因疫苗的制备及预防牙周炎的实验研究
2.1真核表达质粒的构建及其在哺乳动物细胞中的表达
取纯化的真核表达载体PcDNA3.1(+)及pGEM一T/kgl，cd酶切、回
收、连接，转化至E.colt XL一10感受态细胞中，对阳性菌落提取质粒
并进行酶切鉴定，并鉴定插入片断的方向，以构建人岁，cd真核表达质
粒PcDNA3.1+/k即cd。重组质粒构建成功后采用脂质体Lipofectamine
2000介导的瞬时转染贴壁细胞的方法，瞬时转染COS7细胞，然后用
间接免疫荧光、Westem blot两种法证实重组质粒PcDNA3.1+/kgl，cd
在哺乳动物细胞中的表达情况。结果表明:重组质粒PcDNA3 .1(+)/
人{〔牙尸cd转染COS7细胞后目的基因能够在细胞内正确转录、翻译、表
达，表达的蛋白位于胞质中，可与抗KGPed蛋白的抗体特异性结合。
从而证实目的基因可在哺乳动物细胞中正确翻译并表达，为下一步利
用pcDNA3 .1+/k群，cd作为基因疫苗免疫动物提供了依据。
2.2 PcDNA3.l+l棍，cd免疫原性研究
以纯化的KGPed蛋白和PcDNA3.1+/k召夕cd经股四头肌注射、领
下腺区皮下注射两种途径免疫BALB/c小鼠，然后收集唾液和血清样
品，通过ELASA法检测各组唾液中slgA型及血清中IgG型抗KGPed
抗体水平;并以免疫组化和间接免疫荧光观察重组蛋白KGPed在免
疫部位的定位表达。方差分析显示:实验组唾液中slgA型及血清中
IgG型抗体水平明显高于阴性对照组和空白对照组(p<0.01)，蛋白
组抗体水平要高于重组质粒组(p<0.05)，而阴性对照组和空白对照
组唾液中slgA型及血清中IgG型抗体水平均无显著性差异(p>0.01)。
第5页
第四军医大学博士学位论文
一?
Recent evidence strongly suggests that the primary pathogen responsible for chronic progressive periodontal disease is Porphyromonas gingivalis. The gingipains, a group of cysteine proteases produced by P. gingivalis, have received considerable attention. The gingipains are the important virulence factors of P. gingivalis. The gingipain K (Kgp) is one of them. The initial translation product is large precursor, which is composed of four functional regions (the signal peptide, the NH2-terminal prosequence, the mature proteinase domain, and the COOH-terminal hemagglutinin domain). The nucleotide sequence of the kgp DNA covers 6.4kbp. The nucleotide sequence (6,366 nucleotides) includes the complete coding region and parts of the 5'-and 3'-noncoding regions. The open reading frame consisting of 5,169 nucleotides was found to encode 1,723 amino acid residues with a calculated mass of 218 kDa. The amino acid sequence suggests that the first 22 amino acid residues containing a hydrophovic amino acid cluster may re
present a signal peptide. The hydropathy of the the NH2-terminus of the precursor is high enough for it to function as a signal sequence translocating the protein
across the membrane. The next 206-residue sequence is considered to be an NH2-terminal propetid. The amino acid sequence of the mature enzyme was found to start with the 229th Asp residue and to end with the putative site of the 717th Arg residue. The remaining COOH-terminal portion is thought to be the COOH-terminal prosequence, which contains the hemagglutinin-related sequence starting with the 738th Ala residue, identical to the NH2-terminal sequence of P. gingivalis.
In the present study, we assessed whether the KGPcd could be vaccine candidate for prevention of oral bone loss in a rat model. Used the molecular biological techniques, gingipain K catalytic domain (kgpcd) was cloned, expressed in E.coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Thus mass recombinant proteins were obtained. Then the anti-KGPcd antibody was prepared to proceed the following study. At the same time, the eukaryotic expression recombined plasmid of pcDN A3.1+/kgpcd was constructed. Sprague-Dawley (SD) rats were vaccinated with eukaryotic expression plasmid by the route of submandibular gland-targeted injection. The specific immune responses and their protection against periodontitis were observed by ELASA and HE. So, the immunization effects were verified, paving the way for the further study.
This paper consists of the following two parts:
1 Cloning, expression of the gingipain K catalytic domain (kgpcd) gene from Porphyromonas gingivalis and purification of the recombined KGPcd protein and Preparation of anti-KGPcd antibody
1.1 Cloning and sequencing of kgpcd gene from Porphyromonas gingivalis
In the study, P. gingivalis was routinely cultured and its genomic DNA was isolated as templates by bacteria genomic DNA isolation kit.
The desired DNA products encoding gingipain K. catalytic domain were obtained from the total genomic DNA by PCR with the the designed specific primers. The PCR products, ie, these objective gene segments (1467bp), were inserted into cloning vector of pGEM-T easy Vector and the inserting plasmids were transformed into E.coli XL-10 and had the blue-white screening. The positive clones, ie, the white clones, were analyzed by restriction endonuclease mapping and DNA sequencing. The results showed that the sequence of kgpcd were consistent with that of the P. gingivalis 381 in GeneBank.
1.2 Expression of recombinant gingipain K catalytic domain gene in E.coli BL21 (DE3)
The gene fragment encoding kgpcd was inserted into prokaryotic expression vector pET-16b in which foreign gene is controlled by T7 promoters. The recombinant plasmid pET-16b/kgpcd was transformed into E.coli BL21 (DE3) and induced by IPTG in order to express the fusion protein His (Histidine)-KGPcd. The expressed fusion protein was purified by Ni-NTA affinity chromatography and then refolded by dilution and dialysis. After induction, a new anticipated 56kDa
本课题为研究基因免疫在预防牙周炎中的作用，采用分子生物学技术，对P.gingivalis KGPcd进行了克隆、原核表达和纯化，并制备了抗KGPcd抗体；同时构建KGPcd真核表达质粒，并免疫SD大鼠，观察其在防治牙周炎中的作用，实验结果表明：KGPcd可作为基因候选疫苗，有减缓牙槽骨吸收的作用，这为今后进一步研究预防牙周炎的基因疫苗奠定了实验基础，为控制牙周炎的发生提供一种新型的预防方法。本研究分为以下二部分：
第四军医大学博十学位论文
lpgingivalisKGpcd的基因克隆、表达、纯化及抗体制备
1.Ik即cd基因克隆及其序列分析
采用细菌基因组DNA抽提试剂盒从尸gi眼ivalis ATCC 33277中
提取DNA组，然后利用PCR技术，从尸gtngivalis ATCC 33277 DNA
组中扩增人邵，cd的基因片段(1467bp)，将所得的基因片段与克隆载
体pGEM一T easy Vector连接，转化大肠杆菌E.coli XL一10并进行蓝白
筛选，随机挑选数个克隆，提取质粒DNA，通过限制性酶切和核普
酸序列分析鉴定。结果表明:其序列与GeneBank核酸数据库中收录
的尸gl’ngl’vall’s 381的切，cd的序列完全一致。因此通过PCR方法从
尸gl’ngtvalis ATCC 33277基因组中成功获得了及霎，cd基因片段。
1.2 KGPcd在大肠杆菌中的表达
将编码KGPed的基因片段克隆到原核表达载体pET-16b上，使
其受控于T7启动子，构建表达质粒pET- 1 6b/k即cd，以大肠杆菌E. coli
BL21(DE3)为宿主菌，用IPTG诱导其表达，优化IPTG诱导浓度与诱
导时间，并进行蛋白表达形式分析，然后进行大量诱导表达，表达产
物用镍金属鳌合柱纯化，采用稀释加透析方法进行复性处理。再以抗
His标签抗体通过认飞stem blot检测纯化的蛋白。结果表明:经IPTG
诱导后，工程菌在SDS一PAGE上出现一条新蛋白带，分子量与预期大
小一致(约56kDa)。在IPTG不同浓度，不同时间的诱导条件下，对
融合蛋白表达量影响不大，表达量差异不明显。表达形式分析证实表
达的融合蛋白主要以不溶形式存在于包涵体中。经裂菌、洗涤后，SM
尿素溶解，过Ni一SePhrose亲和层析并经透析复性。结果获得了高纯
度融合蛋白His一KGPed，其分子量与预期相符。Westem blot结果也进
一步证实纯化的蛋白为His一KGPed融合蛋白。
1.3抗KGped抗体的制备
以重组、纯化的KGPed蛋白为抗原，与等体积弗氏佐剂充分乳
化后，免疫新西兰大白兔，制备KGPcd的多克隆抗体，将获得的多
第4页
第四军医大学博士学位论文
克隆抗血清初步纯化，并用ELASA法检测抗体效价，用认乞Stem blot
鉴定抗血清的特异性。结果表明:抗体效价为1:6400，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。多克隆抗
KGPcd抗体的获得为后续研究奠定了基础。
2 KG子七d基因疫苗的制备及预防牙周炎的实验研究
2.1真核表达质粒的构建及其在哺乳动物细胞中的表达
取纯化的真核表达载体PcDNA3.1(+)及pGEM一T/kgl，cd酶切、回
收、连接，转化至E.colt XL一10感受态细胞中，对阳性菌落提取质粒
并进行酶切鉴定，并鉴定插入片断的方向，以构建人岁，cd真核表达质
粒PcDNA3.1+/k即cd。重组质粒构建成功后采用脂质体Lipofectamine
2000介导的瞬时转染贴壁细胞的方法，瞬时转染COS7细胞，然后用
间接免疫荧光、Westem blot两种法证实重组质粒PcDNA3.1+/kgl，cd
在哺乳动物细胞中的表达情况。结果表明:重组质粒PcDNA3 .1(+)/
人{〔牙尸cd转染COS7细胞后目的基因能够在细胞内正确转录、翻译、表
达，表达的蛋白位于胞质中，可与抗KGPed蛋白的抗体特异性结合。
从而证实目的基因可在哺乳动物细胞中正确翻译并表达，为下一步利
用pcDNA3 .1+/k群，cd作为基因疫苗免疫动物提供了依据。
2.2 PcDNA3.l+l棍，cd免疫原性研究
以纯化的KGPed蛋白和PcDNA3.1+/k召夕cd经股四头肌注射、领
下腺区皮下注射两种途径免疫BALB/c小鼠，然后收集唾液和血清样
品，通过ELASA法检测各组唾液中slgA型及血清中IgG型抗KGPed
抗体水平;并以免疫组化和间接免疫荧光观察重组蛋白KGPed在免
疫部位的定位表达。方差分析显示:实验组唾液中slgA型及血清中
IgG型抗体水平明显高于阴性对照组和空白对照组(p<0.01)，蛋白
组抗体水平要高于重组质粒组(p<0.05)，而阴性对照组和空白对照
组唾液中slgA型及血清中IgG型抗体水平均无显著性差异(p>0.01)。
第5页
第四军医大学博士学位论文
一?
Recent evidence strongly suggests that the primary pathogen responsible for chronic progressive periodontal disease is Porphyromonas gingivalis. The gingipains, a group of cysteine proteases produced by P. gingivalis, have received considerable attention. The gingipains are the important virulence factors of P. gingivalis. The gingipain K (Kgp) is one of them. The initial translation product is large precursor, which is composed of four functional regions (the signal peptide, the NH2-terminal prosequence, the mature proteinase domain, and the COOH-terminal hemagglutinin domain). The nucleotide sequence of the kgp DNA covers 6.4kbp. The nucleotide sequence (6,366 nucleotides) includes the complete coding region and parts of the 5'-and 3'-noncoding regions. The open reading frame consisting of 5,169 nucleotides was found to encode 1,723 amino acid residues with a calculated mass of 218 kDa. The amino acid sequence suggests that the first 22 amino acid residues containing a hydrophovic amino acid cluster may re
present a signal peptide. The hydropathy of the the NH2-terminus of the precursor is high enough for it to function as a signal sequence translocating the protein
across the membrane. The next 206-residue sequence is considered to be an NH2-terminal propetid. The amino acid sequence of the mature enzyme was found to start with the 229th Asp residue and to end with the putative site of the 717th Arg residue. The remaining COOH-terminal portion is thought to be the COOH-terminal prosequence, which contains the hemagglutinin-related sequence starting with the 738th Ala residue, identical to the NH2-terminal sequence of P. gingivalis.
In the present study, we assessed whether the KGPcd could be vaccine candidate for prevention of oral bone loss in a rat model. Used the molecular biological techniques, gingipain K catalytic domain (kgpcd) was cloned, expressed in E.coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Thus mass recombinant proteins were obtained. Then the anti-KGPcd antibody was prepared to proceed the following study. At the same time, the eukaryotic expression recombined plasmid of pcDN A3.1+/kgpcd was constructed. Sprague-Dawley (SD) rats were vaccinated with eukaryotic expression plasmid by the route of submandibular gland-targeted injection. The specific immune responses and their protection against periodontitis were observed by ELASA and HE. So, the immunization effects were verified, paving the way for the further study.
This paper consists of the following two parts:
1 Cloning, expression of the gingipain K catalytic domain (kgpcd) gene from Porphyromonas gingivalis and purification of the recombined KGPcd protein and Preparation of anti-KGPcd antibody
1.1 Cloning and sequencing of kgpcd gene from Porphyromonas gingivalis
In the study, P. gingivalis was routinely cultured and its genomic DNA was isolated as templates by bacteria genomic DNA isolation kit.
The desired DNA products encoding gingipain K. catalytic domain were obtained from the total genomic DNA by PCR with the the designed specific primers. The PCR products, ie, these objective gene segments (1467bp), were inserted into cloning vector of pGEM-T easy Vector and the inserting plasmids were transformed into E.coli XL-10 and had the blue-white screening. The positive clones, ie, the white clones, were analyzed by restriction endonuclease mapping and DNA sequencing. The results showed that the sequence of kgpcd were consistent with that of the P. gingivalis 381 in GeneBank.
1.2 Expression of recombinant gingipain K catalytic domain gene in E.coli BL21 (DE3)
The gene fragment encoding kgpcd was inserted into prokaryotic expression vector pET-16b in which foreign gene is controlled by T7 promoters. The recombinant plasmid pET-16b/kgpcd was transformed into E.coli BL21 (DE3) and induced by IPTG in order to express the fusion protein His (Histidine)-KGPcd. The expressed fusion protein was purified by Ni-NTA affinity chromatography and then refolded by dilution and dialysis. After induction, a new anticipated 56kDa
引文
1．杨禾．牙龈卟啉菌的蛋白酶及其在牙周病病理机制中的作用于．国外医学口腔医学分册，1999，26(6)：326-328
2. Curtis MA. Analysis of the protease and adhesin domains of the PrpR1 of Porphyromonas gingivalis. J Periodont Res, 1997, 32: 133-139
3. Nakayama K, Kadowaki T, Okamoto K, et al. Construction and characterization of argigine-specific cysteine proteinase (Arg-gingipain)-deficient mutants of Porphyromonas gingivalis. Evidence for significant contribution of Arg-gingipain to virulence. J Biol Chem, 1995,270(40): 23619-23626
4. Pavloff N, Pemberton PA, Potempa J, et al. Molecular cloning and characterization of Porphyromonas gingivalis lysine-specific gingipain. J Biol Chem, 1997, 272(3):1595-1600
5. Okamoto K, Kadowaki T, Nakayama K, et al. Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-gingipain) in Porphyromonas gingivalis: structural relationship with arginine-specific cysteine proteinase (Arg-gingipain). J Biochem, 1996, 120:398-406
6. Fletcher HM, Schemkein HA, Macrina FL. Cloning and characterization of a new protease gene (prtH) from Porphyromonas gingivalis. Infect Immun, 1994,62:4279-4286
7. Kirszbaum L, Sotiropoulos C, Jackson C, et al. Complete nucleotide sequence of a gene prtR of Porphyromonas gingivalis W50 encoding a 132kDa protein contains an arginige-specific thiol endopeptidase domain and a haemagglutinin domain. Biochem biophys Res
Commun, 1995,207:424-431
8. Aduse-Opoku J, Muir J, Slaney JM, et al. Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50. Infect Immun,1995, 63(12): 4744-4754
9. Pavloff NJ, Potempa RN, Pike V , et al. Molecular cloning and structural characterization of the Arg-gingipain proteinase of Porphyromonos gingivalis. Biosynthesis as a proteinase-adhesin polyprotein. J Biol Chem, 1995, 270:1007-1010
10. Rusch S, Kendall DA. Transport of an export-defective protein by a highly hydrophobic signal peptide. J Biol Chem, 1994, 269:1242-1248
11. Salmond GPC, PJ Reeves. Membrane traffic wardens and protein secretion in Gram-negative bacteria. Trends Biochem Sci, 1993, 18:7-12
12．汪喻忠，刘建国．变形链球菌表面蛋白的结构和功能．牙体牙髓牙周病学杂志，2000，10(3)：174-176
13. Chen T, H Dong, R Yong, et al. Pleiotropic pigmentation mutants of Porphromonas gingivalis. Microb Pathog, 2000,28:235-242
14. Chen W, HK Kurmitsu. Molecular mechanism for the endotaneous generation of pigmentless Porphromonas gingivalis mutants. Infect Immun, 1999,67:4926-4930
15. Lewis JP, JA Kawson, JC Hannis, et al. Hemoglobinase activity of the lysine gingipain protease (KGP) of Porphromonas gingivalis W83. J Bacteriol, 1999, 181:4905-4913
16. Simpson W, Wang CY, Mikolajczyk-Pawlinsks J, ea al. Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphromonas gingivalis. Infect Immun,
1999,67(10): 5012-5020
17. Lantz MS, RW Rowland, LM Switalski, et al. Interactions of Bacteroides gingivalis with fibrinogen. Infect Immun,1986,54: 654-658
18. Larjava H, VJ Uitto, M Haapasalo, et al. Fibronectin fragmentation induced by dental plaque and bacteroides gingivalis. Scand J Dent Res, 1987,95:308-314
19. Pike RN, J Potempa, WncGraw, et al. Characterization of the binding activities of proteinase-adhesin complexes from Porphromonas gingivalis. J Bacteriol, 1996, 178:2876-2882
20. Scott CF, EJ Whitaker, BF Hammond et al. Purification and characterization of a potent 70-kDa thiol lysyl-proteinase (Lys-gingipain) from Porphromonas gingivalis that cleaves kininogens and fibrnogen. J Biol Chem, 1993, 268:7935-7942
21. Uitto VJ, Haapasalo M, Laakso T, et al. Degradation of basement membrane collagen by proteases from some anaerobic oral microorganisms. Oral Microbiol Immunol, 1998,3(3): 97-102
22. Carlsson J, BF herrmann, JF Hoffing, et al. Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis. Infect Immun, 1984,43:644-648
23. Sundqvist G, A Rengston, J Carlsson. Generation and degradation of the complement fragment C_(5a) in human serum by Bacteroides gingivalis. Oral Microbiol Immunol, 1988, 3:103-107
24. Wingrove JA, RG Discipio, Z Chen, et al. Activation of complement omponents C3 and C5 by a cysteine proteinase (gingipain-1) from Porphromonas (Bacteroides) gingivalis. J Biol Chem, 1992, 267:18902-18907
25. Jageis MA, J Travis, J Potempa, et al. Proteolytic inactivation of the
leukocyte C_(5-)receptor by proteinases derived from Porphromonas gingivalis. Infect Immun, 1996, 64:1984-1991
26. Kadowaki T, M Yoneda, K Okamoto, et al. Purification and characterization of a novel arginine-specific cysteine proteinase (Argingipain) involved in the pathogenesis of periodontal disease from the culture supernatant of Porphromonas gingivalis. J Biol Chem, 1994, 269:21371-21378
27. Fishburn CS, JM Slaney, RJ Carman, et al. Degradation of plasma proteins by the trypsin-like enzyme of Porphromonas gingivalis and inhibition of protease activity by a serine protease inhibitor of human plasma. Oral Microbiol Immunol, 1991, 6:209-215
28. Gregory RL, KE Kirn, JC Kindle, et al. Immunoglobulin-degrading enzymes in localized juvenile periodontitis. J Periodontal Res, 1992,27:176-183
29. Chen Z, Casiano CA, Fletcher HM. Protease-active extracellular protein preparations from Porphyrornonas gingivalis W83 induce N-cadherin proteolysis, loss of cell adhesion, and apoptosis in human epithelial cells. J Periodontol, 2001, 72(5):641-650
30. Genco CA, BM Odusanya, J Potempa, et al. A peptide domain on gingipain R which confers immunity against Porphromonas gingivalis infection in mice. Infect Immun, 1998, 66:4108-4114
31. O'brien-Simpson NM, RA Paolini, EC Reynolds. RgpA-Kgp peptide-based immunogens provide protection against Porphromonas gingivalis challenge in a murine lesion model. Infect Immun, 2000, 68:4055-4063
32. Yonezawa H, kazuyuki I, Oluda K. Arg-gingipain A DNA vaccine induces protective immunity against infection by Porphromonas gingivalis in a murine model. Infect Immun, 2001,69(5):2858-2864
33. Westerlund B, Korhonen TK. Bacterial proteins binding to mammalian extracellular matrix. Mol Microbiol, 1993, 9:687-694
34. Dame JB, William JL, McCutchan TF, et al. Structure of gene encoding the immunodominant surface antigen on the sporozoite of the hman malarial parasite Plasmodium falciparum. Science, 1984, 225:593-599
35. Williamson MP.The structure and function of proline-rich regions in proteins. J Biochem, 1994, 297:249-260
36. Pavloff N, J Potempa, RN Pike, et al. Molecular cloning and structural characterization of the Arg-gingipain proteinase of Porphromonas gingivalis. J Bio Chem, 1995,270:1007-1010
37. O'Brien S, CL black, PS Bhogal, et al. Serum IgG and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis in adult periodontitis. Infect Immun, 2000, 68(5):2704-2712
38. Cardin AD, HJ Weintraub. Molecular modeling of proteinglycosaminoglycan interactions. Arteriosclerosis, 1989,9:21-32
39. Vysa AA, JJ Pan, HV Patel, et al. Analysis of binding of cobra cardiotoxins to heparin reveals a new beta-sheet heparin-binding structural motif. J Biol chem, 1997, 272:9661-9670
2. Curtis MA. Analysis of the protease and adhesin domains of the PrpR1 of Porphyromonas gingivalis. J Periodont Res, 1997, 32: 133-139
3. Nakayama K, Kadowaki T, Okamoto K, et al. Construction and characterization of argigine-specific cysteine proteinase (Arg-gingipain)-deficient mutants of Porphyromonas gingivalis. Evidence for significant contribution of Arg-gingipain to virulence. J Biol Chem, 1995,270(40): 23619-23626
4. Pavloff N, Pemberton PA, Potempa J, et al. Molecular cloning and characterization of Porphyromonas gingivalis lysine-specific gingipain. J Biol Chem, 1997, 272(3):1595-1600
5. Okamoto K, Kadowaki T, Nakayama K, et al. Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-gingipain) in Porphyromonas gingivalis: structural relationship with arginine-specific cysteine proteinase (Arg-gingipain). J Biochem, 1996, 120:398-406
6. Fletcher HM, Schemkein HA, Macrina FL. Cloning and characterization of a new protease gene (prtH) from Porphyromonas gingivalis. Infect Immun, 1994,62:4279-4286
7. Kirszbaum L, Sotiropoulos C, Jackson C, et al. Complete nucleotide sequence of a gene prtR of Porphyromonas gingivalis W50 encoding a 132kDa protein contains an arginige-specific thiol endopeptidase domain and a haemagglutinin domain. Biochem biophys Res
Commun, 1995,207:424-431
8. Aduse-Opoku J, Muir J, Slaney JM, et al. Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50. Infect Immun,1995, 63(12): 4744-4754
9. Pavloff NJ, Potempa RN, Pike V , et al. Molecular cloning and structural characterization of the Arg-gingipain proteinase of Porphyromonos gingivalis. Biosynthesis as a proteinase-adhesin polyprotein. J Biol Chem, 1995, 270:1007-1010
10. Rusch S, Kendall DA. Transport of an export-defective protein by a highly hydrophobic signal peptide. J Biol Chem, 1994, 269:1242-1248
11. Salmond GPC, PJ Reeves. Membrane traffic wardens and protein secretion in Gram-negative bacteria. Trends Biochem Sci, 1993, 18:7-12
12．汪喻忠，刘建国．变形链球菌表面蛋白的结构和功能．牙体牙髓牙周病学杂志，2000，10(3)：174-176
13. Chen T, H Dong, R Yong, et al. Pleiotropic pigmentation mutants of Porphromonas gingivalis. Microb Pathog, 2000,28:235-242
14. Chen W, HK Kurmitsu. Molecular mechanism for the endotaneous generation of pigmentless Porphromonas gingivalis mutants. Infect Immun, 1999,67:4926-4930
15. Lewis JP, JA Kawson, JC Hannis, et al. Hemoglobinase activity of the lysine gingipain protease (KGP) of Porphromonas gingivalis W83. J Bacteriol, 1999, 181:4905-4913
16. Simpson W, Wang CY, Mikolajczyk-Pawlinsks J, ea al. Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphromonas gingivalis. Infect Immun,
1999,67(10): 5012-5020
17. Lantz MS, RW Rowland, LM Switalski, et al. Interactions of Bacteroides gingivalis with fibrinogen. Infect Immun,1986,54: 654-658
18. Larjava H, VJ Uitto, M Haapasalo, et al. Fibronectin fragmentation induced by dental plaque and bacteroides gingivalis. Scand J Dent Res, 1987,95:308-314
19. Pike RN, J Potempa, WncGraw, et al. Characterization of the binding activities of proteinase-adhesin complexes from Porphromonas gingivalis. J Bacteriol, 1996, 178:2876-2882
20. Scott CF, EJ Whitaker, BF Hammond et al. Purification and characterization of a potent 70-kDa thiol lysyl-proteinase (Lys-gingipain) from Porphromonas gingivalis that cleaves kininogens and fibrnogen. J Biol Chem, 1993, 268:7935-7942
21. Uitto VJ, Haapasalo M, Laakso T, et al. Degradation of basement membrane collagen by proteases from some anaerobic oral microorganisms. Oral Microbiol Immunol, 1998,3(3): 97-102
22. Carlsson J, BF herrmann, JF Hoffing, et al. Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis. Infect Immun, 1984,43:644-648
23. Sundqvist G, A Rengston, J Carlsson. Generation and degradation of the complement fragment C_(5a) in human serum by Bacteroides gingivalis. Oral Microbiol Immunol, 1988, 3:103-107
24. Wingrove JA, RG Discipio, Z Chen, et al. Activation of complement omponents C3 and C5 by a cysteine proteinase (gingipain-1) from Porphromonas (Bacteroides) gingivalis. J Biol Chem, 1992, 267:18902-18907
25. Jageis MA, J Travis, J Potempa, et al. Proteolytic inactivation of the
leukocyte C_(5-)receptor by proteinases derived from Porphromonas gingivalis. Infect Immun, 1996, 64:1984-1991
26. Kadowaki T, M Yoneda, K Okamoto, et al. Purification and characterization of a novel arginine-specific cysteine proteinase (Argingipain) involved in the pathogenesis of periodontal disease from the culture supernatant of Porphromonas gingivalis. J Biol Chem, 1994, 269:21371-21378
27. Fishburn CS, JM Slaney, RJ Carman, et al. Degradation of plasma proteins by the trypsin-like enzyme of Porphromonas gingivalis and inhibition of protease activity by a serine protease inhibitor of human plasma. Oral Microbiol Immunol, 1991, 6:209-215
28. Gregory RL, KE Kirn, JC Kindle, et al. Immunoglobulin-degrading enzymes in localized juvenile periodontitis. J Periodontal Res, 1992,27:176-183
29. Chen Z, Casiano CA, Fletcher HM. Protease-active extracellular protein preparations from Porphyrornonas gingivalis W83 induce N-cadherin proteolysis, loss of cell adhesion, and apoptosis in human epithelial cells. J Periodontol, 2001, 72(5):641-650
30. Genco CA, BM Odusanya, J Potempa, et al. A peptide domain on gingipain R which confers immunity against Porphromonas gingivalis infection in mice. Infect Immun, 1998, 66:4108-4114
31. O'brien-Simpson NM, RA Paolini, EC Reynolds. RgpA-Kgp peptide-based immunogens provide protection against Porphromonas gingivalis challenge in a murine lesion model. Infect Immun, 2000, 68:4055-4063
32. Yonezawa H, kazuyuki I, Oluda K. Arg-gingipain A DNA vaccine induces protective immunity against infection by Porphromonas gingivalis in a murine model. Infect Immun, 2001,69(5):2858-2864
33. Westerlund B, Korhonen TK. Bacterial proteins binding to mammalian extracellular matrix. Mol Microbiol, 1993, 9:687-694
34. Dame JB, William JL, McCutchan TF, et al. Structure of gene encoding the immunodominant surface antigen on the sporozoite of the hman malarial parasite Plasmodium falciparum. Science, 1984, 225:593-599
35. Williamson MP.The structure and function of proline-rich regions in proteins. J Biochem, 1994, 297:249-260
36. Pavloff N, J Potempa, RN Pike, et al. Molecular cloning and structural characterization of the Arg-gingipain proteinase of Porphromonas gingivalis. J Bio Chem, 1995,270:1007-1010
37. O'Brien S, CL black, PS Bhogal, et al. Serum IgG and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis in adult periodontitis. Infect Immun, 2000, 68(5):2704-2712
38. Cardin AD, HJ Weintraub. Molecular modeling of proteinglycosaminoglycan interactions. Arteriosclerosis, 1989,9:21-32
39. Vysa AA, JJ Pan, HV Patel, et al. Analysis of binding of cobra cardiotoxins to heparin reveals a new beta-sheet heparin-binding structural motif. J Biol chem, 1997, 272:9661-9670