【作者】 张凤秋；

【导师】 杨连甲；

【作者基本信息】 第四军医大学 ， 口腔基础医学， 2004， 博士

【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. coliBL21(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Lipofectamine2000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?更多还原

【Abstract】 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 represent 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 proteinacross 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 antibody1.1 Cloning and sequencing of kgpcd gene from Porphyromonas gingivalisIn 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更多还原