【作者】 朱翠明；

【导师】 汪世平；

【作者基本信息】 中南大学 ， 病原生物学， 2012， 博士

【摘要】 肺炎支原体(Mycoplasma pneumoniae, Mp)感染不仅可引起支原体肺炎、气管炎、支气管炎、哮喘等呼吸道疾病,还可导致脑膜脑炎、心肌炎、肾炎、动脉粥样硬化、冠心病等肺外感染和并发症。Mp感染多见于学龄儿童及青少年,是儿童社区获得性肺炎最主要的病原体,其发病率逐年上升,占10%-30%,流行期间为30%～50%,且可引起地区性和世界性流行。虽然大环内酯类等抗生素可控制Mp感染,但耐药菌株的不断增多给临床治疗带来了困难。因此,利用Mp免疫优势抗原基因研制疫苗,是预防和控制Mp感染的关键。P1粘附素是Mp最主要的粘附蛋白和免疫优势抗原,其中和抗体能阻止80%以上的Mp粘附到宿主细胞。由此,P1蛋白是最具潜力的Mp候选疫苗。但由于p1基因中含有RepMP4和RepMP2/3两个高度变异的重复序列,且基因中含有21个"UGA"密码子,克隆和表达p1全基因非常困难。本文分析了p1基因的变异情况,选取了P1蛋白第1125-1395位氨基酸免疫优势表位区(P1C蛋白),研究其生物学活性和其所构建的核酸疫苗的免疫活性及白细胞介素2(interleukin2, IL-2)和大肠杆菌不耐热肠毒素B亚单位(B subunit of Escherichia coli heat-labile enterotoxin, LTB)对p1c核酸疫苗的免疫佐剂效应。目的：检测儿童呼吸道感染患者中分离的159例Mp临床分离株的基因型,分析p1基因的变异情况,确定p1基因的保守序列。构建含Mp P1基因第3373-4185位核苷酸(p1c)的原核表达载体pGEX6p-2//plc,纯化表达产物免疫BALB/c,制备多克隆抗体(pAb);检测其pAb的滴度及对Mp粘附HeLa细胞的抑制作用。构建真核表达载体pcDNA3.1(+)/pklc (pPlC)、pcDNA3.1(+)/plc-IL-2(pPlC-IL-2)和pcDNA3.1(+)/LTB-plc (pLTB-P1C),通过肌肉注射和鼻内接种方式免疫小鼠,检测疫苗所诱发的特异性体液免疫和细胞免疫应答水平；用Mp经呼吸道感染疫苗免疫后的小鼠,观察肺组织病理变化和支气管灌洗液中Mp菌落计数,分析疫苗接种小鼠对Mp感染的免疫保护作用,为研制Mp核酸疫苗提供实验依据。方法：1.复苏Mp临床株,分离培养单个菌落并进行PCR鉴定。通过聚合酶链式反应-限制性片段长度多态性分析对159例Mp临床株进行p1基因分型,并检测p1变异。2.设计特异性引物,PCR扩增p1基因第3373～4185位核苷酸(p1c基因),构建pGEX6p-2/plc原核细胞表达重组体,用PCR介导的定点突变将p1c基因中的“TGA”突变成“TGG”。重组体经IPTG诱导在E.coli BL21中表达融合蛋白P1C-GST,用高亲和力GST Resin亲和柱纯化P1C-GST蛋白,研究P1C-GST蛋白对HeLa细胞的粘附活性；将纯化的PIC-GST蛋白免疫BALB/c鼠,制备多克隆抗体(pAb),研究PIC-GST pAb对Mp的粘附抑制作用。ELISA分析PIC-GST蛋白的免疫原性和抗原性。3.将p1c基因亚克隆至真核表达载体构建pPIC重组体,并将p1c基因与细胞因子佐剂IL-2基因和黏膜佐剂LTB基因分别通过一段特殊核苷酸序列(GAT CCG AGA GTA CCG AGC)连接,构建pP1C-IL-2和pLTB-P1C双基因融合表达重组体。将pP1C和pP1C-IL-2经肌肉注射和鼻内接种方式免疫小鼠,pLTB-PIC鼻内途径免疫小鼠。用ELISA法检测血清和支气管灌洗液中特异性抗体(IgG、IgA)的水平、IgG抗体亚类和IFN-γ、IL-4水平,用MTT法检测脾淋巴细胞特异性增殖反应,以分析疫苗诱导的体液免疫应答和细胞免疫应答水平。4.plc单基因或融合基因疫苗分别免疫小鼠后,将Mp经呼吸道感染小鼠,检测Mp呼吸道攻击后小鼠肺组织病理学改变及支气管灌洗液中Mp的菌落形成单位,以分析疫苗的免疫保护效果。结果：1.159例Mp临床标本中,142例为Mp Ⅰ型(89.31%),17例为Mp Ⅱ型(10.69%)；PCR-RFLP法共检测出21例临床突变株,其中10株为Mp V2c突变株；3株为V2a突变株；7株为V1a突变。研究发现了一株新型V2型突变株Mp100(命名为V2d突变株),其p1基因RepMP4区最接近309(V2a), RepMP2/3区域中有142bp(2772-2913)序列完全同源重组了一段p1基因以外的RepMP2/3-J序列(606517-606658)。2. PCR扩增出约813bp的目的片段；构建了P1C蛋白原核表达重组体pGEX6p-2/p1c,通过PCR介导的定点突变成功地将P1C基因中的TGA突变为TGG。原核表达重组体在E.coli中表达出一相对分子量(Mr)约为66kD的目的蛋白,该目的蛋白在菌体细胞内主要以可溶性形式存在。经高亲和力GST Resin纯化柱纯化,得纯度达95%以上的重组蛋白。3.用纯化的P1C-GST免疫BALB/c小鼠,制备了pAb,抗体效价均在1：4000以上,且能与Mp感染患者血清发生特异性结合反应。4.粘附试验发现P1C对HeLa细胞有粘附活性,粘附抑制试验显示P1C的pAb可抑制Mp对HeLa细胞的粘附,抑制强度与抗体水平呈正相关,抗体滴度越高,抑制作用越强。5.plc单基因疫苗免疫组、p1c-IL-2双基因融合疫苗免疫组小鼠经肌注和鼻饲免疫后,随着接种次数的增多血清抗体水平逐渐升高。第一次免疫后第2w,小鼠血清中可检测到特异性抗体,第6w特异性抗体水平显著升高,第8w抗体水平基本稳定,双基因融合疫苗免疫组血清抗体水平较单基因免疫组高,两种均与对照组有显著性差异(P<0.01)。检测抗体亚类发现单基因免疫组IgG1及IgG2a抗体均较高,而双基因融合疫苗免疫组IgG2a升高更为显著。融合疫苗经鼻饲免疫所诱生的呼吸道局部粘膜抗体显著高于单基因免疫组(P<0.05),肌注免疫组小鼠未产生明显的黏膜抗体。p1c单基因疫苗免疫组、p1c-IL-2双基因融合疫苗免疫组支气管灌洗液中IFN-γ和IL-4水平较对照组显著增高(P<0.05),且双基因疫苗组较单基因疫苗组分泌的IFN-γ和IL-4增高更显著(P<0.05)。疫苗免疫组小鼠脾淋巴细胞增殖反应均较对照组强。肌注组小鼠所诱生的血清IgG抗体滴度和淋巴细胞增殖均强于鼻饲组(P<0.05)。6.用Mp攻击各免疫组小鼠,plc-IL-2双基因融合疫苗免疫组小鼠和p1c单基因疫苗组小鼠的肺间质炎症明显减轻,病变区域较局限,肺泡间隔轻度增宽,淋巴细胞、浆细胞浸润减少。两组的肺组织炎症病理评分明显低于对照组；p1c单基因组和plc-IL-2双基因疫苗免疫组小鼠支气管灌洗液中Mp的菌落数较对照组明显减少,p1c-IL-2双基因疫苗免疫组小鼠的Mp菌落数最少。但两组肺组织病理评分和菌落计数差异无显著性(P>0.05)。7.LRB-p1c双基因融合疫苗组小鼠肺组织炎症减轻,肺组织病理评分和Mp菌落数均低于对照组,但Mp菌落数较ple单基因疫苗减少,但差异无显著性。LTB-p1c组小鼠支气管灌洗液中的血清总抗体、sIgA和支气管灌洗液和血清中IFN-γ、IL-4水平明显高于p1c核酸疫苗组(P<0.05)；8.鼻饲接种LTB-plc免疫组小鼠与plc-IL-2疫苗组相比,支气管灌洗液中的IFN-γ和IgA显著增高,但两组小鼠肺组织炎症和Mp菌落计数差异无显著性(P>0.05)。结论：1.159株Mp临床株主要为Ⅰ型。21株p1基因发生突变,Ⅱ型突变率高于I型。发现一株新的p1基因突变株(V2d)。2.P1C融合蛋白有粘附活性,并具有较好的免疫原性和抗原性。3.plc、LTB-plc和plc-IL-2核酸疫苗均能刺激机体产生高水平的特异性免疫应答水平和免疫保护作用。4.LTB-plc和plc-IL-2核酸疫苗较p1c单基因疫苗诱导更强的特异性免疫应答水平和免疫保护作用。4.plc核酸疫苗鼻饲免疫组小鼠支气管灌洗液产生高水平的IgA,但血清IgG水平低于肌注组,在感染早期发挥更强的作用。肌注组结果相反。更多还原

【Abstract】 Mycoplasma pneumoniae is the causative agent of atypical pneumonia and is also responsible for other respiratory tract infections such as tracheobronchitis, bronchiolitis, croup, and less severe upper respiratory tract infections in older children and young adults. Cardiovascular disease and neurological diseases are serious nonrespiratory symptoms associated with M. pneumoniae infection. It has been estimated that between10and30%of pneumonia cases that occur in the endemic period and that up to30and50%of all cases that occur in the epidemic period are caused by M. pneumoniae. Traditionally, M. pneumoniae infection is controlled by the use of antibiotics. However, the increased prevalence of antimicrobial-resistant strains of M. pneumo-niae increases the risk of reinfection. Accordingly, the prevention of atypical pneumonia through vaccination is needed.The P1protein is considered to be the major ligand mediating attachment of virulent M. pneumoniae to the host cell membrane. An earlier study showed that pretreatment of M. pneumoniae with antiserum directed against P1blocked cytadherence to hamster tracheal rings by up to80%. As the polymorphism regions RepMP4and RepMP2/3of p1gene are variablility and the P1gene contains an open reading frame of4,881nucleo-tides and contains21UGA codons that code for tryptophan, it is very difficulted to express the P1protein in Escherichia coli (E.coli) and mammalian cells as UGA codes for a stop codon. In this study, we studied the p1gene genotyping and the variation of p1gene to determined the conserved sequence of p1gene, chose the biological characteristics of M.pneumoniae P1immundominant region (1125-1395aa, P1C protein) and explored the use of a DNA vaccine encoding P1C. ObjectivesTo determine the conserved sequence of p1gene, the p1gene genotyping and variation was studied with the159M. pneumoniae clinical samples which were isolated from pediatric patients with respiratory tract infection. The prokaryotic expression vector pGEX6p-2/plc with M. pneumoniae p1adhesin immundominant region gene (3374-4185nucleotide, plc gene) was constructed and the recombinant protein was expressed in E.coli BL21(DE3). The purified P1C-GST protein was used to immunize the BALB/c mice and obtain polyclonal antibody. Antibody was used for adhesion and inhibition assay. The immunogenicity or antigenicity of P1C was analyzed. The pcDNA3.1(+)/plc (named pP1C) monogenic nucleic acid vaccine and pcDNA3.1(+)/plc/IL-2(named pP1C-IL-2) fused gene vaccine were contructed and immunogenicity were analyzed in BALB/c mice immunized by intramuscular injection and intranasal innoculation. pcDNA3.1(+)//LTB-plc (named pLTB-P1C) fused gene vaccine was innoculated by intranasal route. An animal model for M.pneumoniae infection was established using BALB/c mice, the humoral and cellular immune response in mice induced by the vaccines and the pathological changes of lung tissue were investigated. This study may provide experimental evidence for the development of high performance and new-style M.pneumoniae nucleic acid vaccine.Methods1. M. pneumoniae clinical isolates were revived and passaged in broth medium, and then cultured in agar medium for single colony which was further characterized by PCR. p1genotyping and the variation of p1gene were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).2. P1adhesin immundominant region gene (3374-4185nucleotide, plc gene) was amplified by PCR from the genome of M. pneumoniae and cloned into pGEX6p-2after digestion with BamH I and EcoR I. A codon TGA encoding Tryptophan was changed into TGG by site-directed mutagenesis. The mutagenized recombinant expression vector pGEX6p-2/plc was transformed into E.coli BL21and induced to express PIC-GST with IPTG. The obtained fusion protein, which was purified by high affinity GST Resin, was analyzed by SDS-PAGE and Western blot. The purified protein was used to immune BALB/c mice. Then the polyclonal antibody against the purified protein was acquired and used for adhesion and inhibition assay. ELISA was used to analyze the immunogenicity and antigenicity of P1C.3. Plc gene was subcloned to pcDNA3.1(+)(named pcD) eukaryotic plasmid. The plc gene and interleukin2(IL-2) gene or B subunit of E.coli heat-labile enterotoxin (LTB) gene was fused with a linker by recombination PCR to construct pP1C-IL-2or pLTB-P1C eukaryotic expression vectors. After verifying that the P1C and P1C-IL-2antigens could be expressed in HeLa cells by Western-blot,6-week-old BALB/c mice were immunized with pP1C-IL-2or pP1C or pcDNA3.1(+)/IL-2(named pIL-2) or pcD or PBS buffer intramuscularly and nasally at2-week interval for three times, while pLTB-P1C was vaccined nasally. ELISA was used for the quantitative detection of the P1C-specific total antibodies and IgG1, IgG2a isotypes, IgA the cytokines IFN-y and IL-4in the sera and bronchoalveolar lavage fluids of BALB/c mice. The proliferation response of spleen cells was detected by MTT assay.4. Mice were inoculated by plc gene or its fusion gene respectively. Mice were exsanguinated and lungs were removed for histological examination after M.pneumoniae challenge inoculation to analyze the immune. Serial10-fold dilutions of BAL fluids were immediately cultured on SP4agar plates, quantification was performed by counting colonies on plated specimens and expressed as log10CFU per milliliter.Results1.159cases of clinical samples in PCR-RFLP indicated that142samples (89.31%) were classified as typel,17samples (10.69%)were classified as type2.21clinical variarable samples were identified by the PCR-RFLP. Among the M.pneumoniae mutants,10were V2c strains,3were V2a strains and7isolates of the point mutants of type1. A new V2d mutant strain was found in the study, which contained a novel variable region of142bp between nucleotides (nt)2772-2913and these variable regions showed sequence homology with the RepMP2/3-j (606517-606658) elements beyond the p1gene.2. The, p1c gene fragment (813bp) was successfully amplified. The prokaryotic expression recombinant pGEX-6P-2/plc was successfully constructed. The codon TGA encoding Tryptophan was changed into TGG by site-directed mutagenesis. A soluble fusion protein with molecular weight about66kDa was obtained after expression and purification. The result of Western blot showed that P1C-GST could be recognized by the monoclonal mouse anti-M.pneumoniae sera.3. The polyclonal antibody against the purified protein was acquired, the titers of the specific antibodies were above1:4000. ELISA and western blot showed the PIC-GST protein could react with the M.pneumoniae infective sera.4. P1C protein could attach to the HeLa cells and the polyclonal antibody against the P1C protein could inhibit the binding of M. pneumoniae to the HeLa cells.5. Immunization with pP1C by the intramuscular route induced a moderate antibody response, and the response was better than those obtained by nasal immunization (P<0.01). IgG1and IgG2a were the dominant subclasses. While antibody titers of mice immunized with pPlC-IL-2were markly increased and mainly of the IgG2a subclass. Nasal-immunized animals exhibited significantly higher levels (P<0.05) of anti-P1C IgA in bronchoalveolar lavage (BAL) fluids than intramuscular-immunized animals, and there was no specific antibody in the BAL fluids of mice immunized with pcD or PBS. The pP1C and pPlC-IL-2groups immunized by intramuscular injection produced significant levels (P<0.05) of IFN-y and IL-4in BAL fluids and sera than those inoculated by nasal route, and only small numbers of nonspecific IFN-y and IL-4secreting cells were detected in the control groups. The stimulation index of pP1C group or pP1C-IL-2group was higher than that of control group (P<0.05).6. M.pneumoniae inoculated Mock-vaccinated or pcDNA3.1-treated mice consisted of peribronchial and perivascular mononuclear infiltrates, but there was no intrabronchial exudate or parenchymal pneumonia (neutrophilic alveolar infiltrate). The histopathologic scores of pP1C and pP1C-IL-2-vaccinated animals were significantly decreased. The infective control mice, which inoculated with sterile SP4broth, had an HPS of0-1. There was no significant difference in the number of M. pneumoniae in BAL Fluids cultures grown from the pP1C-IL-2immunized mice compared with cultures from the pP1C inoculated group.7. The pLTB-P1C immunizations elicited high levels of IgA and IFN-y, while the proliferation response of spleen cells had no difference from pP1C vaccinated mice. When the mice were challenged intranasally with107CFU M.pneumoniae strain (M129), the LTB-plc fusion DNA vaccine conferred significantly better protection than controls (P<0.05), as characterized by lighter inflammation, lower HPS values and lower detectable number of M.pneumoniae strain. The M.pneumoniae strains in BAL fluids of pLTB-P1C immunized mice is lower than pP1C immunized mice. These results indicate that the pLTB-P1C DNA vaccine can efficiently improve protective efficacy against M.pneumoniae infection, and effectively attenuate development of Mycoplasmal pneumomae in mice.Conclusions1.159M.pneumoniae clinical isolates maily for type1strains and21mutants were detected, the mutatuon rate of type2is higher than type1. A new p1mutant strain V2d was discovered.2. P1C protein was shown to be cytadherent, strong immunogenicity and antigenicity.3. Strong cellular immunity and humoral immunity responses could be induced by all pP1C, pP1C-IL-2and pLTB-P1C DNA vaccines in BALB/c mice.4. Both pP1C-IL-2vaccine and pLTB-P1C vaccines could induce more powerful immune response and immune protection than pP1C vaccine.5. Intranasal route of pP1C vaccine Induced higher sera IgG, lower BAL fluids IgA in BALB/c mice than intramuscular immuned mice, and effected mainly in Late stage of M.pneumoniae infection. While the latter was opposite.更多还原