幽门螺杆菌外膜蛋白Omp26重组耻垢分枝杆菌活载体疫苗的研究
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摘要
背景:幽门螺杆菌(Helicobacter pylori, Hp)为定植于人胃黏膜的一种微需氧、螺旋状的革兰阴性杆菌,已被公认为是慢性胃炎、胃十二指肠溃疡的重要病因,并与胃癌、胃黏膜相关性淋巴样组织(MALT)淋巴瘤的发生密切相关,1994年被世界卫生组织定为I类致癌原(grade I carcinogen)。由于Hp的感染率极高,预防和治疗Hp感染成为消化领域和医学微生物界的研究热点。目前常用的治疗方案是抗生素加质子泵抑制剂或/及铋剂的“三联”、“四联”疗法,但药物治疗存在一定的不良反应,停药后易复发,治疗费用较高,患者依从性差及耐药菌株的不断增加等问题使其发展受到限制,而疫苗将是预防和治疗Hp感染最经济有效的方法。Hp蛋白疫苗的免疫原性较低,只有与各种强效的免疫佐剂一起接种时才具有保护作用,但这些佐剂存在较大的毒副作用而限制了其进一步发展,而重组活载体疫苗可有效解决这一难题。Hp活载体疫苗为预防和治疗Hp感染提供了一种新的策略。
耻垢分枝杆菌(Mycobacterium smegmatis,M.s)是一类来源于土壤的快速生长型分枝杆菌, 1～3 h繁殖一代,耻垢分枝杆菌既是一种非致病菌又是一种有效的细胞免疫佐剂,随着研究的深入,很多外源蛋白在耻垢分枝杆菌中获得了表达,并具有较好的免疫效果。这为研究Hp活载体疫苗提供了新的途径。
Omp26是Hp的一种外膜蛋白(Outer membrane protein,Omp),分子量为26 000,不仅可作为检测Hp的一种标志性抗原,而且具有免疫原性。由于小分子(特别是Mr为18 000和26 000)的Omp,不仅对胃癌及胃癌高发人群的筛选具有重要的价值,而且可用其免疫动物或人,清除体内的Hp和防止Hp的感染。
基于上述背景,本研究将Hp Omp26基因克隆入大肠杆菌-分枝杆菌穿梭表达载体中,然后将重组载体通过电穿孔导入M.s,筛选稳定表达Omp26基因的重组M.s菌株;将绿色荧光蛋白质粒电穿孔入构建好的重组M.s中,灌胃免疫小鼠后观察其在小鼠体内外的稳定性及毒副作用;然后将重组疫苗株灌胃免疫BALB/c小鼠或Hp感染BALB/c小鼠,在动物模型中评价重组M.s活载体疫苗预防和治疗Hp感染的效果,并对免疫预防和治疗机理进行探讨。
本研究主要分为四个部分:
第一部分:幽门螺杆菌Omp26重组耻垢分枝杆菌活载体疫苗株的构建
目的:采用基因工程技术构建幽门螺杆菌外膜蛋白Omp26大肠杆菌-分枝杆菌穿梭表达载体,并在耻垢分枝杆菌中进行表达,筛选稳定表达幽门螺杆菌Omp26的重组耻垢分枝杆菌活载体疫苗株,为进一步探讨其防治Hp感染奠定基础。
方法:利用PCR技术,以pET32a-Omp26为模板扩增Omp26基因片段,PCR产物胶回收后与载体pMD18-T连接,通过PCR、限制性酶切分析和测序鉴定阳性重组载体,然后将测序正确的重组载体经kpnI和NotI双酶切后亚克隆至大肠杆菌-分枝杆菌穿梭表达载体pLA71、pLA73、pJMas和pJHsp70中,构建分泌型和非分泌型大肠杆菌-分枝杆菌穿梭表达载体,利用电穿孔方法将其转入M.s中,构建重组耻垢分枝杆菌活载体疫苗株(rM.s),通过PCR、限制性酶切分析鉴定所构建的rM.s疫苗株的正确性,SDS-PAGE及Western blot对其表达的蛋白进行分析。
结果:采用PCR方法从pET32a-Omp26中扩增出约594bp的DNA片段,目的片段与pMD18-T载体连接后测序结果示插入的基因片段为Hp Omp26编码基因,由594bp组成,与GenBank中菌株HP AY033499序列同源性达98.8%。将测序正确的重组T载体分别亚克隆入质粒pLA71,pLA73,pJMas, pJHsp70中,构建分泌性和非分泌性穿梭表达载体, PCR和酶切鉴定穿梭表达载体构建成功。将表达载体利用电穿孔的方法导入M.s中,经卡那霉素抗性筛选、限制性酶切分析和PCR鉴定rM.s株构建成功。重组质粒pPL71-Omp26,pPL73-Omp26在M.s中表达的蛋白可分泌到细菌培养上清中,Western blotting在细胞培养上清中能检测到Omp26。重组质粒pJMas-Omp26和pJHsp70- Omp26由于缺乏信号肽,所表达的蛋白仅存在于细菌沉淀中。
结论:成功构建了重组大肠杆菌-分枝杆菌穿梭表达质粒pPL71-Omp26,pPL73-Omp26,pJMas-Omp26和pJHsp70- Omp26;成功构建了稳定表达幽门螺杆菌Omp26的重组耻垢分枝杆菌活载体疫苗株。
第二部分:幽门螺杆菌Omp26重组耻垢分枝杆菌活载体疫苗株体内外稳定性及安全性研究
目的:研究幽门螺杆菌Omp26耻垢分枝杆菌活载体疫苗株在小鼠体内外的稳定性及在小鼠体内的定植,并对疫苗的安全性进行评价。
方法:将绿色荧光蛋白质粒电转化入构建好的重组耻垢分枝杆菌活载体疫苗中,经体外传代培养,随机挑取20个菌落观察荧光表达及提取质粒判断重组质粒的稳定性;灌胃免疫BALB/c小鼠,免疫4周和8周后处死小鼠,对小鼠各内脏器官行组织学检查,并对各组织内细菌进行活菌计数及质粒提取,以判断重组耻垢分枝杆菌活载体疫苗体内的稳定性及安全性。
结果:体外稳定性研究结果表明重组耻垢分枝杆菌活载体疫苗在体外具有高度稳定性,在有卡那霉素抗性存在和无选择性压力情况下均可连续传代20次而不发生质粒丢失。应用rM.s灌胃免疫BALB/c小鼠后无明显的毒副作用出现,重组活载体疫苗可稳定地存在于胃、脾脏、肺和肝脏。
结论:绿色荧光蛋白为观测rM.s在体内的定植提供了更为直观的手段。rM.s在体内外具有高度的稳定性,无明显毒副作用。
第三部分:重组耻垢分枝杆菌活载体疫苗预防幽门螺杆菌感染的研究
目的:通过动物模型评价重组耻垢分枝杆菌活载体疫苗预防幽门螺杆菌感染的效果,明确其预防幽门螺杆菌感染的作用机制。
方法:选择标准化实验动物SPF级BALB/c小鼠构建Hp定植模型,将2×107 CFUs重组M.s疫苗(pPL73-Omp26)灌胃免疫BALB/c小鼠,同时设PBS对照组、M.s空菌组,免疫4w后,各组处死一批小鼠,取血清、胃、脾组织待用;余下的小鼠用Hp SS1攻击2次,4w后处死小鼠,行快速尿素酶试验, Hp定量培养,组织病理学检查以评价疫苗预防胃黏膜Hp感染的免疫保护作用。MTT检测脾淋巴细胞增殖;RT-PCR检测小鼠胃粘膜和脾组织中Th1细胞因子(IFN-γ、IL-2、IL-12)与Th2细胞因子(IL-4、IL-6、IL-10)的表达; ELISA检测血清Hp特异性IgG, IgA,IgG1和IgG2a抗体水平。
结果:rM.s(pPL73-Omp26)疫苗组胃黏膜中Hp数量明显低于PBS对照组和空菌组, rM.s(pPL73-Omp26)疫苗组不仅可以降低Hp的定植,而且能减轻Hp造成的小鼠胃黏膜的局部慢性炎症反应。用ConA和Hp抗原刺激后rM.s疫苗组小鼠脾淋巴细胞明显增殖。免疫后BALB/c小鼠特异性抗体显示rM.s(pPL73-Omp26)疫苗组诱导血清Hp特异性抗体IgG、IgA、IgG1和IgG2a水平均升高,以IgG2a占优势。RT-PCR结果示,疫苗灌胃免疫后4周,空菌组和重组疫苗组小鼠胃和脾淋巴细胞IL-2, IFN-γ表达量显著增加,PBS对照组无IL-2, IFN-γ表达。受Hp攻击后4周对照组和疫苗组小鼠胃组织和脾淋巴细胞IFN-γ,IL-2和IL-4均有不同程度的的表达,PBS对照组出现以IFN-γ为主的增生,空菌组出现以IL-2为主的增生,IFN-γ和IL-2水平显著高于rM.s疫苗组(P<0.05);而rM.s疫苗组则出现以Th2细胞(IL-4)为主的增生,IL-4水平显著高于PBS对照组和空菌对照组(P<0.05),各实验组Hp攻击前后均未见IL-12, IL-10,IL-6的表达。
结论: rM.s疫苗经灌胃免疫BLAB/c小鼠后不仅能降低Hp定植,而且能减轻小鼠胃黏膜的局部慢性炎症反应,对Hp感染有明显的预防保护效果。该疫苗的作用机制可能是诱导Th1和Th2两种细胞平衡型免疫应答。
第四部分:重组耻垢分枝杆菌活载体疫苗治疗幽门螺杆菌感染的研究
目的:通过动物模型评价重组耻垢分枝杆菌活载体疫苗对幽门螺杆菌感染小鼠的治疗效果,明确其治疗幽门螺杆菌感染的作用机制。
方法:建立幽门螺杆菌感染BALB/c小鼠动物模型,感染4周后将2×107 CFUs重组M.s(pPL73-Omp26)疫苗灌胃免疫幽门螺杆菌感染BALB/c小鼠,对照组用PBS或M.s空菌,免疫4w后处死小鼠,行胃组织快速尿素酶试验, Hp定量培养,组织病理学检查以评价疫苗对胃黏膜Hp感染的免疫治疗作用。MTT检测淋巴细胞增殖;RT-PCR检测小鼠胃粘膜和脾组织中Th1细胞因子(INF-γ、IL-2、IL-12)与Th2细胞因子(IL-4、IL-6、IL-10)的表达; ELISA检测血清Hp特异性抗体IgG, IgA,IgG1和IgG2a水平。
结果:rM.s(pPL73-Omp26)疫苗治疗组小鼠胃黏膜中Hp数量明显低于感染对照组和空菌组, rM.s(pPL73-Omp26)疫苗不仅可以降低Hp的定植,而且能减轻Hp造成的小鼠胃黏膜的局部慢性炎症反应。治疗后四周ConA刺激后各实验组脾淋巴细胞均有不同程度的增殖,与感染对照组和空菌对照组相比,rM.s(pPL73-Omp26)疫苗治疗组小鼠脾淋巴细胞增殖更明显(P<0.05)。治疗后BALB/c小鼠特异性抗体检测示rM.(spPL73-Omp26)疫苗治疗组小鼠血清Hp特异性抗体IgG, IgA,IgG1和IgG2a均明显高于感染对照组和空菌组(P<0.001)。RT-PCR证实,各实验组胃和脾组织IFN-γ,IL-2,IL-4均有不同程度的表达,但rM.s(pPL73-Omp26)疫苗治疗组IFN-γ,IL-2,IL-4水平明显高于感染对照组和空菌组,各实验组均未见IL-6,IL-12和IL-10的表达。
结论:成功建立了BALB/c小鼠的Hp感染模型, rM.s(pPL73-Omp26)疫苗经灌胃免疫Hp感染BLAB/c小鼠能明显降低Hp定植,减轻胃黏膜的炎症反应,对Hp感染有一定的治疗作用。其免疫治疗机理可能是rM.s产生以Th1细胞和Th2细胞协同作用的保护性免疫应答。
Background: Helicobacter pylori (H. pylori) is a spiral-shaped, gram-negative bacterium. H. pylori induces chronic inflammation of the stomach mucosa, causing chronic gastritis and peptic ulcer. Moreover, H. pylori infection is related to gastric mucosa-associated lymphoid tissue lymphoma and to an increased risk of gastric cancer,it has therefore been designated as a grade I carcinogen by the World Health Organization in 1994. Due to the high prevalence of H. pylori, the prevention and treatment of H. pylori infection has become a hot spot in the field of medical micro-organisms and gastroenterology. The current treatment against H. pylori infection consists of a combination therapy with two different antibiotics together with a proton pump inhibitor or/and bismuth.However, there are many drawbacks associated with this treatment such as antibiotic resistance, recurrence, reinfection and high cost. So, Vaccination of humans could be an effective and economic approach to prevent and treat H. pylori infection all over the world. However, the protein vaccines of H. pylori have poor immunogenicity, most vaccines need strong mucosal adjuvant to induce protective immune. But mucosal adjuvant have strong toxic side effects, which limit its further development. A recombinant live vector vaccine which provide a new way for the study of H. pylori vaccines can be an effective solution to this problem .
Mycobacterium smegmatis(M. smegmatis) is a species of rapid growing mycobacteria from soil. It can propagate one generation every 1-3 hours. It is a non-pathegent bacterium and effective cell immunity adjuvant. With the development of molecular biology and genetic engineering technique, many exogenous proteins can be expressed in M. smegmatis, and had good immune effect .It provides a new way for live vector vaccine of H. pylori.
Omp26 is an outer membrane protein of H. pylori, its molecular weight is 26000, The Omp26 not only can be used as an antigen for detecting H. pylori infection, but also have immunogenicity. The Omp with small molecules (in particular, Mr 18 000 and 26 000) not only have an important value for screening gastric cancer and gastric cancer with high-risk population, and can induce immune response in animal or human to eradicate and prevent H. pylori infection.
In this study, the Omp26 gene of H. pylori were cloned into the E.coli–mycobacterium shuttle vector , the recombinant vectors were transformed into M. smegmatis by electroporation, the positive strains of the recombinant M. smegmatis(rM.smegmatis) were identified by restriction map、PCR and western blotting. Plasmid pEGFP was transformed into rM.smegmatis by electroporation,the stability and toxic side effect of rM.smegmatis vaccine were observed. The rM.smegmatis vaccine strains were orally administrated to the BALB/c mice or H. pylori-infected BALB/c mice with gastric tube, the prophylactic or therapeutic effect against H. pylori and immune mechanism of the rM.smegmatis were investigated as a fundament for the development of H. pylori vaccine. H. pylori live vector vaccine provide a new method for preventing and treating H. pylori infection.
This research can be divided into four parts:
Part 1: Construction of recombinant M. smegmatis live vector vaccine expressing outer membrane protein 26kDa antigen of H. pylori
Objective: To construct the E.coli–mycobacterium shuttle expression vector of outer membrane protein 26kDa antigen (Omp26) of Helicobacter pylori and express in M. smegmatis by gene-engineering, and screen recombinant M. smegmatis live vector vaccine expressing outer membrane protein 26kDa antigen of H. pylori, which would lay a foundation for prophylaxis and therapy of H. pylori infection.
Methods: Omp26 gene was amplified from pET32a-Omp26 by PCR and cloned into plasmid pMD18-T, after identification by PCR ,enzyme digestion analysis and sequencing, Omp26 was subcloned into E.coli-mycobacterium shuttle vector ( pLA71,pLA73,pJMas, pJHsp70) that were digested with KpnI and NotI resulting in E.coli-mycobacterium secretive expressing and non-secretive expressing shuttle plasmid. The recombinant vectors were transformed into M. smegmatis by electroporation. The rM. smegmatis was identified by restriction map and PCR, and the expressing product was detected by SDS-PAGE and western blotting.
Results: 594 bp DNA fragment was amplified from pET32a-Omp26 by PCR, sequencing showed that the target gene was Omp26 at length of 594 bp. As compared with gene HP AY033499 in GenBank, cloned Omp26 gene sequence homology was up to 98.8%. Omp26 was subcloned into E.coli-mycobacterium shuttle vector, E.coli-mycobacterium secretive expressing and non-secretive expressing shuttle plasmid were successfully constructed. The recombinant vectors were transformed into M. smegmatis by electroporation, then the rM. smegmatis were identified through kan gene resistance screening, restriction map, PCR technique. Omp26 were secreted into culture supernatant in pPL71-Omp26 and pPL73-Omp26 with signal peptide sequence, and this secreted protein could be recognized by anti-H. pylori antiserum. Otherwise, the protein was expressed in a non-secreted way in the pJMas-Omp26 and pJHsp70- Omp26 due to lack of the coding region of the signal peptide.
Conclusion: Recombinant shuttle plasmid pPL71-Omp26, pPL73-Omp26, pJMas-Omp26 and pJHsp70- Omp26 were successfully constructed . Recombinant M. smegmatis strains expressing Omp26 of H. pylori were successfully constructed.
Part 2: Stability , security and colonization of rM. smegmatis In vitro and vivo
Objective: To study the stability of rM. smegmatis in vitro and vivo, security and colonization in vivo.
Methods: The pEGFP vector was then transformed into the rM. smegmatis by electroporation. Twenty colonies were randomly picked to observe fluorescence and take swift extracting plasmid to determine the stability of the recombinant plasmid. The mice were sacrificed 4 weeks and 8 weeks after orally immunized with rM. smegmatis, stability and safety of rM. Smegmatis were evaluated by histology, viable bacterial counts and plasmid extraction.
Results: The stability of the rM.semgmatis in vitro was studied, the result showed that the rM. semgmatis is stable when it growed with and without Kan resistence . It could contiune propagate for 20 generation. The rM. semgmatis could stably colonize in the gastric mucosa, spleen, lung and liver for 4 weeks after immunization by intragastric administration, and no complications were observed.
Conclusion: Green fluorescent protein could be used as a molecular marker in the investigation of screening recomninant vaccine, rM. smegmatis is a particularly attractive vector for the delivery of heterologous antigens with high stability and low toxicity.
Part 3: The prophylactic effect of rM. semgmatis live vector vaccine against H. pylori infection
Objective: To evaluate the prophylactic effect of rM. semgmatis against H. pylori infection in animal model, and study the mechanism of prophylactic protection.
Methods: 2×107 CFUs of rM. smegmatis strain were orally administrated to the BALB/c mice with gastric tube. At the same time, the PBS and M. smegmatis were set as the control groups, respectively. 4 weeks after immunization a batch of mice in each group were killed, and their serum, stomach and spleen were kept for future use. The remaining were attacked twice by H. pylori SS1 and then killed 4 weeks later. The immunoprotection effect of the gastric mucosa against H. pylori infection were evaluated by the rapid urea assay, H. pylori quantitative cultivation and histological examination. Multiplication of mouse spleen lymphocyte was detected by MTT; The expression of cytokines in stomach and spleen were analyzed by RT-PCR 4 weeks after immunity and challenge. The levels of the H. pylori specific serum IgG, IgA, IgG1 and IgG2a were detected by ELISA analysis.
Results: The number of H. pylori in rM. Smegmatis(pPL73-Om26) were much lower than that of the PBS and the M. smegmatis groups. Meanwhile, the reduction of H. pylori Colonization and decrease of chronic gastritis were observed in rM. Smegmatis(pPL73-Om26). Oral immunization with rM. Smegmatis(pPL73-Om26) have significant proliferation of spleen lymphocyte after the stimulation of ConA and H. pylori antigens. The specific serum IgG , IgA, IgG1 and IgG2a antibodies were significantly induced with Oral immunization of rM. smegmatis(pPL73-Om26). RT-PCR of cytokines revealed that M. smegmatis and rM. smegmatis(pPL73-Om26) group had relatively high levels of mRNA for IFN-γand IL-2, but the levels of IFN-γand IL-2 in PBS group were negative 4 weeks after immunity. After attacked by H. pylori, the levels of IFN-γand IL-2 mRNA expression in rM. smegmatis group were significantly lower than that of the PBS and M.smegmatis group (P<0.05). While the level of IL-4 mRNA expression in rM. smegmatis group were significantly higher than that of the PBS and M. smegmatis group (P<0.05). Other cytokines such as IL-6, IL-12 and IL-10 were not detected in all groups.
Conclusion: The rM. smegmatis by intragastric administration not only can reduce the H. pylori colonization, but also relieve the gastric inflammation reaction and prevent H. pylori infection. The mechanism of prophylactic protection was that rM. smegmatis vaccine induced Th1 and Th2 balanced immune response.
Part 4: The therapeutic effect of rM. semgmatis live vector vaccine against H. pylori infection
Objective: To evaluate the therapeutic effect of rM. semgmatis against H. pylori infection in animal model, and study the mechanism of therapeutic protection.
Methods: H. pylori infected BLAB/c mice model was constructed, four weeks after infection, 2×107 CFUs of rM. smegmatis(pPL73-Omp26) or M. smegmatis were orally administrated to the H. pylori infected mice, using PBS as negative control. Mice in each group were killed four weeks after immunization, the therapeutic protection against H. pylori infection were evaluated by the rapid urea test, H. pylori quantitative cultivation and histological examination. Multiplication of mouse spleen lymphocyte was detected by MTT; The concentrations of Th1 cytokines (IFN-γ, IL-2, IL-12) and Th2 cytokines (IL-4, IL-6, IL-10) in stomach and spleen were assessed by RT-PCR. The levels of the H. pylori specific serum IgG, IgA, IgG1 and IgG2a were detected by ELISA analysis.
Results: Oral therapeutic immunization with rM. smegmatis (pPL73-Omp26) induced a significant reduction in the bacterial load in the stomachs of H. pylori-infected mice, which was comparable to the reduction induced by oral immunization with PBS or M. smegmatis. And the gastric histology of mice infected by H. pylori revealed less severe inflammatory infiltration and gastric damage. Oral immunization with rM. Smegmatis(pPL73-Omp26) have significant proliferation of spleen lymphocyte after the stimulation of ConA ,which was comparable to the proliferation induced by oral immunization with PBS or M. smegmatis .The levels of specific serum IgG , IgA, IgG1 and IgG2a antibodies were significantly increased after oral immunization with rM. smegmatis (pPL73-Omp26) (p< 0.001). RT-PCR of cytokines revealed that mice immunized with rM. smegmatis (pPL73-Omp26) resulted in increased levels of IFN-γ,IL-2 and IL-4 in the stomach and spleen lymphocyte compared to control infected mice (p < 0.05), the cytokines IL-6,IL-12 and IL-10 were not detected in all groups.
Conclusion: H. pylori infected BLAB/c mice model was successfully constructed in this study. The rM. smegmatis by intragastric administration could reduce the H. pylori colonization, relieve the gastric inflammation and treat H. pylori infection.These results showed that the rM. smegmatis vaccine could induce Th1 and Th2 cell-mediated immune responses, and boost antigen-specific Th 1/ Th 2 balanced type humoral immune response.
耻垢分枝杆菌(Mycobacterium smegmatis,M.s)是一类来源于土壤的快速生长型分枝杆菌, 1～3 h繁殖一代,耻垢分枝杆菌既是一种非致病菌又是一种有效的细胞免疫佐剂,随着研究的深入,很多外源蛋白在耻垢分枝杆菌中获得了表达,并具有较好的免疫效果。这为研究Hp活载体疫苗提供了新的途径。
Omp26是Hp的一种外膜蛋白(Outer membrane protein,Omp),分子量为26 000,不仅可作为检测Hp的一种标志性抗原,而且具有免疫原性。由于小分子(特别是Mr为18 000和26 000)的Omp,不仅对胃癌及胃癌高发人群的筛选具有重要的价值,而且可用其免疫动物或人,清除体内的Hp和防止Hp的感染。
基于上述背景,本研究将Hp Omp26基因克隆入大肠杆菌-分枝杆菌穿梭表达载体中,然后将重组载体通过电穿孔导入M.s,筛选稳定表达Omp26基因的重组M.s菌株;将绿色荧光蛋白质粒电穿孔入构建好的重组M.s中,灌胃免疫小鼠后观察其在小鼠体内外的稳定性及毒副作用;然后将重组疫苗株灌胃免疫BALB/c小鼠或Hp感染BALB/c小鼠,在动物模型中评价重组M.s活载体疫苗预防和治疗Hp感染的效果,并对免疫预防和治疗机理进行探讨。
本研究主要分为四个部分:
第一部分:幽门螺杆菌Omp26重组耻垢分枝杆菌活载体疫苗株的构建
目的:采用基因工程技术构建幽门螺杆菌外膜蛋白Omp26大肠杆菌-分枝杆菌穿梭表达载体,并在耻垢分枝杆菌中进行表达,筛选稳定表达幽门螺杆菌Omp26的重组耻垢分枝杆菌活载体疫苗株,为进一步探讨其防治Hp感染奠定基础。
方法:利用PCR技术,以pET32a-Omp26为模板扩增Omp26基因片段,PCR产物胶回收后与载体pMD18-T连接,通过PCR、限制性酶切分析和测序鉴定阳性重组载体,然后将测序正确的重组载体经kpnI和NotI双酶切后亚克隆至大肠杆菌-分枝杆菌穿梭表达载体pLA71、pLA73、pJMas和pJHsp70中,构建分泌型和非分泌型大肠杆菌-分枝杆菌穿梭表达载体,利用电穿孔方法将其转入M.s中,构建重组耻垢分枝杆菌活载体疫苗株(rM.s),通过PCR、限制性酶切分析鉴定所构建的rM.s疫苗株的正确性,SDS-PAGE及Western blot对其表达的蛋白进行分析。
结果:采用PCR方法从pET32a-Omp26中扩增出约594bp的DNA片段,目的片段与pMD18-T载体连接后测序结果示插入的基因片段为Hp Omp26编码基因,由594bp组成,与GenBank中菌株HP AY033499序列同源性达98.8%。将测序正确的重组T载体分别亚克隆入质粒pLA71,pLA73,pJMas, pJHsp70中,构建分泌性和非分泌性穿梭表达载体, PCR和酶切鉴定穿梭表达载体构建成功。将表达载体利用电穿孔的方法导入M.s中,经卡那霉素抗性筛选、限制性酶切分析和PCR鉴定rM.s株构建成功。重组质粒pPL71-Omp26,pPL73-Omp26在M.s中表达的蛋白可分泌到细菌培养上清中,Western blotting在细胞培养上清中能检测到Omp26。重组质粒pJMas-Omp26和pJHsp70- Omp26由于缺乏信号肽,所表达的蛋白仅存在于细菌沉淀中。
结论:成功构建了重组大肠杆菌-分枝杆菌穿梭表达质粒pPL71-Omp26,pPL73-Omp26,pJMas-Omp26和pJHsp70- Omp26;成功构建了稳定表达幽门螺杆菌Omp26的重组耻垢分枝杆菌活载体疫苗株。
第二部分:幽门螺杆菌Omp26重组耻垢分枝杆菌活载体疫苗株体内外稳定性及安全性研究
目的:研究幽门螺杆菌Omp26耻垢分枝杆菌活载体疫苗株在小鼠体内外的稳定性及在小鼠体内的定植,并对疫苗的安全性进行评价。
方法:将绿色荧光蛋白质粒电转化入构建好的重组耻垢分枝杆菌活载体疫苗中,经体外传代培养,随机挑取20个菌落观察荧光表达及提取质粒判断重组质粒的稳定性;灌胃免疫BALB/c小鼠,免疫4周和8周后处死小鼠,对小鼠各内脏器官行组织学检查,并对各组织内细菌进行活菌计数及质粒提取,以判断重组耻垢分枝杆菌活载体疫苗体内的稳定性及安全性。
结果:体外稳定性研究结果表明重组耻垢分枝杆菌活载体疫苗在体外具有高度稳定性,在有卡那霉素抗性存在和无选择性压力情况下均可连续传代20次而不发生质粒丢失。应用rM.s灌胃免疫BALB/c小鼠后无明显的毒副作用出现,重组活载体疫苗可稳定地存在于胃、脾脏、肺和肝脏。
结论:绿色荧光蛋白为观测rM.s在体内的定植提供了更为直观的手段。rM.s在体内外具有高度的稳定性,无明显毒副作用。
第三部分:重组耻垢分枝杆菌活载体疫苗预防幽门螺杆菌感染的研究
目的:通过动物模型评价重组耻垢分枝杆菌活载体疫苗预防幽门螺杆菌感染的效果,明确其预防幽门螺杆菌感染的作用机制。
方法:选择标准化实验动物SPF级BALB/c小鼠构建Hp定植模型,将2×107 CFUs重组M.s疫苗(pPL73-Omp26)灌胃免疫BALB/c小鼠,同时设PBS对照组、M.s空菌组,免疫4w后,各组处死一批小鼠,取血清、胃、脾组织待用;余下的小鼠用Hp SS1攻击2次,4w后处死小鼠,行快速尿素酶试验, Hp定量培养,组织病理学检查以评价疫苗预防胃黏膜Hp感染的免疫保护作用。MTT检测脾淋巴细胞增殖;RT-PCR检测小鼠胃粘膜和脾组织中Th1细胞因子(IFN-γ、IL-2、IL-12)与Th2细胞因子(IL-4、IL-6、IL-10)的表达; ELISA检测血清Hp特异性IgG, IgA,IgG1和IgG2a抗体水平。
结果:rM.s(pPL73-Omp26)疫苗组胃黏膜中Hp数量明显低于PBS对照组和空菌组, rM.s(pPL73-Omp26)疫苗组不仅可以降低Hp的定植,而且能减轻Hp造成的小鼠胃黏膜的局部慢性炎症反应。用ConA和Hp抗原刺激后rM.s疫苗组小鼠脾淋巴细胞明显增殖。免疫后BALB/c小鼠特异性抗体显示rM.s(pPL73-Omp26)疫苗组诱导血清Hp特异性抗体IgG、IgA、IgG1和IgG2a水平均升高,以IgG2a占优势。RT-PCR结果示,疫苗灌胃免疫后4周,空菌组和重组疫苗组小鼠胃和脾淋巴细胞IL-2, IFN-γ表达量显著增加,PBS对照组无IL-2, IFN-γ表达。受Hp攻击后4周对照组和疫苗组小鼠胃组织和脾淋巴细胞IFN-γ,IL-2和IL-4均有不同程度的的表达,PBS对照组出现以IFN-γ为主的增生,空菌组出现以IL-2为主的增生,IFN-γ和IL-2水平显著高于rM.s疫苗组(P<0.05);而rM.s疫苗组则出现以Th2细胞(IL-4)为主的增生,IL-4水平显著高于PBS对照组和空菌对照组(P<0.05),各实验组Hp攻击前后均未见IL-12, IL-10,IL-6的表达。
结论: rM.s疫苗经灌胃免疫BLAB/c小鼠后不仅能降低Hp定植,而且能减轻小鼠胃黏膜的局部慢性炎症反应,对Hp感染有明显的预防保护效果。该疫苗的作用机制可能是诱导Th1和Th2两种细胞平衡型免疫应答。
第四部分:重组耻垢分枝杆菌活载体疫苗治疗幽门螺杆菌感染的研究
目的:通过动物模型评价重组耻垢分枝杆菌活载体疫苗对幽门螺杆菌感染小鼠的治疗效果,明确其治疗幽门螺杆菌感染的作用机制。
方法:建立幽门螺杆菌感染BALB/c小鼠动物模型,感染4周后将2×107 CFUs重组M.s(pPL73-Omp26)疫苗灌胃免疫幽门螺杆菌感染BALB/c小鼠,对照组用PBS或M.s空菌,免疫4w后处死小鼠,行胃组织快速尿素酶试验, Hp定量培养,组织病理学检查以评价疫苗对胃黏膜Hp感染的免疫治疗作用。MTT检测淋巴细胞增殖;RT-PCR检测小鼠胃粘膜和脾组织中Th1细胞因子(INF-γ、IL-2、IL-12)与Th2细胞因子(IL-4、IL-6、IL-10)的表达; ELISA检测血清Hp特异性抗体IgG, IgA,IgG1和IgG2a水平。
结果:rM.s(pPL73-Omp26)疫苗治疗组小鼠胃黏膜中Hp数量明显低于感染对照组和空菌组, rM.s(pPL73-Omp26)疫苗不仅可以降低Hp的定植,而且能减轻Hp造成的小鼠胃黏膜的局部慢性炎症反应。治疗后四周ConA刺激后各实验组脾淋巴细胞均有不同程度的增殖,与感染对照组和空菌对照组相比,rM.s(pPL73-Omp26)疫苗治疗组小鼠脾淋巴细胞增殖更明显(P<0.05)。治疗后BALB/c小鼠特异性抗体检测示rM.(spPL73-Omp26)疫苗治疗组小鼠血清Hp特异性抗体IgG, IgA,IgG1和IgG2a均明显高于感染对照组和空菌组(P<0.001)。RT-PCR证实,各实验组胃和脾组织IFN-γ,IL-2,IL-4均有不同程度的表达,但rM.s(pPL73-Omp26)疫苗治疗组IFN-γ,IL-2,IL-4水平明显高于感染对照组和空菌组,各实验组均未见IL-6,IL-12和IL-10的表达。
结论:成功建立了BALB/c小鼠的Hp感染模型, rM.s(pPL73-Omp26)疫苗经灌胃免疫Hp感染BLAB/c小鼠能明显降低Hp定植,减轻胃黏膜的炎症反应,对Hp感染有一定的治疗作用。其免疫治疗机理可能是rM.s产生以Th1细胞和Th2细胞协同作用的保护性免疫应答。
Background: Helicobacter pylori (H. pylori) is a spiral-shaped, gram-negative bacterium. H. pylori induces chronic inflammation of the stomach mucosa, causing chronic gastritis and peptic ulcer. Moreover, H. pylori infection is related to gastric mucosa-associated lymphoid tissue lymphoma and to an increased risk of gastric cancer,it has therefore been designated as a grade I carcinogen by the World Health Organization in 1994. Due to the high prevalence of H. pylori, the prevention and treatment of H. pylori infection has become a hot spot in the field of medical micro-organisms and gastroenterology. The current treatment against H. pylori infection consists of a combination therapy with two different antibiotics together with a proton pump inhibitor or/and bismuth.However, there are many drawbacks associated with this treatment such as antibiotic resistance, recurrence, reinfection and high cost. So, Vaccination of humans could be an effective and economic approach to prevent and treat H. pylori infection all over the world. However, the protein vaccines of H. pylori have poor immunogenicity, most vaccines need strong mucosal adjuvant to induce protective immune. But mucosal adjuvant have strong toxic side effects, which limit its further development. A recombinant live vector vaccine which provide a new way for the study of H. pylori vaccines can be an effective solution to this problem .
Mycobacterium smegmatis(M. smegmatis) is a species of rapid growing mycobacteria from soil. It can propagate one generation every 1-3 hours. It is a non-pathegent bacterium and effective cell immunity adjuvant. With the development of molecular biology and genetic engineering technique, many exogenous proteins can be expressed in M. smegmatis, and had good immune effect .It provides a new way for live vector vaccine of H. pylori.
Omp26 is an outer membrane protein of H. pylori, its molecular weight is 26000, The Omp26 not only can be used as an antigen for detecting H. pylori infection, but also have immunogenicity. The Omp with small molecules (in particular, Mr 18 000 and 26 000) not only have an important value for screening gastric cancer and gastric cancer with high-risk population, and can induce immune response in animal or human to eradicate and prevent H. pylori infection.
In this study, the Omp26 gene of H. pylori were cloned into the E.coli–mycobacterium shuttle vector , the recombinant vectors were transformed into M. smegmatis by electroporation, the positive strains of the recombinant M. smegmatis(rM.smegmatis) were identified by restriction map、PCR and western blotting. Plasmid pEGFP was transformed into rM.smegmatis by electroporation,the stability and toxic side effect of rM.smegmatis vaccine were observed. The rM.smegmatis vaccine strains were orally administrated to the BALB/c mice or H. pylori-infected BALB/c mice with gastric tube, the prophylactic or therapeutic effect against H. pylori and immune mechanism of the rM.smegmatis were investigated as a fundament for the development of H. pylori vaccine. H. pylori live vector vaccine provide a new method for preventing and treating H. pylori infection.
This research can be divided into four parts:
Part 1: Construction of recombinant M. smegmatis live vector vaccine expressing outer membrane protein 26kDa antigen of H. pylori
Objective: To construct the E.coli–mycobacterium shuttle expression vector of outer membrane protein 26kDa antigen (Omp26) of Helicobacter pylori and express in M. smegmatis by gene-engineering, and screen recombinant M. smegmatis live vector vaccine expressing outer membrane protein 26kDa antigen of H. pylori, which would lay a foundation for prophylaxis and therapy of H. pylori infection.
Methods: Omp26 gene was amplified from pET32a-Omp26 by PCR and cloned into plasmid pMD18-T, after identification by PCR ,enzyme digestion analysis and sequencing, Omp26 was subcloned into E.coli-mycobacterium shuttle vector ( pLA71,pLA73,pJMas, pJHsp70) that were digested with KpnI and NotI resulting in E.coli-mycobacterium secretive expressing and non-secretive expressing shuttle plasmid. The recombinant vectors were transformed into M. smegmatis by electroporation. The rM. smegmatis was identified by restriction map and PCR, and the expressing product was detected by SDS-PAGE and western blotting.
Results: 594 bp DNA fragment was amplified from pET32a-Omp26 by PCR, sequencing showed that the target gene was Omp26 at length of 594 bp. As compared with gene HP AY033499 in GenBank, cloned Omp26 gene sequence homology was up to 98.8%. Omp26 was subcloned into E.coli-mycobacterium shuttle vector, E.coli-mycobacterium secretive expressing and non-secretive expressing shuttle plasmid were successfully constructed. The recombinant vectors were transformed into M. smegmatis by electroporation, then the rM. smegmatis were identified through kan gene resistance screening, restriction map, PCR technique. Omp26 were secreted into culture supernatant in pPL71-Omp26 and pPL73-Omp26 with signal peptide sequence, and this secreted protein could be recognized by anti-H. pylori antiserum. Otherwise, the protein was expressed in a non-secreted way in the pJMas-Omp26 and pJHsp70- Omp26 due to lack of the coding region of the signal peptide.
Conclusion: Recombinant shuttle plasmid pPL71-Omp26, pPL73-Omp26, pJMas-Omp26 and pJHsp70- Omp26 were successfully constructed . Recombinant M. smegmatis strains expressing Omp26 of H. pylori were successfully constructed.
Part 2: Stability , security and colonization of rM. smegmatis In vitro and vivo
Objective: To study the stability of rM. smegmatis in vitro and vivo, security and colonization in vivo.
Methods: The pEGFP vector was then transformed into the rM. smegmatis by electroporation. Twenty colonies were randomly picked to observe fluorescence and take swift extracting plasmid to determine the stability of the recombinant plasmid. The mice were sacrificed 4 weeks and 8 weeks after orally immunized with rM. smegmatis, stability and safety of rM. Smegmatis were evaluated by histology, viable bacterial counts and plasmid extraction.
Results: The stability of the rM.semgmatis in vitro was studied, the result showed that the rM. semgmatis is stable when it growed with and without Kan resistence . It could contiune propagate for 20 generation. The rM. semgmatis could stably colonize in the gastric mucosa, spleen, lung and liver for 4 weeks after immunization by intragastric administration, and no complications were observed.
Conclusion: Green fluorescent protein could be used as a molecular marker in the investigation of screening recomninant vaccine, rM. smegmatis is a particularly attractive vector for the delivery of heterologous antigens with high stability and low toxicity.
Part 3: The prophylactic effect of rM. semgmatis live vector vaccine against H. pylori infection
Objective: To evaluate the prophylactic effect of rM. semgmatis against H. pylori infection in animal model, and study the mechanism of prophylactic protection.
Methods: 2×107 CFUs of rM. smegmatis strain were orally administrated to the BALB/c mice with gastric tube. At the same time, the PBS and M. smegmatis were set as the control groups, respectively. 4 weeks after immunization a batch of mice in each group were killed, and their serum, stomach and spleen were kept for future use. The remaining were attacked twice by H. pylori SS1 and then killed 4 weeks later. The immunoprotection effect of the gastric mucosa against H. pylori infection were evaluated by the rapid urea assay, H. pylori quantitative cultivation and histological examination. Multiplication of mouse spleen lymphocyte was detected by MTT; The expression of cytokines in stomach and spleen were analyzed by RT-PCR 4 weeks after immunity and challenge. The levels of the H. pylori specific serum IgG, IgA, IgG1 and IgG2a were detected by ELISA analysis.
Results: The number of H. pylori in rM. Smegmatis(pPL73-Om26) were much lower than that of the PBS and the M. smegmatis groups. Meanwhile, the reduction of H. pylori Colonization and decrease of chronic gastritis were observed in rM. Smegmatis(pPL73-Om26). Oral immunization with rM. Smegmatis(pPL73-Om26) have significant proliferation of spleen lymphocyte after the stimulation of ConA and H. pylori antigens. The specific serum IgG , IgA, IgG1 and IgG2a antibodies were significantly induced with Oral immunization of rM. smegmatis(pPL73-Om26). RT-PCR of cytokines revealed that M. smegmatis and rM. smegmatis(pPL73-Om26) group had relatively high levels of mRNA for IFN-γand IL-2, but the levels of IFN-γand IL-2 in PBS group were negative 4 weeks after immunity. After attacked by H. pylori, the levels of IFN-γand IL-2 mRNA expression in rM. smegmatis group were significantly lower than that of the PBS and M.smegmatis group (P<0.05). While the level of IL-4 mRNA expression in rM. smegmatis group were significantly higher than that of the PBS and M. smegmatis group (P<0.05). Other cytokines such as IL-6, IL-12 and IL-10 were not detected in all groups.
Conclusion: The rM. smegmatis by intragastric administration not only can reduce the H. pylori colonization, but also relieve the gastric inflammation reaction and prevent H. pylori infection. The mechanism of prophylactic protection was that rM. smegmatis vaccine induced Th1 and Th2 balanced immune response.
Part 4: The therapeutic effect of rM. semgmatis live vector vaccine against H. pylori infection
Objective: To evaluate the therapeutic effect of rM. semgmatis against H. pylori infection in animal model, and study the mechanism of therapeutic protection.
Methods: H. pylori infected BLAB/c mice model was constructed, four weeks after infection, 2×107 CFUs of rM. smegmatis(pPL73-Omp26) or M. smegmatis were orally administrated to the H. pylori infected mice, using PBS as negative control. Mice in each group were killed four weeks after immunization, the therapeutic protection against H. pylori infection were evaluated by the rapid urea test, H. pylori quantitative cultivation and histological examination. Multiplication of mouse spleen lymphocyte was detected by MTT; The concentrations of Th1 cytokines (IFN-γ, IL-2, IL-12) and Th2 cytokines (IL-4, IL-6, IL-10) in stomach and spleen were assessed by RT-PCR. The levels of the H. pylori specific serum IgG, IgA, IgG1 and IgG2a were detected by ELISA analysis.
Results: Oral therapeutic immunization with rM. smegmatis (pPL73-Omp26) induced a significant reduction in the bacterial load in the stomachs of H. pylori-infected mice, which was comparable to the reduction induced by oral immunization with PBS or M. smegmatis. And the gastric histology of mice infected by H. pylori revealed less severe inflammatory infiltration and gastric damage. Oral immunization with rM. Smegmatis(pPL73-Omp26) have significant proliferation of spleen lymphocyte after the stimulation of ConA ,which was comparable to the proliferation induced by oral immunization with PBS or M. smegmatis .The levels of specific serum IgG , IgA, IgG1 and IgG2a antibodies were significantly increased after oral immunization with rM. smegmatis (pPL73-Omp26) (p< 0.001). RT-PCR of cytokines revealed that mice immunized with rM. smegmatis (pPL73-Omp26) resulted in increased levels of IFN-γ,IL-2 and IL-4 in the stomach and spleen lymphocyte compared to control infected mice (p < 0.05), the cytokines IL-6,IL-12 and IL-10 were not detected in all groups.
Conclusion: H. pylori infected BLAB/c mice model was successfully constructed in this study. The rM. smegmatis by intragastric administration could reduce the H. pylori colonization, relieve the gastric inflammation and treat H. pylori infection.These results showed that the rM. smegmatis vaccine could induce Th1 and Th2 cell-mediated immune responses, and boost antigen-specific Th 1/ Th 2 balanced type humoral immune response.
引文
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