【作者】 余光清；

【导师】 李雍龙；

【作者基本信息】 华中科技大学 ， 病原生物学， 2006， 博士

【副题名】重组杆状病毒用于日本血吸虫DNA疫苗的研究

【摘要】 目前血吸虫DNA疫苗在抗血吸虫疫苗研究中占据着主导地位。质粒载体是最常用的DNA疫苗载体，由于质粒转染效率较低，影响了编码抗原的有效表达，导致机体不能诱导足够有效的免疫应答，因此难以取得理想的抗血吸虫感染的保护效果。同时质粒载体的容量较小可能会影响到大片段抗原分子和多价疫苗的应用。如何增强DNA疫苗的免疫效果已成为当前抗血吸虫疫苗研究的重点。病毒载体中的哺乳动物病毒载体如痘苗病毒和腺病毒载体也可用于构建DNA疫苗，虽然哺乳动物病毒载体克服了质粒的转染效率和容量的不足，但由于自身的免疫原性和体内存在针对这些病毒载体的抗体，应用上也受到了限制。因此发展新的疫苗载体必将有助于血吸虫疫苗的研究。而杆状病毒属于昆虫病毒，动物体内缺乏预先存在的抗体。杆状病毒载体除具备常用病毒载体的优势外，还具有低毒、安全、制备简单和易于得到高滴度的病毒粒子等优点，这些为杆状病毒开发成DNA疫苗的载体提供了理论基础。在伪狂犬病病毒、丙型肝炎病毒和鼠疟疾伯氏疟原虫研究中，重组杆状病毒介导的外源基因的表达也能引起机体的特异性免疫反应，这些为杆状病毒开发成DNA疫苗的载体提供了实验依据。此外，DNA疫苗和蛋白疫苗联合免疫也可以提高DNA疫苗的免疫效果。本研究选用日本血吸虫26 kDa谷胱甘肽S-转移酶(Sj26)作为抗原分子，一方面探讨杆状病毒作为日本血吸虫DNA疫苗载体的可行性；另一方面观察DNA疫苗和蛋白疫苗联合免疫的协同作用。一、日本血吸虫Sj26重组质粒DNA疫苗和重组蛋白疫苗联合免疫的保护作用研究目的：观察含有增强型绿色荧光蛋白(enhanced green fluorescent protein，EGFP)的真核表达载体pEGFP-N3构建的重组质粒pEGFP-Sj26的体外表达、免疫保护效应及其与日本血吸虫26 kDa谷胱甘肽S-转移酶重组蛋白(rSj26 GST)联合免疫对小鼠抗日本血吸虫感染的保护作用。方法：PCR扩增Sj26基因并连入T载体进行序列测定，正确重组的质粒命名为pT1-Sj26。再将Sj26基因定向克隆入含有EGFP的真核表达载体pEGFP-N3，构建重组质粒pEGFP-Sj26。pEGFP-Sj26体外转染幼仓鼠肾(baby hamster kidney，BHK)细胞，用荧光显微镜和蛋白质印迹法(Western blot)检测蛋白表达。60只BALB／c小鼠随机分为A、B、C、D、E 5组，每组12只，每只小鼠均免疫3次，间隔2周。A组为PBS对照组，B组为pEGFP-N3组，C组为pEGFP-Sj26组，D组为rSj26 GST组，E组为pEGFP-Sj26+rSj26 GST联合免疫组。联合免疫组先用pEGFP-Sj26免疫2次，再用rSj26 GST加强免疫1次。以上各组均于末次免疫后2周进行攻击感染，感染45d后剖杀，计数虫荷和肝内虫卵数，计算减虫率和肝组织中每条雌虫减卵率。结果：PCR扩增产物连入T载体测序结果显示Sj26基因与文献报道的序列一致性为100％。重组质粒pEGFP-Sj26构建正确并成功转染BHK细胞，荧光显微镜下观察到绿色荧光表达，Western blot证实了pEGFP-Sj26能够表达Sj26-EGFP融合蛋白。动物保护性实验显示，pEGFP-Sj26和rSj26 GST联合免疫组的减虫率为50.8％，显著高于pEGFP-Sj26单独免疫组(28.0％)和rSj26 GST单独免疫组(25.5％)。联合免疫组、rSj26 GST组和pEGFP-Sj26组减卵率分别为32.7％、33.0％和20.6％。与PBS对照组相比，联合免疫组和rSj26 GST单独免疫组肝组织中每条雌虫产卵数差异有显著性(P＜0.01)，pEGFP-Sj26单独免疫组差异无显著性(P＞0.05)。结论：含有增强型绿色荧光蛋白的重组质粒pEGFP-Sj26能成功地在哺乳动物细胞中表达；重组质粒DNA疫苗(pEGFP-Sj26)免疫对小鼠具有保护作用；pEGFP-Sj26和重组蛋白疫苗(rSj26 GST)联合免疫可以增强小鼠抗日本血吸虫感染的保护作用。二、重组杆状病毒介导的日本血吸虫Sj26 DNA疫苗和重组蛋白疫苗联合免疫的保护作用研究目的：构建日本血吸虫26 kDa谷胱甘肽S-转移酶(Sj26)重组杆状病毒并观察其在哺乳动物细胞中的表达；探讨杆状病毒作为日本血吸虫DNA疫苗载体的可行性；观察重组杆状病毒介导的Sj26 DNA疫苗和重组蛋白(rSj26 GST)疫苗联合免疫对小鼠抗日本血吸虫感染的保护作用。方法：PCR扩增Sj26基因并连入T载体进行序列测定，正确重组的质粒命名为pT2-Sj26。Sj26基因插入改建的杆状病毒供体质粒(pFBDGC)CMV-IE启动子下游，构建重组供体质粒pFBDGC-Sj26并进行酶切和PCR扩增鉴定。重组供体质粒pFBDGC-Sj26转化DH10Bac，提取重组Bacmid DNA(AcCMVSj26)，并采用PCR鉴定是否发生转座。AcCMVSj26转染Sf9细胞，收获重组杆状病毒vAcCMVSj26，提取vAcCMVSj26基因组DNA并进行PCR鉴定，终点稀释法测定重组病毒滴度。重组杆状病毒vAcCMVSj26转导转染幼仓鼠肾(baby hamster kidney，BHK)细胞，Western blot检测Sj26基因的表达。动物实验分两部分，第一部分用重组杆状病毒vAcCMVSj26肌肉注射免疫BALB／c小鼠，并设空病毒(vAcBW3)和攻击感染对照(Grace培养基)，每只小鼠免疫3次，间隔2周。第二部分联合免疫组分别在第0、2周用vAcCMVSj26免疫2次，第4周再用重组蛋白rSj26 GST加强免疫1次，并设单独免疫vAcCMVSj26和rSj26 GST对照。所有免疫小鼠均于末次免疫后2周进行攻击感染，感染45 d后剖杀，计数虫荷和肝内虫卵数，计算减虫率和肝组织中每条雌虫减卵率。结果：PCR扩增产物连入T载体测序结果显示Sj26基因与文献报道的序列一致性为100％；酶切和PCR鉴定重组供体质粒pFBDGC-Sj26构建正确。PCR证实了pFBDGC-Sj26和AcMNPV Bacmid在大肠杆菌中发生了同源重组，形成了重组AcCMVSj26。AcCMVSj26转染Sf9细胞后，产生了重组杆状病毒vAcCMVSj26并在Sf9细胞中复制、表达绿色荧光蛋白，PCR进一步鉴定重组杆状病毒含有Sj26基因。终点稀释法测定vAcCMVSj26的滴度为1.2×10~8 TCID／ml。vAcCMVSj26体外转导BHK细胞后，Western blot证实重组杆状病毒能够在BHK细胞中表达Sj26 GST蛋白。动物保护性实验显示，vAcCMVSj26免疫小鼠后获得了28.3％的减虫率和23.6％的减卵率，与空病毒和攻击感染对照组比较，差异有显著性。vAcCMVSj26和rSj26 GST联合免疫组的减虫率为44.0％，明显高于vAcCMVSj26单独免疫组(22.7％)和rSj26 GST单独免疫组(25.6％)。联合免疫组、rSj26 GST组和vAcCMVSj26组减卵率分别为25.5％、33.0％和23.9％，与PBS对照组相比，肝组织中每条雌虫产卵数差异有显著性(P＜0.05或0.01)。结论：成功构建了Sj26重组杆状病毒；Sj26重组杆状病毒能够在哺乳动物细胞中表达Sj26 GST蛋白；杆状病毒可以作为日本血吸虫DNA疫苗的载体；重组杆状病毒介导的Sj26 DNA疫苗(vAcCMVSj26)和重组蛋白(rSj26 GST)疫苗联合免疫可以产生协同保护作用。课题的特色和创新点：1)用含有增强型绿色荧光蛋白的真核表达载体pEGFP-N3构建了日本血吸虫26 kDa谷胱甘肽转移酶(Sj26)的重组质粒DNA疫苗(pEGFP-Sj26)。本研究重组的质粒能够借助报告基因来显示目的基因Sj26在体外哺乳动物细胞中的表达情况。2)重组质粒pEGFP-Sj26免疫小鼠后能产生一定的抗日本血吸虫感染的保护作用。3)首次构建了以杆状病毒为载体的日本血吸虫DNA疫苗并证实其在哺乳动物细胞中能有效表达。4)首次证明了重组杆状病毒介导的DNA疫苗能产生一定的抗日本血吸虫感染的保护作用。5)首次运用重组杆状病毒介导的DNA疫苗和蛋白疫苗联合免疫，并证实二者联合免疫能产生协同保护作用。更多还原

【Abstract】 Currently, DNA vaccine remains the highest priority of vaccine against schistosome. Two general classes of gene-based vaccines are being developed. One is based on the use of plasmid DNA encoding the target antigen. However, the use of plasmid DNA as a gene transfer vehicle for immunization is somewhat limited by the low transduction efficiency that hampers its immunogenic potential. To overcome this problem, numerous strategies are being developed that use electrical stimulation, microparticles, adjuvants, and costimulatory molecules to enhance both the gene transfer capabilities and the intrinsic immunogenicity of plasmid DNA. Mainwhile, the capacity of plasmid vector limits the application of a high molecular weight antigen or multi-antigen DNA vaccine, which is the emphasis to enhance the immune efficacy of anti-schistosome vaccine.The other is based on viral vector. Viruses have highly evolved structures that enable them to bind to cells and deliver genes into the cells that they infect. Thus, it is reasonable to consider using live virus vaccines to induce cytolytic T lymphocytes to treat a disease in which cellular immunity is an absolute requirement. To this end, a variety of viral vectors, primarily based on poxvirus and adenovirus, have been tested, with encouraging results. However, these vectors are themselves antigenic and can cause inflammation, which may constitute a liability for their use as vehiclesfor vaccination. An additional drawback is that existing immunity due to previous infection (as in the case of adenovirus) or to vaccination (smallpox) may limit the potency of the vaccine by clearing the viral vector before it can infect cells and deliver its payload of antigen genes. Thus, there is a need to identify unrelated viral vectors that function efficiently as gene transfer vehicles for immunization whose efficacy is not limited by problems related to toxicity or existing immunity.In recent years, a number of features make baculovirus as attractive potential gene therapy vector. In contrast to other commonly used viral vectors, baculoviruses have the unique property of replicating in insect cells while being incapable of initating a replication cycle and producing infectious virus in mammalian cells, it shows a good biosafety and the baculovirus have absence of pre-existing Abs against of baculovirus in animals. Another this vector can efficiently transduct a variety of cell lines without any apparent cytopathic effect, even at a high multiplicity of infection (MOI). The advantage of baculovirus was also highlighted by the considerably high gene delivery efficiency compared to plasmid tranfection. Mainwhile, manufacture of large quantities of relatively high-titre stock can be easily achieved, and very large insert sizes can be accommodated. The baculovirus as a vector for vaccination was used in pseudorabies virus, hepatitis C virus (HCV) and rodent malaria Plasmodium berghei, which the expression of foreign gene mediated by recombinant baculovirus could elicit antigen-specific immune response in animal experiment. These provide the laboratorial basis that the baculovirus was exploited as vaccine carrier.Furthermore, the co-immunization with DNA vaccine and protein vaccine can enhance protective efficacy. In the present study, we evaluated the feasibility of using baculovirus as a vaccine carrier, the gene encoding 26 kDa glutathione S-transferase of Schistosoma japonicum (Sj26), which is one of promising vaccine candidates selected by WHO, was used as antigen molecular. Whether the synergetic effect of the co-immunization with DNA vaccine and protein vaccine was also observed.The main contents of this thesis include two parts.Part 1: Protective Efficacy of Co-immunization with Sj26 DNA and Recombinant Protein Vaccine against Schistosoma japonicum in MiceObjective: 1) To observe the expression of recombinant plasmid pEGFP-Sj26 with enhanced green fluorescent protein in mammalian cells in vitro and protective immunity of pEGFP-Sj26 DNA vaccine. 2) To study the protective efficacy of co-immunization with pEGFP-Sj2 DNA vaccine and recombinant Sj26 protein (rSj26 GST) vaccine against Schistosoma japonicum in mice.Methods: The Sj26 gene was amplified from the plasmid pGEX-3X by PCR and the product of PCR was ligated with pGEM-T Easy vector. The positive clones were screened and identified by endonuclease digestion and sequencing, the correct recombinant plasmid was named pT1-Sj26. Then the Sj26 gene was cloned into eukaryotic expression vector pEGFP-N3 with enhanced green fluorescence protein (EGFP). The recombinant plasmid pEGFP-Sj26 was transfected into baby hamster kidney (BHK) cells by using cationic lipids (lipofectamine). Both fluorescent microscope and western blot were employed to identify the expressed products. Sixty BALB/c mice were divided randomly into five groups, each consisting of 12 mice, which were immunized three times. The co-immunization group was primed with plasmid pEGFP-Sj26, boosted 2 weeks later and immunized with rSj26 GST 4 weeks later. Two weeks after last immunization, each mouse was challenged with 40±l of cercariae of Schistosoma japonicum Chinese strain. On day 45 post-infection, the mice were sacrificed and perfused from portal vein, then the number of worms and liver eggs were counted. Percent of worm reduction and percent reduction in liver eggs in co-immunization group were compared with that in pEGFP-Sj26 group and rSj26 GST group.Results: Sequence analysis revealed that the full length of Sj26 gene was 654 bp, and showed 100% identical to the sequence reported in literature. The recombinantplasmid pEGFP-Sj26 was successfully constructed, and could be transfected BHK cells in vitro. In BHK cells transfected with the recombinant plasmid pEGFP-Sj26, the bright green fluorescence was observed under fluorescent microcopy, and western blot confirmed that sera from rabbit immunized with rSj26 GST could recognize the band with Mr 53 kDa.Percent of worm reduction in co-immunized group was 50.8%, it was significantly higher than that in pEGFP-Sj26 group (28.0%, P<0.01) and rSj26 GST group (25.5%, P<0.01). Percent of liver egg reduction in co-immunized group, rSj26 GST group and pEGFP-Sj26 group were 32.7%, 33.0% and 20.6% respectively, and percent of liver egg reduction in the former two groups were significantly higher than that in control group (P<0.01), but the difference between pEGFP-Sj26 group and control group was not significant.Conclusion: 1) The recombinant plasmid pEGFP-Sj26 with enhanced green fluorescent protein has been constructed successfully. 2) Eukaryotic expression plasmid pEGFP-Sj26 was expressed successfully in mammalian cells in vitro. 3) pEGFP-Sj26 DNA vaccine could induce partial protective immunity in BALB/c mice. 4) Compared with only Sj26 DNA vaccine or recombinant protein vaccine, the co-immunization with Sj26 DNA vaccine and recombinant protein could enhance protective efficacy in BALB/c mice.Part 2: Protective Efficacy of Co-immunization with Recombinant Baculovirus Carrying Sj 26 DNA and Recombinant Protein Vaccine against Schistosoma japonicum in MiceObjective: 1) To construct the recombinant baculovirus carrying Schistosoma japonicum 26 kDa glutathione S-transferase (Sj26). 2) To explore the feasibility of using baculovirus as DNA vaccine carrier. 3) To study the protective efficacy of recombinant baculovirus carrying Sj26 DNA vaccine. 4) To study the protectiveefficacy of co-immunization with recombinant baculovirus carrying Sj26 DNA vaccine and recombinant protein (rSj26 GST) vaccine against Schistosoma japonicum in mice.Methods: The Sj26 gene was amplified from the plasmid pGEX-3X by PCR and the product of PCR was ligated with pGEM-T Easy vector. The positive clones were screened and identified by endonuclease digestion and sequencing, the correct recombinant plasmid was named pT2-Sj26. Then the Sj26 gene was inserted into the downstream of CMV-IE promoter of donor plasmid pFBDGC, and transformed into DH10Bac, then the recombinant bacmid AcCMVSj26 was isolated and its transposition was confirmed by PCR. The AcCMVSj26 was transfected into Sf9 cells, green fluorescent was observed under fluorescent microscope. The recombinant baculovirus vAcCMVSj26 was harvested and then identified by PCR, and the titer of vAcCMVSj26 was determined by end-point dilution. After the BHK cells transducted with recombinant baculovirus, the expression of Sj26 GST was identified by western blot.The independent vaccination trials were performed. All animals were inoculated three times with two weeks interval. In trial 1, BALB/c mice were immunized with recombinant baculovirus vAcCMVSj26 by intramuscular injection, baculovirus vAcBW3 and Grace medium as control. In trial 2, the co-immunization group was primed with vAcCMVSj26, boosted 2 weeks later and immunized with rSj26 GST 4 weeks later and only vAcCMVSj26 or rSj26 GST as control. Two weeks after last immunization, each mouse in two trials was challenged with 40±1 of cercariae of Schistosoma japonicum Chinese strain. On day 45 post-infection, the mice were sacrificed and perfused from portal vein, then the number of worms and liver eggs were counted. Percent of worm reduction and percent reduction in liver eggs were calculated.Results: Sequence analysis revealed that the full length of Sj26 gene was 654 bp,and showed 100% identical to the sequence reported in literature. The recombinant donor plasmid pFBDGC-Sj26 was successfully constructed, and PCR confirmed that the gene of interest had transposed to the recombinant bacmid AcCMVSj26. When AcCMVSj26 was transfected into Sf9 cells, green fluorescent was observed under fluorescent microscope during 4-7 days post-transfection, it showed that recombinant baculovirus vAcCMVSj26 had generated. The Sj26 gene in vAcCMVSj26 was confirmed by PCR, and the final titer of vAcCMVSj26 was 1.2×10~8 TCID/ml. The results of western blot showed that the Sj26 GST was expressed in BHK cells transfected with recombinant baculovirus vAcCMVSj26.Animal protection experiment shows in trial 1, vAcCMVSj26 vaccination elicited a 28.3% reduction in worm burdens and 23.6% reduction in liver eggs per female, compared to mice immunized with vAcBW3 baculovirus and Grave medium control. The effect of co-immunization was evaluated in trial 2, in which, percent of worm reduction in co-immunized group was 44.0%, it was significantly higher than that in vAcCMVSj26 group (22.7%, P<0.01) and rSj26 GST group (25.6%, P<0.01). Percent of liver egg reduction in co-immunized group, rSj26 GST group and vAcCMVSj26 group were 25.5%, 33.0% and 23.9% respectively, and it showed the significant difference with PBS control.Conclusion: 1) The recombinant baculovirus carrying Schistosoma japonicum 26 kDa glutathione S-transferase (Sj26) was constructed successfully. 2) The recombinant baculovirus carrying Sj26 were expressed successfully in mammalian cells in vitro. 3) The baculovirus can be used as DNA vaccine carrier. 4) The co-immunization with recombinant baculovirus carrying Sj26 DNA vaccine and recombinant protein vaccine against Schistosoma japonicum in mice could similarly enhance protective efficacy in BALB/c mice.更多还原