【作者】 尹荣兰；

【导师】 张乃生；

【作者基本信息】 吉林大学 ， 临床兽医学， 2009， 博士

【摘要】 本研究利用分子生物学、细菌学、免疫学及基因工程和DNA重组技术等理论和方法,以研制安全有效的奶牛乳腺炎金黄色葡萄球菌基因工程疫苗为目的,基于金黄色葡萄球菌的基因组结构、感染与致病以及免疫反应等特点,甄选具有主要抗原活性的纤黏连结合蛋白A(FnbA)配基结合区基因和凝集因子A(ClfA)及其活性基因作为研究对象,通过PCR获得目的基因,利用原核表达系统制备了可溶性的重组蛋白,纯化后进行了小鼠免疫试验;以安全性真核表达载体pVAX1为基础,扩增牛白介素18基因(bIL18)作为基因佐剂,构建了系列单独表达或共表达pFnbA、ClfA和bIL18基因的侯选重组核酸疫苗质粒,开展了小鼠免疫试验研究。同时,通过不同疫苗组合、添加佐剂以及免疫-增强(Prime-boost)等策略探讨了联合免疫在临床应用的可行性。运用血清ELISA抗体检测、Th1/Th2类细胞因子含量测定、T细胞增殖实验等方法对免疫小鼠的各项指标进行综合评价,结果表明,所构建候选疫苗均能诱导机体产生特异的细胞免疫和体液免疫应答,其中3种蛋白类疫苗主要以体液免疫为主;5种DNA疫苗二者兼有;添加壳聚糖佐剂组具有更强的免疫诱导作用;以DNA疫苗首免、重组蛋白加强免疫刺激机体产生的免疫应答水平优于单独应用同种疫苗进行的免疫。上述研究为奶牛乳腺炎金黄色葡萄球菌新型基因工程疫苗的研制提供了原始数据,建立了技术平台,为有效免疫和临床应用研究奠定了坚实基础。更多还原

【Abstract】 Mastitis is one of the most important and costly disease in dairy cow herds resulted in huge economic loss worldwide. The occurrence and prevalence of the disease threatens human healthy as well as public safety. During the last decade, numerous attempts of developing different methods to prophylaxis or therapy the disease have been carried out. But, there still has no effective solutions. So, it’s urgent to develop new methods or strategies to prevent and control the disease better.Many factors associated with mastitis in cows. Among those, microorganisms’infection is regard as the main reason. Staphylococcus aureus (S. aureus) has been identified as one of the most important etiological agents. Therefore, it has been shown to focus on the research against S. aureus is a very significant direction.Vaccination was regarded as the most effective and expected methods to eradicate infectious disease spread. The traditional vaccines include whole-organism vaccines, capsular polysaccharide (CP) vaccines, toxic vaccines, and so on. Although several successful stimulations of immune responses and decrease of clinical symptoms have been made, unfortunately, commercially available vaccines have shown limited efficacy under field conditions. With the development of genetic engineering, molecular biology, genetics as well as immunology, more researchers focus on new vaccines investigation because of its merits such as safety, easy to control, etc. New vaccines against S. aureus also made certain progress, especially with regard to genetic engineering subunit vaccine and DNA vaccine.Genetic engineering subunit vaccine is constructed by inserting the major protective antigen gene into an expression plasmid, and then expressed through different expression systems (such as prokaryotic or eukaryotic cells, etc.). After prepared and purified, the protein was vaccinated as immunogen so as to stimulate the body inducing immune response. Because the vaccine contains only effective immune structure of the pathogen, when stimulate immune protection at the same time ensuring the safety, it shows good perspective. But there still have some problems need to pay an attention including small quantities of antigens, inadequate and poor immunogenicity, etc. Therefore, increasing antigen epitopes to enhance immunogenicity is the major direction. Deoxyribonucleic acid (DNA) vaccination, also named nucleic immunization or genetic immunization, has presently been developed as a simple and efficient technique to prevent many current infectious diseases. This technique is a novel vaccine technology that uses naked DNA injection to induce immune responses different from those traditional peptide or protein vaccines. It has been shown that DNA immunogens could stimulate both the cellular and/or humoral arms of the immune system. In addition, DNA vaccines are stable, safe, simple to manipulate and test, easy to store and transport, low cost and reproducible large scale production. All these unique features make it well suitable as an immunization or immune therapy strategy.In addition, the protective immune effects of vaccines have important relations with vaccination strategies. Previous studies have shown that it is not ideal when immuned with a single antigen vaccine or the same vaccine injected several times. However, combination of different forms of vaccine expressed the same epitope or the addition of endogenous or exogenous gene adjuvants will induce stronger immune response. It has been proved to be an effective immune strategy that recombinant DNA (rDNA) vaccines primed, followed by a boosting of recombinant virus or proteins in AIDS research field.Therefore, the study was conducted to develop new genetic vaccines against S. aureus infection in cow. Based on the genomic structure of S. aureus, infection and pathogen mechanism, and characteristics of immune response, and make full use of current molecular biology, bacteriology, immunology, genetic engineering and recombinant DNA technology, the research was carried out to investigate anti-cow mastitis of S. aureus genetic engineering subunit vaccine and DNA vaccine. Firstly, the genomic DNA (gDNA) was extracted from S. aureus isolated by our laboratory. Then the main antigenic genes of S. aureus containing clumping factor A (cClfA), the A region of ClfA (pClfA) and ligand binding regeion of fibronectin-binding protein A (pFnbA) were cloned successfully by polymerase chain reaction (PCR), respectively. The results of homology comparison and protein secondary structure prediction and analysis indicated that the sequence of cloned gene was very conservative, and contained major antigenic epitopes. It laid a solid foundation for constructing ideal vaccines.Secondly, based on the prokaryotic expression vector pET-28a(+), the recombinant expression plasmids pET-cClfA, pET-pClfA and pET-pFnbA were constructed. Through IPTG induction, SDS-PAGE and western blot analysis showed that the proteins were expressed successfully. And the target products existed mainly in soluble form. After optimizing the induction conditions, the optimal induction conditions were determined. The induced temperature is the same 37℃. But the other conditions were different from each other. Under the optimal induction condition, the highest products of pET-cClfA, pET-pClfA and pET-pFnbA account for 30.06%, 31.69% and 58.02% of the total bacterial proteins respectively. They can reach approximately a yield of 0.46mg/mL, 0.48mg/mL and 1.12mg/mL respectively. The proteins were purified by Ni+ metal chelate affinity chromatography. Thin-layer gel scanning analysis showed that the purity of three recombinant protein reached more than 95%. This is helpful to the further animal experiment.Thirdly, after preparation of recombinant proteins, mice immune experiment was carried out. To evaluate humoral responses, the antigen-specific antibodies were detected using indirect enzyme-linked immunosorbent assay (ELISA). Compared with the negative control, antibody titers increased following vaccines administrations and dynamic trend of antibody titer is basically the same. Vaccination with cClfA induced more antibody production than the others did individually (p<0.05). T-lymphocyte proliferation assay with the MTT method was performed to evaluate cellular immune responses elicited by the proteins. The results showed that antigen-specific T-lymphocyte proliferation responses in mice were induced a little but significant comparable (p>0.05). Cytokine levels were evaluated using the mouse Th1/Th2 ELISA Ready-SET-Go! kit (eBioscience). The concentrations of four cytokines increased to various extents in the vaccine groups compared with controls. Secretion levels of IL2 and IFN-γwere statistically comparable except for cClfA protein groups. However, the IL4 and IL10 levels were remarkably increased. This result illustrates that protein vaccines were tend to induce humoral immunity responses.Fourthly, several potential DNA vaccine candidates pV-cClfA, pV-pClfA, pV-pFnbA, pV-pFnbA-pClfA and pV-pFnbA-pClfA-bIL18 were constructed sucessfully containing the cClfA、pClfA、pFnbA with or without the bIL18 gene based on the eukaryotic expression vector pVAX1. The ability of the recombinant DNA plasmids to express their antigens were evaluated in HeLa transfected cells. The result showed that the plasmids successfully expressed their target exogenous genes and the proteins could be recognized by antibodies against S. aureus through indirect immunofluorescent assay (IFA). BALB/c mice were immunized by the recombinant plasmids separately. The result manifested that all the DNA vaccines could stimulate mice to generate specific humoral and cellular immune responses. The vaccine groups including fusion genes (pV-pFnbA-pClfA) induced stronger immune responses than one gene (pV-pClfA or pV-pFnbA). This result illustrated that a combination of diverse antigens was responsible for high antibody induction. And pV-pFnbA-pClfA-bIL18 vaccine groups induced highest cellular immune responses. The outcomes illustrated that IL18 is a good adjuvant.Lastly, in the basis of the above-mentioned immune experiment, combined immune strategies as well as effective immune methods were carried out in this paper. The results showed that all the combined immune groups can generate specific antibodies against surface proteins of S. aureus, and T-lymphocyte proliferation responses were induced as well as Th1/Th2 type cytokine levels were increased to some extend. The immunization strategy of mixing two plasmids encoding different genes is better than fusing the two genes in one plasmid. Immunization with bIL18 or chitosan can induce more cellular response. rDNA vaccines primed twice, followed by a boosting of proteins, could induce higher humoral immunity. All the above findings indicated: the DNA vaccine containing fusion antigen and bIL18 gene is a good candidate vaccine; both mixed immunity and prime-boost immune method are effective immune strategy; and chitosan is a good adjuvant which is expected to apply to S. aureus vaccine. At the same time, it will be expected to achieve better immune effect when combined and integrated the above immunization strategies together.It is the first time to application ClfA and FnbA genes of S. aureus to construct subunit vaccine and DNA vaccine, and application bIL18, C1fA and FnbA gene to construct complex antigen DNA vaccine. Also, the systematic immune animal experiments were carried out to examine the different immunization strategy in this paper.Thus, the study will provide new breakthrough ideas, accumulate raw data, and establish technology platforms for cow mastitis S. aureus genetic engineering vaccine research. Furthermore, the research will lay the groundwork for effective immunization and pre-clinical research of S. aureus vaccine, and provide a good basis for the adoption of a scientific and rational comprehensive prevention and control measures to restrict the occurrence of mastitis cows.更多还原