鸡毒支原体遗传转化方法的建立及应用

发布时间：2018-03-29 09:48

本文选题：鸡毒支原体　切入点：F36株　出处：《华中农业大学》2017年硕士论文

【摘要】：鸡毒支原体(Mycoplasma gallisepticum,MG)是引起鸡慢性呼吸道疾病的重要病原。国内的控制与预防策略倾向于使用F36弱毒疫苗株。MG F36株对鸡安全,具有良好的呼吸道定植能力,能够诱导粘膜免疫,持续产生免疫保护。为了开发MG F36株的应用潜力,我们拟构建MG基因操作系统,尝试以MG F36株作为载体,表达其他呼吸道疾病的免疫抗原基因,为探索MG载体新型疫苗奠定基础。目前,我们获得如下结果:1.利用大肠杆菌的Lac Z基因作为报告基因,融合MG S6株粘附蛋白Gap A启动子,借助转座子p MT85,建立鸡毒支原体遗传转化方法。操作简便无需特殊仪器及耗材,转化效率也能满足实验要求。通过优化的电转化法/PEG转化法,成功将转座子插入MG F36株和MG S6株的基因组中。转化后的MG阳性菌株能够在庆大霉素抗性平板上生长,菌落形态呈油煎蛋样,带有明显蓝色。重复数次转化操作后,最终的到了包含600个突变体的转座子库。随机挑选12株通过FM-4液体培养基培养传代,并用PCR检测验证突变体的稳定。结果在无抗性培养基上,连续传至25代,均能扩增出转座子上的庆大霉素抗性基因,说明转座子在鸡毒支原体中有良好的稳定性。2.以p MD-18T为骨架,先后克隆并插入约1800bp的MG长复制区(Long ori C)和808bp的MG短复制区(Short ori C)以及庆大霉素抗性基因,构建大肠杆菌-鸡毒支原体的穿梭质粒。同时也将Lac Z基因表达盒克隆至穿梭质粒上,构建报告质粒,检验穿梭质粒的表达效率。用穿梭质粒和报告质粒分别转化F36株,并涂布含庆大霉素抗性的平板。结果在抗性平板上长出了阳性菌落,而用报告质粒转化后长出的菌落也能够产生蓝斑,而且在抗性条件下能够传代。但再用无抗性X-gal培养基检测,大部分的菌落不能够产生蓝斑,这说明菌体中的质粒不稳定,在传代过程中会丢失。3.克隆禽流感病毒H5亚型的HA1基因,并在3’端加入Flag标签,与Gap A启动子融合,克隆到转座子载体p MT85中构建了H5_HA1的表达质粒。转化鸡毒支原体F36株后,筛选到了2个阳性菌株。经PCR和Western blot鉴定后,阳性菌株均表达了HA1蛋白,表达的HA1具有一定的免疫学活性。这为后续进一步以鸡毒支原体为载体的多联活疫苗研发奠定了坚实的基础。
[Abstract]:Mycoplasma gallisepticum (MG) is an important pathogen of chronic respiratory diseases in chickens. The domestic control and prevention strategies tend to use F36 attenuated vaccine strain. MGF36 strain is safe for chicken, has good respiratory tract colonization ability, and can induce mucosal immunity. In order to develop the application potential of MG F36 strain, we intend to construct MG gene operating system and try to express the immune antigen gene of other respiratory diseases by using MG F36 strain as vector. At present, we obtained the following results: 1. Using the Lac Z gene of E. coli as reporter gene, we fused Gap A promoter of MG S6 strain adhesion protein. With the help of transposon pMT85, a genetic transformation method of Mycoplasma gallus was established. The method was simple and easy to operate without special instruments and consumables, and the efficiency of transformation could meet the experimental requirements. The transposon was successfully inserted into the genome of MG F36 and MG S6 strains. The transformed MG positive strain was able to grow on gentamicin resistant plate. Finally, the transposon library containing 600 mutants was obtained. Twelve strains were randomly selected for passage on FM-4 liquid medium, and the stability of the mutants was verified by PCR test. The gentamicin resistance gene on transposon was amplified, indicating that transposon has good stability in mycoplasma. 2. Using p MD-18T as skeleton, The MG-long ori C of about 1800bp, short ori C of 808bp and gentamicin resistance gene were cloned and inserted. At the same time, the Lac Z gene expression box was cloned into the shuttle plasmid, the reporter plasmid was constructed, and the expression efficiency of the shuttle plasmid was tested. The shuttle plasmid and the report plasmid were transformed into F36 strains, respectively. And coated with gentamicin resistant plates, positive colonies were grown on the resistant plates, and colonies transformed with reporter plasmids also produced locus coeruleus. However, most colonies could not produce locus coeruleus, which indicated that the plasmids in the bacteria were unstable. During passage, the HA1 gene of H5 subtype of avian influenza virus was cloned, and the Flag tag was added at the 3'end, fused with Gap A promoter, and cloned into the transposon vector p MT85 to construct the expression plasmid of H5_HA1. Two positive strains were screened and identified by PCR and Western blot. All the positive strains expressed HA1 protein. The expressed HA1 has a certain immunological activity, which lays a solid foundation for the further research and development of multiplex live vaccine based on Mycoplasma gallus.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.62

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