本文選題：雞毒支原體　切入點：F36株　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：雞毒支原體(Mycoplasma gallisepticum,MG)是引起雞慢性呼吸道疾病的重要病原。國內(nèi)的控制與預(yù)防策略傾向于使用F36弱毒疫苗株。MG F36株對雞安全,具有良好的呼吸道定植能力,能夠誘導(dǎo)粘膜免疫,持續(xù)產(chǎn)生免疫保護。為了開發(fā)MG F36株的應(yīng)用潛力,我們擬構(gòu)建MG基因操作系統(tǒng),嘗試以MG F36株作為載體,表達其他呼吸道疾病的免疫抗原基因,為探索MG載體新型疫苗奠定基礎(chǔ)。目前,我們獲得如下結(jié)果:1.利用大腸桿菌的Lac Z基因作為報告基因,融合MG S6株粘附蛋白Gap A啟動子,借助轉(zhuǎn)座子p MT85,建立雞毒支原體遺傳轉(zhuǎn)化方法。操作簡便無需特殊儀器及耗材,轉(zhuǎn)化效率也能滿足實驗要求。通過優(yōu)化的電轉(zhuǎn)化法/PEG轉(zhuǎn)化法,成功將轉(zhuǎn)座子插入MG F36株和MG S6株的基因組中。轉(zhuǎn)化后的MG陽性菌株能夠在慶大霉素抗性平板上生長,菌落形態(tài)呈油煎蛋樣,帶有明顯藍色。重復(fù)數(shù)次轉(zhuǎn)化操作后,最終的到了包含600個突變體的轉(zhuǎn)座子庫。隨機挑選12株通過FM-4液體培養(yǎng)基培養(yǎng)傳代,并用PCR檢測驗證突變體的穩(wěn)定。結(jié)果在無抗性培養(yǎng)基上,連續(xù)傳至25代,均能擴增出轉(zhuǎn)座子上的慶大霉素抗性基因,說明轉(zhuǎn)座子在雞毒支原體中有良好的穩(wěn)定性。2.以p MD-18T為骨架,先后克隆并插入約1800bp的MG長復(fù)制區(qū)(Long ori C)和808bp的MG短復(fù)制區(qū)(Short ori C)以及慶大霉素抗性基因,構(gòu)建大腸桿菌-雞毒支原體的穿梭質(zhì)粒。同時也將Lac Z基因表達盒克隆至穿梭質(zhì)粒上,構(gòu)建報告質(zhì)粒,檢驗穿梭質(zhì)粒的表達效率。用穿梭質(zhì)粒和報告質(zhì)粒分別轉(zhuǎn)化F36株,并涂布含慶大霉素抗性的平板。結(jié)果在抗性平板上長出了陽性菌落,而用報告質(zhì)粒轉(zhuǎn)化后長出的菌落也能夠產(chǎn)生藍斑,而且在抗性條件下能夠傳代。但再用無抗性X-gal培養(yǎng)基檢測,大部分的菌落不能夠產(chǎn)生藍斑,這說明菌體中的質(zhì)粒不穩(wěn)定,在傳代過程中會丟失。3.克隆禽流感病毒H5亞型的HA1基因,并在3’端加入Flag標簽,與Gap A啟動子融合,克隆到轉(zhuǎn)座子載體p MT85中構(gòu)建了H5_HA1的表達質(zhì)粒。轉(zhuǎn)化雞毒支原體F36株后,篩選到了2個陽性菌株。經(jīng)PCR和Western blot鑒定后,陽性菌株均表達了HA1蛋白,表達的HA1具有一定的免疫學(xué)活性。這為后續(xù)進一步以雞毒支原體為載體的多聯(lián)活疫苗研發(fā)奠定了堅實的基礎(chǔ)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.62

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