雞毒支原體套式PCR檢測(cè)方法的建立及其mgc2基因的原核表達(dá)
發(fā)布時(shí)間：2019-06-21 13:11
【摘要】：雞毒支原體(Mycoplasma Gallisepticum,MG)感染是危害養(yǎng)禽業(yè)重要的呼吸道傳染病之一,由于MG通常與其他呼吸道病原混合感染,給臨床MG感染的診斷帶來困難。為探討MG黏附蛋白的反應(yīng)原性,建立一種特異性好、敏感性高的診斷MG感染的方法,本研究以肉雞雞毒支原體感染為研究對(duì)象,分離鑒定病原并建立了套式PCR診斷方法;同時(shí)對(duì)黏附蛋白mgc2進(jìn)行克隆與表達(dá)分析。方法:采集發(fā)生自然感染的呼吸道疾病的肉雞的肺臟、氣囊、氣管分泌物,實(shí)驗(yàn)室進(jìn)行病原的分離培養(yǎng),通過對(duì)病原的初步鑒定(L型細(xì)菌鑒定、菌落形態(tài)觀察、染色鏡檢、紅細(xì)胞吸附試驗(yàn))、PCR擴(kuò)增與測(cè)序等方法進(jìn)行MG病原鑒定;并對(duì)目前常用的5對(duì)引物(gapA-F/gapA-R、16s rRNA-F/16s rRNA-R、mgc2-F1/mgc2-R1、mgc2-F2/mgc2-R2、LP-F/LP-R)進(jìn)行靈敏性比較,篩選出靈敏度最高的引物建立套式PCR檢測(cè)方法,同時(shí)對(duì)其特異性、敏感性進(jìn)行檢測(cè);最后對(duì)MG的黏附蛋白mgc2進(jìn)行克隆與原核表達(dá),采用Western blot分析是否具有反應(yīng)原性。結(jié)果:(1)MG分離與鑒定:試驗(yàn)共分離出36株MG菌株。其中有3株來自MG單純感染病例,從3~20日齡的肉雞場(chǎng)分離得到,33株來自MG混合感染病例,從21~40日齡的肉雞場(chǎng)分離得到。(2)MG套式PCR檢測(cè)結(jié)果:通過比較5對(duì)引物對(duì)MG的靈敏度,可得以mgc2基因設(shè)計(jì)的引物靈敏度最高,能檢測(cè)出的MG的最低濃度為57.6 pg/μL,用此引物建立的套式PCR檢測(cè)方法能夠擴(kuò)增基因片段大小為300 bp,與GenBank收錄的相關(guān)序列同源性為98%,而與大腸桿菌、沙門氏菌、雞滑液囊支原體均無交叉反應(yīng),能夠檢測(cè)出DNA的最小量為0.18 fg/μL。通過對(duì)安徽、山東省部分地區(qū)疑似MG的50只病死雞進(jìn)行檢測(cè),陽性檢出率為80%。(3)雞毒支原體的黏附蛋白mgc2原核表達(dá)結(jié)果:將mgc2基因序列中一個(gè)編碼Trp的密碼子TGA成功突變?yōu)門GG,突變后的mgc2基因克隆到原核表達(dá)載體pET-30a(+)中,構(gòu)建了重組質(zhì)粒pET/mgc2,經(jīng)IPTG誘導(dǎo),成功地在大腸桿菌BL21(DE3)宿主菌中高效表達(dá)分子量約為39.6 kDa的融合蛋白。Western blot分析表明該融合蛋白具有反應(yīng)原性。結(jié)論:(1)建立的MG套式PCR檢測(cè)方法特異性強(qiáng)、敏感性高。(2)成功表達(dá)了有良好反應(yīng)原性的黏附蛋白mgc2。
[Abstract]:Mycoplasma gallisepticum (Mycoplasma Gallisepticum,MG) infection is one of the important respiratory infectious diseases in poultry industry. Because MG is usually mixed with other respiratory pathogens, it is difficult to diagnose MG infection in clinic. In order to investigate the reaction genicity of MG adhesion protein and establish a method for the diagnosis of MG infection with good specificity and high sensitivity, the pathogen was isolated and identified by using Mycoplasma gallisepticum infection in broilers, and the diagnostic method of PCR was established. At the same time, the adhesion protein mgc2 was cloned and expressed. Methods: the lung, airbag and trachea exudates of broilers with naturally infected respiratory diseases were collected and cultured in the laboratory. The pathogens were identified by preliminary identification (L-form bacteria identification, colony morphology observation, staining microscopic examination, red blood cell adsorption test), PCR amplification and sequencing, etc.). The pathogen was identified by the methods of preliminary identification of the pathogen (L-form bacteria identification, colony morphology observation, staining microscopic examination, red blood cell adsorption test MG amplification and sequencing, etc.). The sensitivity of five pairs of primers (gapA-F/gapA-R,16s rRNA-F/16s rRNA-R,mgc2-F1/mgc2-R1,mgc2-F2/mgc2-R2,LP-F/LP-R) was compared, and the primers with the highest sensitivity were selected to establish a nest PCR detection method, and its specificity and sensitivity were detected at the same time. Finally, the adhesion protein mgc2 of MG was cloned and expressed in prokaryotic cells. Western blot was used to analyze whether the adhesion protein mgc2 was reactive or not. Results: (1) isolation and identification of MG: a total of 36 strains of MG were isolated. Among them, 3 strains were isolated from MG simple infection cases, 33 strains from MG mixed infection cases, and 33 strains from 21 鈮，
本文編號(hào)：2504102
[Abstract]:Mycoplasma gallisepticum (Mycoplasma Gallisepticum,MG) infection is one of the important respiratory infectious diseases in poultry industry. Because MG is usually mixed with other respiratory pathogens, it is difficult to diagnose MG infection in clinic. In order to investigate the reaction genicity of MG adhesion protein and establish a method for the diagnosis of MG infection with good specificity and high sensitivity, the pathogen was isolated and identified by using Mycoplasma gallisepticum infection in broilers, and the diagnostic method of PCR was established. At the same time, the adhesion protein mgc2 was cloned and expressed. Methods: the lung, airbag and trachea exudates of broilers with naturally infected respiratory diseases were collected and cultured in the laboratory. The pathogens were identified by preliminary identification (L-form bacteria identification, colony morphology observation, staining microscopic examination, red blood cell adsorption test), PCR amplification and sequencing, etc.). The pathogen was identified by the methods of preliminary identification of the pathogen (L-form bacteria identification, colony morphology observation, staining microscopic examination, red blood cell adsorption test MG amplification and sequencing, etc.). The sensitivity of five pairs of primers (gapA-F/gapA-R,16s rRNA-F/16s rRNA-R,mgc2-F1/mgc2-R1,mgc2-F2/mgc2-R2,LP-F/LP-R) was compared, and the primers with the highest sensitivity were selected to establish a nest PCR detection method, and its specificity and sensitivity were detected at the same time. Finally, the adhesion protein mgc2 of MG was cloned and expressed in prokaryotic cells. Western blot was used to analyze whether the adhesion protein mgc2 was reactive or not. Results: (1) isolation and identification of MG: a total of 36 strains of MG were isolated. Among them, 3 strains were isolated from MG simple infection cases, 33 strains from MG mixed infection cases, and 33 strains from 21 鈮，
本文編號(hào)：2504102
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