鸡毒支原体套式PCR检测方法的建立及其mgc2基因的原核表达
发布时间：2019-06-21 13:11
【摘要】：鸡毒支原体(Mycoplasma Gallisepticum,MG)感染是危害养禽业重要的呼吸道传染病之一,由于MG通常与其他呼吸道病原混合感染,给临床MG感染的诊断带来困难。为探讨MG黏附蛋白的反应原性,建立一种特异性好、敏感性高的诊断MG感染的方法,本研究以肉鸡鸡毒支原体感染为研究对象,分离鉴定病原并建立了套式PCR诊断方法;同时对黏附蛋白mgc2进行克隆与表达分析。方法:采集发生自然感染的呼吸道疾病的肉鸡的肺脏、气囊、气管分泌物,实验室进行病原的分离培养,通过对病原的初步鉴定(L型细菌鉴定、菌落形态观察、染色镜检、红细胞吸附试验)、PCR扩增与测序等方法进行MG病原鉴定;并对目前常用的5对引物(gapA-F/gapA-R、16s rRNA-F/16s rRNA-R、mgc2-F1/mgc2-R1、mgc2-F2/mgc2-R2、LP-F/LP-R)进行灵敏性比较,筛选出灵敏度最高的引物建立套式PCR检测方法,同时对其特异性、敏感性进行检测;最后对MG的黏附蛋白mgc2进行克隆与原核表达,采用Western blot分析是否具有反应原性。结果:(1)MG分离与鉴定:试验共分离出36株MG菌株。其中有3株来自MG单纯感染病例,从3~20日龄的肉鸡场分离得到,33株来自MG混合感染病例,从21~40日龄的肉鸡场分离得到。(2)MG套式PCR检测结果:通过比较5对引物对MG的灵敏度,可得以mgc2基因设计的引物灵敏度最高,能检测出的MG的最低浓度为57.6 pg/μL,用此引物建立的套式PCR检测方法能够扩增基因片段大小为300 bp,与GenBank收录的相关序列同源性为98%,而与大肠杆菌、沙门氏菌、鸡滑液囊支原体均无交叉反应,能够检测出DNA的最小量为0.18 fg/μL。通过对安徽、山东省部分地区疑似MG的50只病死鸡进行检测,阳性检出率为80%。(3)鸡毒支原体的黏附蛋白mgc2原核表达结果:将mgc2基因序列中一个编码Trp的密码子TGA成功突变为TGG,突变后的mgc2基因克隆到原核表达载体pET-30a(+)中,构建了重组质粒pET/mgc2,经IPTG诱导,成功地在大肠杆菌BL21(DE3)宿主菌中高效表达分子量约为39.6 kDa的融合蛋白。Western blot分析表明该融合蛋白具有反应原性。结论:(1)建立的MG套式PCR检测方法特异性强、敏感性高。(2)成功表达了有良好反应原性的黏附蛋白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，
本文编号：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，
本文编号：2504102
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