摘要
猪支原体肺炎(Mycoplasma Pneumoniae of Swine,MPS)是由猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)引起猪以咳嗽和气喘为主要症状的慢性接触性呼吸道传染病。主要症状及表现为咳嗽、气喘、饲料的利用率低,病猪生长十分缓慢,甚至生长停滞。该病以发病率高,死亡率低为其特点。而在临床上检测感染猪,仍面临困难。猪肺炎支原体的表面膜蛋白是该菌主要的免疫抗原,对猪肺炎支原体感染的诊断有重要意义。
本研究通过制备抗猪肺炎支原体高免血清,运用辛酸-硫酸铵盐析和阴离子交换层析技术,获得高活性、高电泳纯的IgG。再将纯化IgG与CNBr-activated Sepharose4B偶联制得抗Mhp—免疫亲和层析柱,以提取和纯化通过TritonX-100结合KI破碎的支原体细胞所获得的猪肺炎支原体膜蛋白。纯化的膜蛋白抗原经SDS-PAGE电泳分析,结果显示,纯化后的抗原蛋白分子的纯度显著提高,仅呈现66KD,48KD,46KD,40KD,32KD和24KD的6条清晰的蛋白条带,而未纯化的膜蛋白条带有11条,分子量范围在20KD-100KD之间。
本研究采用纯化的膜蛋白抗原建立了猪肺炎支原体的间接ELISA检测方法。试验确定的最适ELISA工作条件为:包被纯化抗原蛋白浓度5ug/ml,血清稀释度为1:80,封闭液为1.5%BSA,封闭时间120min,抗原抗体作用时间90min,酶标二抗SPA(1:1000稀释)作用时间为60min,阴阳性界限OD值为0.289。
分别用纯化抗原和粗提抗原包被ELISA板,对比二者的检测结果发现,纯化抗原对样品的阳性检出率比粗提抗原提高了7.5%。用建立的标准猪肺炎支原体ELISA检测方法与中国兽医药品监察所研制的IHA试剂盒同时检测50份血清样品,二者总的符合率为92%。对临床送检的150份不同年龄段的猪血清样品,用该ELISA方法检测,结果显示,断奶前仔猪Mhp感染率为44.2%,保育猪为41.2%,育肥猪为17.9%。检测结果说明,猪肺炎支原体在猪群中广泛存在,也证明了建立的猪肺炎支原体ELISA检测方法在临床诊断中具有重要的实用价值。
Mycoplasma Pneumoniae is the etiological agent of Mycoplasma Pneumoniae of swine (MPS), which is a chronic respiratory disease of swine. MPS is characterized by cough, sneezing, high morbidity rates, low mortality rates, retarded growth, poor food conversion. The clinical suspected pigs were often difficult to make confirmed diagnosis. The membrane protein of Mycoplasma Pneumoniae is the main immunogens and it has important significance to diagnose the infection of MPS.
The high activity and purity IgG had obtained from the anti-Mhp serum antibody which was purified by using caprylic acid and ammonium sulphate (CA-AS), and anion-exchange chromatography. The immunoadsorbent affinity chromatography of anti-Mhp were prepared by coupling the IgG to CNBr-activated Sepharose 4B. Membrane proteins which were extracted from Mycoplasma Pneumoniae by TritonX-100 and KI, adsorbed by such immunoadsorbent could be purified. The result of SDS-PAGE shows that the purified membrane proteins have high-purity that the molecular weights were 66KD, 48KD, 46KD, 40KD, 32KD and 24KD. The unpurified membrane proteins have low- purity and the range of molecular weights was 20KD to 100KD.
An indirect enzyme-linked immunosorbent assay for detecting antibody Mycoplasma Pneumoniae was established by using purified membrane proteins as antigen for coating. Through repeatedly test and confirmed the optimization test conditions: the coating concentration of antigen was 5ug/ml, the optimum dilution of the tested serum should be 1:80, the sealed buffer was 1.5% BSA and blocking for 120 min, the action between antigen and antibody lasted for 90min, the optimum action period for the binding of antigen with enzyme-signed SPA which were diluted by 1:1000 was 60min, samples with an optical density (OD) value≥0.289 were scored positive base on the ELISA test.
Coating the unpurified and purified proteins respectively in ELISA test, the results showed that the positive detection rate in sera from clinically pigs was raised to 7.5% by using purified membrane protein. 92% of agreement was obtained between our new ELISA and commercial IHA kit by simultaneously detecting 50 serum samples. The assay was further applied to detect 150 clinically collected at random serum samples. The detection rates in sera from postweaning piglets, growing pigs, finishing pigs were 44.2%, 41.2% and 17.9%. The result showed the prevalence of Mycoplasma Pneumoniae in pig herds and the significance of the new indirect ELISA in clinical diagnosis of MPS.
引文
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