应用重组抗原建立检测牛传染性胸膜肺炎的间接ELISA方法  

Development of an Indirect Enzyme-Linked Immunosorbent Assay for Contagious Bovine Pleuropneumonia Using Recombination Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC

作　　者：辛九庆[1] 高玉龙[1] 李媛[1] 王砚范[1] 钱爱东[2] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所/国家牛传染性胸膜肺炎参考实验室,哈尔滨150001 [2]吉林农业大学动物科技学院,长春130118

出　　处：《中国农业科学》2006年第1期181-186,共6页Scientia Agricultura Sinica

基　　金：国家"十五"科技攻关重大专项基金项目(2002BA518A04)

摘　　要：目的丝状支原体丝状亚种SC(MycoplasmamycoidessubspmycoidesSC,MmmSC)是引起牛传染性胸膜肺炎(contagiousbovinepleuropneumonia,CBPP)的病原体。成熟的脂蛋白LppQN端是亲水性的,在自然感染和人工感染时,特异性免疫反应产生早,持续时间长,抗体滴度高,因此有理由相信脂蛋白LppQN端片段将有可能成为一种理想的CBPP诊断抗原。方法由于TGA密码子在支原体中编码色氨酸(Trp),而在细菌中则为终止密码子,故利用一步重叠延伸PCR突变方法体外诱变中国生产抗原用毒株HVRIⅩ的lppQ基因进行原核表达,利用重组抗原建立间接ELISA方法。结果序列分析表明,将lppQ基因第198位的A成功突变为G,并将突变后的脂蛋白LppQN端基因片段插入原核表达载体pET32a的多克隆位点,构建了原核表达载体。重组菌经诱导后,表达出了分子量约为42kD、带有6个组氨酸标签的重组融合蛋白,纯化后蛋白质纯度高达95%,Westernblot结果显示,重组蛋白具有很好的免疫活性。将纯化的蛋白质稀释至0.35μg·ml-1,包被酶标反应板,优化反应条件,P/N为4.8(0.934/0.193)。应用TG-ROC统计软件分析确定阴阳性血清临界OD值为0.376,敏感性为95.8%(46/48),特异性为98.9%(161/163)。应用间接ELISA和补体结合试验(CFT)对来自3个省自治区3817头份牛血清进行了检测。结论Kappa值为0.63,两种方法存在中、高度一致性。[Objective] Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The N-terminal domain of the mature lipoprotein LppQ is hydrophobic, and it induces a strong, specific, early and persistent immune response in naturally and experimentally infected animals. People have good reason to believe N-terminal fragment of lipoprotein LppQ will become a kind of ideal CBPP diagnosis antigen. [Method] Mycoplasma-specific TGA （Trp） codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain was mutated with a one-step overlapping extension PCR, and was expressed in E. coll. An indirect enzyme-hnked immunosorbent assay was estabhshed by using recombination lipoprotein LppQ [Result] Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42kD was purified by Ni-NTA His.Bind purification kit and the purity up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35μg·ml^-1, and coated to microtiter ELISA plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 （0.934/0.193）, the sensitivity to be 95.8% （46/48）, and the specificity to be 98.9% （161/163）. A total of 3 817 bovine serum samples were detected by indirect ELISA and CFT. [Conclusion] The Kappa value is 0.63, which is middle or high agreemen between the two methods.

关 键 词：牛 牛传染性胸膜肺炎 脂蛋白LppQ 突变 ELISA 

分 类 号：S858.23[农业科学—临床兽医学]