A型副鸡嗜血杆菌血凝素基因原核表达及间接ELISA方法的建立及应用
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摘要
原核表达A型副鸡嗜血杆菌(Haemophilus paragallinarum serotype A,Hpgserotype A)的血凝素蛋白HA,并以HA蛋白为包被抗原,建立血清间接ELISA检测方法。克隆Hpg HA基因,将其克隆至原核表达载体pET-28a(+)中进行重组HA蛋白的诱导表达,对表达产物进行纯化,并进行Western blot鉴定;用纯化后的HA蛋白作为包被抗原,优化反应条件,建立血清间接ELISA检测方法,并进行特异性、灵敏度和重复性试验,最后对临床样品进行检测。原核表达获得大小约为37ku的重组HA蛋白;Western blot结果表明重组蛋白具有良好的特异性和抗原反应性。Hpg间接ELISA优化反应条件为:抗原包被量每孔500ng,血清以1∶200倍稀释作用1h,酶标二抗以1∶2 500倍稀释作用1h,TMB显色时间为10min。在该优化条件下,OD_(450)≥0.34为阳性,反之为阴性;特异性试验结果表明对鸡新城疫病毒、禽流感病毒、沙门菌、金黄色葡萄球菌、多杀性巴氏杆菌和大肠埃希菌阳性血清检测均为阴性;对85份阳性血清进行检测,敏感性为98.8%;重复性试验结果表明,批内和批间OD_(450)值的变异系数分别1.5%~3.7%和6.5%~8%。用本试验建立的方法对临床上215份鸡血清样品进行检测,阳性率为25.1%。说明建立的ELISA检测方法可用于Hpgserotype A抗体水平的检测。
After prokaryotic expression of Haemophilus paragallinarum serotype A HA protein,and using the HA protein as the antigen,the indirect ELISA detection method was established.Hpgserotype A HA protein gene was cloned into E.coli expression vector pET-28 a(+)in this study to induce the expression of recombinant HA protein.HA protein was purified and tested by Western blot.Using the recombinant HA protein as the antigen,the indirect ELISA detection method was established to detect the level of Hpg antibody in serum.Specificity,sensitivity and reproducibility tests were carried out,and the clinical samples were tested.The recombinant protein with the size of 37 ku was obtained.Western blot showed that the purified HA protein had good specificity and antigen reactivity.The optimal reaction conditions were established as follows:the coating amount of antigen was 500 ng per hole,closed 1 h;the serum dilution was1∶200,reacting 1 h;the second antibody dilution was 1∶2 500,reacting 1 h;the TMB reacting time was10 min.Criteria for determining the negative and positive results of serum samples were OD_(450)≥0.34 and OD_(450)<0.34,respectively.Specificity tests showed that the results of Newcastle disease virus,avian influenza virus,Salmonella spp,Staphylococcus aureus,Pasteurella multocida,Escherichia coli positive sera were negative.85 positive sera were tested and the sensitivity was 98.8% .The repeatability test showed that the coefficient of variation of OD_(450) between the same batch and different batch were 1.5% -3.7% and 6.5% -8% ,respectively.The clinical 215 serum samples were tested using established system and the positive rate was 25.1% .The established system in this test can be used for clinical detection of serum antibody and epidemiological investigation of Hpgserotype A.
After prokaryotic expression of Haemophilus paragallinarum serotype A HA protein,and using the HA protein as the antigen,the indirect ELISA detection method was established.Hpgserotype A HA protein gene was cloned into E.coli expression vector pET-28 a(+)in this study to induce the expression of recombinant HA protein.HA protein was purified and tested by Western blot.Using the recombinant HA protein as the antigen,the indirect ELISA detection method was established to detect the level of Hpg antibody in serum.Specificity,sensitivity and reproducibility tests were carried out,and the clinical samples were tested.The recombinant protein with the size of 37 ku was obtained.Western blot showed that the purified HA protein had good specificity and antigen reactivity.The optimal reaction conditions were established as follows:the coating amount of antigen was 500 ng per hole,closed 1 h;the serum dilution was1∶200,reacting 1 h;the second antibody dilution was 1∶2 500,reacting 1 h;the TMB reacting time was10 min.Criteria for determining the negative and positive results of serum samples were OD_(450)≥0.34 and OD_(450)<0.34,respectively.Specificity tests showed that the results of Newcastle disease virus,avian influenza virus,Salmonella spp,Staphylococcus aureus,Pasteurella multocida,Escherichia coli positive sera were negative.85 positive sera were tested and the sensitivity was 98.8% .The repeatability test showed that the coefficient of variation of OD_(450) between the same batch and different batch were 1.5% -3.7% and 6.5% -8% ,respectively.The clinical 215 serum samples were tested using established system and the positive rate was 25.1% .The established system in this test can be used for clinical detection of serum antibody and epidemiological investigation of Hpgserotype A.
引文
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[6]李峰,苗立中,沈志强.一株A型副鸡嗜血杆菌的分离鉴定[J].家禽科学,2014(10):46-48.
[7]张培君,苗得园,龚玉梅,等.B型副鸡嗜血杆菌的分离鉴定[J].中国预防兽医学报,2003,25(1):56-58.
[8]苗得园,张培君.副鸡嗜血杆菌免疫鸡阻断ELlSA抗体曲线的观察[J].中国兽医杂志,2000,26(1):18-19.
[9]苗得园,张培君,龚玉梅,等.Dot_ELISA检测副鸡嗜血杆菌血清抗体的研究[J].中国预防兽医学报,2000,22(1):52-54.
[10]黄金海,王英珍,丁伯良,等.间接ELISA检测鸡传染性鼻炎抗体的研究[J].中国预防兽医学报,2001,23(6):454-457.
[11]王雪敏,梁玉荣,吕学泽,等.副鸡嗜血杆菌基因文库的构建与筛选[J].中国兽医科学,2012(4):368-372.
[2]Hobb R I,Tseng H J,Downes J E,et al.Molecular analysis of a haemagglutinin of Haemophilus paragallinarum.[J].Microbiology,2002,148(7):2171-9.
[3]Fernandez R P,Garcia-Delgado G A,Ochoa P,et al.Characterization of Haemophilus paragallinarumisolates from Mexico.[J].Avian Pathol,2000,29(5):473-6.
[4]Poernomo S,Rafiee M.Characterisation of isolates of Haemophilus paragallinarum from Indonesia[J].Australian Vet J,2000,78(11):759-62.
[5]王宏俊,张培君,龚玉梅,等.A型副鸡嗜血杆菌血凝素基因的克隆表达及其产物的生物学活性鉴定[J].中国兽医科学,2006,36(10):60-60..
[6]李峰,苗立中,沈志强.一株A型副鸡嗜血杆菌的分离鉴定[J].家禽科学,2014(10):46-48.
[7]张培君,苗得园,龚玉梅,等.B型副鸡嗜血杆菌的分离鉴定[J].中国预防兽医学报,2003,25(1):56-58.
[8]苗得园,张培君.副鸡嗜血杆菌免疫鸡阻断ELlSA抗体曲线的观察[J].中国兽医杂志,2000,26(1):18-19.
[9]苗得园,张培君,龚玉梅,等.Dot_ELISA检测副鸡嗜血杆菌血清抗体的研究[J].中国预防兽医学报,2000,22(1):52-54.
[10]黄金海,王英珍,丁伯良,等.间接ELISA检测鸡传染性鼻炎抗体的研究[J].中国预防兽医学报,2001,23(6):454-457.
[11]王雪敏,梁玉荣,吕学泽,等.副鸡嗜血杆菌基因文库的构建与筛选[J].中国兽医科学,2012(4):368-372.