PCR检测感染牛血液中边缘无浆体DNA

以边缘无浆体表面保护抗原的ｍｓｐｌβ基因设计和合成的１对２０ｍｅｒ寡核苷酸作为引物，用耐热的Ｔａｇ－ＤＮＡ聚合酶经５０个循环扩增边缘无浆体模板，扩增产物直接用凝胶电泳检测，并用标准分子量确定。结果表明，只有用边缘无浆体ＤＮＡ模板进行扩增时，扩增产物才能生成，而以血液原虫，如牛巴贝斯虫、双芽巴贝斯虫、锥虫和牛白细胞ＤＮＡ作模板扩增，无扩增产物生成。ＰＣＲ检测边缘无浆体的灵敏度可达约３００个无浆体的ＤＮＡ。边缘无浆体ＰＣＲ适用于牛边缘无浆体病的病因鉴定和检测     

边缘无浆体MSP5蛋白基因的克隆及原核表达

参照已发表的边缘无浆体主要表面蛋白5（MSP5）基因的核苷酸序列,设计了1对特异性引物,以边缘无浆体基因组DNA为模板,采用PCR技术扩增获得了MSP5蛋白基因.将其克隆到pGEM-T Easy载体,并进行测序分析.结果表明,克隆的MSP5基因与GenBank上登录的Florida株MSP5蛋白基因的序列同源性达98.6%,编码氨基酸的同源性为99.0%,并且该序列包含有完整的开放阅读框,大小为633 bp.将该基因亚克隆入原核表达载体pGEX-4T-1,构建了重组原核表达载体.将其转化到DH5a宿主菌中,用IPTG进行诱导表达,实现了融合表达,表达产物的分子质量为45 ku.Western-blot分析表明,此表达产物能够被抗边缘无浆体阳性血清所识别.     

实时荧光PCR检测牛边缘无浆体方法的建立及应用

根据牛边缘无浆体表面蛋白4的保守基因序列设计特异引物AMOC9/AMOC5、AMOC10/AMOC12和特异性探针MP,首次建立了牛边缘无浆体的实时荧光PCR检测方法,检测DNA的最低限度为200 fg。对中央无浆体、绵羊无浆体、牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫和伊氏锥虫进行检测,无荧光检测信号。本研究用所建立的方法检测采自江苏和哈尔滨的180份抗凝血（奶牛和肉牛）,其阳性率为8.9%。结果表明,建立的实时荧光PCR检测牛边缘无浆体的方法具有较高的特异性和敏感性,可用于牛边缘无浆体病的流行病学调查、检疫和监测。     

快速凝集试验与补体结合试验检测牛边缘无浆体感染的效果对比

从１３８份血清样品的比较试验结果显示，快速凝集成试验（ＲＣＡ）比补结合试验（ＣＦ）检测边缘无浆体感染的敏感性高（８８．９％：８１．５％），假阴性率低（１１．１％：１８．５％），两者都具有良好的特异性和预测性，检测阳性符合率高，快速凝集试验对一次感染牛的阳性时间更长久（３０３天：９２天）。     

奶牛无浆体病PCR诊断方法的建立及应用

利用边缘无浆体(Anaplasma marginale)高度保守的msp5基因,建立了奶牛无浆体病PCR诊断方法。特异性试验表明与牛巴贝斯虫、双芽巴贝斯虫、温氏附红细胞体、大肠杆菌、金黄色葡萄球菌和牛白细胞DNA无交叉反应;敏感性试验表明可检测到约200个感染红细胞。利用该方法对黑龙江省西部多个养牛场送检的140份血样进行检测,其中曾出现过高热、气喘、流涎和贫血等临床症状的"无名高热"奶牛血样95份、无临床症状奶牛血样45份,结果有上述临床症状奶牛PCR血样阳性率为44.2%,无症状奶牛血样PCR阳性率为13.3%。从而证实无浆体是引起奶牛"无名高热"的病原之一。     

边缘无浆体MSP5基因序列分析及其融合蛋白的纯化

参照已发表的主要表面蛋白5（MSP5）基因的核苷酸序列,设计了一对特异性引物,以边缘无浆体基因组DNA为模版。采用PCR技术扩增获得了MSP5基因;将其克隆到pGEM—TEasy载体,并进行测序分析,结果表明,克隆的MSP5基因与GenBank上登录的Florida株MSP5基因的序列同源性达98．6％,编码氨基酸的同源性为99％,并且该序列包含有完整的开放阅读框,大小为633bp。将该基因亚克隆入原核表达载体pGEX-4T-1,构建了重组原核表达载体。将其转化到DH5a宿主茵中,用IPTG进行诱导表达,实现了融合表达。表达产物的分子质量为45ku。Western blot分析表明,此表达产物能够被抗边缘无浆体阳性血清所识别。通过裂解、洗涤、变性、复性等方法对包涵体蛋白进行处理,获得的纯化产物浓度为1mg／mL。     

快速凝集试验与补体结合试验检测牛边缘无浆体感染的效果对比

从138份血清样品的比较试验结果显示，快速凝集试验（RCA）比补体结合试验（CF）检测边缘无浆体感染的敏感性高（88．9％：81．5％），假阴性率低（11．1％：18．5％），两者都具有良好的特异性和预测性，检测阳性符合率高，快速凝集试验对一次感染牛的持续检出阳性时间更长久（303天：92天）     

抗牛边缘无浆体MSP5单克隆抗体的制备

为制备抗牛边缘无浆体膜表面蛋白(MSP5)单克隆抗体(MAb),以原核表达的重组MSP5蛋白(rMSP5)免疫BALB/c鼠,应用常规杂交瘤技术获得2株能稳定分泌特异性MAb的杂交瘤细胞株(1D8和2F3).间接ELISA检测腹水效价分别为5.49×105和7.83×104,亚类鉴定其重链为分别为IgG2b和1gG2a,轻链均为K型;ELISA叠加试验表明这2株MAb识别的抗原位点相同或相近;Western blot结果显示2株MAb均能与rMSP5发生反应.特异性抗MSP5的MAb的获得,为牛边缘无浆体检测奠定了基础.     

Comparison of a competitive inhibition ELISA and the card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle

OBJECTIVE: To compare a recently developed recombinant MSP-5 competitive inhibition ELISA with a card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in Australian cattle. MATERIALS AND METHODS: The ELISA was compared with the card agglutination test using 208 sera from cattle in Anaplasma-free herds, 86 sera from cattle experimentally infected with A marginale or A centrale and 757 sera from cattle in areas endemic for A marginale. RESULTS: The specificity of the ELISA, based on testing 208 sera from cattle in Anaplasma-free areas, was 99.5%, and the sensitivities for detection of antibodies to A marginale and A centrale in sera from the experimentally infected cattle were 98.0% and 100%, respectively. For the same sets of sera, the specificity of the card agglutination test was 98.6% and the sensitivities for detection of antibodies to A marginale and A centrale were 98.0% and 100%, respectively. For the 757 sera collected from cattle in areas endemic for A marginale, the agreement between the ELISA and the card agglutination test depended on the positive threshold selected for the ELISA. The maximum achievable agreement was 91.5% (kappa = 0.73; 95% confidence interval 0.66, 0.79). CONCLUSION: We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs.     

蓝舌病病毒重组VP7蛋白单克隆抗体制备及竞争ELISA检测方法的建立

为了建立蓝舌病(BT)的血清学诊断方法,本研究利用原核表达的蓝舌病病毒(BTV)血清型12型VP7纯化蛋白免疫BALB/c小鼠,制备2株单克隆抗体(MAb),分别命名为BTV-2D10和BTV-4H7。IFA试验表明,2株MAb均能与BTV 24个血清型发生特异性反应,而与茨城病病毒(IBAV)、中山病病毒(CV)、赤羽病病毒(AKAV)、牛病毒性腹泻病毒(BVDV)、牛传染性鼻气管炎病毒(IBRV)、牛轮状病毒(BRV)、牛肠道病毒(BEV)、牛呼肠孤病毒(RV)及口蹄疫病毒(FMDV)无交叉反应,表明2株MAb均为BTV群特异性抗体。采用重组表达的VP7蛋白作为包被抗原建立的竞争ELISA方法证明,BTV-4H7 MAb对不同血清型BTV阳性血清具有良好的阻断效果,而对AKAV、IBAV、BRV和FMDV阳性血清无阻断作用。本研究建立的竞争ELISA方法与IDEXX公司的试剂盒检测包括65份已知背景血清和322份采自广西省的山羊血清样品,检测结果符合率分别达100%和98%。该竞争ELISA方法的建立为BTV抗体的监测提供了安全、快速、准确的技术手段。     

Anaplasma marginale inactivated vaccine: dose titration against a homologous challenge

The present study was performed to dose-titrate an Anaplasma marginale experimental immunogen derived from partially purified initial bodies from three geographically different Mexican strains. Three five-bovine groups were inoculated twice on days zero and 21 with A. marginale initial bodies equivalent to 1.5×10¹⁰ (group I), 3×10¹⁰ (group II) or 6×10¹⁰ (group III) infected erythrocytes mixed with STDCM® adjuvant. A similar group served as non-vaccinated controls. All four groups were challenged with 1×10⁸ infected erythrocytes from a donor cow with an increasing rickettsemia of strain MEX-15 on day 87 post-vaccination. The prepatent period was very similar for all four groups. All five non-vaccinated controls presented typical acute anaplasmosis syndrome reaching a mean of 30.9% rickettsemia and a loss of 73.4% in the packed cell volume (PCV). Two of five controls died of acute anaplasmosis. Within the vaccinated groups only one animal (group II) suffered acute disease and died. Although all the other vaccinated animals were free of clinical signs, they developed very low rickettsemias (3.2, 3.8 and 4.3%) and PCV losses of 49.9, 47.8, and 49.3% for groups I, II and III. The starting mean weight was very similar for all four groups. All animals lost weight following challenge but losses for groups I and II were lower and significantly different from group IV losses (P0.1). Although there were no significant differences among vaccinated groups, group III was more severely affected. Taken altogether, these results show a 93.3% protection against both illness and death for all groups; and 100% protection for groups I and III, and 80% for group II.     

P49重组抗原检测旋毛虫抗体的问接ELISA方法建立

以纯化的本地毛形线虫P49排泄分泌(ES)重组蛋白包被微量反应板,建立检测旋毛虫抗体的间接ELISA方法,并确定了最适反应参数:抗原包被浓度4μg/mL;血清稀释度1:100,作用时间90 min;酶标二抗稀释度1:10 000,作用时间60min;最适封闭液为5 g/L聚乙烯醇(PVA);最适稀释液为含20 g/L PEG6000的PBS;底物最佳显色时间15 min.ELISA体系评价结果表明,该方法重复性好,灵敏度高,且可用于不同种株旋毛虫感染的P49血清抗体检测.     

Evaluation of a competitive ELISA for antibody detection against avian influenza virus

Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity.     

牛γ干扰素单克隆抗体的制备与抗原捕获ELISA检测方法的建立

为了建立一种简便、快速、可靠和经济的牛γ干扰素(BOIFN-γ)的免疫学检测方法,本研究通过杂交瘤技术建立了2株抗rBoIFN-γ单克隆抗体(MAb)2G6和5F9.Western blot、抗病毒活性阻断实验表明2株MAb与rBoIFN-γ有高亲和活性.相加EUSA表明2G6和5F9识别rBoIFN-γ不同的抗原表位.利用2G6作为捕获抗体,5F9-HEP作为检测抗体建立的抗原捕获ELlSA方法可特异性的检测不同系统表达的rBoIFN-γ,而且与rBoIFN-a、rBoIFN-β不发生交叉反应.该方法的建立为抗原捕获ELISA定量检测BoIFN-γ试剂盒的研制奠定了基础.     

猪蓝耳病病毒抗体双抗原夹心ELISA方法的建立及初步应用

根据PRRSV VR-2332株序列设计了1对扩增PRRSV N蛋白基因的特异性引物,从PRRSV北方株感染细胞中提取总RNA,通过RT-PCR获得长约372 bp的N蛋白编码基因片段。将其克隆到pGEX-6p-1质粒,构建了原核表达载体pPRRS-N。重组基因在大肠杆菌中表达出相对分子质量约为41 000的融合蛋白,目的蛋白表达量约占菌体蛋白的28.5%。利用此重组融合N蛋白建立了一种检测PRRSV特异性抗体的双抗原夹心ELISA,并通过与商品化试剂盒的应用比较对本方法进行了系统评价。分析了来自北京、山东、河南、河北4省区13个养猪场的260份血清,结果表明,本方法的敏感性和特异性分别为93.5%和86.7%,与IDEXX PRRSV抗体检测试剂盒检测结果的符合率达到91.5%。