新疆地区流产奶牛新孢子虫病流行病学研究及NcSRS2基因原核表达载体的构建
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摘要
新孢子虫病(Neosporosis)是由犬新孢子虫(N.caninum)寄生在宿主体内引起多种家畜共患的原虫病,此病对牛的危害尤为严重,主要造成母牛流产、产死胎以及新生犊牛的运动神经系统疾病,给畜牧业造成了巨大的经济损失。迄今为止,还没有防治新孢子虫病的疫苗和有效药物,对新孢子虫病的早期确诊是目前控制该病发生和蔓延的重要手段。我国对该领域的研究起步较晚,但近年来逐渐系统和深入。为更好地了解新疆地区流产奶牛新孢子虫病的感染情况,从奶牛主要产区选定15个地区共20个较大规模牛场采集了419头流产史奶牛的血清,应用VMRD公司生产的进口ELISA试剂盒对其进行了抗体检测,并用chahan B建立的rELISA方法进行了复检。ELISA试剂盒检测结果表明,在419份被检血清中有43份为阳性,新孢子虫阳性率为10.26%,各个地区的牛场阳性率在0-50%之间,不同地区流产奶牛新孢子虫病阳性率差异显著(P<0.05);各个年龄段流产奶牛阳性率差异不显著(P>0.05);不同胎次的流产奶牛阳性率差异显著(P<0.05)。rELISA方法检测结果表明,419份血清中有47份阳性,阳性率为11.22%,说明新疆地区存在新孢子虫的感染。
根据GenBank上发表的新孢子虫NcSRS2基因序列,设计合成一对引物,以新孢子虫标准虫株为模板,PCR方法扩增NcSRS2基因,与pMD18-T Simple载体连接并转化到DH5α宿主菌中,提取质粒,酶切后并测序验证,获得阳性重组质粒pMD18-T-NcSRS2t。阳性质粒pMD18-T-NcSRS2t和表达载体pGEX-4T-3质粒,经EcoRⅠ酶切后分别回收,连接,转化到表达菌E.coli BL21,提取质粒,酶切后并测序验证,将构建的重组载体命名为pGEX-4T-NcSRS2t。阳性菌株在IPTG诱导下表达,经SDS-PAGE电泳分析表明,表达产物与预期大小相符,约60.2kDa,为建立新孢子虫病免疫学诊断方法及研制低成本、高特异性的新孢子虫病诊断试剂盒奠定基础。
Neosporosis is a protozoan disease caused by Neospora caninum, an intracellular parasite that infects various species of livestock, which has brought serious detrimental effects on cattle. It is primarily causative agent of abortion, stillbirth and motor nervous system disease of nascent calves in cattle and has brought about huge economic loss to the livestock industry. Currently, there is no effective drug and vaccines for control of Neosporosis, so it is important to develop an early, accurate diagnostic method for controlling Neosporosis. The research on this field in China is relatively late, but has been studied deeply and systematically in recent years. The prevalence of Neosporosis in dairy cattle in Xinjiang of China was investigated. In total, 419 serum samples taken from aborting dairy cattle from 20 cattle farms of 15 regions in Xinjiang were examined for Neospora caninum infection by ELISA kit and rELISA methods by chahan B et al. Of the 419 samples, 43 samples were positive by ELISA kit and the serum antibody-positive rate was 10.26%. Neospora caninum serum antibody-positive rate in all cattle farms was 0 to 50% and there were no significant difference (P>0.05)in the antibody-positive rate between different ages of abortion dairy cattle sera, but there were significant difference(P<0.05)between different regions and different times of abortion dairy cattle sera. Of the 419 samples, 47 samples were positive by rELISA and the serum antibody-positive rate was 11.22%. Show that the existence of the Xinjiang region of Neospora infection.
Based on the NcSRS2 gene sequence of Neospora caninum in the GenBank, the specific primers containing EcoRⅠenzyme digestion sites were designed. NcSRS2 gene was amplified by PCR using genomic DNA and inserted into pMD18-T Simple vector, then transformed into the competent E.coli DH5α. The positive recombinant pMD18-T-NcSRS2t and the expression vector pGEX-4T-3 were digested by restriction endonuclease digestion, then transformed into the competent E.coli BL21. Finally a recombinant plasmid named pGEX-4T-NcRSR2t was obtained, and was identified by restriction enzyme analysis and sequencing. Positive strains were induced with IPTG. The results of SDS-PAGE showed that the protein was 60.2kDa in size. The recombinant NcSRS2 protein will be used for further studies in diagnosis and immunity against Neospora caninum infection.
根据GenBank上发表的新孢子虫NcSRS2基因序列,设计合成一对引物,以新孢子虫标准虫株为模板,PCR方法扩增NcSRS2基因,与pMD18-T Simple载体连接并转化到DH5α宿主菌中,提取质粒,酶切后并测序验证,获得阳性重组质粒pMD18-T-NcSRS2t。阳性质粒pMD18-T-NcSRS2t和表达载体pGEX-4T-3质粒,经EcoRⅠ酶切后分别回收,连接,转化到表达菌E.coli BL21,提取质粒,酶切后并测序验证,将构建的重组载体命名为pGEX-4T-NcSRS2t。阳性菌株在IPTG诱导下表达,经SDS-PAGE电泳分析表明,表达产物与预期大小相符,约60.2kDa,为建立新孢子虫病免疫学诊断方法及研制低成本、高特异性的新孢子虫病诊断试剂盒奠定基础。
Neosporosis is a protozoan disease caused by Neospora caninum, an intracellular parasite that infects various species of livestock, which has brought serious detrimental effects on cattle. It is primarily causative agent of abortion, stillbirth and motor nervous system disease of nascent calves in cattle and has brought about huge economic loss to the livestock industry. Currently, there is no effective drug and vaccines for control of Neosporosis, so it is important to develop an early, accurate diagnostic method for controlling Neosporosis. The research on this field in China is relatively late, but has been studied deeply and systematically in recent years. The prevalence of Neosporosis in dairy cattle in Xinjiang of China was investigated. In total, 419 serum samples taken from aborting dairy cattle from 20 cattle farms of 15 regions in Xinjiang were examined for Neospora caninum infection by ELISA kit and rELISA methods by chahan B et al. Of the 419 samples, 43 samples were positive by ELISA kit and the serum antibody-positive rate was 10.26%. Neospora caninum serum antibody-positive rate in all cattle farms was 0 to 50% and there were no significant difference (P>0.05)in the antibody-positive rate between different ages of abortion dairy cattle sera, but there were significant difference(P<0.05)between different regions and different times of abortion dairy cattle sera. Of the 419 samples, 47 samples were positive by rELISA and the serum antibody-positive rate was 11.22%. Show that the existence of the Xinjiang region of Neospora infection.
Based on the NcSRS2 gene sequence of Neospora caninum in the GenBank, the specific primers containing EcoRⅠenzyme digestion sites were designed. NcSRS2 gene was amplified by PCR using genomic DNA and inserted into pMD18-T Simple vector, then transformed into the competent E.coli DH5α. The positive recombinant pMD18-T-NcSRS2t and the expression vector pGEX-4T-3 were digested by restriction endonuclease digestion, then transformed into the competent E.coli BL21. Finally a recombinant plasmid named pGEX-4T-NcRSR2t was obtained, and was identified by restriction enzyme analysis and sequencing. Positive strains were induced with IPTG. The results of SDS-PAGE showed that the protein was 60.2kDa in size. The recombinant NcSRS2 protein will be used for further studies in diagnosis and immunity against Neospora caninum infection.
引文
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