牛星状病毒神经型与肠道型ORF2蛋白原核表达及间接ELISA的建立与初步应用

Prokaryotic expression of bovine astrovirus neural and intestinal ORF2 protein and establishment and preliminary application of indirect ELISA

通过GenBank中登录号(神经型BufAstV-GX-NN-ORF2-14、肠道型BufAstV-GX-NN-ORF2-12)合成神经型ORF2-14-1与肠道型ORF2-12-1基因序列,运用PCR技术扩增神经型ORF2-14与肠道型ORF2-12目的片段的基因并克隆到pET-32a(+)载体,成功构建了重组质粒神经型pET-32a-ORF2-14与肠道型pET-32a-ORF2-12,经鉴定正确后转化BL21(DE3)感受态细胞中成功诱导表达,纯化后的重组蛋白利用SDS-PAGE、Western blot进行鉴定。以纯化的神经型pET-32a-ORF2-14与肠道型pET-32a-ORF2-12重组蛋白为诊断抗原,成功建立牛星状病毒(bovine astrovirus,BoAstV)神经型与肠道型的间接ELISA抗体检测方法。运用建立的间接ELISA对2019-2020年广西部分地区采集的712份牛血清进行BoAstV抗体的检测,结果显示肠道型共检出170份抗体阳性血清,BoAstV抗体阳性率为23%;神经型共检出124份抗体阳性血清,BoAstV抗体阳性率为17%;其中,2种型共同感染阳性率占总份数10%。结果表明,本试验成功建立了对BoAstV的神经型与肠道型分型检测方法,为临床上对BoAstV分型检测提供良好的检测方法打下基础。

The gene sequences of neural type ORF2-14-1 and intestinal type ORF2-12-1 were synthesized according to their gene sequences in GenBank(neurotype BufAstV-GX-NN-ORF2-14,intestinal type BufAstV-GX-NN-ORF2-12),the gene fragment of neural type ORF2-14 and intestinal type ORF2-12 were amplified by PCR technology and cloned into pET-32 a(+)vector,and the recombinant plasmid pET-32 a-ORF2-14 and pET-32 a-ORF2-12 were successfully constructed.The recombinant plasmid was identified and transformed into BL21(DE3)competent cells to induce expression.The purified recombinant protein was identified by SDS-PAGE and Western blot.Using the purified ORF2-14 and ORF2-12 recombinant proteins as diagnostic antigens,an indirect ELISA for the neural and intestinal types of bovine astrovirus(BoAstV)was successfully established.The established indirect ELISA was used to detect BoAstV antibodies in 712 bovine sera collected from parts of Guangxi Province from 2019to 2020.The results showed that a total of 170antibody-positive sera were detected to be intestinal type,and the positive rate of BoAstV antibody was 23%.A total of 124antibody-positive sera were detected to be neural type,and the positive rate of BoAstV antibody was 17%;the positive rate of co-infection of the two types accounted for 10%.The results showed that the detection method of neurotype and intestinal type BoAstV was successfully established,which laid a good foundation for clinical detection of BoAstV.