【作者】 贾洪林； 仇华吉； 李国新； 朱庆虎； 李娜； 童光志；

【Author】 JIA Honglin, QIU Huaji, LI Guoxin, ZHU Qinghu, LI Na, TONG Guangzhi (National Key Laboratory of Veterinary Biotechnology, Harbin Veterianry Research Institute, CAAS, Harbin 150001)

【机构】 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室；

【摘要】 猪瘟(classical swine fever,CSF)是由猪瘟病毒(classical swine fever virus,CSFV)引起的一种高度传染性和致死性疾病。为建立与以猪瘟病毒囊膜糖蛋白E2为基础的标记疫苗相配套的鉴别诊断试验,本研究克隆了猪瘟病毒石门系强毒株Erns基因和E2基因主要抗原编码区,同时分别将其在大肠杆菌中进行了高效表达,并建立了基于重组蛋白抗原的Erns-ELISA和E2-ELISA诊断方法。设计并合成跨越CSFV Erns基因和E2基因主要抗原编码区序列的特异性引物,采用RT-PCR方法从CSFV石门系强毒株基因组RNA中扩增出0.7kb的完整Erns基因片段,从含有E2基因的重组质粒pcCDST中扩增出0.4kb的部分E2基因片段,分别将其克隆于原核表达载体pPROEXTMHTc和pPROEXTMHTb中,经酶切和PCR鉴定后,再进行序列测定。结果表明,Erns基因编码区核苷酸序列长度为723bp,编码240个氨基酸残基;部分E2基因片段的核苷酸序列长384bp,编码128个氨基酸残基,分别与GenBank中收录的CSFV石门系强毒株(GenBank accession No.AF092448)对应序列一致。阳性菌经IPTG诱导后获得高效表达,表达产物经SDS-PAGE分析,结果显示,表达的重组Erns融合蛋白和截短的重组E2融合蛋白分子量分别为31ku和19ku左右,分别占菌体总蛋白的27.6%和33.5%。Western blotting分析表明,表达的融合蛋白均具有免疫反应性。将表达的融合蛋白分别进行亲和层析纯化,得到纯度很高的目的蛋白。用纯化的Erns融合蛋白作为包被抗原建立了Erns-ELISA,经优化,抗原最适包被量为0.15μg,血清工作浓度为1:100倍稀释,阳性判定标准为OD490(?)0.3。经敏感性试验、特异性试验、阻断试验和重复性试验表明,本研究建立的Erns-ELISA简便、敏感、特异、稳定。通过对90份血清进行平行盲测,Erns-ELISA与E2-ELISA的符合率高达96.7%,与基于病毒抗原的Dot-ELISA的符合率达77.5%。总之,本研究高效表达了猪瘟病毒Erns与部分E2基因,建立了Erns-ELISA和E2-ELISA,可以作为基于E2蛋白的猪瘟标记疫苗的配套鉴别诊断技术,用于今后的猪瘟扑灭计划。更多还原

【Abstract】 Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by classical swine fever virus (CSFV). In order to establish differential diagnostic assays accompanying marker or DIVA vaccines against CSF, Erns gene and partial E2 gene containing major antigenic regions were amplified by PCR from the genomic RNA of CSFV Shimen and the recombinant plasmid pcDST containing CSFV Shimen E2 gene, respectively, using two pairs of primers specific for Erns and partial E2 genes based on the published sequence of CSFV Shimen strain (GenBank accession No. AF092448). The amplified fragments were then cloned into prokaryotic expression vectors pPROEXTMHTc and pPROEXTMHTb, creating pPROC-Erns and pPROB-E2, respectively. The resulting recombinant plasmids were subjected to sequencing after identification by restriction analysis and PCR. The cloned Erns gene was shown to encode 240 amino acid (aa) residues, and the truncated E2 gene encode 128 aa residues. Both of the sequences are identical to the corresponding sequences of CSFV Shimen strain deposited in GenBank.His-tagged recombinant proteins were expressed with high efficiency in E. coli transformed by pPROC-Erns and pPROB-E2 (harboring Erns and E2 6xHis fusion genes, respectively) after induction with IPTG The recombinant fusion proteins have apparent molecular weights of about 31 and 19 ku, respectively, as indicated by SDS-PAGE analysis, and showed specific immunoreactivity with anti-CSFV sera in Western blotting.The fusion proteins were purified by RoboPopTM Ni-NTA His·Bind Purification Kit. An indirect ELISA (Erns-ELISA) was established using the Erns fusion protein as coating antigen. The optimal coating amount was determined to be 0.15 μg of Erns antigen, and the serum dilution was optimalized at a dilution of 1:100. The established Erns-ELISA was shown to be sensitive, specific and reproducible, and has a good agreement with E2-ELISA based on the recombinant fusion E2 protein (96.7%) and Dot-ELISA based on whole viral antigens (77.5%).In summary, Erns and truncated E2 genes were efficiently expressed in E. coli, and Erns-ELISA and E2-ELISA were developed based on the recombinant fusion proteins. The Erns-ELISA combined with E2-ELISA can be used as differential diagnostic assays accompanying marker or DIVA vaccines against CSF to differentiate animals infected with wild-type viruses (E2-positive and Erns-positive) from animals vaccinated with E2-based marker vaccines (E2-positive but Erns-negative).更多还原