传染性胰腺坏死病毒VP3基因的分段表达及其抗原表位区域分析

Expression of overlapping fragments of IPNV VP3 gene and analysis of antigenic epitope domains

采用PCR方法克隆了传染性胰腺坏死病毒（IPNV）VP3四段相互重叠的基因片段L1、L2、L3和L4,将PCR产物分别连接到原核表达载体pGEX-6P1和pET32a上,经酶切、PCR、测序鉴定,获得重组质粒pGEX-6P1-VP3（L1）、pET32a-VP3（L2）、pGEX-6P1-VP3（L3）和pGEX-6P1-VP3（L4）;将其分别转化感受态菌Rosetta和BL21,经1.0mmol/L的IPTG诱导,分别得到大小为32 kD、26 kD、30 kD和31 kD的目的蛋白。经Western-blo-ting分析,4段目的蛋白中,只有VP3（L1）和VP3（L4）基因片段所表达的融合蛋白能与IPNV阳性血清反应,说明VP3蛋白的免疫优势区域集中在该蛋白的1~70和143~206氨基酸之间。该融合蛋白可以作为诊断抗原用于建立ELISA诊断方法。本实验为进一步精确定位VP3蛋白抗原表位奠定了基础。

Four overlapping fragments L1,L2,L3 and L4 of VP3 gene of infectious pancreatic necrosis virus（IPNV） were amplified by PCR.PCR products were cloned into the expression vector pGEX-6P1 and pET32a.The recombinant plasmids pGEX-6P1-VP3（L1）,pET32a-VP3（L2）,pGEX-6P1-VP3（L3） and pGEX-6P1-VP3（L4） were identified by digestion,PCR and sequencing,and transformed into E.coli Rosetta and BL21 cells.The target proteins of 32 kD,26 kD,30 kD and 31 kD were obtained by induction using IPTG at 1.0 mmol/L.The VP3（L1） and VP3（L4） protein reacted with IPNV positive serum in Western-blotting,indicating that the immunodominant region of the VP3 protein was located in 1~70 and 143~206 amino acid sequence of VP3 protein.The VP3 recombinant fusion protein can be used as the specific diagnosis antigen for ELISA assay.This study will serve as the basis for further precise identification of VP3 protein epitope.