【摘要】 为制备新型鸭呼肠孤病毒（NDRV）σC蛋白的单克隆抗体（MAb）并鉴定其识别的抗原表位，本研究采用原核系统表达重组GST-σC蛋白（rGST-σC）并采用谷胱甘肽树脂纯化，经SDS-PAGE检测该蛋白的表达与纯化效果。利用构建的并经测序鉴定正确的真核表达质粒pCAGGS-HA-σC转染HEK293T细胞，24 h后采用Western blot检测重组HA-σC蛋白（rHA-σC）的表达。将rGST-σC免疫6周龄BALB/c小鼠3次，3免7 d后采用间接ELISA检测小鼠血清中的抗体效价。取血清效价最高的免疫小鼠脾细胞与SP2/0细胞融合后，通过间接ELISA筛选稳定分泌NDRVσC蛋白MAb的杂交瘤细胞并经4次克隆纯化。以rGST-σC作为包被抗原，采用间接ELISA测定杂交瘤细胞上清的抗体效价，并利用试剂盒鉴定MAb的亚类。SDS-PAGE结果显示，在60 ku处出现目的条带且该蛋白在上清液中表达。纯化结果显示，在60 ku处出现单一目的条带。Western blot结果显示，在35 ku处出现特异性条带。表明rGST-σC和r HA-σC均获得了表达。间接ELISA结果显示，免疫3次后小鼠血清抗体效价最高达1:25 600。间接ELISA结果显示，筛选的杂交瘤细胞上清中的MAb效价可达1:12 800，且该MAb为IgG1亚类，轻链为κ链。表明获得1株NDRVσC蛋白的MAb 10F6。将重组质粒pCAGGS-HA-σC转染HEK293T细胞，24 h后分别通过Western blot和间接免疫荧光试验（IFA）鉴定MAb的反应性。结果显示，分别在35 ku处出现特异性条带和特异性绿色荧光，而对照细胞无该条带和绿色荧光。表明MAb 10F6的反应性较强。通过对σC蛋白随机截短（σC-1～σC-7）并经原核系统表达，利用Western blot初步鉴定MAb 10F6识别的抗原表位；分别向初步鉴定的抗原表位N端和C端以单个氨基酸为步移，由公司合成9条多肽（C1～C9）作为包被抗原，采用间接ELISA进一步鉴定MAb 10F6识别的抗原表位。Western blot结果显示，MAb 10F6仅与σC-2反应后出现特异性条带，而与σC-1和σC-3～σC-7反应后均无特性条带，即MAb 10F6识别的抗原表位位于aa72～aa77。间接ELISA结果显示MAb 10F6与C4、C5、C6和C7均呈强阳性反应，这几条多肽均含⁷³LSSRV⁷⁷。表明MAb 10F6识别的最小线性表位为⁷³LSSRV⁷⁷。综上，本研究制备了MAb 10F6，其反应性较强，并鉴定了MAb的抗原表位，为NDRV检测方法的建立及σC蛋白在病毒感染中的作用研究奠定了基础。更多还原

【Abstract】 To prepare the monoclonal antibody（MAb） against sigmaC（σC） protein of novel duck reovirus（NDRV） and identify the antigenic epitope recognized by the MAb, prokaryotic expression system and glutathione resin were used to express and purify the NDRV recombinant GST-σC proteins（rGST-σC）, and the expression and purificaiton of rGST-σC were analyzed by SDS-PAGE.The reconstructed plasmid pCAGGS-HA-σC that was correctly identified by sequencing was transfected into HEK293T cells. At24 hours post-transfection, the expression of recombinant HA-σC protein（rHA-σC） was identified by Western blot. Six-week-old BALB/c mice were immunized with purified rGST-σC three times. Seven days after the third immunization, indirect ELISA was applied to detect antibody titers in mouse sera. Spleen cells from the mouse with the highest serum antibody titer were fused with SP2/0 myeloma cells. Hybridoma cell line that steadily secreted MAb against the NDRV σC protein was screened by indirect ELISA and subjected four rounds of cloning purification. Using rGST-σC as the coating antigen, the antibody titer in hybridoma cell culture supernatants was determined by indirect ELISA. The MAb subclasses was tested by commercialized kit. The SDS-PAGE results showed that the target protein band, which appeared at 60 ku, was expressed in the supernatant, and the purified protein displayed a single band at 60 ku. The Western blot result revealed a specific band at 35 ku. These results indicated that both r GST-σC and rHA-σC were successfully expressed. The results of indirect ELISA showed that the highest serum antibody titer of mice after the third immunization was 1:25600. This mouse was then selected for boost immunization, followed by fusion with SP2/0 cells and screening for hybridoma cells stably secreting MAb against the NDRV σC protein. The indirect ELISA results showed that the antibody titer in hybridoma cell culture supernatant was 1:12800, and the MAb belonged to IgG1 subtype with κ chain. One hybridoma cell line secreting MAb against NDRV σC protein was obtained and named 10F6. The reconstructed plasmid p CAGGS-HA-σC was transfected into HEK293T cells. At 24 hours post-transfection, the reactivity of MAb was identified by Western blot and indirect immunofluorescence assay（IFA）. Results showed that a specific band appeared at 35 ku, and the specific green fluorescence was observed;whereas there was no band and green fluorescence observed in the control cells. The above results indicated that the MAb 10F6 had strong reactivity. The random truncated σC proteins（σC-1-σC-7） were expressed by prokaryotic expression system. Western blot was used to preliminary identify the antigenic epitope recognized by MAb 10F6. Based on the result, nine peptides（C1-C9）, which moved stepwise with one amino acid towards epitope N-terminus or C-terminus, were synthesized. The peptides were used as the coating antigen inindirect ELISA to further identify the antigenic epitope recognized by MAb 10F6. The Western blot result showed that onlyσC-2, but not σC-1 and σC-3-σC-7, displayed the specific band after reacting with MAb 10F6, suggesting that the antigenic epitope recognized by MAb 10F6 was located at aa72-aa77. The indirect ELISA results showed that MAb 10F6 exhibited astrong positive reaction with C4, C5, C6, and C7, all of which contained⁷³LSSRV⁷⁷. These results indicated that the minimal linear epitope recognized by MAb 10F6 was⁷³LSSRV⁷⁷. To conclude, the MAb 10F6 with strong reactivity was prepared, and the antigentic epitope recognized by the MAb was identified in this study. These findings provide the basis for the development of NDRV detection method and facilitate the further studies on the NDRV σC protein in viral infection.更多还原