高致病性猪繁殖与呼吸综合征病毒M蛋白CD_4^+ T细胞表位优势区的鉴定

Identification of CD_4^+ T cell epitope dominance region in M protein of porcine reproductive and respiratory syndrome virus

采用RT-PCR方法扩增猪繁殖与呼吸障碍综合征病毒(PRRSV)临床分离株的M基因(525 bp),通过设计4对引物,把M蛋白分成4段K1(78-112)、K2(98-131)、K3(118-153)、K4(139-174),分别扩增相应基因M1、K1、K2、K3和K4,连接pET-32a(+)表达载体,经鉴定后转化Escherichia coliBL21,经IPTG诱导表达,SDS-PAGE和Western-blot检测结果显示,分别在相对分子质量30.8×103和24.4×103位置出现目的条带,重组蛋白均可与阳性血清发生特异反应。将蛋白M1免疫BALB/c小鼠,于第三次免2周后从小鼠脾脏分离淋巴细胞,分别用K1、K2、K3、K4蛋白进行体外刺激。流式细胞仪检测分泌IFN-γ细胞因子的T细胞的频率,K1蛋白刺激组显著高于其他组和对照组(P≤0.01)。结论:M1蛋白K1(78-112)段为T细胞表位优势区。

The M gene of porcine reproductive and respiratory syndrome virus（PRRSV）was amplified by RT-PCR and then the truncated M1 and K1（78-112）,K2（98-131）,K3（118-153）and K4（139-174）protein genes were amplified by PCR and cloned into a prokaryotic expression vector pET-32a（＋）.The recombinant plasmids pET-M1,K1,K2,K3,K4 were transformed into Escherichia coli BL21 cells.The target proteins of 30.8×103 and 24.4×103 were expressed with IPTG induction and showed a strong reaction to the PRRSV positive serum in Western-blot.BALB/c mice were immunized with purified M1 protein,setting PRRSV as the control.Lymphotytes suspension was prepared from spleens two weeks after the third immunity and stimulated with K1,K2,K3 and K4 protein in vitro.The IFN-γ producing cell in CD＋3 CD＋4 cell subsets was determined and analyzed by flow cytometry（FCM）.Quantities of IFN-γ producing cell in CD＋3 CD＋4 cell in K1 protein stimulated group was higher than in other groups.The result demonstrated that K1 protein contain immunodominant CD＋4 T cell epitopes.