摘要： 为了更好防治由出血性大肠杆菌O157∶ H7引起的疾病,尝试研制基因工程疫苗,其中亚单位疫苗时具有很大优势.构建表达tir和stx1b的融合基因,将tir基因中间295个氨基酸残基(Tir295)与stx1b亚基基因72个氨基酸串联构建pGEX-stx2b-tir-stx1b重组质粒,将其转化于BL21(DE3),用IPTG进行诱导表达,经SDS-PAGE电泳检测,该融合蛋白获得了高效表达,目的蛋白表达量占菌体总蛋白含量的30%左右.重组蛋白纯化后皮下途径免疫小鼠,能够诱导高滴度(105)的IgG,鼻腔内途径免疫小鼠不仅能够诱导高滴度(104)的IgG,还能诱导高达102的IgA.二免后攻击50LD50的EHEC O157∶ H7,相对于对照组,免疫组粪便中排菌量明显降低、排菌时间明显缩短.结果表明:该融合蛋白免疫原性良好,并且由Tir、Stx2b和Stx1b三部分抗原组成,可刺激机体产生针对转位紧密素受体(Tir)和志贺毒素(Stx)的抗体,在EHEC O157亚单位疫苗设计或单克隆抗体抗制备中具有重要价值.
关键词:
EHEC O157:H7,
转位紧密素受体,
志贺毒素,
融合基因,
免疫原性
Abstract: In order to prevent and cure EHEC O157∶ H7，genetically engineering vaccine，especially subunit vaccine， has significant dominance over others. To construct tir-stx fusion gene，the DNA fragments encoding middle 295 amino acids(Tir295) of tir and 72 amino acids(stx72) of stx1b and stx2b were fused in turn in frame with the multiple cloning sites of pGEX and the recombinant plasmid of pGEX-stx2b-tir-stx1b was transferred to host cells E. coli BL21 strain (DE3) . Then，the protein was induced and expressed with induction of IPTG. The expression quantities and style of fusion protein were determined by SDS-PAGE and thin layer scanning results showed that amount of expressed fusion protein was 30% in total of bacteria protein. Purified recombinant Stx2B-Tir-Stx1B protein can elicit high titer IgG (105) by subcutaneous immunization BALB / c mice，also，e licit high titer IgG (104) and IgA (102) by intra nasal immunization. Moreover， compared to non-immunized mice still shed EHEC O157∶ H7 on 14th day，immunized mice shed no bacteria in the fecal samples 8 day later after challenged with O157∶ H7. All results shows that stx2b-tir-stx1b protein has good antigenicity， and tir295 ，and stxb72 antigens can evoke immune responses against tir295 and stxb72， reseptively. The tir-stxb fusion protein expressed in E.coli could offer an important ingredient that should be consider in construction of recombinant EHEC O157 vaccine or preparation of experimental material for the monoclonal antibody.
Key words:
EHEC O157∶ H7,
Tir,
Stx,
Fusion gene,
Immunogenicity
中图分类号:
张雪寒, 何孔旺, 卢维彩, 赵攀登, 温立斌, 李彬, 郭容利, 王小敏, 倪艳秀, 周俊明, 俞正玉, 茅爱华, 吕立新. 出血性大肠杆菌O157: H7 Stx2B-Tir-Stx1B多价融合蛋白表达及免疫原性研究[J]. 华北农学报, 2010, 25(4): 180-185. doi: 10.7668/hbnxb.2010.04.038.
ZHANG Xue-han, HE Kong-wang, LU Wei-cai, ZHAO Pan-deng, WEN Li-bin, LI Bin, GUO Rong-li, WANG Xiao-min, NI Yan-xiu, ZHOU Jun-ming, YU Zheng-yu, MAO Ai-hua, LU Li-xin. Expression of Fusion Protein Stx2B-Tir-Stx1B from Enterohemorrhagic Escherichia coli O157: H7 and Its Immunogenicity Analysis in BALB/c Mice[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2010, 25(4): 180-185. doi: 10.7668/hbnxb.2010.04.038.