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目的:克隆转位紧密黏附素受体细胞骨架偶联蛋白(TccP)基因,表达纯化TccP,制备其多克隆抗体并研究该抗体在EHEC O157∶H7黏附宿主细胞模型中的应用。方法:从EHEC O157∶H7 Sakai菌株基因组中克隆得到tccP基因,构建重组表达质粒pET28a-TccP。将此质粒转化大肠杆菌BL21(DE3)后用异丙基-D-硫代半乳糖苷(IPTG)诱导表达。经纯化分离后,免疫新西兰大白兔,制备其多克隆抗体,并用ELISA法和Western blot法对其进行鉴定。使用兔抗TccP多克隆抗体对EHEC O57∶H7黏附宿主细胞模型中的TccP分子进行定位研究。结果:获得了TccP cDNA,与GenBank报道的序列一致,并成功构建了高效原核表达质粒pET28a-TccP;Western blot结果表明,经IPTG诱导,在大肠杆菌BL21(DE3)中表达了相对分子质量(Mr)约37 000的目的蛋白。表达产物N端氨基酸序列测序结果与GenBank公布的基因序列推定的氨基酸序列完全一致。所制备的兔抗TccP多克隆抗体经Western blot法测定可与Mr37 000目的蛋白发生特异性结合反应;免疫荧光检测发现TccP聚集在EHEC O157∶H7黏附处细胞膜的下方。结论:成功构建了pET28a-TccP载体,获得了高纯度的重组TccP及其特异性多克隆抗体。
OBJECTIVE: To clone and translocate TCCP gene and express purified TccP to prepare its polyclonal antibody, and to study its application in EHEC O157: H7 adhesion cell model. Methods: The tccP gene was cloned from the genome of EHEC O157: H7 Sakai strain and the recombinant expression plasmid pET28a-TccP was constructed. The plasmid was transformed into E. coli BL21 (DE3) and induced with isopropyl-D-thiogalactoside (IPTG). After purified and isolated, New Zealand white rabbits were immunized and their polyclonal antibodies were prepared and identified by ELISA and Western blot. TccP molecules in the EHEC O57: H7 adhesion host cell model were targeted using a rabbit anti-TccP polyclonal antibody. Results: The TccP cDNA was obtained, which was consistent with the sequence reported in GenBank. The prokaryotic expression plasmid pET28a-TccP was successfully constructed. The result of Western blot showed that the recombinant plasmid pET28a-TccP was expressed in E. coli BL21 (DE3) ) About 37 000 of the target protein. The N-terminal amino acid sequence of the expression product was completely consistent with the putative amino acid sequence of the gene sequence published in GenBank. The prepared rabbit anti-TccP polyclonal antibody could specifically react with Mr37 000 protein as determined by Western blot. Immunofluorescence showed that TccP aggregated below the membrane of EHEC O157:H7. CONCLUSION: The pET28a-TccP vector was successfully constructed and highly purified recombinant TccP and its specific polyclonal antibody were obtained.