摘要
目的获得高纯度、高免疫原性的破伤风毒素C片段(TTC)蛋白。方法以PCR从破伤风杆菌质粒DNA中扩增TTC基因片段,将其插入载体pET43.1a(+)并导入大肠杆菌BL21(DE3)plysS中表达。用Ni2+-NTA琼脂糖柱纯化融合蛋白,经凝血酶酶切,再用Ni2+-NTA琼脂糖柱分离目的蛋白。SDS-PAGE和免疫印迹分析目的蛋白的相对分子质量和抗原性,动物免疫实验鉴定其免疫原性。结果获得1373bp的TTC基因片段,构建成重组表达载体pET43.1a(+)-TTC并在大肠杆菌中表达。TTC-Nus融合蛋白的表达量占菌体总蛋白的22%,酶切后TTC蛋白可与载体蛋白有效分离,获得纯度为95.5%的TTC蛋白,该蛋白可被破伤风抗毒素识别;将TTC蛋白免疫小鼠后,免疫血清可与破伤风毒素特异性结合,三次免疫后其抗体效价可达1∶25600。结论所获TTC蛋白纯度高,免疫原性好,为研究体内外抗原提呈、免疫应答提供了良好的模式抗原。
Objective To obtain highly purified tetanus toxin fragment C （TTC） with good immunogenicity. Methods The gene fragment encoding TTC was amplified from Clostridium tetani plasmid DNA by PCR, inserted into the vector pET43, la （＋） and expressed in E. coli BL21 （DE3）plysS. After purification using Ni^2＋-chelate affinity chromatography, the expressed fusion protein was digested by thrombin and the resultant TTC protein was purified with Ni^2＋-chelate affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purifed TTC protein was then used to immunize mice to test its immunogenecity. Results The 1373-bp gene fragment encoding TTC was obtained, and the constructed recombinant expression vector pET43.1a （＋）-TTC was successfully expressed in E. coli BL21 （DE3）plysS. SDS-PAGE identified a recombinant fusion protein with relative molecular mass （Mr） of 117 000, which accounted for 22% of the total bacterial protein. The TTC protein with Mr of 50 000 was obtained after purification of the thrombin digestion products of the fusion protein, with a purity reaching 95.5%. Both the fusion protein and TTC protein could be recognized by anti-tetanus toxin antibody as shown by Western blotting. The titer of the anti-serum from mice immunized with the TTC protein was 1：25 600, and the anti-serum could specifically bind to tetanus toxin. Conclusion Highly purified and immunogenetic TTC protein has been successfully obtained, which provides a good model antigen for studying antigen presentation and immune responses in vivo.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2008年第5期731-735,共5页
Journal of Southern Medical University
基金
广东省自然科学基金研究团队项目(015003)~~
关键词
破伤风毒素C蛋白
基因克隆
重组表达
免疫原性
模式抗原
tetanus toxin fragment C
gene cloning
recombinant expression
immunogenicity
model antigen
