破伤风毒素C端片段与心肌营养素-1融合蛋白表达、纯化与靶向神经元逆向运输及神经营养活性鉴定

Expression, purification of tetanus toxin C fragment/cardiotrophin-1 recombinant fusion protein, target delivery to CNS and neurotrophy biology ability

目的在BL21（DE3）大肠杆菌中表达破伤风毒素C端片段（TFC）与心肌营养素（CT）-1融合蛋白并纯化。探讨TTC转导结构域对CT-1的靶向神经元逆向运输活性与CT．1的神经营养活性。方法异丙基-B—D硫代半乳糖苷（IPTG）诱导BL2（DE3）大肠杆菌表达CT-1／TYC融合蛋白，SDS-聚丙烯酰胺凝胶电泳（SDS—PAGE）与WB法鉴定融合蛋白表达。制备SD大鼠坐骨神经损伤模型，分别经神经再生室、肌肉注射给药，自由放养1周后处死，4％多聚甲醛灌注固定，取脊髓L4～L6腰膨大标本，冰冻切片，免疫组织化学检测。制备新生6～7d龄sD大鼠坐骨神经损伤模型，肌肉注射CT-1／TTC融合蛋白，自由放养1周后处死，4％多聚甲醛灌注固定，取L4～L6腰膨大标本，冰冻切片，尼氏染色计数脊髓前角运动神经元。结果原核表达目的蛋白CT-1／TTC表达量占全菌蛋白总量的15％，以可溶性蛋白为主。通过谷胱甘肽S-转移酶（GST）标签亲和纯化获得2.7g／L的CT-1／TTC重组融合蛋白。免疫组织化学于脊髓腰膨大检测到CT-1／TTC融合蛋白。坐骨神经损伤后，脊髓L4-L6腰膨大前角运动神经元计数较CT-1／TTC融合蛋白处理组减少，存活率下降。结论基因重组CT-1／TTC蛋白在大肠杆菌中获得了表达和纯化，所获得的融合蛋白有靶向性脊髓神经元细胞的活性和对损伤后运动神经元的神经营养活性。

Objective Expression, purification of tetanus toxin C fragment/cardiotrophin-1 recombinant fusion protein （CT-1/TTC） in BL21 （DE3 ） E. coli, examined whether tetanus toxin C fragment mediate the cardiotrophin-1 target delivery to the central nervous system and the cardiotrophin-1 has the neurotrophic ability. Methods Induction by IPTG, the fusion protein was expressed and then purified by GST affinity agarose. The interest protein was viewed by SDS-PAGE, further characterized by Western Blot. Rat sciatic nerve transected model was selected. Using drug by nerve-regeneration-chamber and intramuscular injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained with immunohistochemistry method. And select the new born SD rat sciatic nerve transected model, using CT-1/TTC fusion protein by muscle injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained by Nissl＇s staining. Results After induction, the fusion protein was about 15% of the total protein and the soluble part was predominant. Purified by GST fusion protein column, the interest protein＇s concentration is 2.7 g/L. The CT-1/TTC fusion protein was found in lumbar intumescentia by immunohistochemistry method. And after sciatic nerve transected, the numbers of cornu anterius medullae spinalis motoneurons in L4-L6 segments, compared to CT-1/TTC protein grope, have a lower survival rate. Conclusions The recombinant CT-1/TTC protein can be expressed and purified in BL21 （ DE3 ） E. coli. This fusion protein has two biological activities of targeting delivery to central nervous system and protecting the cornu anterius medullae spinalis motoneurons.