破伤风毒素C片段在乳链菌内的表达

目的：构建破伤风毒素C片段(TETC)乳链菌表达载体，并验证其生物活性。方法: 将采用基因拼接方法获得的TETC基因从测序正确的质粒pBS-TETC上以Sal I、Hind Ⅲ切下，分别定向连接到以Xho I、 Hind Ⅲ酶切的质粒分泌表达的乳链菌表达载体pNBC1000和膜锚定表达的乳链菌表达载体pNBC2000。电穿孔转化乳链菌，分别提取分泌蛋白及膜锚定蛋白，通过蛋白电泳及Western-blot检测蛋白定位表达情况。结果：构建了分泌表达及锚定表达TETC的乳链菌表达载体pNBCL1002与pNBCL2002，电穿孔转化乳链菌获得了分泌表达及膜锚定表达TETC的乳链菌，SDS-PAGE分析显示分泌表达的TETC大小约为57.8kDa，表达量占总体蛋白的8.06%、上清蛋白的9.82%，锚定表达的TETC大小约为73.5 kDa的表达蛋白，表达量占总体蛋白的10.7%,膜蛋白的17.9%； Western-blot检测显示所表达的TETC均可与破伤风抗毒素血清发生免疫印迹。结论：成功构建了TETC乳链菌表达载体，并在乳链菌内表达了TETC，并初步验证了表达的TETC的免疫反应性，这一结果可能为进一步研究生物佐剂及防治破伤风疫苗奠定了基础。

Expression of tetanus toxin C fragment in Lactococcus lactis

To expreess the tetanus toxin C fragment(TETC) and test its bioactivity in lactococcus lactis. Methods The gene of the tetanus toxin C fragment acquired by the PCR based gene assembly was cut from the plasmid pBS-TETC, then cloned into the lactococcus lactis secreted expression system pNBC1000 and cell wall anchored expression system pNBC2000 to form pNBC1002 and pNBC2002, the confirmed sequence was cut and ligated to pTRKH2 a shuttle vector for E. coli and Lactococcus Lactis to generate pNBCL1002 and pNBCL2002. pNBCL1001 and pNBCL2002 were transformed into Lactococcus Lactis by electroporation. Protein electrophoresis and Western-Blot were used to detect the expession of TETC and its bioactivity. Results The TETC expression vectors were constructed successfully. The secretory form of TETC had a weight of　57.8kDa and the portion of recombinant protein was over 8.06% of the total cell protein, and over 9.82% of the supernatant protein. The cell anchored form of TETC had a weight of　73.5 kDa, with the recombinant protein portion of 10.7% over the total cell protein and 17.9% over the cell wall protein. All the forms of TETC could be deteced with the tetanus antitoxin by western blotting. Conclusion The recombinant lactococcus lactis expressing the TETC may lay a foundation for the study of biology adjuvant and vaccine against tetanus.