破伤风毒素C片段在乳链菌内的表达

摘要

Objective To construct tetanus toxin C fragment ( TETC ) expression vector with lactococcus lactis.Methods TETC gene was acquired from the PCR based gene assembly plasmid pBS - TETC with Sal Ⅰ and Hind Ⅲ restriction enzymes, and subsequently cloned into the Lactococcus lactis secreted expression system pNBC1OOO and cell wall anchored expression system pNBC2000 to form pNBC1002 and pNBC2002. The expression systems were transformed into Lactococcus lactis by electroporation. Protein electrophoresis and Western - blot were used to detect the expression and bioactivity of TETC. Results The TETC expression vector was constructed successfully. The 57. 8 kD secretory TETC accounted for 8. 06％ of the total cell proteins, and 9. 82％ of the supernatant proteins. The 73. 5 kD anchored TETC accounted for 10. 7％ of the total cell proteins. and 17. 9％ of the cell wall proteins. Both forms of TETC were detected with the tetanus antitoxin hy Western - blot. Conclusion The recombinant TETC expressing Lactococcus lactis provides foundation for future study of biologic adjuvant and vaccine against tetanus.%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57.8 kD,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 kD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TETC,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基础.