破伤风毒素C片段在乳链菌内的表达  

Expression of tetanus toxin C fragment in Lactococcus lactis

作　　者：杨晓强[1] 赵亚刚[2] 孙大勇[2] 姜泊[3] 陈学清[4] 

机构地区：[1]中国人民解放军75600部队医院综合科 [2]广州军区广州总医院消化内科 [3]南方医科大学南方医院消化内科 [4]广州医学院第一附属医院消化内科

出　　处：《广东医学》2011年第13期1644-1647,共4页Guangdong Medical Journal

基　　金：国家自然科学基金自由申请面上项目(编号:30570839);广东省自然科学基金资助项目(编号:05004735);广东省教育部产学研结合项目(编号:2009B090300354)

摘　　要：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SalⅠ、HindⅢ切下,分别定向连接到以XhoⅠ、HindⅢ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。Objective To construct tetanus toxin C fragment （TETC） expression vector with lactocoeeus lactis. Methods TETC gene was acquired from the PCR based gene assembly plasmid pBS - TETC with Sal Ⅰand Hind Ⅲ restriction enzymes, and subsequently cloned into the Lactococcus laetis secreted expression system pNBC1000 and cell wall anchored expression system pNBC2000 to form pNBC1002 and pNBC2002. The expression systems were transformed into Lactoeoccus lactis by electroporation. Protein eleetrophoresis 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 supematant 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 by Western - blot. Conclusion The recombinant TETC expressing Lactococeus lactis provides foun- dation for future study of biologic adjuvant and vaccine against tetanus.

关 键 词：破伤风毒素C片段 乳链菌 生物佐剂 疫苗 

分 类 号：R392-33[医药卫生—免疫学]