梅毒螺旋体重组蛋白Tp0608的表达与鉴定

Expression and Identification of Recombinant Protein Tp0608 of Treponema Pallidum

目的表达梅毒螺旋体(Tp)重组蛋白Tp0608(rTp0608)并鉴定其抗原性,为深入探讨其在梅毒血清学诊断中的价值奠定基础。方法 PCR扩增Tp0608全长基因,构建原核表达重组体pET-28a(+)/Tp0608,转化宿主菌诱导表达rTp0608,Ni-NTA亲和层析法纯化蛋白,SDS-PAGE分析蛋白表达形式与纯度,Western blot鉴定rTp0608的抗原性。结果原核表达重组体pET-28a(+)/Tp0608在宿主菌内经诱导表达了分子量大约为38 kDa的重组蛋白,以包涵体表达形式为主,纯化蛋白的纯度>95%;Western blot结果显示纯化蛋白能被梅毒患者混合血清特异性识别。结论 PET-28a(+)表达载体高效表达了rTp0608,该重组抗原有良好的抗原性。

Objective To express and identify antigenicity of recombinant protein Tp0608 （rTp0608） of Treponema pallidum （Tp） , providing a basis for further investigation of its significance in syphilis serodiagnosis. Methods Full length of Tp0608 gene was amplified by using PCR. The prokaryotie expression recombinant pET - 28a（ ＋ ）/Tp0608 was constructed and transformed into E. eoli BI21 to express rTp0608 ,and rTp0608 was purified with Ni-NTA affinity chroma- tography. Expression and purity of rTp0608 were analysed by SDS-PAGE. Antigenicity of rTp0608 was tested by Western blot. Results The prokaryotie recombinant pET -28a（ ＋ ）/Tp0608 was constructed successfully and an approximate 38 KDa recombinant protein was expressed efficiently as inclusion body in E. coli and the protein purity was over 95%. West- ern blot indicated that the purified proteins were able to be recognized specifically by sera from syphilis patients. Conclu- sions The efficiently expressed recombinant Tp0608 has good antigenicity.