结核分枝杆菌保护性肽表位KLIANNTRV多聚体重组表达及鉴定

Recombinant expression of the multimer peptide derived from Mycobacterium tuberculosis antigens

目的重组表达结核分枝杆菌保护性肽表位KLIANNTRV二聚体、四聚体、八聚体,以进一步探讨其免疫效应。方法选取结核分枝杆菌抗原表位KLIANNTRV,引入Th表位PADRE及木马肽序列(RKKRRQRRR),并以RVKR序列作为接头,分别合成结核分枝杆菌保护性肽表位KLIANNTRV二聚体、四聚体、八聚体全基因序列,经PCR扩增后分别插入融合表达载体pGEX-4T-1,将重组质粒转入大肠杆菌BL21并以IPTG诱导表达,进一步经GST亲合层析柱纯化,经SDS-PAGE和Western-blot鉴定重组表达蛋白。结果成功构建了结核分枝杆菌保护性表位KLIANNTRV二聚体、四聚体、八聚体的表达质粒,并经IPTG诱导表达了大小约为20000、32000、54000的融合蛋白。结论结核分枝杆菌保护性肽表位KLIANNTRV多聚体的重组表达为进一步开发结核疫苗提供有价值的依据。

Objective To express recombinant mulfimer peptides from of Mycobacterium tuberculosis （Mtb） in E. coli. Methods The DNA sequence encoding dimer, tetramer, and octamer of peptide KLIANNTRV derived from Mtb were synthesized, and then linked with PADRE, Trojan peptide, and furin-sensitive linker RVKR, respectively. These genes were amplified by polymerase chain reaction （PCR）, inserted into the expression vector pGEX-4T-1 and transformed into E. Coli BL21 （DE3）. After induced by isopropylthio-β-D-galactoside （IPTG）, the recombinant protein was confirmed by SDS-PAGE and Western blotting. Results The recombinant Mr 20 000, 32 000, 54 000 multimer peptide proteins were expressed and confirmed by SDS-PAGE and Western blotting. Conclusion The recombinant muhimer peptides may provide the basis for developing novel TB vaccines.