毕赤酵母对不同细菌抗原的差异性表达

Differential bacterial gene expression by Pichiapastoris

利用毕赤酵母研究真核细胞对不同的原核细菌可溶性蛋白抗原的差异性表达。用PCR技术扩增3种不同的结核杆菌抗原基因Ag85a、ESAT6、以及rdESAT6和1种肺炎链球菌抗原基因PsaA。将它们分别连接到pPic9k载体中,在毕赤酵母中进行表达。然后采用离子交换柱或镍柱(Ni-NTA)分别纯化这4种可溶性蛋白抗原。在这些表达的原核可溶性抗原中,肺炎球菌的PsaA蛋白抗原表达量最高,约200 mg/L;然而,3种结核杆菌抗原的表达量差异性不大,rdESAT6约2 mg/L,Ag85a约1 mg/L,而ESAT6低于0.5 mg/L且不稳定。另外,ESAT6和rdESAT6蛋白分别有不同程度的糖基化现象。在相似的条件下,不同的原核抗原在毕赤酵母系统中的表达在难易程度与产量等方面存在很大的差异姓。

The present study aims to obtain soluble bacterial antigens expressed by Pichiapastori and to probe the differential gene expression of prokaryotic antigens in eukaryotic cells. The genes ag85a, esat6, and rdesat6 from Mycobacterium tuberculosis and the gene of psaa fromStreptococcus pneumoniae were am- plified by polymerase chain reaction individually and then cloned into the vector pPic9k. The gene expression of the four antigens. Ag85a, ESAT6, rdESAT6 and PsaA were induced in Pichiapastoris and the four secreted proteins were purified by using either ion exchange or Ni-NTA column. The highest protein expression level in Pichiapastoris, about 200 mg/L, was obtained by cloning the psaa gene of Streptococcus pneurnoniae into the pPic9k vector. Minor difference was shown in the expression of the three different genes of Mycobacteriurn tuberculosis, ag85a, esat6, and rdesat6. The protein expression levels of rdESAT, Ag85a and ESAT6 were 2 rag/L,1 mg/L,and under 0.5 mg/L respectively. In addition,N-linked glycosy lation was found in both ESAT6 and rdESAT6 proteins. The difference of the protein expression of the genes of Mycobacterium tuberculosis was minor. However, N-linked glycosylation was found in both ES- AT6 and rdESAT6 proteins,not the Ag85a protein in Pichiapastoris expression System.