hTERT主要HLA-A2.1相关基因的表达

Expression of HLA-A2.1 Restricted hTERT Gene in Pichia pastoris

为了研究hTERT的表达特性及意义 ,根据hTERT基因中与HLA_A2 1分子相关性较强的几个 9mer肽段合成引物 ,通过RT_PCR技术分别对 14例RCC组织和 2例肿瘤细胞株进行了扩增 ,扩增片段大小为 30 6bp (小片段 )和 10 0 2bp (大片段 ) ,结果有 13例癌肿组织表达hTERT基因小片段 ,1例组织不表达hTERT基因 ;Hela细胞和 786_0肾腺癌细胞株均表达hTERT ;对大片段进行克隆 ,酵母表达 ,构建了pPICZαA_hTERT重组表达载体和PichiapastorisX33 hTERT酵母工程菌 ;SDS_PAGE电泳显示毕赤酵母能分泌表达 39kD的目标蛋白。得出 ,大部分RCC肿瘤组织和细胞表达hTERTmRNA ,而正常组织则不表达 ,提示hTERT可作为通用的抗肿瘤治疗尤其是免疫治疗的靶点。

In order to understand the immune and biological significance of expression of hTERT mRNA, primers were synthesized based on hTERT cDNA sequences which contains several coding regions for HLA-A2\^1 restricted peptides, and the hTERT mRNA levels were assessed in 14 RCC tissues and 2 tumor cell lines, with RT-PCR, the obtained hTERT gene fragment was cloned and expressed in a \%Pichia\% yeast expression system. The findings showed that hTERT mRNA was expressed in 13 RCC tissues, not expressed in 1 RCC tissue and adjacent normal tissues; Hela and 786-0 cell lines expressed hTERT mRNA; The target fragment was constructed recombinant expression vector of pPICZαA-hTERT and expressed in yeast engineering strain of \%Pichia pastoris\% X33/hTERT; SDS-PAGE showed a 39 000 protein band in the supernatant of\% Pichia\% medium. These results identified that hTERT mRNA was expressed in most RCC tissues and tumors cell lines, and suggested hTERT as a potentially important and widely applicable target for anticancer immunotherapeutic strategies.