降低mRNA翻译起始区的稳定性原核非融合表达HAb18GEF

Non-fused Expression of HAb18GEF by Reducing Stability of Translational Initiation Region in mRNA

为在大肠杆菌中非融合表达肝癌相关抗原HAb18G胞外区片段 (HAb18GEF) ,将HAb18GEF基因的cDNA插入原核表达载体pET2 1a +。通过计算机辅助设计 ,对重组的HAb18GEF pET2 1a +的mRNA翻译起始区 (TIR)的二级结构和密码子偏性同时进行预测。结果发现其存在稳定的茎环结构和许多稀有密码子。通过优化二级结构和优化密码子偏性二种策略分别来降低HAb18GEF pET2 1a +的mRNA翻译起始区 (TIR)的稳定性。在不改变氨基酸序列的前提下 ,利用密码子的简并性 ,通过非连续定点突变实现这两种优化。将突变前后的重组子经酶切鉴定和测序验证后 ,转化感受态JM10 9 DE3宿主菌后 ,随机挑菌 3 7℃下用IPTG诱导表达。SDS PAGE、间接ELISA、Westernblot和细胞分级分离法分析这些重组子的诱导表达情况。RNAdotblot对比分析优化前后目的基因mRNA的量。结果证明 ,成功地构建了HAb18GEF pET2 1a +及其二种优化突变体。仅优化TIR区二级结构或仅优化TIR区密码子偏性均能实现HAb18GEF蛋白的非融合表达 ,而未优化的重组子不表达任何HAb18GEF。非融合表达产物在大肠杆菌中主要以包涵体形式存在 ,高达 2 9 3 %。由于过表达和细胞渗漏 ,培养基和周质腔中也可检测到少许的HAb18GEF。优化二级结构和优化密码子偏性二种策略的HAb18GEF的非融合?

To express the extracellular fragement of hepatoma associated antigen HAb18G(HAb 18GEF) in E.coli efficiently in a non-fusing way, the cDNA of HAb18GEF gene was inserted into prokaryotic expression vector pET21a+. The secondary struct ure and codon adaptation of translational initiation region (TIR, from-30 to +3 9) in mRNA of recombinant vector HAb18GEF/pET21a+ was predicted simultaneousl y by computer-aided design. Stable Stem-Loop structures and many low-usage co dons were detected in mRNA-TIR of non-optimized recombinant HAb18GEF/pET21a+ vector. The stability of mRNA-TIR in recombinant HAb18GEF/pET21a+ vector was reduced with following methods: (1) optimization of secondary structure (2) opt imization of codon adaptation. These optimization were realized by non-continua l site-directed mutagenesis without changing any amino acid sequence in TIR. Af ter being checked through restriction endonuclease digestion and confirmed throu gh nucleotide sequencing, the pre-optimized and post-optimized recombinant vec tors were transformed into competent E.coli JM109-DE3 The resulted recomb inant clones were selected randomly and induced by IPTG at 37℃. The induced pro duction of these recombinants was analyzed by SDS-PAGE, indirect ELISA, Western blot, and cell fractionation assay. The amount of HAb18GEF mRNA was also detec ted by RNA dot blot between pre-optimized recombinant and post-optimized recom binant. The results revealed that recombinant non-fused vectors HAb18GEF/pET21a + were successfully constructed and optimized in the secondary structure and cod on adaptation of TIR respectively. The HAb18GEF was expressed efficiently in a n on-fusing way in recombinant E.coli by secondary structure optimization or codon adaptation optimization. Whereas, no expression of HAb18GEF was detected i n pre-optimized recombinants. The non-fused expression products-HAb18GEF, mai nly as inclusion body in E.coli, yielded highly above 29 3%,. A trait of ex pression HAb18GEF was also detected both in intermembrane space and in culture m edium due to over-expression and cell leakage. Difference in non-fused express ion level of HAb18GEF between secondary structure optimization and codon adaptat ion optimization was negligible. No difference in amount of transcribed mRNA of HAb18GEF between the pre-optimized and the post-optimized recombinants was det ected. To sum up, it's feasible to express hepatoma associated antigen HAb18GEF in a non-fusing way by reducing the stability of TIR in mRNA.