Zhang Si-He, Xing Jin-Liang, Yao Xi-Ying, Chen Zhi-Nan
Cell Engineering Research Center, Fourth Military Medical University, Xi' an 710032, China.
Sheng Wu Gong Cheng Xue Bao. 2004 Mar;20(2):175-80.
To express the extracellular fragement of hepatoma associated antigen HAbl8G(HAb18GEF) in E. coli efficiently in a non-fusing way, the cDNA of HAb18GEF gene was inserted into prokaryotic expression vector pET21a + . The secondary structure and codon adaptation of translational initiation region (TIR, from-30 to + 39) in mRNA of recombinant vector HAb18GEF/ pET21a + was predicted simultaneously by computer-aided design. Stable Stem-Loop structures and many low-usage codons 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) optimization of codon adaptation. These optimization were realized by non-continual site-directed mutagenesis without changing any amino acid sequence in TIR. After being checked through restriction endonuclease digestion and confirmed through nucleotide sequencing, the pre-optimized and post-optimized recombinant vectors were transformed into competent E. coli JM109-DE3. The resulted recombinant clones were selected randomly and induced by IPTG at 37 degrees C. The induced production of these recombinants was analyzed by SDS-PAGE, indirect ELISA, Western blot, and cell fractionation assay. The amount of HAb18GEF mRNA was also detected by RNA dot blot between pre-optimized recombinant and post-optimized recombinant. The results revealed that recombinant non-fused vectors HAb18GEF/pET21a + were successfully constructed and optimized in the secondary structure and codon adaptation of TIR respectively. The HAb18GEF was expressed efficiently in a non-fusing way in recombinant E. coli by secondary structure optimization or codon adaptation optimization. Whereas, no expression of HAb18GEF was detected in pre-optimized recombinants. The non-fused expression products-HAb18GEF, mainly as inclusion body in E. coli, yielded highly above 29.3%. A trait of expression HAb18GEF was also detected both in intermembrane space and in culture medium due to over-expression and cell leakage. Difference in non-fused expression level of HAb18GEF between secondary structure optimization and codon adaptation optimization was negligible. No difference in amount of transcribed mRNA of HAb18GEF between the pre-optimized and the post-optimized recombinants was detected. 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.
为了以非融合方式在大肠杆菌中高效表达肝癌相关抗原HAbl8G的细胞外片段（HAb18GEF），将HAb18GEF基因的cDNA插入原核表达载体pET21a+。通过计算机辅助设计同时预测重组载体HAb18GEF/pET21a+ mRNA中转录起始区域（TIR，从-30至+39）的二级结构和密码子适应性。在未优化的重组HAb18GEF/pET21a+载体的mRNA-TIR中检测到稳定的茎环结构和许多低使用率密码子。采用以下方法降低重组HAb18GEF/pET21a+载体中mRNA-TIR的稳定性：（1）二级结构优化；（2）密码子适应性优化。这些优化通过非连续定点诱变实现，而不改变TIR中的任何氨基酸序列。经限制性内切酶消化检查并经核苷酸测序确认后，将优化前和优化后的重组载体转化入感受态大肠杆菌JM109-DE3。随机选择所得重组克隆并在37℃用IPTG诱导。通过SDS-PAGE、间接ELISA、Western印迹和细胞分级分离试验分析这些重组体的诱导产物。还通过RNA斑点印迹检测优化前重组体和优化后重组体之间HAb18GEF mRNA的量。结果表明，成功构建了重组非融合载体HAb18GEF/pET21a+，并分别在TIR的二级结构和密码子适应性方面进行了优化。通过二级结构优化或密码子适应性优化，HAb18GEF在重组大肠杆菌中以非融合方式高效表达。然而，在优化前的重组体中未检测到HAb18GEF的表达。非融合表达产物-HAb18GEF在大肠杆菌中主要以包涵体形式存在，产量高达29.3%以上。由于过表达和细胞渗漏，在膜间空间和培养基中也检测到HAb18GEF的表达特性。二级结构优化和密码子适应性优化之间HAb18GEF的非融合表达水平差异可忽略不计。在优化前和优化后的重组体之间未检测到HAb18GEF转录mRNA量的差异。总之，通过降低mRNA中TIR的稳定性以非融合方式表达肝癌相关抗原HAb18GEF是可行的。
