重组sTNFα-R1蛋白同义密码突变库的构建及筛选

Construction and Screening of Recombination sTNFα-R1 Protein Synonymous Mutation Library

目的:以重组肿瘤坏死因子-α受体1(sTNFα-R1)的基因序列为模板,构建同义密码突变库,在不改变原氨基酸序列的同时筛选获得目标蛋白产量提高的突变菌株。方法:分段设计并合成同义密码突变兼并引物,通过优化PCR反应条件,构建目的基因的完整同义密码突变库。突变库经双酶切插入到pET11(b)载体上,并电转至E.coli BL21(DE3)细胞中。阳性克隆经PCR鉴定后,细胞表达目标蛋白,应用SDS-PAGE等方法分析目标蛋白产量。结果:目的基因同义突变库的PCR扩增产物可见330bp的特征条带,突变库测序结果显示整个基因序列的第三位碱基发生预期的同义突变;完成了445株克隆筛选,针对其中产量高于对照组的45株克隆进行测序,获得42株基因序列不同但氨基酸序列未发生变化的克隆菌株,其同义密码突变阳性率高达93%;选择其中2株初筛产量较高的菌株进行放大验证,结果显示7#突变株的包涵体收率及目标蛋白相对百分含量明显高于对照株,其每升发酵液的目标蛋白总产量比对照株提高2.44倍,405#突变株比对照株提高1.44倍。结论:通过优化PCR反应条件成功构建了目标基因全序列的同义密码突变库,采用同义密码突变库技术途径,在不改变原氨基酸序列的前提下可以快速优化蛋白表达产量。

Objective： Construct a synonymous mutation library with recombinant tumor factor-α receptor 1（sTNFα-R1） gene as a template, and increase the production of the target protein by screening the synonymous mutation library. Methods： Design and synthesize the degenerate primer fragments for the synonymous mutation library. PCR reaction conditions were optimized chemically. The complete sequence of the mutation library of sTNFα-R1 gene was constructed by PCR with the primer fragments and linked with vector pET11b. The constructed recombinant plasmid pET11b-sTNFα-R1 was transformed to E.coli BL21 （DE3） with penicillin-resistant gene mutation. The positive clones were identified by colony PCR and induced by IPTG. Finally, application method such as SDS-PAGE was used to analyze the yield of the target protein. Results： The length of amplified sTNFα-R1 gene was about 330bp. Sequencing result showed that the third base of amino acid has mutated. Screen 445 clones and sequence 45 clones whose productivity has increased. It was proved that the gene sequences of 42 strains were different but their amino acid sequences were the same. The molecular mass of the expressed recombinant protein was about 13kDa. The productivity of the target protein of mutant 7# has increased 2.44 times than that of the original sequence. At the same time, the productivity of the target protein of mutant 405# has increased 1.44 times. Conclusion： The synonymous mutation library of the target gene was constructed successfully by optimizing the PCR condition. Using synonymous mutation library technology, the protein production can be optimized quickly without changing the original amino acid sequence.