乳头瘤病毒表位融合蛋白的基因定点诱变

Site-Directed Mutagenesis of HPV Multi-Epitope Fusion Protein

目的定点诱变热休克蛋白-乳头瘤病毒表位融合蛋白的基因中编码半胱氨酸的密码子,解决下游纯化工作中产生多聚体蛋白的问题。方法在对蛋白质序列进行计算机表位预测及生物学特性分析的基础上,利用PCR的定点诱变技术,使原始基因序列中编码半胱氨酸的TGC突变为编码甘氨酸的GGC,克隆并表达了该基因编码的蛋白质。结果经克隆、测序后证实已成功获得突变基因,其编码的融合蛋白经纯化后没有多聚体的产生。结论利用PCR定点诱变技术对基因进行突变,在不改变蛋白质抗原性质的基础上,优化蛋白质序列,提高蛋白质纯化质量和效率。

Objective To solve the purification problem derived from cysteine residue in primary structure of recombinant protein by site-directed mutagenesis. Methods Codon TGC (cysteine) was mutated to GGC ( glycine) through site-directed mutagenesis PCR and the selection of the mutated site was based on computer-assisted epitope prediction. Results Gene mutation has been successfully achieved, as verified by sequencing. The fusion protein was purified successfully without formation of polymers. Conclusion The efficiency and quality of protein purification has been greatly improved without modification of the antigenicity of proteins through site-directed mutagenesis.