关键词: 人溶菌酶, 牛乳蛋白基因, 密码子优化, 荧光素酶检测, 实时荧光定量PCR, Western-blot

Abstract:

【Objective】The aims of present study were to investigate the codon usage bias of seven milk protein genes in cattle mammary gland, to optimaize human lysozyme gene partially and totally based on codon usage bias of milk protein genes, and to evaluate the codon optimized effect of human lysozyme in multiple cells, so as to provide theoretical basis for increasing recombinant human lysozyme expression and developing new high-effect and safe recombinant human lysozyme. 【Method】The codon usage bias and high/low frequency codons of bovine major milk protein genes were confirmed through bioinformatics analysis by CodonW and EMBOSS software. Human lysozyme gene was partially (22 codons in the translation start region, named LYZop22) and totally (named LYZop) optimized according to the codon usage frequency of bovine major milk protein genes. The human lysozyme-luciferase fusion expression vector (pGL3-LYZcw/op22/op) and overexpression vector (pcDNA-LYZcw/op22/op) were constructed and transfected to cattle mammary epithelial cells (BMEC), cattle fibroblasts (BFFC) and mouse mammary epithelial cells for codon optimization effect evaluation by luciferase, real-time qPCR and western blot methods. 【Result】 The cattle milk protein genes prefer GC end, which the GC3s content was 0.537±0.062. However, the human lysozyme gene prefer AT end, and the GC3s content was 0.407. Moreover, differences in codon usage bias between casein protein genes and whey protein genes were found by cluster analysis. Five high frequency codons (RSCU>1.5) and seven low frequency codons (RSCU<0.5) were found in the bovine milk protein genes.The human lysozyme gene was optimized according to the bovine milk protein codon usage bias and evaluated by multiple cells transfection. The luciferase result showed that, the codon optimization could significantly increase the human lysozyme expression, which were 1.48-fold (P<0.01) and 1.30-fold (P>0.05) increased in BMEC and BFFC of LYZop22, respectively. While, 2.2-fold and 2.44-fold increase in the BMEC and BFFC of LYZop, respectively. Real-time qPCR result showed that, the mRNA levels of human lysozyme were increased in BMEC (2.08-fold, P<0.05) and BFFC (1.5-fold, P>0.05) of LYZop22. While the significantly increase in mRNA level of human lysozyme were found in the BMEC (17.8-fold , P<0.01) and BFFC (22-fold, P<0.01) of LYZop, the totally codon optimized type. In addition, there was a positive correlation between mRNA level and mRNA second structure stability. Western-blot result showed that, the expression of human lysozyme was effectively increased both in partially and totally codon optimized human lysozyme gene in the BMEC. Moreover, the effect on human lysozyme expression in totally codon optimization type was better than partially codon optimization type. 【Conclusion】 The codon usage bias and high/low frequency codons of bovine major milk protein genes were obtained, and the codon optimization of human lysozyme gene according bovine major milk protein gene codon usage bias could significantly improve the mRNA and protein level of human lysozyme, which would lay the foundation for producing recombinant human lysozyme effectively by bioreactor.

Key words: human lysozyme, cattle milk protein genes, codon optimization, luciferase analysis, real-time qPCR, Western-blot