SARS-CoV-2刺突蛋白基因密码子偏好性与进化分析

Codon Bias and Evolutionary Analysis of SARS-CoV-2 Spike Protein Gene

新型冠状病毒(SARS-CoV-2)属于冠状病毒家族新成员,其刺突蛋白(spike protein,S-蛋白)是侵染人体并决定其入侵宿主的亲和能力及组织特异性的包膜蛋白,研究S-蛋白基因的密码子偏好性不仅有利于重组表达,也利于分析其进化规律。利用Codon W和SPSS等在线服务器及软件,首先分析SARS-CoV-2等冠状病毒的S-蛋白基因的密码子偏好性,随后以SARS-CoV-2的S-蛋白基因为重点,比较其与大肠埃希菌、酵母和λ噬菌体等模式生物的密码子使用频率。结果显示,冠状病毒S-蛋白基因密码子的总GC和GC3s含量均小于0.4,更偏向以A/T编码,ENc为44.15,密码子偏好性整体较弱。28个密码子的偏好性较强(RSCU>1),3个密码子的偏好性极强(RSCU>2),UAA为最优密码子。ENc-plot结果显示,大部分基因的ENc距ENc期望值较近,表明冠状病毒密码子偏好性主要受到碱基突变的影响。相较于CDS序列,基于RSCU值的聚类更适合于冠状病毒的系统发育研究。综合ENc比值与聚类结果,SARS-CoV-2与SARS的亲缘关系最近,可能起源于同一祖先冠状病毒。酿酒酵母是SARS-CoV-2较好的外源表达系统,但仍需密码子优化。根据密码子优化结果,成功实现了S-蛋白RBD区域的原核表达。以上研究结果为S-蛋白基因的优化改造,提高其表达效率提供参考。

SARS-CoV-2 belongs to the family of coronavirus,its spike protein(S-protein)is an envelope protein that invades human body and thus determines its affinity and tissue specificity.Study on codon bias of its S-protein gene is conducive not only to its recombinant expression but also to analyze its evolutionary law.In this paper,CodonW and SPSS and other on-line server and soft ware were firstly used to analyze the S-protein codon bias of spike genes from SARS-CoV-2 and other coronaviruses,and then compare the codon usage frequency of bias spike gene of SARS-CoV-2 with those model organisms like E.coli,Saccharomyces cerevisiae,andλbacteriophage etc.The results showed that low total content of both of GC and GC3s all under 0.4 indicated that coronavirus was preferable to be encoded by A/T,ENc at 44.15,their codon bias of spike gene was integrally weak.28 codons have stronger bias(RSCU>1),3 codons have very strong codon bias(RSCU>2),UAA was the most excellent codon.The result of ENc-plot showed that most genes were near to the expectation of ENc,suggested that the formation of codon bias of coronavirus was resulted from the effect of mutation.AS compared with CDS sequence,cluster based on RSCU value was fit for phylogenetic analysis of coronavirus.Summarizing the ENc-plot ratio and clustering result,SARS-CoV-2 and SARS were suggested to possess closest genetic relationship and probably originate from one ancestral coronavirus.And S.cerevisiae was a better exogenous expression system for SARS-CoV-2 than other model organisms,despite need for further codon optimization.Finally,according to the results of codon optimization,RBD domain of S-protein was successfully expressed procaryotically.The results of this study have laid a foundation for the optimization reformation of S-protein gene and improve its expression efficiency.