Analysis Of Codon Bias Of Highly Pathogenic Coronavirus And Construction Of Recombinant Plasmid For Eukaryotic Expression Of S Protein

ObjectsCodon bias analysis has become a hot issue and frontier in multidisciplinary research.In the field of medical virology,codon bias analysis is often used to understand the evolutionary relationship between different strains,to clarify the genetic characteristics between them,and to provide an important theoretical basis for disease prevention and control and vaccine development.This study analyzed the codon bias of six human coronaviruses?HCoV: SARS-CoV,MERS-CoV,HCoV-229 E,HCoV-NL63,HCoV-OC43,HCoV-HKU1?in order to understand the evolutionary relationship between them;Furthermore,the codon bias of highly pathogenic coronavirus?SARS-CoV,MERS-CoV?between different hosts and their evolutionary relationships were analyzed to clarify the genetic characteristics of highly pathogenic coronavirus,providing important theoretical basis for its prevention and control.At the same time,we used molecular cloning technology to construct eukaryotic expression recombinant plasmids of S protein?SARS-S,SARS-S1,SARS-RBD,MERS-S1,MERS-RBD?of different lengths of highly pathogenic coronavirus,with a view to further S Protein expression and functional studies lay a certain theoretical foundation.MethodsWe downloaded the required coronavirus gene coding sequence?CDS?from NCBI,saved the truncated protein coding sequence using the Lasergene subroutine EditSeq,and used the EMBOSS subroutine CUSP to calculate the codon frequency value;CodonW calculated the codons ENC,GC,GC3 S,RSCU value.Codon preference was measured using ENC and RSCU indicators;ENC-Plot,Neutrality Plot,Parity Rule 2 plot analyses,and cluster analysis were used to analyze the influencing factors of codon preference and the evolutionary relationship between them.At thesame time,we used molecular cloning technology to construct eukaryotic expression recombinant plasmids of S protein?SARS-S,SARS-S1,SARS-RBD,MERS-S1,MERS-RBD?of different lengths of highly pathogenic coronavirus.Results1.The results of six HCoV codon bias analysis showed that the average values of ENC of each of the six HCoV proteins were close to 61;the bias codon between the six HCoV proteins was found by RSCU average analysis?RSCU>1?.And non-preferred codons?RSCU<1?,In which each HCoV-preferred codon is dominated by codons ending in A and U;ENC-Plot results show that the six HCoV proteins are almost in similar regions and far from the standard curve;Among the six HCoVs,the slope?b?is smaller except for the larger slope of HCoV-NL63?0.7267?;.The odd-even rule analysis showed that most of the genes fell to the lower left.In addition,based on the codon bias clustering results of the structural protein S and the non-structural protein ORF1 ab showed that HCoV-OC43,HCoV-NL63,and HCoV-229 E are aggregated together on the S protein;and HCoV-HKU1,SARS-CoV,MERS-CoV is clustered together;ORF1ab results showed that SARS-CoV,HCoV-NL63,HCoV-229 E,HCoV-OC43,HCoV-HKU1 are grouped together,and MERS-CoV is grouped together.2.The comparison of SARS-CoV and SARSr-CoV codon preference and cluster analysis showed that the average number of effective codons?ENC?of each protein was close to 61;The number of preferred codons?RSCU>1?of each protein encoded by SARS-CoV is between 20-27,and the number of preferred codons?RSCU>1?of each protein encoded by SARSr-CoV is between 17-30.And both are mainly codons ending in A and U;Among them,AUU and ACU are the preferred codons for the 12 proteins of SARS-CoV,while GGG,UGG,CUG,AUC and AUG are non-biased codons.In addition,CUU is a preferred codon for the 12 proteins of SARSr-CoV,while GGG,UGG,AGC,and CGG are non-biased codons;ENC-Plot results show that the two are almost in similar areas and far from the standard curve;Neutrality Plot showed that the slopes of both S and ORF1 a of SARS-CoV and SARSr-CoV were small;the odd-even rule analysis showed that most of the genes fell to the lower left.Cluster analysis based on codon bias showed that on S protein,SARSr-CoV/RSYN2013/Yunnan,SARSr-CoV/RS/WIVI1/Yunnan/2013,SARSr-CoV/Rs/WIV16/Yunnan/2013 are most closely related to the evolution of SARS-CoV.The five newly reported SARSr-CoVs,RS7327,RS9401,RS4084,RS4231,and RS4874,can also be grouped together with SARS-CoV;the newly discovered RSSAR1,RS4255,AS6526,RS4237,RS4247,and Rf4092 CoV is clusteredwith SARSr-CoV found in South China?Hong Kong,Guangxi?;Yunnan strains YNLF₃1C,YNLF₃4C,RS3367 and Southwest China?Guizhou,Guangxi?,Central China?Hubei?,North China?Hebei?,Northeast?Jilin?.The discovered virus strains were clustered together.At the same time,Shanxi and Shaanxi,which are geographically adjacent,were grouped together.The cluster analysis of the non-structural protein ORF1 a found that the SARSr-CoV found in Yunnan bats was clustered with SARS-CoV,which can infect humans and civet cats.At the same time,the branch also includes strains from Guizhou,Guangxi,and Hubei.The SARSr-CoV strain reported by Hong Kong differs greatly from SARS-CoV and is clustered together with a strain of Hubei.The SARSr-CoVs in Shanxi,Shaanxi,Hebei,Hubei,and Jilin in central and northern China are clustered together.3.MERS-CoV codon bias analysis and cluster analysis showed that the average ENC of Human-MERS-CoV and Camel-MERS-CoV were close to 61;The number of preferred codons?RSCU>1?of each protein encoded by Human-MERS-CoV is between 29 and 31,and the number of codons?RSCU>1?of each protein encoded by Camel-MERS-CoV is 29-32,and both are mainly codons ending in A and U;Among them,UUU,UUG,CUU,AUU,UCU,CCU,CAU,AAU,GAA,CGU,AGU,AGG,GGU are the preferred codons for the 11 proteins of Human-MERS,while UUC,CUC,CUA,AUC,GUC,GUA,UCC,CCC,ACC,ACG,GCC,UAC,CAC,GAC,UGC,CGC,CGA,AGC are non-bias codons shared by 11 proteins of Human-MERS.In addition,UUU,UUA,UUG,AUU,GUG,UCU,UCA,CCU,CCA,ACU,ACA,GCU,GCA,UAU,CAA,GAA,UGU,AGU,AGA,AGG,GGU are Camel-MERS-CoV 11 proteins share a preferred codon,and UUC,CUC,CUA,AUC,AUG,GUC,UCG,CCC,CCG,ACC,ACG,GCC,GCG,UAC,UAG,UCC,CAC,CAG,GAG,UGC,UGG,CGC,CGA,CGG,AGC,GGG are non-bias codons shared by 11 proteins of Camel-MERS-CoV;ENC-Plot results show that the two are almost in similar areas and far from the standard curve;Neutrality Plots showed that the slopes of each of Human-MERS-CoV and Camel-MERS-CoV proteins were small.The odd-even rule analysis showed that most of the genes fell to the lower left.The results of cluster analysis showed that the Camel-MERS-CoV preference was similar to that of Human-MERS-CoV in 2012-2018,and the epidemic strains in which the two were clustered were observed on almost every protein.The codon bias of the MERS-CoV ORF8 b protein is relatively stable and aggregated into a single group.However,from the internal analysis of the epidemic time,it was found that the 2017Camel-MERS-CoV epidemic strain was clustered in many internal proteins?E,ORF1 a,ORF1ab,ORF4 a,ORF5,ORF8b?from other years?2012-2016?.However,from the internal analysis of the epidemic time,it was found that the 2017 Camel-MERS-CoV epidemic strain was clustered in many internal proteins?E,ORF1 a,ORF1ab,ORF4 a,ORF5,ORF8b?from other years?2012-2016?a farther location;among them,among the two proteins E and ORF1 ab,which are well clustered with the 2011Bat-MERS-CoV;At the same time,Camel-MERS-CoV was combined with Human-MERS-CoV on S,M and ORF4 b proteins in 2017.In Human-MERS-CoV,except for ORF1 ab and ORF8 b,the 2018 Human-MERS-CoV and Camel-MERS-CoV?2012-2016?have very similar clustering relationships;Among the ORF4 b proteins of MERS-CoV,in 2015-2017,Human-MERS-CoV and Camel-MERS-CoV were homogenized in each year.4.In this study,we used molecular cloning technology to successfully construct eukaryotic expression recombinant plasmids of highly pathogenic coronavirus S proteins?SARS-S,SARS-S1,SARS-RBD,MERS-S,MERS-S1,MERS-RBD?.Conclusion1.The codon bias of the six HCoVs was weak,and the preferred codons were mainly terminated by A and U.The six were affected by the dual factors of self-mutation and natural selection during the evolution process,and were mainly based on natural selection.In the third base of amino acid coded by four codons,the frequency of C and T?pyrimidine?is higher than that of G and A?purine?,and there is a significant bias in their frequency.Among which highly pathogenic coronavirus?SARSr-CoV,MERS-CoV?,both pyrimidine and purine are used,while ordinary HCoV?HCoV-229 E,HCoV-NL63,HCoV-HKU1,HCoV-OC43?hardly use G and A?purine?.The results of cluster analysis based on codon preference showed that the clustering relationship of the six HCoV was different from the phylogenetic tree based on their genome.2.The codon bias of SARS-CoV and SARSr-CoV is weak,and the preferred codons are mainly at the end of A and U.Both of them are influenced by self-mutation and natural selection,among which natural selection is dominant.In the third base of amino acid coded by four codons,the frequency of C and T?pyrimidine?is higher than that of G and A?purine?,and there is a significant bias in their frequency.The codon preference of SARSr-CoV is related to their geographic location,and SARSr-CoV in similar regions is similar.SARS-CoV in Yunnan bats may be an important natural gene pool of SARS-CoV and SARS-CoV in other areas.The possibility of SARS-CoV trans-species infection in Yunnan needs our attention.3.The codon preference of Human-MERS-CoV and Camel-MERS-CoV is weak,andthe codons preferred by them are mainly at the end of A and U.Both of them are influenced by self-mutation and natural selection,and natural selection is the dominant factor in the evolution of human-MERS-CoV and Camel-MERS-CoV.PR2 analysis showed that the frequency of codon 3 C and T?pyrimidine?was higher than that of G and A?purine?,and the frequency of codon 3 C and T?pyrimidine?was significantly biased.At the same time,cluster analysis based on codon bias shows that the preference of Human-MERS-CoV is similar to that of Camel-MERS-CoV,and their evolutionary relationship is similar.In 2017,Camel-MERS-CoV may have mutated.In the past three years,human-MERS-CoV and Camel-MERS-CoV have very similar clustering relationships,while ORF8 b and human-MERS-CoV clustered separately,showing relatively stable codon bias clustering characteristics.Therefore,ORF4 b and ORF8 b may be the key proteins for the prevention and control of MERS.4.The recombinant eukaryotic expression plasmids of different lengths of S protein?SARS-S,SARS-S1,SARS-RBD,MERS-S,MERS-S1,MERS-RBD?of highly pathogenic coronavirus were successfully constructed by molecular cloning technology,which laid a theoretical foundation for further study of S protein expression and function and laid a theoretical foundation for further study of S protein expression and function.