| In the past few decades,the world has faced multiple outbreaks of new and recurring infectious diseases,which have posed a serious threat to global health and the global economy,and seriously endangered human health.Most of the pathogens of new and recurring infectious diseases are caused by arboviruses,such as West Nile virus,dengue fever virus,Bunia virus,Zika virus and African swine fever virus,which are all important to global public health.Has had a major impact,which requires us to quickly intervene and prevent and treat infections.In recent years,most of the new and recurring infectious diseases in my country are mainly arboviruses,including Zika virus and African swine fever virus.Arboviruses,as an important part of new and recurring infectious diseases,have seriously threatened people's lives.And physical health,the prevention and control situation are becoming increasingly severe.Obviously,we need a multifaceted approach to cope with the emergence of new and recurring infectious diseases,including crossdisciplinary and cross-biological integration.Research on different levels from molecules to pathogens to ecosystems can give us more opportunities to deal with the threats of epidemics and pandemics.The virus needs to replicate its genome to complete its life cycle,thereby producing new virus particles.Therefore,how to regulate the replication and transcription process of the genome after entering the host cell is very important.Using various methods to study virus replication and transcription from different levels will become an important theoretical basis for the prevention of related viral diseases.This thesis is based on the Bayesian-Markov chain molecular evolution method to analyze the molecular evolution of the Zika virus NS5 gene of the recurrent infectious disease.At the same time,the X-ray diffraction method is used to analyze the three-dimensional structure of the African swine fever virus D250 R protein.Virology and bioinformatics are cross-combined to understand the life activities of viruses from a new perspective.Part One Molecular Evolution of Zika Virus NS5 Gene.Zika virus is a global infectious disease,manifested as headache and fever.Pregnant women infected with it can cause symptoms of microcephaly in newborns.At present,there is no corresponding specific treatment method after infection.The virus was first discovered in 1947.In 2015,many newborn babies with microcephaly appeared in Brazil,which attracted the attention of the World Health Organization.Due to the current development of sequencing technology,the continuous in-depth study of viruses at the molecular level has promoted the rapid development of bioinformatics technology.The use of big data calculation methods to analyze biological-related problems has become a new trend.Using Bayesian method to construct the largest credible branch tree can get more accurate and efficient results.The method is to make the results more accurate by selecting the most suitable nucleotide substitution model,and to further standardize the data by calculating the posterior probability of the data.Because of the various advantages of the Bayesian method,this method has been widely used in the analysis of various viruses.In Zika virus,we selected relatively conservative genes for evolutionary analysis.Due to another outbreak of Zika virus in 2015,it is particularly important to explore the genetic characteristics and evolutionary trends of the new outbreak.In this study,a total of 108 full-length nucleotide and amino acid sequences of the NS5 gene of Zika virus were collected in the Genbank database.After sequence alignment,saturation detection,recombination analysis,model selection,etc.,they will be processed.The sequence of the software uses the Bayesian method to construct the largest credible branch tree,and analyzes the effective number of the population.At the same time,for these sequences,the potential glycosylation modification sites were predicted,and the potential modification sites were tried to be explored.After analysis,it was found that the nucleotide similarity of the NS5 gene of the 108 strains of Zika virus was 95.4%-100%,and no potential modification sites were found in the prediction of glycosylation modification sites.According to the analysis of the constructed phylogenetic tree,Zika virus from 1947 to 2017 was divided into two genotypes: African type and Asian type.Its population dynamics curve shows a trend of first decline and then rise in 2015.The mutation rate of the three codons encoding NS5 gene was 0.3695,0.1596 and 2.4709.This shows that the NS5 gene is relatively conserved during the spread of Zika virus.Part 2 Study on protein structure of African swine Fever virus D250RAfrican swine fever virus is the only species in the family of African swine fever.Its infectivity and mortality are very high.The mortality rate of domestic pigs within ten days after being infected with the virus can be close to 100%.A case of African swine fever was first detected in my country in August 2018.As there is currently no specific vaccine or specific treatment for this virus,the spread of this virus has had a serious impact on the global pig industry.African swine fever virus is a large icosahedral DNA virus with a genome ranging from 170 kb to 190 kb in length,capable of encoding 150 to 200 proteins.At present,there are few studies on the function and structure of the protein encoded by the African swine fever virus.However,existing studies have shown that the protein encoded by the D250 R gene has the function of a decapping enzyme.It can recognize m RNA,degrade its cap structure,and affect protein translation.In his research,it has not been found that there are other proteins with the same function in the virus genome.Therefore,analyzing its three-dimensional structure is very important for further understanding the molecular mechanism of its enzyme activity.In this study,the D250 R gene was cloned into the expression vector p ET-15 b,and the E.coli expression system was used to express the protein in large quantities.A nickel column affinity chromatography and gel filtration chromatography are used to obtain protein samples with high purity and uniform properties.After optimizing and screening the growth conditions of protein crystals by the sitting drop method,the protein crystals that can be diffracted were finally obtained by sitting drop method at 16?,0.1 M Sodium cacodylate p H 5.6,14.5% w/v PEG 4000 conditions.Crystal,the three-dimensional structure of D250 R protein was analyzed by singlewavelength anomalous scattering method.From the structure,it can be seen that the protein contains 11 ? helices and 7 ? folds.The results show that D250 R is mainly divided into N-terminal domain,Nudix Motif and C-terminal domain in structure.Its Nudix Motif consists of 23 amino acid residues and is located near the C-terminal domain.At the same time,the two RNA binding surfaces of the protein were inferred based on the surface electrostatic potential of the structure,which provided a structural basis for the following functional studies. |
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