Genome Analysis And Cloning And Expression Of Virulence Gene Of Chlamydia Strains In China

Chlamydia is a kind of obligate intracellular parasitic bacteria.Two-phase development cycle makes its infection mechanism unique,resulting in various diseases difficult to control.The development cycle of Chlamydia is that elementary body?EB?adsorbs on cells firstly and then enters into them by endocytosis.EB develops into reticulate body?RB?after the host cell wrapping EB into vacuoles.RB has no infection but can breed by the form of bipartition.Chlamydia completes a round of growth and development after RB differentiating to EB.Chlamydia psittaci?Cps?belongs to the Chlamydia genus,widely distributed and infects poultry or livestock particularly by latent infection.Human beings can be infected Cps through respiratory tract,aerosol,skin,mucosa,etc.,and mild symptoms are similar to common cold symptoms,but severe symptoms of systemic poisoning can occur,and even death.Due to the lack of typical symptoms and specific diagnostic measures,Cps infection in clinical has misdiagnosis and missed diagnosis frequently.In addition,Cps has been included in the Convention on Prohibion of Biological Weapons in the field of biological warfare agents and biological terrorists because of its easy infection and high pathogenicity.Chlamydia trachomatis?Ct?also belongs to the Chlamydia genus and human beings are the only host of Ct,which mostly results in self-limited disease,and repeated or persisted infection can cause infertility,ectopic gestation,blindness and other serious diseases.Social problems of developing and developed countries respectively are the infection of blinding trachoma and urogenital system.WHO aims at eliminating blinding trachoma in 2020,and many countries have taken WHO SAFE strategies and achieve significant progress,but whether achieving sustainable elimination of trachoma target in 2020 is still unknown.Cps and Ct respectively contain 9 and 15 omp A genotypes.There are certain host infections in different types of chlamydia.With the recent development of whole-genome sequencing technology,foreign countries report more and more Cps and Ct genome sequencing analysis,deepening the understanding of coevolution of these two kinds of pathogens in different regions and hosts.Genome comparative analysis also provides an important tool for the identification of novel virulence genes.As a kind of pathogen which lacks of research,there are few studies on Cps or Ct genomics and the identification of virulence genes in China.1.The first part of my paper analyzes the whole genome sequence of Cps strain CG1 and Ct strain QH111 L isolated in China for the first time,which is of great significance for the further understanding of the evolution of Cps and Ct in China.1.1 Whole genome sequencing and analysis of abortion strain CG1 form Cps.The preliminary study found that China's Cps CG1 strains can cause sheep abortion.Type C strains form Cps in China also were isolated from cows,pigs and other mammals,ducks and other poultry,however the reports of foreign Cps type-C bacteria had only been found in waterfowl.It's different.Genome sequencing of CG1 strain was carried out by the two generation sequencing technology,and compared with the genome of C strain GR9.The results showed that,compared with the GR9 strain,the SNP mutation sites of CG1 mostly concentrated in a fixed area of about 23Kb?B598₀269-B598₀288?.BLAST analysis of the region of CG1 revealed that the 8 open reading frames were highly homologous to the Chlamydia abortus,which may explain why CG1 can infect mammals?sheep?and cause abortion in sheep.With the exception of a gene sequence of Chlamydia abortus in CG1,the genome comparative analysis shows that the B598₀681 gene of CG1 had a nonsense mutation encoding a putative inclusion membrane protein.1.2 Whole genome sequencing and analysis of Ct QH111 L strain.QH111L's ompA genotype is type B,which is highly homologous to the ompA sequence of the B/Tunis-864 strain,and the codon of the ninety-first amino acid has been found only in UGT strains,which include type LGV,type F and type G etc.,indicating that the ompA of QH111 L has characteristics of UGT type.Phylogenetic analysis showed that QH111 L belongs to the typical Ct branch of the eye,between type B and type C,but its genetic distance is far away from other B or C of Ct.Comparing the genome sequences of QH111 L with B/TZ1A828 isolated from Tanzania,found that there are 22 ORF which have more than 10 SNP.Including 9 ORF and 12 ORF were the highest homology with type C and type UGT respectively,that means chromosome sequence of QH111 L having type C and type UGT characteristics.The plasmid sequences of Ct were highly conserved.There were only limited SNP's differences between the plasmids of different Ct isolates.The QH111L's plasmid which belongs to the typical eye-type branch of Ct,but the pgp2 and pgp5 gene containing a UGT-type SNP loci respectively,indicating that the coevolution exist in plasmid and chromosome of QH111 L,matched UGT features in the ompA and chromosome sequence.2.The second part of my paper clones and expresses the virulence genes?CT135 homologous gene of Ct and Cps,B598₀681 gene of Cps?,which lays the foundation for the study of their pathogenic mechanisms.2.1 Cloning and expression of CT135 gene of Ct and Cps.In the subculture of type A-K Ct,the CT135 gene is prone to nonsense mutation,which may be related to the adaptive growth of Ct in vitro.In the female genital tract infection model,CT135 mutation was significantly correlated with the infection duration and pathogenicity of Ct,but the expression and localization of the gene has not been reported.One of the main reasons is that the CT135 gene is prone to nonsense mutation in the type A-K gene.However the CT135 gene in the type LGV of Ct remains intact,it may not be expressed normally.The CT135 homology gene is specificity in Chlamydia,and there is no spontaneous nonsense mutation in Cps and other Chlamydia.Through rebuilding the prokaryotic expression vector of CT135 gene?B598₀590?in Cps to express protein and purify the protein,immune mouse for attaining immune serum of polyclonal antibodies.The HeLa229 cells infected with CG1 were fixed with methanol,and the antiserum was used as the first antibody to detect the localization of the protein in the cells.It was found that the protein encoded by B598₀590 gene was stained with the known Inc A in the inclusion body.It was confirmed that the B598₀590 gene encodes the inclusion membrane protein,but its staining characteristic is different from that of Inc A,and it is unevenly distributed on the inclusion body,which may be related with its function.The CT135 gene of Ct was cloned and expressed by the same method as above,and it found that the recombinant protein could not be obtained after several attempts,which was not consistent with the expression of Cps CT135 homologous gene.The technique of qPCR was used to analyze the causes of protein expression in E.coli,and it was found that the amount of CT135 mRNA was decreased significantly after induction,which was not consistent with the expected increase of mRNA.After the deletion of A in the initiation codon ATG of the CT135 gene in the prokaryotic expression vector,the results of qPCR experiment showed that the amount of CT135 was greatly increased.This suggests that the low level of CT135 protein may be involved in the stability regulation of mRNA.Finally,we found that the recombinant protein could be obtained by the fusion of GST protein in the expression vector CT135 C-terminal,which laid the foundation for the preparation of anti CT135 antibody.2.2 Cloning and expression of Cps B598₀681 gene.In vitro culture,the CT135 gene of Ct is prone to mutation.There is a lack of study on genome stability of Cps,and we find that B598₀681 has a nonsense mutation by analyzing genomic sequence of the Cps CG1 strain,which may be related to subculture because the gene is only mutated in the partial Cps isolates.The gene also encodes a hypothetical species-specific inclusion membrane protein,which is already recombined expressed and purified.Summary: The genomics study of Cps CG1 and Ct QH111 L strains is of great importance to deepen the understanding of their adaptive evolution,and prevent and control two kinds of Chlamydia in China,as well as provides the direction for the further study of their adaptive evolutionary mechanisms.Cps and Ct CT135 homologous genes and cloning and expression of Cps B598₀681 gene lay a solid foundation for the further study of their function and pathogenesis.