国内衣原体流行株的基因组分析与毒力基因克隆表达

发布时间：2018-07-05 01:07

本文选题：CT135 + 鹦鹉热衣原体　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：衣原体(Chlamydia)是一类专性细胞内寄生的细菌,二相发育周期使其感染机制独特,引发的各类疾病难以控制。衣原体的发育周期为原体(elementary body,EB)首先吸附细胞并通过细胞吞饮作用进入细胞,宿主细胞包裹EB形成空泡后EB发育为网状体(reticulate body,RB),后者无感染性但可通过二分裂形式进行繁殖,RB分化为EB后衣原体完成一轮生长发育。鹦鹉热衣原体(Chlamydia psittaci,Cps)属于衣原体属,分布广泛,尤其是家禽家畜自然感染普遍,多以潜伏性感染为主。人类可通过呼吸道-气溶胶、皮肤、粘膜等途径引起感染,轻症类似感冒症状,而重症可出现全身中毒症状,甚至死亡。由于缺乏典型症状和诊断措施,Cps感染在临床上误诊漏诊众多。另外,由于其易传播性以及高致病性,Cps已被列入国际《禁止生物武器公约》中的经典生物战剂和生物恐怖剂。沙眼衣原体(Chlamydia trachomatis,Ct)也属于衣原体属,人是Ct的唯一宿主,Ct导致的疾病多为自限性疾病,反复感染或长期感染导致的炎症反应可使宿主患不孕、异位妊娠、失明等严重疾病[1]。其中致盲性沙眼和泌尿生殖系统的感染分别是发展中国家和发达国家的一种社会问题。世界卫生组织旨在2020年消灭致盲性沙眼[2],许多国家为了消灭沙眼采取WHO的SAFE策略并取得了显著进步[3],然而十多年后是否完成可持续地消灭沙眼的目标仍是未知数。Cps和Ct分别包含9种和15种omp A基因型,不同型的不同衣原体种存在一定的宿主和组织感染嗜性。由于全基因组测序技术的快速发展,近年来国外报道了很多Cps和Ct基因组测序分析结果,加深了对这两种病原体在不同地区与不同宿主共进化的认识。基因组的比较分析也为发现鉴定衣原体新型毒力基因提供了重要工具。作为一类缺乏研究的病原体,来自国内的Cps或Ct基因组学报道及在此基础上的毒力基因鉴定研究还很有限。1论文第一部分,首次分析了国内Cps羊流产株CG1和眼型Ct分离株QH111L的全基因组序列,这对加深理解Cps和Ct在我国的进化有重要意义。1.1 Cps羊流产株CG1的全基因组序列测定与分析。前期研究发现,我国的Cps CG1流行株可以导致羊流产,Cps C型菌也在我国的牛、猪等哺乳动物和鸭等禽类中被分离到,这与国外Cps C型菌只在水禽类动物中被发现的报道不一致[4]。采用二代测序技术对CG1株进行基因组重测序,比对分析重测序序列结果和国外发表的C型菌GR9株的基因组序列。结果发现,与GR9株相比,CG1的SNP变异位点多数集中于一段约23kb的固定区域(B598_0269-B598_0288)。将CG1的该区域进行BLAST分析,发现12个开放阅读框均与流产衣原体高度同源,这或许可以用来解释CG1可以感染哺乳动物(羊),并导致羊流产的原因。除了发现CG1中存在一段流产衣原体的基因序列,在基因组对比分析中我们还发现了CG1的B598_0681基因发生了无义突变,该基因编码一个假定的包涵体膜蛋白。1.2眼型Ct分离株QH111L的全基因组测序与分析。QH111L为omp A基因B型,与B/Tunis-864株的omp A序列高度同源,只在LGV、F和G等UGT型菌株的omp A中被发现的密码子改变出现在第91位氨基酸上,说明QH111L的omp A有UGT型特征。染色体全长序列的系统进化发育分析表明,QH111L属于典型的眼型Ct分支,介于B和C型菌的分支之间,但与其它B或C型Ct的遗传距离较远。将QH111L与坦桑尼亚分离株B/TZ1A828的染色体序列进行比对,发现22个ORF存在10个以上SNP差异,其中的9个ORF和12个ORF分别与C型菌和UGT型的同源性最高,说明QH111L的染色体序列具有C型和UGT型特征。Ct的质粒序列高度保守,不同Ct分离株的质粒间仅存在有限的SNP差异。QH111L质粒属于典型的眼型Ct分支,但其编码的pgp2和pgp5基因分别含有一个UGT型SNP位点,这说明QH111L的质粒与染色体存在共进化,与在omp A和染色体序列中发现UGT型特征相一致。2论文第二部分,对衣原体毒力基因(Ct和Cps的CT135同源基因、Cps的B598_0681基因等)进行克隆表达,为研究它们的致病机制奠定基础。2.1 Ct和Cps的CT135基因克隆表达。A-K型Ct在传代培养时,CT135基因易发生无义突变,该基因可能与Ct的体外适应性生长有关。在雌鼠生殖道感染模型上,CT135变异与Ct的感染持续时间、致病能力等显著相关[5],但该基因的表达、定位等仍没有报道。一个主要原因是A-K型衣原体在培养时CT135基因易发生无义突变,而在LGV型Ct中CT135基因虽保持完整但很可能无法正常表达。CT135同源基因为衣原体科特异性,在Cps等其它衣原体中该基因不存在自发的无义突变现象。通过重构Cps CT135同源基因(B598_0590)的原核表达载体并诱导表达蛋白,切胶纯化方式纯化目的蛋白,免疫小鼠制备血清多抗。对感染CG1的He La229细胞进行甲醇固定,用抗血清作一抗,免疫荧光实验观察蛋白在细胞中的定位。发现B598_0590基因编码的蛋白在包涵体膜上浓染,这与已知的Inc A蛋白类似,证实了B598_0590基因编码包涵体膜蛋白,但其染色特征与Inc A存在差异,在包涵体上呈现不均匀分布,这可能与其功能有关。采用相同的方法克隆表达Ct CT135基因,多次尝试后发现不能获得重组蛋白,这与Cps CT135同源基因的表达结果不一致。用q PCR技术在E.coli中分析了蛋白未能表达的原因,发现诱导后CT135 m RNA的量出现大幅下降,与m RNA量上调的预期不符。将原核表达载体中CT135基因的起始密码子ATG中的A删除,q PCR实验发现诱导后CT135 m RNA的量大幅提高。这提示,低水平表达的CT135蛋白可能参与调节自身m RNA的稳定性。最后我们发现,在表达载体中CT135的C端融合GST可获得重组蛋白,为制备抗CT135抗体奠定了基础。2.2 Cps B598_0681基因的克隆表达。在体外传代培养时,Ct的CT135基因易发生变异。Cps的基因组稳定性缺乏研究,我们上面通过对Cps CG1株的基因组测序分析发现B598_0681发生了无义突变,这可能与传代培养相关,因为该基因只是在部分Cps分离株中发生了变异。B598_0681同样编码一个假定的科特异性包涵体膜蛋白,目前已完成了该蛋白的重组表达和纯化。总结:对Cps CG1株和Ct QH111L株的基因组学研究对加深理解两种衣原体在我国的适应性进化、对防控两种衣原体病有重大意义,也为深入研究它们的适应性进化机制提供了方向。Cps和Ct CT135同源基因、及Cps B598_0681的克隆表达为深入研究它们的功能和致病机理奠定了基础。
[Abstract]:Chlamydia (Chlamydia) is a kind of specific intracellular parasitic bacteria. The two phase development cycle makes the infection mechanism unique and the various diseases are difficult to control. The development cycle of Chlamydia is elementary body (EB), which first adsorb cells and enter cells through cell swallowing, and the host cells encapsulate EB to form vacuoles and develop into nets after EB. Reticulate body (RB), the latter is non infectious but can propagate in the form of two division, and the Chlamydia trachomatis can grow and develop after RB differentiation. The Chlamydia Chlamydia (Chlamydia psittaci, Cps) belongs to the chlamydia, which is widely distributed, especially in poultry domestic animals, mostly by latent infection. Human beings can breathe through respiration. Cps infection, such as aerosols, skin, mucous membrane and other ways of infection, light symptoms similar to cold symptoms, and severe symptoms and even death. Due to the lack of typical symptoms and diagnostic measures, Cps infection is misdiagnosed clinically as numerous. In addition, because of its susceptibility to transmission and high pathogenicity, Cps has been included in the International Convention on biological weapons prohibition. Classic biological warfare agents and bioterrorism agents. Chlamydia trachomatis (Ct) also belongs to the chlamydia, human is the only host of Ct, and most of the diseases caused by Ct are self limiting diseases. The inflammatory reaction caused by repeated infection or long-term infection can cause the host to suffer from infertility, ectopic pregnancy, blindness and other serious diseases, such as the blind trachoma of [1].. And the infection of the genitourinary system is a social problem in developing and developed countries. WHO aims to eliminate blindness [2] in 2020. Many countries have made significant progress in WHO's SAFE strategy to eliminate trachoma, but the goal of completing the sustainable elimination of the trachoma after more than 10 years is still still not. The number of.Cps and Ct contains 9 and 15 OMP A genotypes. Different types of Chlamydia have a certain host and tissue infection. Due to the rapid development of whole genome sequencing technology, a lot of Cps and Ct genome sequencing results have been reported in recent years, and the two pathogens in different regions and different hosts have been deepened. The comparative analysis of genomes also provides an important tool for the discovery and identification of the new virulence genes of Chlamydia. As a kind of lack of research pathogens, the Cps or Ct genomics reports from domestic and on the basis of the research on virulence gene identification are also limited in the first part of the.1 paper, and the first analysis of the abortion of domestic Cps sheep The whole genome sequence of strain CG1 and eye type Ct isolate QH111L, which is of great significance for understanding the evolution of Cps and Ct in our country, the whole genome sequencing and analysis of.1.1 Cps sheep abortion strain CG1. Earlier studies found that the Cps CG1 epidemic of our country could cause the abortion of sheep, Cps C type bacteria were also in our cattle, pigs and other mammals and ducks and so on. The class was separated from the reports that the foreign Cps C bacteria were found only in the waterfowl animals. [4]. was sequenced by two generation sequencing technology for the genome of CG1 strain, compared with the results of the re sequencing and the genome sequence of the GR9 strain of the C strain of the C bacteria published abroad. The results showed that the SNP variation loci of CG1 were most set in comparison with the GR9 strain. In a fixed area of about 23KB (B598_0269-B598_0288), the BLAST analysis of the region of CG1 found that 12 open reading frames were all highly homologous to Chlamydia abortus, which may be used to explain the causes of CG1 to infect mammals (sheep) and cause abortion in sheep. In addition to the discovery of a gene sequence of a Chlamydia abortion in CG1 In the genome comparative analysis, we also found that the B598_0681 gene of CG1 has a nonsense mutation. The gene encodes a hypothetical inclusion body membrane protein.1.2 eye type Ct isolate QH111L. The whole genome sequencing and analysis of.QH111L is the B type of the OMP A gene, which is highly homologous to the OMP A sequence of the B/Tunis-864 strain. The mutation of the codon found in OMP A appears on the ninety-first bit amino acid, indicating that the OMP A of QH111L has a UGT type characteristic. The phylogenetic analysis of the complete sequence of chromosomes indicates that QH111L belongs to the typical branch of the eye type Ct, which is between the branches of the B and C bacteria, but is far away from the other B or C type. Compared with the chromosome sequence of the isolated B/TZ1A828, it was found that there were more than 10 SNP differences in 22 ORF, of which 9 ORF and 12 ORF were the highest homology with C and UGT, indicating that the sequence of the chromosome sequence of QH111L was highly conserved by the C type and UGT type characteristic.Ct, and that there was only a limited difference between the plasmids of the isolates from the Ct isolates. The.QH111L plasmids belong to the typical Ct branch of the eye type, but the pgp2 and pgp5 genes encoded by the plasmid contain a UGT SNP locus, which indicates that the QH111L plasmid and the chromosome are coevolution, and the second part of the.2 paper is consistent with the discovery of the UGT type in OMP A and the chromosome sequence. The B598_0681 gene of PS, etc., was cloned and expressed in order to study the pathogenesis of.2.1 Ct and Cps, and the CT135 gene of Cps was cloned and expressed as.A-K Ct in the passage culture, and the CT135 gene was easily mutated. This gene may be related to the adaptive growth of Ct in vitro. In the female mouse reproductive tract infection model, CT135 variation and Ct infection The expression and location of the gene are not reported, but the gene expression and location of the gene are still not reported. One of the main reasons is that the CT135 gene of Chlamydia A-K is easily mutated during culture, while the CT135 gene in LGV Ct is intact but may not normally express the.CT135 homologous gene as the Chlamydia heterosexual, in Cps and so on. It does not have spontaneous nonsense mutation in the Chlamydia gene. By reconstructing the prokaryotic expression vector of Cps CT135 homologous gene (B598_0590) and inducing the expression protein, purify the target protein and immunize mice to prepare the serum polyclonal antibody. The He La229 cells infected with CG1 are immobilized with the antiserum and the immunofluorescence is used. It is found that the protein encoded by the B598_0590 gene is concentrated on the inclusion body membrane, which is similar to the known Inc A protein, which confirms that the B598_0590 gene encodes the inclusion body membrane protein, but its dyeing characteristics are different from the Inc A, and the inhomogeneous distribution on the inclusion bodies may be related to its function. The same may be related to its function. The Ct CT135 gene was cloned and expressed. After several attempts, it was found that the recombinant protein could not be obtained. This was not consistent with the expression of Cps CT135 homologous gene. The reason for the failure to express the protein was analyzed by Q PCR technology in E.coli, and it was found that the amount of CT135 m RNA decreased significantly after the induction, which was not consistent with the expectation of M RNA. The A deletion in the starting codon ATG of the CT135 gene in the body was deleted, and the Q PCR experiment found that the amount of CT135 m RNA was greatly increased after the induction. This suggests that the low level expressed CT135 protein may be involved in regulating the stability of the m RNA of its own m. The cloning and expression of the basic.2.2 Cps B598_0681 gene. The genomic stability of the CT135 gene of Ct is vulnerable to the lack of genomic stability in the Ct CT135 gene. We found that the B598_0681 mutation has occurred in B598_0681 by sequencing the genome of the Cps CG1 strain, which may be related to the passage culture, because the gene is only part of the Cps. The variant.B598_0681 also encodes a hypothetical specific inclusion body membrane protein, which has now completed the recombinant expression and purification of the protein. The genomics of the Cps CG1 strain and the Ct QH111L strain is important for understanding the adaptive evolution of two chlamydia in China and the prevention and control of two chlamydia. It also provides the direction.Cps and Ct CT135 homologous genes for their adaptive evolutionary mechanism, and the cloning and expression of Cps B598_0681 lays the foundation for the in-depth study of their function and pathogenesis.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R374

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