本文選題：CT135 + 鸚鵡熱衣原體��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：衣原體(Chlamydia)是一類專性細(xì)胞內(nèi)寄生的細(xì)菌,二相發(fā)育周期使其感染機(jī)制獨特,引發(fā)的各類疾病難以控制。衣原體的發(fā)育周期為原體(elementary body,EB)首先吸附細(xì)胞并通過細(xì)胞吞飲作用進(jìn)入細(xì)胞,宿主細(xì)胞包裹EB形成空泡后EB發(fā)育為網(wǎng)狀體(reticulate body,RB),后者無感染性但可通過二分裂形式進(jìn)行繁殖,RB分化為EB后衣原體完成一輪生長發(fā)育。鸚鵡熱衣原體(Chlamydia psittaci,Cps)屬于衣原體屬,分布廣泛,尤其是家禽家畜自然感染普遍,多以潛伏性感染為主。人類可通過呼吸道-氣溶膠、皮膚、粘膜等途徑引起感染,輕癥類似感冒癥狀,而重癥可出現(xiàn)全身中毒癥狀,甚至死亡。由于缺乏典型癥狀和診斷措施,Cps感染在臨床上誤診漏診眾多。另外,由于其易傳播性以及高致病性,Cps已被列入國際《禁止生物武器公約》中的經(jīng)典生物戰(zhàn)劑和生物恐怖劑。沙眼衣原體(Chlamydia trachomatis,Ct)也屬于衣原體屬,人是Ct的唯一宿主,Ct導(dǎo)致的疾病多為自限性疾病,反復(fù)感染或長期感染導(dǎo)致的炎癥反應(yīng)可使宿主患不孕、異位妊娠、失明等嚴(yán)重疾病[1]。其中致盲性沙眼和泌尿生殖系統(tǒng)的感染分別是發(fā)展中國家和發(fā)達(dá)國家的一種社會問題。世界衛(wèi)生組織旨在2020年消滅致盲性沙眼[2],許多國家為了消滅沙眼采取WHO的SAFE策略并取得了顯著進(jìn)步[3],然而十多年后是否完成可持續(xù)地消滅沙眼的目標(biāo)仍是未知數(shù)。Cps和Ct分別包含9種和15種omp A基因型,不同型的不同衣原體種存在一定的宿主和組織感染嗜性。由于全基因組測序技術(shù)的快速發(fā)展,近年來國外報道了很多Cps和Ct基因組測序分析結(jié)果,加深了對這兩種病原體在不同地區(qū)與不同宿主共進(jìn)化的認(rèn)識�；蚪M的比較分析也為發(fā)現(xiàn)鑒定衣原體新型毒力基因提供了重要工具。作為一類缺乏研究的病原體,來自國內(nèi)的Cps或Ct基因組學(xué)報道及在此基礎(chǔ)上的毒力基因鑒定研究還很有限。1論文第一部分,首次分析了國內(nèi)Cps羊流產(chǎn)株CG1和眼型Ct分離株QH111L的全基因組序列,這對加深理解Cps和Ct在我國的進(jìn)化有重要意義。1.1 Cps羊流產(chǎn)株CG1的全基因組序列測定與分析。前期研究發(fā)現(xiàn),我國的Cps CG1流行株可以導(dǎo)致羊流產(chǎn),Cps C型菌也在我國的牛、豬等哺乳動物和鴨等禽類中被分離到,這與國外Cps C型菌只在水禽類動物中被發(fā)現(xiàn)的報道不一致[4]。采用二代測序技術(shù)對CG1株進(jìn)行基因組重測序,比對分析重測序序列結(jié)果和國外發(fā)表的C型菌GR9株的基因組序列。結(jié)果發(fā)現(xiàn),與GR9株相比,CG1的SNP變異位點多數(shù)集中于一段約23kb的固定區(qū)域(B598_0269-B598_0288)。將CG1的該區(qū)域進(jìn)行BLAST分析,發(fā)現(xiàn)12個開放閱讀框均與流產(chǎn)衣原體高度同源,這或許可以用來解釋CG1可以感染哺乳動物(羊),并導(dǎo)致羊流產(chǎn)的原因。除了發(fā)現(xiàn)CG1中存在一段流產(chǎn)衣原體的基因序列,在基因組對比分析中我們還發(fā)現(xiàn)了CG1的B598_0681基因發(fā)生了無義突變,該基因編碼一個假定的包涵體膜蛋白。1.2眼型Ct分離株QH111L的全基因組測序與分析。QH111L為omp A基因B型,與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中被發(fā)現(xiàn)的密碼子改變出現(xiàn)在第91位氨基酸上,說明QH111L的omp A有UGT型特征。染色體全長序列的系統(tǒng)進(jìn)化發(fā)育分析表明,QH111L屬于典型的眼型Ct分支,介于B和C型菌的分支之間,但與其它B或C型Ct的遺傳距離較遠(yuǎn)。將QH111L與坦桑尼亞分離株B/TZ1A828的染色體序列進(jìn)行比對,發(fā)現(xiàn)22個ORF存在10個以上SNP差異,其中的9個ORF和12個ORF分別與C型菌和UGT型的同源性最高,說明QH111L的染色體序列具有C型和UGT型特征。Ct的質(zhì)粒序列高度保守,不同Ct分離株的質(zhì)粒間僅存在有限的SNP差異。QH111L質(zhì)粒屬于典型的眼型Ct分支,但其編碼的pgp2和pgp5基因分別含有一個UGT型SNP位點,這說明QH111L的質(zhì)粒與染色體存在共進(jìn)化,與在omp A和染色體序列中發(fā)現(xiàn)UGT型特征相一致。2論文第二部分,對衣原體毒力基因(Ct和Cps的CT135同源基因、Cps的B598_0681基因等)進(jìn)行克隆表達(dá),為研究它們的致病機(jī)制奠定基礎(chǔ)。2.1 Ct和Cps的CT135基因克隆表達(dá)。A-K型Ct在傳代培養(yǎng)時,CT135基因易發(fā)生無義突變,該基因可能與Ct的體外適應(yīng)性生長有關(guān)。在雌鼠生殖道感染模型上,CT135變異與Ct的感染持續(xù)時間、致病能力等顯著相關(guān)[5],但該基因的表達(dá)、定位等仍沒有報道。一個主要原因是A-K型衣原體在培養(yǎng)時CT135基因易發(fā)生無義突變,而在LGV型Ct中CT135基因雖保持完整但很可能無法正常表達(dá)。CT135同源基因為衣原體科特異性,在Cps等其它衣原體中該基因不存在自發(fā)的無義突變現(xiàn)象。通過重構(gòu)Cps CT135同源基因(B598_0590)的原核表達(dá)載體并誘導(dǎo)表達(dá)蛋白,切膠純化方式純化目的蛋白,免疫小鼠制備血清多抗。對感染CG1的He La229細(xì)胞進(jìn)行甲醇固定,用抗血清作一抗,免疫熒光實驗觀察蛋白在細(xì)胞中的定位。發(fā)現(xiàn)B598_0590基因編碼的蛋白在包涵體膜上濃染,這與已知的Inc A蛋白類似,證實了B598_0590基因編碼包涵體膜蛋白,但其染色特征與Inc A存在差異,在包涵體上呈現(xiàn)不均勻分布,這可能與其功能有關(guān)。采用相同的方法克隆表達(dá)Ct CT135基因,多次嘗試后發(fā)現(xiàn)不能獲得重組蛋白,這與Cps CT135同源基因的表達(dá)結(jié)果不一致。用q PCR技術(shù)在E.coli中分析了蛋白未能表達(dá)的原因,發(fā)現(xiàn)誘導(dǎo)后CT135 m RNA的量出現(xiàn)大幅下降,與m RNA量上調(diào)的預(yù)期不符。將原核表達(dá)載體中CT135基因的起始密碼子ATG中的A刪除,q PCR實驗發(fā)現(xiàn)誘導(dǎo)后CT135 m RNA的量大幅提高。這提示,低水平表達(dá)的CT135蛋白可能參與調(diào)節(jié)自身m RNA的穩(wěn)定性。最后我們發(fā)現(xiàn),在表達(dá)載體中CT135的C端融合GST可獲得重組蛋白,為制備抗CT135抗體奠定了基礎(chǔ)。2.2 Cps B598_0681基因的克隆表達(dá)。在體外傳代培養(yǎng)時,Ct的CT135基因易發(fā)生變異。Cps的基因組穩(wěn)定性缺乏研究,我們上面通過對Cps CG1株的基因組測序分析發(fā)現(xiàn)B598_0681發(fā)生了無義突變,這可能與傳代培養(yǎng)相關(guān),因為該基因只是在部分Cps分離株中發(fā)生了變異。B598_0681同樣編碼一個假定的科特異性包涵體膜蛋白,目前已完成了該蛋白的重組表達(dá)和純化�？偨Y(jié):對Cps CG1株和Ct QH111L株的基因組學(xué)研究對加深理解兩種衣原體在我國的適應(yīng)性進(jìn)化、對防控兩種衣原體病有重大意義,也為深入研究它們的適應(yīng)性進(jìn)化機(jī)制提供了方向。Cps和Ct CT135同源基因、及Cps B598_0681的克隆表達(dá)為深入研究它們的功能和致病機(jī)理奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R374

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 周玉梅;王智群;孫旭光;;我國北方兩地區(qū)小學(xué)生沙眼衣原體檢測及基因分型研究[J];眼科研究;2007年06期

2 李俊;俞盈;王豪舉;;細(xì)菌Ⅵ型分泌系統(tǒng)的研究進(jìn)展[J];微生物學(xué)報;2011年03期

3 Ningli Wang;Shijing Deng;Lei Tian;;A review of trachoma history in China: research, prevention, and control[J];Science China(Life Sciences);2016年06期

，

本文編號：2098250