【作者】 刘琴；

【导师】 贺兰；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2021， 博士

【摘要】 巴贝斯虫病（Babesiosis）是一类由巴贝斯虫感染引起的人畜共患血液原虫病,其症状主要包括发热、血红蛋白尿、溶血性贫血,严重时可导致宿主死亡,宿主一经感染终生带虫免疫。巴贝斯虫种类繁多,至今报道的已有100多种。犬的巴贝斯虫病主要由吉氏巴贝斯虫（Babesia gibsoni）、犬巴贝斯虫（Babesia canis）、犬韦氏巴贝斯虫（Babesia canis vogeli）和罗氏巴贝斯虫（Babesia canis rossi）等感染引起,在我国以吉氏巴贝斯虫为主。目前尚无针对巴贝斯虫病的商业化检测试剂盒、无可推广的有效疫苗亦无有效的治疗手段,因此对该病的综合防控迫在眉睫。巴贝斯虫感染宿主后,在宿主体内通过裂殖生殖的方式进行无性繁殖。释放的裂殖子入侵更多红细胞,导致宿主红细胞不断破裂,损害宿主循环系统。此过程中,巴贝斯虫分泌一些抗原使得虫体能特异性识别、粘附或入侵宿主红细胞。理论上这类分泌抗原裸露于宿主免疫系统,能引起宿主免疫保护机制,因而一直被认为是优秀的诊断标识或疫苗候选者,这对巴贝斯虫病的诊断和预防具有重要意义。因此,筛选出优质分泌抗原对于探索巴贝斯虫的入侵和致病机制具有重要意义。随着生物信息学发展,基于基因组图谱可批量筛选具有一定特性的蛋白,例如:具有典型分泌特征的分泌蛋白（Secreted protein）或具有GPI锚定位点（Glycosylphosphatidy-linositol-anchored）的GPI蛋白等。而拥有完整的基因组图谱是筛选分泌抗原的前提,本课题克服了收集纯净巴贝斯虫虫体的技术难题,收集了无宿主污染的吉氏巴贝斯虫纯净虫体,通过吉氏巴贝斯虫全基因组测序,完成其全基因组（核外基因组和核基因组）的图谱绘制及分析,成功筛选出基因组上具典型分泌特征的分泌抗原。结合虫体体外培养上清蛋白质质谱分析,最终筛选到一种高丰度分泌抗原并进行了鉴定。主要结果如下:（1）绘制核外（顶质体）基因组图谱:Babesia gibsoni（B.gibsoni）的核外基因组包括线粒体基因组和顶质体基因组,其中线粒体基因组已有报道。本论文以顶质体基因组为对象进行研究,对顶质体基因组进行注释和分析,结果表明B.gibsoni的顶质体基因组由28386 bp（28.4 Kb）环状DNA分子组成,A+T含量高达86.33%,其t RNA数量少于其它巴贝斯虫（23/24）,4个基因簇的基因结构相对保守。系统发育分析显示B.gibsoni与Babesia orientalis（B.orientalis）的顶质体基因组具有高度相关性,并且在进化过程中B.gibsoni顶质体的基因组逐渐趋于“简单化”,只留下部分具有重要功能的基因,少数基因转移至核基因组,最后靶向于顶质体发挥功能。以上研究结果将有助于针对顶质体设计新型抗巴贝斯虫病的药物靶点。（2）绘制核基因组图谱:本论文克服了纯净虫体收集难题,通过改进方法收集了无宿主污染的纯净B.gibsoni虫体,基于B.gibsoni全基因组测序技术,完成B.gibsoni核基因组图谱绘制、注释和分析。结果显示其核基因组大小为7937104 bp（7.94 Mb）,包含4条染色体,约3552个基因。基因组共线性分析表明B.gibsoni与Babesia bovis（B.bovis）有2903个共线性基因,其基因组相关性高于Babesia microti（B.microti）。物种时间树对几种代表性顶复门寄生虫分歧时间进行分析,结果显示B.gibsoni分歧时间早于其他巴贝斯虫,且比较基因组-Venn图显示B.gibsoni有22个独特的COGs,这个基因主要有转运蛋白、分泌蛋白和虫体裂殖子蛋白组成,揭示了B.gibsoni潜在的物种特异性。KEGG分析和GO分析结果显示约73.9%（2641/3572）的基因分别被富集到316个KEGG通路和608个GO功能。（3）筛选并鉴定03g00237是一个高丰度分泌抗原:本研究基于基因组数据预测基因组所有蛋白信号肽、跨膜区等特性筛选出376个分泌蛋白,结合虫体体外培养上清蛋白质质谱分析结果,最终筛选出高丰度分泌蛋白Bg WH₀3g00237（03g00237）作为候选抗原。同时,鉴定出此候选抗原是一种GPI蛋白家族成员GPI1（GPI1-9）。实验结果证明该蛋白是一个高丰度蛋白,具有良好的免疫原性和反应原性,主要定位于虫体胞质,也能被释放至虫体外。（4）构建单基因敲除虫株Δ03g00237:运用基因编辑技术构建靶向03g00237基因的敲除质粒,筛选出2株Δ03g00237单克隆虫株,分别用常规PCR、Southern blot、q PCR、Western blot和IFAT进行鉴定,成功构建了单基因敲除虫株Δ03g00237。通过实验鉴定到03g00237蛋白影响着虫体生长,推测其可能是一种虫体粘附/入侵红细胞相关蛋白。综上所述,本研究绘制了B.gibsoni的基因组图谱,填补了B.gibsoni基因组信息的空缺;筛选并鉴定了一种与虫体生长相关的高丰度蛋白03g00237,此蛋白与一种GPI蛋白家族的其他8个成员序列高度相似,证明该蛋白是一种潜在的诊断标识或药物靶标。本研究结果为吉氏巴贝斯虫的生物学功能探索奠定了基础,也为吉氏巴贝斯虫病诊断标识与药物靶标的研究提供理论基础和科学依据。更多还原

【Abstract】 Babesiosis is a type of blood protozoal disease caused by Babesia.The symptoms of this disease are mainly fever,hemoglobinuria,hemolytic anemia and even death.and more than 100 types have been reported.Babesia gibsoni（B.gibsoni）,Babesia canis（B.canis）,Babesia canis vogeli（B.canis vogeli）and Babesia canis rossi（B.canis rossi）are the main causes of babesiosis in dogs,and the main pathogen in China is B.gibsoni.At present,there is no commercial detection kit for Babesiosis,no effective vaccine to promote and no effective treatment,so the comprehensive prevention and control of babesiosis is urgent.After infection,Babesia spp.reproduce asexually in the host by fission reproduction.The released merozoites invade more red blood cells,resulting in continuous rupture of host red blood cells and damage to the host circulatory system.During this process,Babesia spp.secretes antigens that allow the insect to specifically recognize,adhere to,or invade host red blood cells.In theory,this type of secreted antigen is exposed to the host immune system and can cause the host immune protection mechanism.Therefore,they have been considered as an excellent diagnostic marker or vaccine candidate,which is of great significance for the diagnosis and prevention of Babesiosis.Therefore,screening high-quality secreted proteins is of great significance to explore the invasion and pathogenesis of Babesia.With the development of bioinformatics,high-throughput screening of proteins with certain characteristics based on genomic information,such as secreted proteins with typical secretion characteristics or GPI proteins with Glycosylphosphatidy-linositol-anchored,etc.Complete genomic information is the prerequisite for screening secreted antigen.In this study,the whole genome（extranuclear genome and nuclear genome）of B.gibsoni was sequenced to complete the mapping and analysis of its genome,and the secreted antigen with typical secreted characteristics on the genome was successfully screened.Combined with the protein mass spectrometry analysis of the parasite in vitro culture supernatant,a highly abundant secreted antigen was finally screened and identified.The main results are as follows:（1）Mapping the genome of the extranuclear（apicoplast）: The extranuclear genomes of B.gibsoni include mitochondrial genome and apicoplast genome,of which mitochondrial genome have been reported.In this study,the apicoplast genome of B.gibsoni was studied,and the results showed that the apicoplast genome of B.gibsoni is composed of28386 bp（28.4 Kb）circular DNA molecules,with A+T content as high as 86.33%.The number of t RNA was less than that of other Babesia（23/24）,and the gene structure of the four gene clusters was relatively conserved.Phylogenetic analysis showed that the apicoplast genome of B.gibsoni and B.orientalis are highly similar.In the process of evolution,the apicoplast genome of B.gibsoni tends to be simplified,leaving only some genes with important functions,a few genes are transferred to nuclear genes,and finally targeted to the apicoplast to function.The results of this study will contribute to the design of novel drug targets against Babesiosis based on the apicoplast.（2）Mapping nuclear genome: In this paper,the collection of pure B.gibsoni without host contamination was overcome by an improved method.Complete mapping,annotation and analysis of B.gibsoni nuclear genome based on whole genome sequencing technology.The results showed that the nuclear genome size was about 7937104 bp（7.94 Mb）,containing 4 chromosomes and about 3552 genes.Genomic collinearity analysis showed that B.gibsoni had 2903 collinearity genes with Babesia bovis（B.bovis）,which was higher than that with Babesia microti（B.microti）.Species time tree analysis showed that B.gibsoni had an earlier divergence time than other Babesia.Furthermore,the comparative genome-Venn diagram showed that B.gibsoni had 22 unique COGs,which were mainly composed of transporter secreted proteins and parasite merozoite proteins,revealing the potential species-specificity of B.gibsoni.KEGG analysis and GO analysis showed that about 73.9%（2641/3572）genes were enriched into 316 KEGG pathways and 608 GO functions,respectively.（3）Screening and identifying 03g00237 as a high abundance secreted antigen: In this study,376 secreted proteins were selected based on the characteristics of signal peptides and transmembrane regions of all proteins predicted by genomic information.Combined with the results of protein mass spectrometry analysis of the supernatant of babesia in vitro,the high abundance secreted protein Bg WH₀3g00237（03g00237）was selected as candidate antigen.The candidate antigen was identified as GPI1,a member of the GPI protein family（GPI1-9）.The results showed that the protein was a high abundance protein with good immunogenicity and reactivity.It was mainly located in parasite cytoplasm and could also be secreted into the parasite.（4）C onstruction of single gene knockout strain Δ03g00237: Two monoclonal strains of Δ03g00237 were screened out and identified by conventional PCR,Southern blot,q PCR,Western blot and IFAT,respectively.The single gene knockout strain Δ03g00237 was successfully constructed.The protein 03g00237 was identified to affect the growth ofparasite,and it was speculated that it might be a protein related to parasite adhesion/invasion of red blood cells.In summary,this study mapped the genome of B.gibsoni and filled in the gap of B.gibsoni’s genome information.A high abundance protein 03g00237 associated with parasite growth was screened and identified.This protein is highly similar to 8 other members of the GPI protein family（Bg GPI1-9）,indicating that this protein is a potential diagnostic marker or drug target.The results of this study laid a foundation for the exploration of biological functions of B.gibsoni,and also provided a theoretical basis and scientific basis for the study of diagnostic identification and drug targets of babesiosis.更多还原