云南西部HIV阳性者感染弓形虫的SAG2基因位点研究

发布时间：2018-05-14 07:01

本文选题：HIV阳性 + 弓形虫　；参考：《大理学院》2014年硕士论文

【摘要】：刚地弓形虫是一种专性细胞寄生原虫，可以感染包括人类在内的几乎所有脊椎动物，并在世界范围内传播，引起人兽共患寄生虫病。同时弓形虫是一种机会性致病原虫,免疫功能正常的人感染后常呈隐性感染,但在艾滋病、恶性肿瘤、长期应用免疫抑制剂治疗的患者或其它免疫功能严重受损者，弓形虫是引发致死性病变的主要原因之一。弓形虫病是AIDS最常见的机会性感染疾病之一，也是引起中枢神经系统局灶性占位症状最常见的原因。随着国内艾滋病发病率逐年升高，弓形虫感染引起的相关性疾病发生率也在升高，在世界很多地方还发生过流行，给人类带来巨大的健康威胁和经济上的严重损失，因而受到了越来越大的重视。不同地区或同一地区不同宿主的弓形虫基因型不同。常规聚合酶链反应（polymerase chain reaction PCR）技术及其衍生技术广泛应用于克隆基因、基因测序、基因分型等医学基础和临床研究中。通过运用PCR技术对弓形虫基因进行研究，可将弓形虫基因型分为I型、Ⅱ型和Ⅲ型，I型是公认的强毒株，主要引起先天性弓形虫病。其余两型属弱毒株，Ⅱ型多见于人体感染，Ⅲ型多见于动物感染。弓形虫不同虫株基因组之间存在微小差异，这可能是导致不同虫株之间的毒力、感染力等方面差异的原因，不同基因型的弓形虫虫株对药物的敏感性也不同。通过对弓形虫株基因鉴定和分型为弓形虫群体生物学、流行病学、疾病的防治等提供重要的依据。本实验通过采集云南西部HIV阳性者的血液，对弓形虫IgG和IgM阳性的全血样本进行基因提取，运用巢式PCR和RFLP等技术分析，与弓形虫国际标准株进行比较，对云南西部HIV阳性者血液内分离的弓形虫SAG2基因(241bp、221bp)进行遗传标记的分析，探讨云南西部HIV阳性者感染弓形虫的基因型，为发病机制和感染免疫的深入研究及临床治疗提供依据。 1.研究目的：了解云南西部HIV阳性者感染弓形虫的基本情况，初步分析并确定云南西部HIV阳性者感染弓形虫的主要基因型，从而为云南西部HIV阳性者弓形虫病的防治及其流行病学研究提供重要理论依据。 2.研究方法： 2.1样本收集收集云南省龙陵县、大理市、临沧市艾滋病防治机构的HIV阳性者血液样本，均经蛋白印迹(WB)试验阳性确证。 2.2弓形虫抗体检测将收集到的HIV阳性者血清样本应用酶联免疫吸附试验（ELISA）进行弓形虫IgG和IgM抗体检测，筛选出弓形虫IgG和或IgM阳性的样本。 2.3弓形虫基因提取及扩增、回收、纯化采用全血基因提取试剂盒对HIV阳性者弓形虫IgG和IgM阳性的血液样本进行基因提取；引用国际上比较成熟的引物及巢氏PCR技术对弓形虫SAG2基因进行扩增，经两轮扩增后电泳，全自动凝胶成像分析系统进行分析。对弓形虫SAG2基因扩增阳性的，用凝胶回收试剂盒进行回收、纯化。 2.4酶切用限制性内切酶Sau3AI和HhaI对弓形虫SAG2基因扩增阳性产物进行酶切，酶切后电泳结果与弓形虫标准株进行对比、鉴定，确定基因型。 2.5测序将弓形虫SAG2基因扩增阳性的产物进行回收、纯化后，共挑选7份（1份纯化弓形虫速殖子、6份HIV阳性者血液样本）送到上海立菲测序有限公司进行测序，测序结果与Genbank上注册的弓形虫株基因序列进行对比分析。 3.结果： 3.1血清抗体检测本次检测共392份全血样本，其中弓形虫IgG抗体阳性114份，阳性率为29.1%；弓形虫IgM抗体阳性有13份，阳性率为3.3%。本次检测共挑选出127份弓形虫抗体阳性的HIV阳性者全血样本。 3.2基因扩增及酶切本实验对SAG2基因扩增阳性的120份全血样本进行酶切，电泳成像后分别可见一大小约241bp、221bp的条带，分别经两个酶切后见到目的基因条带，与国际弓形虫标准基因型株（RH株）进行对比。由于本实验室缺乏Ⅱ型和Ⅲ型弓形虫标准株，根据参考文献进行对比分析，结果119份与国际标准株（RH株）一致，只有1份与Ⅲ型一致。 3.3序列分析云南西部HIV阳性者感染弓形虫株SAG2基因测序6份，6份源于随机选择的酶切标本。虽然各弓形虫株SAG2基因之间的差异率非常小，但6份弓形虫株进行基因测序的部分碱基对仍存在缺失、插入或突变。将弓形虫标准株（RH）、云南省HIV阳性者感染弓形虫株（样本）和Genbank注册的弓形虫株基因序列进行对比分析，它们之间的碱基对差异主要有以下几处：241bp的差异：样本3、4、7在第182对碱基处有G的缺失。221bp的差异：样本4在第31对碱基处G变为C，第48对碱基处G变为A，第163对碱基处有T的缺失；样本5在第84对碱基处G变为T，第163对碱基处G变为A；样本6在第163对碱基处G变为C，，C后插入了A；样本2、3、7在第163对碱基处T变为C，第165对碱基处G变为A。可见样本4的碱基变化较多，说明与标准株（RH）基因型不一致，有可能是其它基因型，而样本4酶切结果初步确定为Ⅲ型，恰好与测序结果一致。因此，完全排除了样本4不是I型，要完全确定样本4是否是Ⅲ型有待进一步研究。 4.结论 4.1云南西部HIV阳性者弓形虫感染率高于全国正常人群的平均水平。 4.2初步确定云南西部HIV阳性者感染弓形虫的基因型主要以Ⅰ型为主，Ⅲ型少见，未见基因Ⅱ型和其它基因型。 5.本实验研究的创新点 5.1本实验通过收集云南西部三地区艾滋病防治机构的HIV阳性者血液样本，进行弓形虫SAG2基因提取、扩增、酶切及序列分析，进一步结合弓形虫株B1基因、GRA6基因的研究结果进行综合分析，初步确定云南西部地区HIV阳性者感染弓形虫的基因型。 5.2本实验主要有以下两个方面的创新点： 5.2.1直接从云南西部HIV阳性者全血样本中提取弓形虫基因并成功扩增出弓形虫SAG2基因。 5.2.2从SAG2基因位点进行分析，初步确定云南西部HIV阳性者感染弓形虫的基因型。
[Abstract]:Toxoplasma gondii is a kind of parasitic protozoa, which can infect almost all vertebrates including humans and spread worldwide and cause zoonosis. At the same time, Toxoplasma gondii is an opportunistic pathogenic protozoa. Toxoplasma gondii is one of the main causes of fatal disease with the use of immunosuppressive agents or other severe immune dysfunction. Toxoplasmosis is one of the most common opportunistic infections in AIDS, and is also the most common cause of the central nervous system focal occupying symptoms. The incidence of related diseases caused by Toxoplasma infection is also increasing. It has also been prevalent in many parts of the world. It has brought great health threats and serious economic losses to mankind, and has been paid more and more attention.
The Toxoplasma gondii genotypes of different hosts in different regions or in the same region are different. The conventional polymerase chain reaction (polymerase chain reaction PCR) technology and its derivatization technology are widely used in the medical basis and clinical study of cloned genes, gene sequencing, genotyping, and so on. By using PCR technology to study the Toxoplasma gondii gene, the Toxoplasma can be arcuate The insect genotypes are divided into I, type II and type III type. I type is recognized as a strong virulent strain, which mainly causes congenital toxoplasmosis. The other two types are weak strains, type II is mostly in human infection, type III is mostly found in animal infection. There are small differences between the genomes of different strains of Toxoplasma gondii, which can lead to virulence and infection among different strains of insects. The sensitivity of different genotypes of Toxoplasma gondii strains to drugs is also different. Through the identification of Toxoplasma gondii and typing of Toxoplasma gondii, it provides important basis for the population biology of Toxoplasma gondii, epidemiology, and the prevention and control of the disease. In this experiment, the blood of HIV positive people in Western Yunnan was collected, and the total blood samples positive for Toxoplasma IgG and IgM were found. The genetic markers of Toxoplasma gondii SAG2 (241bp, 221bp), which were isolated from the blood of HIV positive people in Western Yunnan, were compared with the international standard strains of Toxoplasma gondii, and the genetic markers were compared with international standard strains of Toxoplasma gondii in Western Yunnan. The genotypes of Toxoplasma gondii infected by HIV positive people in Western Yunnan were analyzed, and the pathogenesis and infection of immunization were discussed. It provides the basis for the study and clinical treatment.
1. the purpose of the study:
In order to understand the basic situation of the infection of Toxoplasma gondii by HIV positive people in Western Yunnan, the main genotypes of Toxoplasma gondii infected by HIV positive people in Western Yunnan are preliminarily analyzed and determined, which provides an important theoretical basis for the prevention and control of toxoplasmosis and the epidemiological study of HIV positive people in western Yunnan.
2. research methods:
2.1 sample collection
Blood samples of HIV positive persons from AIDS prevention and control institutions in linling County, Dali City, Yunnan Province, were collected and confirmed by Western blot (WB) test.
Antibody detection of 2.2 Toxoplasma gondii
HIV positive serum samples were collected by enzyme linked immunosorbent assay (ELISA) for detection of Toxoplasma gondii IgG and IgM antibody, and the samples of Toxoplasma IgG and IgM positive were screened.
2.3 Toxoplasma gondii gene extraction, amplification, recovery and purification
The whole blood gene extraction kit was used to extract the HIV positive blood samples of Toxoplasma gondii IgG and IgM positive, and the SAG2 gene of Toxoplasma gondii was amplified by international mature primers and nesting PCR technology. After two rounds of amplification, the full-automatic gel imaging analysis system was analyzed. The SAG2 gene of Toxoplasma gondii Kuo Zengyang was analyzed. The gel was recovered and purified by gel Recovery Kit.
2.4 enzyme digestion
The positive products of Toxoplasma gondii SAG2 gene were cut by restriction endonuclease Sau3AI and HhaI. The results were compared with the standard strains of Toxoplasma gondii, and the genotype was identified.
2.5 sequencing
After reclaiming the positive product of Toxoplasma gondii SAG2 gene, 7 copies (1 purified Toxoplasma gondii tachyonus and 6 HIV positive blood samples) were sent to Shanghai Li Fei sequencing Co., Ltd. to be sequenced, and the sequencing results were compared with the gene sequence of the Toxoplasma gondii strain registered on the Genbank.
3. results:
3.1 serum antibody test
A total of 392 whole blood samples were tested, of which 114 were positive of Toxoplasma IgG antibody, the positive rate was 29.1%, and 13 positive of Toxoplasma IgM antibody positive. The positive rate was 3.3%., and the whole blood samples were selected to select 127 HIV positive antibodies positive for Toxoplasma gondii.
3.2 gene amplification and enzyme digestion
In this experiment, 120 whole blood samples with positive SAG2 gene were cut by enzyme. After electrophoretic imaging, the size of 241bp and 221bp was observed, and the target gene bands were seen after two enzymes and compared with the international standard strains of Toxoplasma gondii (RH strain). According to the comparative analysis of references, the results were consistent with 119 international standard strains (RH strain), and only 1 were consistent with type III.
3.3 sequence analysis
The SAG2 gene of Toxoplasma gondii strain infected by HIV in Western Yunnan was sequenced in 6 copies and 6 originates from random selected enzyme cut specimens. Although the difference rate of SAG2 genes between the Toxoplasma gondii strains was very small, the partial base pairs of 6 Toxoplasma gondii strains were still missing, inserted or mutated. The standard strain of Toxoplasma gondii (RH) and the positive sense of HIV in Yunnan Province The gene sequences of Toxoplasma gondii strains (samples) and Genbank registered Toxoplasma strains were compared and analyzed. The differences in base pairs between them were mainly as follows: the difference in 241bp: the difference between the sample 3,4,7 and the G deletion.221bp at 182nd bases: the sample 4 at thirty-first pairs of base G into C, forty-eighth to the base G to A, 163rd to the base There was a loss of T; sample 5 changed into T at eighty-fourth bases at base, 163rd G changed into A at base base, 6 at base base and G changed into C, C inserted A after C; 2,3,7 in 163rd of the base changed into C, and 165th changed into a sample 4. Genotypes, and the results of sample 4 enzyme digestion were preliminarily identified as type III, which coincided with the sequencing results. Therefore, the sample 4 was completely excluded from the I type. It is necessary to fully determine whether the sample 4 is type III is to be further studied.
4. conclusion
4.1 the infection rate of Toxoplasma gondii among HIV positive people in Western Yunnan is higher than that of the national normal population.
4.2 the genotype of Toxoplasma gondii infected by HIV positive in Western Yunnan was mainly type I, and type III was rare. No genotype II and other genotypes were found.
5. innovation points of the experimental research
5.1 by collecting the blood samples from the HIV positive people of AIDS prevention and control institutions in three regions of Western Yunnan, the SAG2 gene of Toxoplasma gondii was extracted, amplified, enzyme cut and sequence analysis. The results of the B1 gene of Toxoplasma gondii and the results of the GRA6 gene were synthetically analyzed to determine the base of the infection of the Toxoplasma gondii in the western region of Western Yunnan. Type.
5.2 this experiment mainly has the following two aspects of innovation:
5.2.1 directly extracted Toxoplasma gondii genes from whole blood samples of HIV positive persons in Western Yunnan and successfully amplified the SAG2 gene of Toxoplasma gondii.
5.2.2 was analyzed from SAG2 gene locus, and the genotype of Toxoplasma gondii infection in HIV positive persons in Western Yunnan was preliminarily determined.

【学位授予单位】：大理学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91;R531.8

【参考文献】