發(fā)布時(shí)間：2018-05-14 07:01

  本文選題：HIV陽(yáng)性 + 弓形蟲(chóng)��； 參考：《大理學(xué)院》2014年碩士論文

【摘要】：剛地弓形蟲(chóng)是一種專(zhuān)性細(xì)胞寄生原蟲(chóng)，可以感染包括人類(lèi)在內(nèi)的幾乎所有脊椎動(dòng)物，并在世界范圍內(nèi)傳播，引起人獸共患寄生蟲(chóng)病。同時(shí)弓形蟲(chóng)是一種機(jī)會(huì)性致病原蟲(chóng),免疫功能正常的人感染后常呈隱性感染,但在艾滋病、惡性腫瘤、長(zhǎng)期應(yīng)用免疫抑制劑治療的患者或其它免疫功能?chē)?yán)重受損者，弓形蟲(chóng)是引發(fā)致死性病變的主要原因之一。弓形蟲(chóng)病是AIDS最常見(jiàn)的機(jī)會(huì)性感染疾病之一，也是引起中樞神經(jīng)系統(tǒng)局灶性占位癥狀最常見(jiàn)的原因。隨著國(guó)內(nèi)艾滋病發(fā)病率逐年升高，弓形蟲(chóng)感染引起的相關(guān)性疾病發(fā)生率也在升高，在世界很多地方還發(fā)生過(guò)流行，給人類(lèi)帶來(lái)巨大的健康威脅和經(jīng)濟(jì)上的嚴(yán)重?fù)p失，因而受到了越來(lái)越大的重視。 不同地區(qū)或同一地區(qū)不同宿主的弓形蟲(chóng)基因型不同。常規(guī)聚合酶鏈反應(yīng)（polymerase chain reaction PCR）技術(shù)及其衍生技術(shù)廣泛應(yīng)用于克隆基因、基因測(cè)序、基因分型等醫(yī)學(xué)基礎(chǔ)和臨床研究中。通過(guò)運(yùn)用PCR技術(shù)對(duì)弓形蟲(chóng)基因進(jìn)行研究，可將弓形蟲(chóng)基因型分為I型、Ⅱ型和Ⅲ型，I型是公認(rèn)的強(qiáng)毒株，主要引起先天性弓形蟲(chóng)病。其余兩型屬弱毒株，Ⅱ型多見(jiàn)于人體感染，Ⅲ型多見(jiàn)于動(dòng)物感染。弓形蟲(chóng)不同蟲(chóng)株基因組之間存在微小差異，這可能是導(dǎo)致不同蟲(chóng)株之間的毒力、感染力等方面差異的原因，不同基因型的弓形蟲(chóng)蟲(chóng)株對(duì)藥物的敏感性也不同。通過(guò)對(duì)弓形蟲(chóng)株基因鑒定和分型為弓形蟲(chóng)群體生物學(xué)、流行病學(xué)、疾病的防治等提供重要的依據(jù)。本實(shí)驗(yàn)通過(guò)采集云南西部HIV陽(yáng)性者的血液，對(duì)弓形蟲(chóng)IgG和IgM陽(yáng)性的全血樣本進(jìn)行基因提取，運(yùn)用巢式PCR和RFLP等技術(shù)分析，與弓形蟲(chóng)國(guó)際標(biāo)準(zhǔn)株進(jìn)行比較，對(duì)云南西部HIV陽(yáng)性者血液內(nèi)分離的弓形蟲(chóng)SAG2基因(241bp、221bp)進(jìn)行遺傳標(biāo)記的分析，探討云南西部HIV陽(yáng)性者感染弓形蟲(chóng)的基因型，為發(fā)病機(jī)制和感染免疫的深入研究及臨床治療提供依據(jù)。 1.研究目的： 了解云南西部HIV陽(yáng)性者感染弓形蟲(chóng)的基本情況，初步分析并確定云南西部HIV陽(yáng)性者感染弓形蟲(chóng)的主要基因型，從而為云南西部HIV陽(yáng)性者弓形蟲(chóng)病的防治及其流行病學(xué)研究提供重要理論依據(jù)。 2.研究方法： 2.1樣本收集 收集云南省龍陵縣、大理市、臨滄市艾滋病防治機(jī)構(gòu)的HIV陽(yáng)性者血液樣本，均經(jīng)蛋白印跡(WB)試驗(yàn)陽(yáng)性確證。 2.2弓形蟲(chóng)抗體檢測(cè) 將收集到的HIV陽(yáng)性者血清樣本應(yīng)用酶聯(lián)免疫吸附試驗(yàn)（ELISA）進(jìn)行弓形蟲(chóng)IgG和IgM抗體檢測(cè)，篩選出弓形蟲(chóng)IgG和或IgM陽(yáng)性的樣本。 2.3弓形蟲(chóng)基因提取及擴(kuò)增、回收、純化 采用全血基因提取試劑盒對(duì)HIV陽(yáng)性者弓形蟲(chóng)IgG和IgM陽(yáng)性的血液樣本進(jìn)行基因提��；引用國(guó)際上比較成熟的引物及巢氏PCR技術(shù)對(duì)弓形蟲(chóng)SAG2基因進(jìn)行擴(kuò)增，經(jīng)兩輪擴(kuò)增后電泳，全自動(dòng)凝膠成像分析系統(tǒng)進(jìn)行分析。對(duì)弓形蟲(chóng)SAG2基因擴(kuò)增陽(yáng)性的，用凝膠回收試劑盒進(jìn)行回收、純化。 2.4酶切 用限制性?xún)?nèi)切酶Sau3AI和HhaI對(duì)弓形蟲(chóng)SAG2基因擴(kuò)增陽(yáng)性產(chǎn)物進(jìn)行酶切，酶切后電泳結(jié)果與弓形蟲(chóng)標(biāo)準(zhǔn)株進(jìn)行對(duì)比、鑒定，確定基因型。 2.5測(cè)序 將弓形蟲(chóng)SAG2基因擴(kuò)增陽(yáng)性的產(chǎn)物進(jìn)行回收、純化后，共挑選7份（1份純化弓形蟲(chóng)速殖子、6份HIV陽(yáng)性者血液樣本）送到上海立菲測(cè)序有限公司進(jìn)行測(cè)序，測(cè)序結(jié)果與Genbank上注冊(cè)的弓形蟲(chóng)株基因序列進(jìn)行對(duì)比分析。 3.結(jié)果： 3.1血清抗體檢測(cè) 本次檢測(cè)共392份全血樣本，其中弓形蟲(chóng)IgG抗體陽(yáng)性114份，陽(yáng)性率為29.1%；弓形蟲(chóng)IgM抗體陽(yáng)性有13份，陽(yáng)性率為3.3%。本次檢測(cè)共挑選出127份弓形蟲(chóng)抗體陽(yáng)性的HIV陽(yáng)性者全血樣本。 3.2基因擴(kuò)增及酶切 本實(shí)驗(yàn)對(duì)SAG2基因擴(kuò)增陽(yáng)性的120份全血樣本進(jìn)行酶切，電泳成像后分別可見(jiàn)一大小約241bp、221bp的條帶，分別經(jīng)兩個(gè)酶切后見(jiàn)到目的基因條帶，與國(guó)際弓形蟲(chóng)標(biāo)準(zhǔn)基因型株（RH株）進(jìn)行對(duì)比。由于本實(shí)驗(yàn)室缺乏Ⅱ型和Ⅲ型弓形蟲(chóng)標(biāo)準(zhǔn)株，根據(jù)參考文獻(xiàn)進(jìn)行對(duì)比分析，結(jié)果119份與國(guó)際標(biāo)準(zhǔn)株（RH株）一致，只有1份與Ⅲ型一致。 3.3序列分析 云南西部HIV陽(yáng)性者感染弓形蟲(chóng)株SAG2基因測(cè)序6份，6份源于隨機(jī)選擇的酶切標(biāo)本。雖然各弓形蟲(chóng)株SAG2基因之間的差異率非常小，但6份弓形蟲(chóng)株進(jìn)行基因測(cè)序的部分堿基對(duì)仍存在缺失、插入或突變。將弓形蟲(chóng)標(biāo)準(zhǔn)株（RH）、云南省HIV陽(yáng)性者感染弓形蟲(chóng)株（樣本）和Genbank注冊(cè)的弓形蟲(chóng)株基因序列進(jìn)行對(duì)比分析，它們之間的堿基對(duì)差異主要有以下幾處：241bp的差異：樣本3、4、7在第182對(duì)堿基處有G的缺失。221bp的差異：樣本4在第31對(duì)堿基處G變?yōu)镃，第48對(duì)堿基處G變?yōu)锳，第163對(duì)堿基處有T的缺失；樣本5在第84對(duì)堿基處G變?yōu)門(mén)，第163對(duì)堿基處G變?yōu)锳；樣本6在第163對(duì)堿基處G變?yōu)镃，，C后插入了A；樣本2、3、7在第163對(duì)堿基處T變?yōu)镃，第165對(duì)堿基處G變?yōu)锳�？梢�(jiàn)樣本4的堿基變化較多，說(shuō)明與標(biāo)準(zhǔn)株（RH）基因型不一致，有可能是其它基因型，而樣本4酶切結(jié)果初步確定為Ⅲ型，恰好與測(cè)序結(jié)果一致。因此，完全排除了樣本4不是I型，要完全確定樣本4是否是Ⅲ型有待進(jìn)一步研究。 4.結(jié)論 4.1云南西部HIV陽(yáng)性者弓形蟲(chóng)感染率高于全國(guó)正常人群的平均水平。 4.2初步確定云南西部HIV陽(yáng)性者感染弓形蟲(chóng)的基因型主要以Ⅰ型為主，Ⅲ型少見(jiàn)，未見(jiàn)基因Ⅱ型和其它基因型。 5.本實(shí)驗(yàn)研究的創(chuàng)新點(diǎn) 5.1本實(shí)驗(yàn)通過(guò)收集云南西部三地區(qū)艾滋病防治機(jī)構(gòu)的HIV陽(yáng)性者血液樣本，進(jìn)行弓形蟲(chóng)SAG2基因提取、擴(kuò)增、酶切及序列分析，進(jìn)一步結(jié)合弓形蟲(chóng)株B1基因、GRA6基因的研究結(jié)果進(jìn)行綜合分析，初步確定云南西部地區(qū)HIV陽(yáng)性者感染弓形蟲(chóng)的基因型。 5.2本實(shí)驗(yàn)主要有以下兩個(gè)方面的創(chuàng)新點(diǎn)： 5.2.1直接從云南西部HIV陽(yáng)性者全血樣本中提取弓形蟲(chóng)基因并成功擴(kuò)增出弓形蟲(chóng)SAG2基因。 5.2.2從SAG2基因位點(diǎn)進(jìn)行分析，初步確定云南西部HIV陽(yáng)性者感染弓形蟲(chóng)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.

【學(xué)位授予單位】：大理學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R512.91;R531.8

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 李雪秋;劉轉(zhuǎn)轉(zhuǎn);殷國(guó)榮;李佩珍;孟曉麗;劉紅麗;;小鼠血清中弓形蟲(chóng)Peroxiredoxin抗原間接ELISA檢測(cè)方法的建立[J];中國(guó)生物制品學(xué)雜志;2010年03期

2 崔黎明;霍威;李淑紅;劉妮;;醫(yī)院病人弓形蟲(chóng)感染血清流行病學(xué)調(diào)查[J];中國(guó)病原生物學(xué)雜志;2008年05期

3 葉玉美,魏德瓊,涂育發(fā),江華;云南省弓形蟲(chóng)病流行病學(xué)調(diào)查研究[J];中國(guó)人獸共患病雜志;2001年02期

4 李文姝,陸惠民,閔太善,黃偉達(dá);弓形蟲(chóng)棒狀體蛋白R(shí)OP2基因的克隆與表達(dá)[J];中國(guó)人獸共患病雜志;2005年10期

5 沈杰;我國(guó)寄生蟲(chóng)學(xué)界對(duì)弓形蟲(chóng)病認(rèn)識(shí)上的飛躍[J];中國(guó)獸醫(yī)寄生蟲(chóng)病;2004年02期

6 殷國(guó)榮;楊亞波;鄭金平;楊建一;侯玉英;單聯(lián)U

本文編號(hào)：1886822