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  本文關(guān)鍵詞： 大理 弓形蟲 HIV陽性 基因擴增 測序　出處：《大理學院》2014年碩士論文　論文類型：學位論文

【摘要】：1研究目的 1.1初步了解云南大理HIV陽性者弓形蟲感染情況。 1.2探討云南大理HIV陽性者感染弓形蟲株B1、GRA6、SAG3基因位點，初步了解云南大理弓形蟲基因型，為進一步的分子遺傳學研究以及弓形蟲病的防制提供資料。 2研究方法 2.1樣本收集 血液樣本由云南大理白族自治州的艾滋病防治機構(gòu)提供，經(jīng)Western-blotting實驗方法驗證HIV陽性者的血液樣本。 2.2弓形蟲IgM與IgG抗體的檢測 采用酶聯(lián)免疫吸附法（ELISA）對HIV陽性者的血液樣本進行弓形蟲IgM與IgG抗體的檢測，篩選弓形蟲IgM與IgG抗體陽性的標本。 2.3弓形蟲基因提取選擇弓形蟲IgM與IgG抗體陽性的全血標本進行弓形蟲基因提取。 2.4基因擴增 采用經(jīng)兩輪擴增的巢式PCR擴增技術(shù)。 2.5基因回收、純化 選擇B1、GRA6、SAG3基因擴增陽性的產(chǎn)物經(jīng)瓊脂糖凝膠電泳成像后，進行基因產(chǎn)物的回收和純化。 2.6基因的限制性內(nèi)切酶酶切（RFLP） B1基因選擇Pm1I與XhoI內(nèi)切酶，GRA6基因選擇MseI內(nèi)切酶，SAG3基因選擇NciI內(nèi)切酶。將純化后產(chǎn)物加入相應的限制性內(nèi)切酶，消化16小時后，進行瓊脂糖凝膠電泳成像。 2.7基因的序列測定 將巢式PCR技術(shù)擴增陽性的B1、GRA6、SAG3基因的產(chǎn)物純化處理后，送基因公司進行序列測定。 3結(jié)果 3.1本次共檢測317份血液樣本，其中HIV陽性感染者的血液樣本293份，正常對照人群樣本24份。云南大理HIV陽性者弓形蟲感染率為36.2%，其中弓形蟲IgG抗體陽性97份，陽性率為33.1%；弓形蟲IgM抗體陽性9份，陽性率為3.1%；健康體檢者IgG抗體陽性率4.2%。 3.2采用巢式PCR成功擴增出B1基因47份、GRA6基因29份、SAG3基因42份。用相應的限制性內(nèi)切酶進行酶切，瓊脂糖凝膠電泳后可見B1基因有500bp左右大小的條帶，GRA6基因有600bp和200bp兩條條帶，SAG3基因有200bp左右大小的條帶。 3.3選擇樣本擴增陽性的弓形蟲B1、GRA6、SAG3基因進行序列測定后，與Genbank上注冊的弓形蟲B1、GRA6、SAG3基因（注冊號分別為AF17987.1、AJ635332.1、JX218225.1）序列進行對比，發(fā)現(xiàn)上述基因的堿基對之間存在少量的差異和缺失，并在弓形蟲SAG3基因序列中的25bp處找到NciI酶切位點(CCGGG)，弓形蟲GRA6基因序列中的146bp和690bp處找到MseI酶切位點(TTAA)。 4結(jié)論 4.1云南大理HIV陽性者弓形蟲感染率為36.2%，高于該地區(qū)正常人群弓形蟲抗體陽性率。 4.2采用巢式PCR成功從云南大理HIV陽性者弓形蟲抗體陽性的全血標本中擴增出B1、GRA6、SAG3基因。 4.3云南大理HIV陽性者感染的弓形蟲B1、GRA6、SAG3基因酶切的凝膠電泳結(jié)果與弓形蟲RH株相同，初步判斷云南大理HIV陽性者感染的弓形蟲基因型以基因I型為主。 4.4云南大理HIV陽性者感染的弓形蟲B1、GRA6、SAG3基因擴增陽性樣本的測序結(jié)果顯示，弓形蟲B1、GRA6、SAG3基因與Genbank上注冊的弓形蟲RH株基因的堿基對之間僅存在少量的差異和缺失,初步判斷云南大理HIV陽性弓形蟲感染者基因型與弓形蟲RH株一致。 4.5云南大理HIV陽性者感染的弓形蟲為基因I型，，未見基因II型、基因III型和和其它基因型。
[Abstract]:1 purpose of research
1.1 preliminarily understand the infection of Toxoplasma gondii in the HIV positive people in Dali, Yunnan.
1.2 of Toxoplasma infection in Yunnan Dali HIV positive strains B1, GRA6, SAG3 loci, a preliminary understanding of genotype in Yunnan Dali Toxoplasma, to provide data for further study on molecular genetics of toxoplasmosis and prevention.
2 research methods
2.1 sample collection
Blood samples were provided by the AIDS prevention and control agency of the Dali Bai Autonomous Prefecture in Yunnan, Yunnan, and the blood samples of the HIV positive were tested by the Western-blotting test.
Detection of IgM and IgG antibodies of 2.2 Toxoplasma gondii
Enzyme-linked immunosorbent assay (ELISA) was used to detect Toxoplasma gondii IgM and IgG antibodies in blood samples of HIV positive persons, and samples of IgM and IgG positive antibodies of Toxoplasma gondii were screened.
2.3 Toxoplasma gondii gene extracts selected Toxoplasma IgM and IgG antibody positive whole blood samples for Toxoplasma gondii gene extraction.
2.4 gene amplification
The nested PCR amplification technique was adopted by two rounds of amplification.
2.5 gene recovery and purification
The products of B1, GRA6, and SAG3 genes were amplified by agarose gel electrophoresis to recover and purify the gene products.
Restriction endonuclease digestion (RFLP) of the 2.6 gene
B1 gene was selected for Pm1I and XhoI endonuclease. GRA6 gene was selected for MseI endonuclease. SAG3 gene was selected for NciI endonuclease. The purified product was added to the corresponding restriction endonuclease and digested for 16 hours, then agarose gel electrophoresis imaging was performed.
Sequencing of 2.7 gene
After purify the product of B1, GRA6, and SAG3 gene by nested PCR technique, the gene company was sequenced.
3 Results
3.1 of the 317 blood samples were detected, the positive HIV infected blood samples of 293 normal controls, 24 samples of Yunnan. Dali HIV positive Toxoplasma infection rate was 36.2%, the Toxoplasma IgG antibody positive 97, positive rate was 33.1%; Toxoplasma IgM antibody positive 9, positive rate 3.1% healthy subjects; the positive rate of IgG antibody 4.2%.
3.2 by nested PCR successfully amplified the B1 gene 47, GRA6 gene 29, SAG3 gene 42. Enzyme digestion with restriction endonuclease and corresponding bands in agarose gel electrophoresis showed B1 gene is about 500bp the size of the GRA6 gene of 600bp and 200bp two bands, band SAG3 gene is about 200bp the size of the.
3.3 samples were positive Toxoplasma B1, GRA6, SAG3 gene sequencing, GRA6 and Genbank on the registration of Toxoplasma gondii B1, SAG3, gene (accession number were AF17987.1, AJ635332.1, JX218225.1) sequence comparison, found that the gene base found small differences and lack of, and 25bp. In the sequence of SAG3 gene of Toxoplasma gondii in finding NciI restriction sites (CCGGG), 146bp and 690bp of Toxoplasma gondii GRA6 gene sequence to find MseI restriction sites (TTAA).
4 Conclusion
4.1 the infection rate of Toxoplasma gondii was 36.2% in the HIV positive people in Dali, Yunnan, which was higher than the positive rate of Toxoplasma gondii antibody in the normal population of the region.
4.2 the B1, GRA6 and SAG3 genes were amplified by nested PCR from the whole blood specimens with positive Toxoplasma gondii antibody positive in Dali, Dali, Yunnan.
4.3, the results of gel electrophoresis of Toxoplasma gondii B1, GRA6 and SAG3 genes infected by HIV positive persons in Dali, Yunnan were the same as that of Toxoplasma gondii RH. It was preliminarily judged that the genotype of Toxoplasma gondii infected by HIV positive people in Yunnan Dali was mainly I genotype.
4.4 Yunnan Dali HIV positive infection of Toxoplasma gondii B1, GRA6 sequencing, SAG3 gene amplification positive samples showed that Toxoplasma B1, GRA6, SAG3 and Genbank on the registered gene of Toxoplasma gondii RH strain gene base there is only a small amount of differences and lack of preliminary judgment between genotype and Toxoplasma gondii RH strain Yunnan Dali HIV positive infection of Toxoplasma gondii.
4.5 the Toxoplasma gondii infected by HIV positive people in Dali, Yunnan, is I type, no gene II, gene type III and other genotypes.

【學位授予單位】：大理學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R531.8

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