云南大理HIV阳性者感染弓形虫B1、GRA6、SAG3基因位点的研究

发布时间：2018-01-19 06:16

  本文关键词： 大理 弓形虫 HIV阳性 基因扩增 测序　出处：《大理学院》2014年硕士论文　论文类型：学位论文

【摘要】：1研究目的 1.1初步了解云南大理HIV阳性者弓形虫感染情况。 1.2探讨云南大理HIV阳性者感染弓形虫株B1、GRA6、SAG3基因位点，初步了解云南大理弓形虫基因型，为进一步的分子遗传学研究以及弓形虫病的防制提供资料。 2研究方法 2.1样本收集 血液样本由云南大理白族自治州的艾滋病防治机构提供，经Western-blotting实验方法验证HIV阳性者的血液样本。 2.2弓形虫IgM与IgG抗体的检测 采用酶联免疫吸附法（ELISA）对HIV阳性者的血液样本进行弓形虫IgM与IgG抗体的检测，筛选弓形虫IgM与IgG抗体阳性的标本。 2.3弓形虫基因提取选择弓形虫IgM与IgG抗体阳性的全血标本进行弓形虫基因提取。 2.4基因扩增 采用经两轮扩增的巢式PCR扩增技术。 2.5基因回收、纯化 选择B1、GRA6、SAG3基因扩增阳性的产物经琼脂糖凝胶电泳成像后，进行基因产物的回收和纯化。 2.6基因的限制性内切酶酶切（RFLP） B1基因选择Pm1I与XhoI内切酶，GRA6基因选择MseI内切酶，SAG3基因选择NciI内切酶。将纯化后产物加入相应的限制性内切酶，消化16小时后，进行琼脂糖凝胶电泳成像。 2.7基因的序列测定 将巢式PCR技术扩增阳性的B1、GRA6、SAG3基因的产物纯化处理后，送基因公司进行序列测定。 3结果 3.1本次共检测317份血液样本，其中HIV阳性感染者的血液样本293份，正常对照人群样本24份。云南大理HIV阳性者弓形虫感染率为36.2%，其中弓形虫IgG抗体阳性97份，阳性率为33.1%；弓形虫IgM抗体阳性9份，阳性率为3.1%；健康体检者IgG抗体阳性率4.2%。 3.2采用巢式PCR成功扩增出B1基因47份、GRA6基因29份、SAG3基因42份。用相应的限制性内切酶进行酶切，琼脂糖凝胶电泳后可见B1基因有500bp左右大小的条带，GRA6基因有600bp和200bp两条条带，SAG3基因有200bp左右大小的条带。 3.3选择样本扩增阳性的弓形虫B1、GRA6、SAG3基因进行序列测定后，与Genbank上注册的弓形虫B1、GRA6、SAG3基因（注册号分别为AF17987.1、AJ635332.1、JX218225.1）序列进行对比，发现上述基因的碱基对之间存在少量的差异和缺失，并在弓形虫SAG3基因序列中的25bp处找到NciI酶切位点(CCGGG)，弓形虫GRA6基因序列中的146bp和690bp处找到MseI酶切位点(TTAA)。 4结论 4.1云南大理HIV阳性者弓形虫感染率为36.2%，高于该地区正常人群弓形虫抗体阳性率。 4.2采用巢式PCR成功从云南大理HIV阳性者弓形虫抗体阳性的全血标本中扩增出B1、GRA6、SAG3基因。 4.3云南大理HIV阳性者感染的弓形虫B1、GRA6、SAG3基因酶切的凝胶电泳结果与弓形虫RH株相同，初步判断云南大理HIV阳性者感染的弓形虫基因型以基因I型为主。 4.4云南大理HIV阳性者感染的弓形虫B1、GRA6、SAG3基因扩增阳性样本的测序结果显示，弓形虫B1、GRA6、SAG3基因与Genbank上注册的弓形虫RH株基因的碱基对之间仅存在少量的差异和缺失,初步判断云南大理HIV阳性弓形虫感染者基因型与弓形虫RH株一致。 4.5云南大理HIV阳性者感染的弓形虫为基因I型，，未见基因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.

【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】：R531.8

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