弓形虫高反应原性抗原的筛选及其初步应用

发布时间：2018-03-06 10:51

本文选题：弓形虫　切入点：GRA7　出处：《吉林大学》2012年硕士论文　论文类型：学位论文

【摘要】：弓形虫病（toxoplasmosis）是一种重要的人兽共患寄生虫病，同时也是一种重要的机会食源性寄生虫病。目前，世界各地人群中，弓形虫感染相当普遍，全世界约有20亿人感染弓形虫。在医学上它对于妊娠妇女及免疫力低下的人而言尤为重要。在我国，自1964年谢天华报道第一例弓形虫病病人以来，陆续发现了弓形虫病病例，这些病例中，胎儿畸形在先天性弓形虫病中居首位[2]。孕妇可经胎盘传染给胎儿，能导致流产、早产、死胎或严重的先天性畸形，免疫力低下的人感染弓形虫有时是致命的[2]。近些年来，许多弓形虫基因被克隆表达，但是很少有人对重组蛋白进行酶联免疫试验。本研究是将从弓形虫ME49株cDNA文库筛选得到基因进行扩增，连接，，转化，测序比对，选出能表达强抗原性GRA7蛋白的GRA7基因进行克隆，表达及纯化重组蛋白GRA7。重组蛋白GRA7与弓形虫天然抗原同时进行ELISA检测，比较分析重组抗原GRA7用于诊断弓形虫病的可行性。首先，通过PCR扩增、连接、转化弓形虫ME49株cDNA文库筛选结果中的46个阳性克隆，测序结果在NCBI（http://blast.ncbi.nlm.nih.gov/Blast.）上与Genebank中的基因进行比对，共获得2个cDNA分子，分别编码ME49株弓形虫致密颗粒蛋白7(GRA7)和棒状体蛋白2（ROP2）的部分片段。然后克隆和表达GRA7基因，通过镍柱纯化可溶性的重组蛋白，作为ELISA诊断包被的重组抗原，同时体外培养弓形虫ME49株，收集、纯化弓形虫速殖子全虫可溶性抗原，获得弓形虫天然抗原，通过间接ELISA法检测分别包被GRA7重组蛋白和弓形虫天然抗原，间接ELISA法检测200份孕妇的血清，重组抗原GRA7检测血清17例为阳性。天然抗原检测200血清显示有19例阳性。阳性率分别为8.5%和9.5%。与天然抗原相比，重组抗原GRA7的检测敏感性为89.5%，有望替代天然抗原作为诊断抗原。
[Abstract]:Toxoplasmosis (toxoplasmosis) is an important zoonotic parasitic disease, but also an important opportunity for foodborne parasitic disease. At present, people around the world, Toxoplasma infection is quite common, there are about 2 billion people worldwide are infected with Toxoplasma gondii. In medicine it is for pregnant women and immunocompromised patients is particularly important. In China, since 1964, Michael Tse reported the first cases of toxoplasmosis have been found since the toxoplasmosis cases, these cases of fetal malformation, congenital toxoplasmosis in pregnant women in the first place [2]. can be transmitted to the fetus through the placenta, can cause miscarriage, premature birth, stillbirth or severe congenital malformations, immunocompromised people infected with Toxoplasma gondii sometimes deadly [2].
In recent years, a lot of Toxoplasma gondii gene was cloned in expression, but few enzyme linked immunosorbent assay of recombinant protein. This study screened gene was amplified from ME49 strain of Toxoplasma gondii cDNA library connection, transformation, sequencing, GRA7 gene expression can choose strong antigenicity of GRA7 protein was cloned, expressed and purified the recombinant protein GRA7. recombinant protein GRA7 of Toxoplasma gondii and natural antigen ELISA was tested at the same time, a comparative analysis of recombinant antigen GRA7 in diagnosis of toxoplasmosis. First of all, was connected through the PCR 46 positive clones of ME49 strain Toxoplasma gondii cDNA library screening results, the sequencing results in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.) compared with Genebank in the gene, received a total of 2 cDNA molecules, respectively encoding Toxoplasma gondii ME49 strain 7 (GRA7) and rhoptry protein 2 (ROP2) and part of the pieces. After cloning and expression of GRA7 gene, the recombinant protein was purified by nickel column as a soluble, ELISA diagnosis of the recombinant antigen, and in vitro culture of Toxoplasma gondii ME49 strain, collection, purification of Toxoplasma gondii tachyzoites: soluble antigen of Toxoplasma gondii, get natural antigen, detected by indirect ELISA method respectively coated GRA7 recombinant protein and bow insect natural antigen, indirect ELISA detection of 200 serum, recombinant antigen to detect serum GRA7 of 17 cases were positive. 200 serum natural antigen detection showed that 19 cases were positive. The positive rate was 8.5% and 9.5%. respectively compared with the natural antigen, the detection sensitivity of the recombinant antigen GRA7 of 89.5%, is expected to replace the natural antigen as a diagnostic antigen.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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