本文選題：弓形蟲　切入點：GRA7　出處：《吉林大學》2012年碩士論文　論文類型：學位論文

【摘要】：弓形蟲�。╰oxoplasmosis）是一種重要的人獸共患寄生蟲病，同時也是一種重要的機會食源性寄生蟲病。目前，世界各地人群中，弓形蟲感染相當普遍，全世界約有20億人感染弓形蟲。在醫(yī)學上它對于妊娠婦女及免疫力低下的人而言尤為重要。在我國，自1964年謝天華報道第一例弓形蟲病病人以來，陸續(xù)發(fā)現(xiàn)了弓形蟲病病例，這些病例中，胎兒畸形在先天性弓形蟲病中居首位[2]。孕婦可經(jīng)胎盤傳染給胎兒，能導致流產(chǎn)、早產(chǎn)、死胎或嚴重的先天性畸形，免疫力低下的人感染弓形蟲有時是致命的[2]。 近些年來，許多弓形蟲基因被克隆表達，但是很少有人對重組蛋白進行酶聯(lián)免疫試驗。本研究是將從弓形蟲ME49株cDNA文庫篩選得到基因進行擴增，連接，，轉化，測序比對，選出能表達強抗原性GRA7蛋白的GRA7基因進行克隆，表達及純化重組蛋白GRA7。重組蛋白GRA7與弓形蟲天然抗原同時進行ELISA檢測，比較分析重組抗原GRA7用于診斷弓形蟲病的可行性。首先，通過PCR擴增、連接、轉化弓形蟲ME49株cDNA文庫篩選結果中的46個陽性克隆，測序結果在NCBI（http://blast.ncbi.nlm.nih.gov/Blast.）上與Genebank中的基因進行比對，共獲得2個cDNA分子，分別編碼ME49株弓形蟲致密顆粒蛋白7(GRA7)和棒狀體蛋白2（ROP2）的部分片段。然后克隆和表達GRA7基因，通過鎳柱純化可溶性的重組蛋白，作為ELISA診斷包被的重組抗原，同時體外培養(yǎng)弓形蟲ME49株，收集、純化弓形蟲速殖子全蟲可溶性抗原，獲得弓形蟲天然抗原，通過間接ELISA法檢測分別包被GRA7重組蛋白和弓形蟲天然抗原，間接ELISA法檢測200份孕婦的血清，重組抗原GRA7檢測血清17例為陽性。天然抗原檢測200血清顯示有19例陽性。陽性率分別為8.5%和9.5%。與天然抗原相比，重組抗原GRA7的檢測敏感性為89.5%，有望替代天然抗原作為診斷抗原。
[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.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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