費昌勇林大盛2014-10-272014-10-272008-06-??http://rportal.lib.ntnu.edu.tw/handle/20.500.12235/7067弓蟲感染在人跟動物都會造成嚴重的後果。弓蟲症是一分佈很廣的人畜共通傳染病，因此在經濟及公共衛生上皆很重要。由於弓蟲症缺乏特殊的臨床症狀，必須要使用診斷技術偵測感染。偵測抗弓蟲抗體，是一非常有用的感染指標。然而，只當測試樣品能辨識弓蟲獨特的表面抗原1(SAG1)時，方能確定診斷。SAG1在各種不同株的弓蟲其結構幾乎完全一樣，因此在弓蟲症診斷上為一非常有用的分子。在本研究，SAG1基因經增幅並進一步純化出具有1,011base pairs之核苷酸，然後連接上已去磷酸化直線形的pET-24b載體，形成具有6,320 base pairs之pET-24b/SAG1重組體。將此重組體植入氯化鈣處理過之Escherichia coli後，表現一63 kilodaltons之蛋白質。利用immunoblotting證實，此重組蛋白質亦可被能辨識SAG1分子之弓蟲感染貓的血清所結合。更進一步測試發現，所有30個能辨識表現的重組蛋白質之陽性血清，在使用此重組蛋白質之kinetics-based enzyme linked immunosorbent assay (ELISA)亦呈陽性。因此，使用此表現的重組蛋白質之ELISA結合了傳統ELISA與費時的immunoblotting兩者之優點，具有非常好的靈敏性與特異性，將成為一調查大量血清樣品之非常有效的工具。此重組蛋白質之可能發展為疫苗使用亦在文中討論。Toxoplasma gondii (T. gondii) infection can cause serious consequences in both humans and animals. Toxoplasmosis is a widespread zoonotic disease and thus is of economic as well as public health importance. Because toxoplasmosis lacks specific clinical signs or syndromes, the application of diagnostic techniques is required to detect the infection. Detection of antibodies to T. gondii has been very useful as an indicator of infection. Nevertheless, no final diagnosis will be made unless a native 30-kilodalton T. gondii-specific surface antigen 1 (SAG1) is recognized by the test samples. SAG1 is highly conserved among various strains of T. gondii and thus is a very useful molecule for diagnosis of toxoplasmosis. In this study, SAG1 gene was amplified and further purified as a 1,011-base pair nucleotide which was then ligated with pET-24b vector as a 6,320-base pair recombinant pET-24b/SAG1. After transformation, a 63-kilodalton protein was expressed in calcium chloride-treated competent Escherichia coli. This recombinant protein could be identified by T. gondii-infected cat sera which also were able to recognize SAG1 molecule by immunoblotting. Furthermore, all 30 sera which could recognize expressed recombinant protein were also positive in kinetics-based enzyme linked immunosorbent assay (ELISA) with recombinant protein. Therefore, ELISA with this expressed recombinant protein, combining the advantages of both traditional ELISA and time consuming immunoblotting, will be a powerful tool for surveying a large number of serum samples with excellent sensitivity and specificity. The potential candidate of this recombinant protein for vaccine development is also discussed.弓蟲重組蛋白質抗體T. gondiiRecombinant proteinAntibodiesSAG1P30