本文選題：沙眼衣原體 + MOMP�。� 參考：《浙江大學》2010年碩士論文

【摘要】： 沙眼衣原體(Ct)是引起沙眼、非淋球菌尿道炎和許多泌尿生殖道疾病的病原微生物。其18個血清型都具有一個主要外膜蛋白(Major Outer MembraneProtein,MOMP),不僅在介導Ct黏附宿主細胞的過程中起重要作用,而且本身作為一種重要的免疫原能刺激機體產生細胞免疫和體液免疫,而ompA基因是編碼Ct MOMP的結構基因。因此,根據ompA基因的特異性,制備具有良好免疫原性的MOMP抗原,對研究Ct的致病機制、臨床快速診斷Ct感染的試劑盒研發(fā)、肽芯片及疫苗制備等有重要意義。 本文分析了Ct 18個血清型ompA基因的堿基序列,B型ompA基因與其它型別相比,其表達產物有2-3個氨基酸的差異,因此確定選用B/TW-5 ompA表位,共62個氨基酸。并根據大腸桿菌的偏好密碼子優(yōu)化、設計并合成堿基序列,使得擬表達的目的基因序列能夠較好地與原核表達系統(tǒng)相匹配,提高表達效率。通過目的基因片段與pET-41a構建重組質粒并轉化大腸桿菌后,IPTG誘導表達,SDS-PAGE分析,得到了分子量約為34KD的重組蛋白,并將選定的表達株放大培養(yǎng),采用柱層析方法純化重組蛋白,確定了結合緩沖液:Na_2H PO_4:NaH_2PO_4 3:2(20mmol/L),8M/L尿素,5mmol/L咪唑,pH8.0;洗脫緩沖液:Na_2H PO_4:NaH_2PO_4 3:2(20mmol/L),8M/L尿素,500mmol/L咪唑,pH8.0,分別用5%,10%,15%,20%,25%,洗脫液梯度洗滌樣品,是比較合適的純化目的蛋白的條件。純化后蛋白用ELISA檢測臨床血清標本,敏感性為85.7%,特異性為87.8%。
[Abstract]:Chlamydia trachomatis (Ct) is a pathogenic microorganism that causes trachoma, non gonococcal urethritis and many genitourinary diseases. Its 18 serotypes have a major outer membrane protein (Major Outer MembraneProtein, MOMP), which not only play an important role in mediating Ct adhesion to host cells, but also as an important immunogenic energy itself. Stimulating the body to produce cellular and humoral immunity, and the ompA gene is a structural gene encoding Ct MOMP. Therefore, the preparation of the MOMP antigen with good immunogenicity based on the specificity of the ompA gene is of great significance for the study of the pathogenesis of Ct, the development of the kit for the rapid diagnosis of Ct infection, the peptide chip and the preparation of the vaccine.
In this paper, the base sequence of 18 serotype ompA genes of Ct is analyzed. Compared with other types, the B ompA gene has a difference in the expression product of 2-3 amino acids. Therefore, the B/TW-5 ompA epitope is selected and a total of 62 amino acids are selected. The base sequence is designed and synthesized according to the preference codon of Escherichia coli to make the target gene sequence of the expressed gene. The column can be well matched with the prokaryotic expression system to improve the expression efficiency. After the recombinant plasmid was constructed with pET-41a, the recombinant plasmid was constructed and transformed into Escherichia coli, IPTG induced expression and SDS-PAGE analysis. The recombinant protein with a molecular weight of about 34KD was obtained, and the selected expression strain was amplified and cultured, and the recombinant protein was purified by column chromatography. The combined buffer solution: Na_2H PO_4:NaH_2PO_4 3:2 (20mmol/L), 8M/L urea, 5mmol/L imidazole, pH8.0, and elution buffer solution: Na_2H PO_4:NaH_2PO_4 3:2 (20mmol/L), 8M/L urea, 20%, 25%, eluent ladder washing samples respectively, are the suitable conditions for the purification of the target protein. The purified protein is used for the purification. The sensitivity and specificity of A were 85.7% and 87.8%. respectively.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R759

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