沙眼衣原体主要外膜蛋白重组质粒的构建、表达和活性鉴定

发布时间：2018-06-26 22:27

本文选题：沙眼衣原体 + MOMP　；参考：《浙江大学》2010年硕士论文

【摘要】： 沙眼衣原体(Ct)是引起沙眼、非淋球菌尿道炎和许多泌尿生殖道疾病的病原微生物。其18个血清型都具有一个主要外膜蛋白(Major Outer MembraneProtein,MOMP),不仅在介导Ct黏附宿主细胞的过程中起重要作用,而且本身作为一种重要的免疫原能刺激机体产生细胞免疫和体液免疫,而ompA基因是编码Ct MOMP的结构基因。因此,根据ompA基因的特异性,制备具有良好免疫原性的MOMP抗原,对研究Ct的致病机制、临床快速诊断Ct感染的试剂盒研发、肽芯片及疫苗制备等有重要意义。本文分析了Ct 18个血清型ompA基因的碱基序列,B型ompA基因与其它型别相比,其表达产物有2-3个氨基酸的差异,因此确定选用B/TW-5 ompA表位,共62个氨基酸。并根据大肠杆菌的偏好密码子优化、设计并合成碱基序列,使得拟表达的目的基因序列能够较好地与原核表达系统相匹配,提高表达效率。通过目的基因片段与pET-41a构建重组质粒并转化大肠杆菌后,IPTG诱导表达,SDS-PAGE分析,得到了分子量约为34KD的重组蛋白,并将选定的表达株放大培养,采用柱层析方法纯化重组蛋白,确定了结合缓冲液:Na_2H PO_4:NaH_2PO_4 3:2(20mmol/L),8M/L尿素,5mmol/L咪唑,pH8.0;洗脱缓冲液:Na_2H PO_4:NaH_2PO_4 3:2(20mmol/L),8M/L尿素,500mmol/L咪唑,pH8.0,分别用5%,10%,15%,20%,25%,洗脱液梯度洗涤样品,是比较合适的纯化目的蛋白的条件。纯化后蛋白用ELISA检测临床血清标本,敏感性为85.7%,特异性为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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R759

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