【作者】 万忠海；

【导师】 朱平；

【作者基本信息】 吉林大学 ， 预防兽医学， 2008， 博士

【摘要】 绿脓杆菌(Pseudomonas aeruginodsa PA)是导致外科手术或烧伤细菌感染死亡的最主要的病原细菌之一。绿脓杆菌外毒素A(PEA)是该菌的主要致病因子之一。PEA分为三个(I~III)功能区,I区与易感细胞识别有关;II区与PEA的“跨膜移位”有关;III区具有细胞毒性作用。PEA的毒性是其I区与细胞表面受体结合后,由受体介导的一系列反应引发的。所以抑制I区与受体的结合就可以阻断毒素的全部毒性作用。为获得绿脓杆菌外毒素A受体结合区蛋白,本研究将PEA受体结合区亚单位基因克隆到表达载体pET28(c)中,构建了原核表达质粒pET-28(c)+B10,表达了重组的绿脓杆菌外毒素A受体结合区蛋白,进行了免疫学实验。进而利用该蛋白免疫Balb/c小鼠制备了抗PEA单克隆抗体,抗PEA(3C6)单克隆抗体经过正辛酸和硫酸铵纯化后,对严重绿脓杆菌感染小鼠进行了疗效实验,效果明显。在此基础上,采用RT-PCR重叠延伸剪接法方法构建了单链抗体基因,并将基因克隆入pET28a中,筛选得到重组表达质粒pET28a-(PEA-1)ScFv。通过表达、变性、复性、纯化之后,进行了PEA的细胞毒性抑制实验。单链抗体在高浓度下能够起到一定的抑制作用。本研究旨在寻求治疗烧伤后绿脓杆菌感染的新方法新途径。更多还原

【Abstract】 Pseudomonas aeruginosa is the main pathogen caused infection after surgery and burn, and extoxin A is one of the principle pathogenic factor of PA. PEA consists of 3 functional domains. One of them, domain I, the receptor-binding subunit plays an important role in the process of PA infection of target cells.An expression plasmid pET-28(c)+B10 was constructed by insertion of the subunit gene from the expression vector pET-20(b)+B10 into pET28(C) downstream of T7lac promoter. After transforming the expression plasmid into the expression hosts BL21(DE3), BL21(DE3) pLysS, the transformed hosts were induced with IPTG. By SDS-PAGE and Western blot analysis of the host protein, the expression of the subunit was detected, and it coμld account for 58% of the total host protein.The mice inocμlated with the expressed PEA subunit 3 times at 0.5mg/mice each time were challenged with 3 LD50 of PEA at 0,7,14,21,28,35 days after the final inocμlation. The mice were 100% protected during at 14 days, 50% protection at 21days and no protection at 28 and 35 days. The principle histopathological lesion was degeneration and necrosis in the liver and kidney cells.Balb/c mice were immunized with the recombinant protein of the receptor binding domain of pseudomonas exotoxin A (PEA) , from the immunize mice were fused with SP2/0 myeloma cells. Positive hybridoma clones were screened by ELISA, and limited dilution method was performed to subclone the positive clones. After three cycles of subcloning, four McAbs against the PEA were obtained, designated as1C4, 1D4, 2E12 and 3C6. The ELISA titers of cμlture supernatant and ascites were 4000-8000 and 25600-51200.The 3C6 McAb had higher neutralization titers, the affinity constant was 8.93×108M-1. he monoclonal antibody gaining may bring the important effect on resisting Pseduomonas-aeruginosa infection.The mice, which infected severely with Pseudomonas aeruginosa, inocμlated with the purified protein of (PEA-1)McAbs 3 times at 0.2ml/mice each time. The mice were 70% protected.The immunoglobμlin VH and VL fragments were obtained from hybridoma mRNA by RT-PCR and were then constructed into scFv fragment. Sequence analysis showed that scFv fragment was 729bp, VH fragment was 351bp encoding 117 amino acids, VL fragment was 333bp encoding 111 amino acids. The scFv fragment was a new gene belonging to the second subclass of mouse immunoglobμlin by comparing with the sequences of mouse antibody variable fragments in Genbank.Construction the recombinant expressing plasmid pET28a-(PEA-1)ScFv and expressed recombinant proteins accumμlated in intracellμlar as inclusion bodies. Analysis of SDS-PAGE resμlts showed that expressed recombinant proteins was 24.4% in total lyses protein of bacteria.For the sake of obtaining recombinant proteins, insoluble form of inclusion bodies were prepared by sonication of the bacterial cells and then subjected to denature in 8 M urea. The denatured protein were purified through Gel filtration chromatography respectively. The resμlt showed that denature proteins was over 80% in purity.The purified protein of (PEA-1)ScFv was used in the experiment of competitive inhibition for cytotoxicity of PEA. 20ng/ml PEA incubated with its sensitive cell lineBHK-21, about 36 hours later, the cytobiological pathological change coμld be found under microscope, but the cell which incubated PEA and renatured protein of (PEA-1)ScFv was the same as the cell incubated without PEA, which was still viability, division and proliferation until all the cell was aging and death. It is indicated that the action of cytotoxicity of PEA may be inhibited by the protein of (PEA-1)ScFv.更多还原