【摘要】： 莢膜多糖(Capsular polysaccharide,CP)是存在于細菌表面的一類特殊物質,是細菌的致病因子之一。對于金黃色葡萄球菌(Staphylococcus aureus,S.aureus)來說,莢膜多糖有11種血清型,其中5型莢膜多糖(CP5)是主要的血清型,它可以使細菌長期定居在粘膜和內皮表面,一旦發(fā)生感染,莢膜多糖通過抗吞噬作用,增強了金葡菌的毒力。金葡菌可以引起包括奶牛乳腺炎在內的多種疾病,金葡菌感染一旦發(fā)生,抗生素治療效果并不明顯,因此建立一種能快速、準確的區(qū)分金葡菌莢膜多糖血清型的診斷方法對預防和治療金葡菌性疾病是十分重要的。 本實驗利用化學試劑十六烷基三甲基溴化銨(CTAB),提取、純化出CP5157.47mg,純度為72.48%,并且用CP5與胎牛血清白蛋白偶聯(lián),制備了人工完全抗原。采用常規(guī)免疫方法,免疫BalB/C小鼠。將免疫后的BalB/C小鼠脾細胞與SP2/0細胞進行融合,進而篩選、克隆,共獲得能穩(wěn)定分泌抗CP5的單克隆抗體雜交瘤細胞2株,分別命名為2F3和5E8。其中2F3分泌抗體亞類為IgG1,5E8分泌的抗體為IgG2a。在對單抗的效價、相對親和力等進行分析的基礎上,選擇單抗2F3用于建立CP5的間接競爭ELISA檢測方法,對間接競爭ELISA方法進行優(yōu)化后,建立了標準曲線,其標準曲線的回歸方程為y=-1.6629x+4.9697,相關系數(shù)R2=0.9901,檢測范圍為62.5～4000ng/ml,最低檢測限為62.5ng/mL。CP5間接競爭ELISA的建立為金葡菌性疾病的快速診斷以及金葡菌莢膜多糖的流行病學調查奠定了物質基礎。
文內圖片：
圖片說明：電鏡下的血清5型金葡菌Figure1.1Type5CapsularPolysaccharidesofStaphylococcusAureusbytransmissionelectronmicroscope
[Abstract]:Capsule polysaccharide (Capsular polysaccharide,CP) is a kind of special substance which exists on the surface of bacteria and is one of the pathogenic factors of bacteria. For Staphylococcus aureus (Staphylococcus aureus,S.aureus), there are 11 serotypes of capsula polysaccharide, among which type 5 capsula polysaccharide (CP5) is the main serotype, which can make bacteria settle on the surface of mucous membrane and endothelial for a long time. Once infection occurs, capsule polysaccharide enhances the virulence of Staphylococcus aureus by anti-phagocytosis. Staphylococcus aureus can cause a variety of diseases, including mastitis in dairy cows. Once Staphylococcus aureus infection occurs, the effect of antibiotics is not obvious. Therefore, it is very important to establish a rapid and accurate diagnostic method to distinguish Staphylococcus aureus capsule polysaccharide serotypes for the prevention and treatment of Staphylococcus aureus. In this experiment, the purity of CP5157.47mg, was 72.48% by (CTAB), extraction with cetyltrimethylammonium bromide, and the artificial complete antigen was prepared by coupling CP5 with fetal bovine serum albumin (FSA). BalB/C mice were immunized by routine immunization. After fusion of immunized BalB/C mouse spleen cells with SP2/0 cells, two hybridoma cells, named 2F3 and 5E8, were obtained, which could stably secrete anti-CP5 monoclonal antibodies. Among them, the antibody secreted by 2F3 is IgG2a., which is secreted by IgG1,5E8. On the basis of analyzing the titer and relative affinity of monoclonal antibody, the monoclonal antibody 2F3 was selected to establish the indirect competitive ELISA detection method of CP5. After optimizing the indirect competitive ELISA method, the standard curve was established. The regression equation of the standard curve was y 鈮，

本文編號：2510089