【作者】 黄艺丹；

【导师】 汪开毓； 郑建；

【作者基本信息】 四川农业大学 ， 基础兽医学， 2010， 硕士

【摘要】 目的豚鼠气单胞菌是一种条件性致病菌：当鱼类抵抗力下降时或鱼体表有损伤时,容易被该菌侵入,反复感染后会导致其致病力增强,可形成很强的传染性。近年来,在我国养殖的南方鲇中发生了一种由豚鼠气单胞菌引起的以体表溃疡为特征的疾病,流行广、发病快、死亡率高,对水产养殖动物危害极大,导致巨大经济损失,严重危害我国渔业及水产业的发展。目前对豚鼠气单胞菌的鉴定主要依赖临床观察、解剖及一般细菌学检查法,操作过程既繁琐又费时,而且准确性不够,因此,建立快速、特异性强的诊断方法对更好防控豚鼠气单胞菌感染对水生养殖动物的危害具有重要的意义。本研究拟利用鱼类致病性豚鼠气单胞菌为抗原,制备抗该菌的单克隆抗体,并对其生物学特性进行了鉴定,在此基础上利用胶体金技术和所制备的单克隆抗体研制能快速检测豚鼠气单胞菌感染的胶体金试纸条,并进行性能测试,从而建立鱼类致病性豚鼠气单胞菌单克隆抗体-胶体金快速检测方法,为水产上豚鼠气单胞菌病的流行监测提供一种便捷的方法,同时也为该细菌引起的疾病检测方法研究积累一些资料。方法以福尔马林灭活的豚鼠气单胞菌按25μg/只、50μg/只分成两个剂量组,皮下多点注射免疫Balb/c小鼠,通过杂交瘤技术制备针对豚鼠气单胞菌的单克隆抗体(McAb),用间接ELISA法对所需的杂交瘤细胞株进行特异性筛选,对筛选到的高特异性的单抗进行分离、纯化及鉴定：先通过饱和硫酸铵法粗提抗体,再用Protein A亲和层析法进一步纯化,然后对单抗进行了包括亚类鉴定、效价、相对亲和力的测定,并通过单克隆抗体的位点相加实验、与豚鼠气单胞菌脂多糖(LPS)的间接ELISA实验对单抗识别的抗原表位进行了初步研究。然后采用胶体金标记技术,通过双抗体夹心法(单克隆抗体—抗原—单克隆抗体),以所得单抗作为包被及标记抗体,制备胶体金-抗体结合物和反应膜,研制豚鼠气单胞菌胶体金快速检测卡,并通过特异性、灵敏度、重复性、稳定性和临床样本的检测进行方法学评价。结果获得了2株可分泌特异性McAb的杂交瘤细胞,并分别命名为3F3和2C9C3。经过鉴定,这2株McAb能够特异性的针对豚鼠气单胞菌,其抗体亚类分别为IgG1型和IgM型；腹水效价分别为10-6和10-5；相对亲和力较高；3F3针对豚鼠气单胞菌脂多糖表位,而2C9C3针对非脂多糖抗原位点。利用本实验制备的McAb建立了以McAb为基础的双抗夹心法膜式胶体金快速检测方法,所研制的豚鼠气单胞菌胶体金快速检测卡具有灵敏度好,最低检测量为1.71×104cfu/ml,特异性高,重复性100%,稳定性好(室温保存一年也不失活),检测时间快(5分钟可判定结果)等优点,对临床样本的检测准确率高,操作简便。结论成功地制备出抗豚鼠气单胞菌的单克隆抗体,并研制了能快速特异检测鱼类豚鼠气单胞菌的胶体金试纸条,为水产上豚鼠气单胞菌的快速鉴定和诊断以及该菌流行的监测提供有力的工具。更多还原

【Abstract】 Objective: Aeromonas caviae (A. caviae) is one of the most prominent pathogenic bacteria which in China has been reported that is harmful to our fresh water aquaculture. As a major causative agent of infections in fish, A. caviae becomes highly pathogenic and infective when the resistance of fish decreases or the fish is injured. In recent years, the outbreak of epizootic ulcerative syndrome associated with A. caviae was reported among the southern catfish. The diseases caused by A. caviae are characterized by rapid invasion, wide prevalence and high mortality, which can cause serious economic loss in fish keeping. At present, the commonly used diagnostic methods for A. caviae depends on clinical observation, dissection and common bacteriology tests that are tedious, time-consuming and have poor accuracy. It is obvious that the establishment of a rapid method with higher specificity is one of the better approaches to prevent and control the infection caused by A. caviae.This research is to prepare A. caviae hybridoma cell lines, analyze the biological characteristics and develop colloidal gold test strips to establish a rapid and accurate diagnostic method for A. caviae in aquacultureMethods: Balb/c mice were immunized with A. caviae killed by formalin as the dosage of 25μg per mouse and 50μg per mouse respectively. The hybridoma cell lines which consistently secreted monoclonal antibody (McAb) against A. caviae were obtained through cell fusion. The specificity of the McAb was analyzed by indirect ELISA. Then the subtypes were identified, the titer and relative affinity constant were measured. Tests were made to identify the antigen epitope of the McAbs by combined experimental site and indirect ELISA with LPS of A. caviae. The McAb conjugating to colloidal gold against A. caviae was established, and its specificity, sensitivity, repeatability, stability and clinic sample test were estimated respectively.Results: From many positive hybridomas which secreted anti-Aeromonas caviae McAbs, two strains of hybridomas were screened out, and designated with 3F3 and 2C9C3. The subtypes of the McAb were IgG1 and IgM. The titer of 3F3 and 2C9C3 McAb produced by ascites fluid were 10-6 and10-5. The McAbs had high relative affinity. The two strains of McAbs targeted to different antigen epitope:3F3 targeted to the LPS of A. caviae, while 2C9C3 targeted to the non-LPS sites of A. caviae. Basing on the McAbs, the rapid detection McAbs conjugating to colloidal gold against A. caviae was developed to detect the thalli antigen of A. caviae specifically. The results demonstrated that this method had high specificity and good repeatability (100%). The minimum detection dose of strips was 1.71×104 cfu/ml. The test results by the strips could be observed within 5 minutes. The strips could be used easily and exactly in clinical test.Conclusion: The results demonstrated that this method had high specificity, sensitivity, stability and repeatability. It could serve as an effective detection measure for clinic, and as the method of the rapid identification for A. caviae in aquaculture and monitoring the epidemic of A. caviae.更多还原