氯霉素单克隆抗体的研制及其特性分析

为了研制氯霉素单克隆抗体,建立氯霉素的简便有效的检测方法.采用碳二亚胺(EDC)方法制备氯霉素免疫抗原和包被抗原,经SDS-PAGE凝胶电泳,三硝基苯磺酸(TNBS)法鉴定表明偶联成功.采用制备的抗原CAP-HS-BSA((CAP-HS,氯霉素琥珀酸酯;BSA,牛血清白蛋白)免疫Balb/c小鼠,通过杂交瘤技术获得了2株抗氯霉素的杂交瘤细胞株,细胞株体外传代和冻存复苏后抗体分泌稳定.经体内诱生法产生腹水,ELISA检测纯化腹水的效价为1:10~6.抗体的亲和常数分别为:1.13×10~(10) L/mol,1.41×10~(10) L/mol.ELISA检测显示这2株单克隆抗体与氯霉素琥珀酸交叉反应率为300%,与其他抗生素及结构类似物的交叉反应小,表明该单抗可满足建立免疫学检测方法的需要和开发应用的要求.     

单环刺螠硫醌氧化还原酶间接竞争ELISA检测方法的建立

硫醌氧化还原酶(Sulfide:quinone oxidoreductase,SQR),是线粒体硫化物代谢的关键酶。本研究以生活在潮间带下区及潮下带中的底栖生物—单环刺螠SQR重组蛋白为材料,建立了单环刺螠SQR间接竞争ELISA检测方法,为定量分析SQR蛋白水平表达奠定了基础。将SQR重组蛋白免疫新西兰大白兔获得多克隆抗体,检测效价高达1:64 000。采用免疫印迹实验,将该抗体与重组蛋白和体壁总蛋白杂交,均得到了单一的条带,表明该抗体特异性好。优化SQR间接竞争ELISA检测条件,得出最佳的抗原包被浓度为250ng/mL,一抗浓度为1∶20 000,二抗浓度为1∶12 000,此条件下建立的标准曲线检测范围为2.5～1 000ng/mL。精确度检测结果显示,批内差异在0.42%～7.27%、批间差异在2.58%～7.78%范围内,表明该条件下精确度具有良好的准确性和重复性。以上结果表明,已成功建立单环刺螠SQR间接竞争ELISA检测方法。     

抗鲶爱德华氏菌多克隆抗体的制备及特性分析

利用2株黄颡鱼"红头病"病原菌——鲶爱德华氏菌的灭活疫苗作为免疫原,免疫新西兰大白兔,制备了高效价的多克隆抗体。通过间接酶联免疫、间接免疫荧光和免疫印迹等技术对多克隆抗体特性进行了分析。测定获得多抗效价分别为1∶215和1∶214,2株病原菌之间存在很强的交叉反应,且2种多抗均与鲶爱德华氏菌、迟钝爱德华氏菌、副溶血弧菌、鳗弧菌、溶藻弧菌、火神弧菌、河流弧菌、创伤弧菌标准菌株存在交叉反应,与粪肠球菌、大肠杆菌、酿脓链球菌、海豚链球菌、杀鲑气单胞菌、嗜水气单胞菌、类产碱假单胞菌标准菌株均无交叉反应。免疫印迹实验结果表明,2株病原菌的主要抗原决定簇相同,分子量分别为61.1,46.6,41.3,28.8和19 kDa。本实验得到了黄颡鱼"红头病"致病菌高效价的多克隆抗体,可以作为初步筛选"红头病"病原菌的工具,为建立快速有效的疾病监测方法和进一步的免疫学防病研究提供了依据,奠定了基础。     

牙鲆病原秦皇岛弧菌的血清型及荧光抗体检验

用分离于病(死)牙鲆(Paralichthys olivaceus L.)的一种新病原弧菌——秦皇岛弧菌(Vibrio qinhuangdaora sp.nov.)的代表菌株(HQ010712-1株),分别制备全菌(OK)及热处理(121℃作用1h)的菌体(O)免疫原,强化免疫接种家兔制备相应抗血清,对供试12株秦皇岛弧菌进行了血清型检定,结果表明均存在同种的K抗原和同种的O抗原(血清同源),其中O抗原具有强免疫原性、K抗原的免疫原性弱但具有强O凝集抑制作用;以相应OK抗血清为第一抗体,以标准的羊抗兔IgG荧光抗体为第二抗体,对12株秦皇岛弧菌的纯培养物及用代表菌株(HQ010712-1)人工感染病死牙鲆的肝组织进行了间接荧光抗体染色,结果显示了特异的荧光反应。     

软骨藻酸多克隆抗体的制备

为建立软骨藻酸(DA)的免疫检测法,本研究采用活泼酯法将DA与载体蛋白KLH(BSA)偶联形成完全免疫抗原(包被抗原),并通过紫外光谱扫描证明偶联成功与否。用完全免疫抗原DA-KLH对新西兰白兔进行免疫,间接ELISA法测定血清效价。实验结果表明偶联成功,经过19周免疫后血清效价达到1:1600,纯化后获得效价为1:800的多克隆抗体,同时也间接证明了合成的完全抗原具有免疫原性,为以后建立免疫分析法奠定了基础。     

免疫粒子群优化在波阻抗反演中的应用

针对标准粒子群优化（PSO）算法易出现早熟而陷入局部最优以及进化后期收敛速度慢等缺陷,引入免疫系统的免疫记忆和抗体浓度选择机制,构造了基于免疫机制的粒子群优化（IPSO）算法,并将其应用到波阻抗反演问题中。免疫记忆能够保留高适应度个体,抗体浓度选择机制进一步保证了粒子的多样性,从而能较好地避免早熟收敛,提高算法的全局搜索能力。对理论模型试算表明,IPSO算法在进行波阻抗反演时不仅收敛速度快,而且具有较高的精确度和抗噪性能。     

大黄鱼肌肉生长抑制素基因原核表达及多克隆抗体的制备

肌肉生长抑制素抑制动物肌肉的生长发育.根据本实验室克隆的大黄鱼（Pseudosciaena cro-cea）肌肉生长抑制素（MSTN）基因编码序列设计引物,用RT-PCR扩增目的片段,构建MSTN-pET-28 a重组表达质粒,将其转化到大肠杆菌BL21上并用1.0 mmol/dm3IPTG诱导表达,SDS-PAGE电泳检测该目的蛋白大小约为44 kDa,与理论值大小符合.用0.25 mol/dm3KCl染色后从凝胶中切取该蛋白且回收.该回收蛋白与弗氏佐剂等量混合后注射ICR小鼠制备多克隆抗体,并用Westernblot检测该抗体.免疫印迹结果在44 kDa的位置上出现棕色条带,表明大黄鱼肌肉生长抑制素的多克隆抗体制备成功.这为今后对肌肉生长抑制素功能的研究奠定了基础.     

Development and application of antibody microarray for white spot syndrome virus detection in shrimp

Detecting white spot syndrome virus (WSSV) in shrimp in high efficiency and veracity is important for disease prevention in aquaculture. Antibody-based microarray is a novel proteomic technology that can meet the requirements. In this study, we developed an antibody microarray for WSSV-detection in a specific and parallel way at multiple samples. First, seven slides each with different modifications were characterized by atomic force microscope, and were compared in the efficiency of immobilizing proteins. Of the seven, 3-dimensional structured agarose gel-modified slides were chosen appropriate for the microarray for having higher signal value and superior spot size. A purified rabbit anti-WSSV antibody was arrayed as the capture antibody of the microarray on the agarose gel-modified slides, and then the microarray slides were incubated in the tissue homogenate of sampled shrimp and the antibody-antigen complex was detected by Cy3-conjugated anti-WSSV monoclonal antibody. The results were measured by a laser chipscanner and analyzed with software. To obtain satisfied fluorescence signal intensity, optimal conditions were searched. The detection limit of the antibody microarray for WSSV is 0.62 μg/mL, with a proven long shelf life for 6 months at 4°C or 8 months at -20°C. Furthermore, concordance between antibody microarray and traditional indirect ELISA reached 100% for WSSV detection. These results suggest that the antibody microarray could be served as an effective tool for diagnostic and epidemiological studies of WSSV.     

A Fast and Indriect Fluorescent Antibody Assay for the Vibro in Large Yeelow Croaker Pseudosciaena Crocea (Richardson)

A fast and indirect fluorescnet antibody assay for the Vibrio alginolyticus and V.Parahaemolyticus infecting the large yellow croaker has been developed.The specific antisera for the two strains of vibrio were prepared with New Zealand rabbit and the antiserum and cross-reactive efficacy was tested by coagulation in tube.It showed that the goat anti-rabbit IgG had been labeled by fuorescence isothiocyanate(FITC).The results showed that positive reactions were 100% for the large yellow croaker Pseudosciaena crocea with typical symptom of vibrio infection,while the positive reaction to the pathogen in healthy yellow croakers reached 40%,but seemed negative for aquaculter water.The results demonstrated that this fast and indirect fluorescent antibody assay can be used not only to test the vibrio pathogen in diseased yellow croaker but also in infected animals with no symptom.     

A Fast and Indirect Fluorescent Antibody Assay for the Vibrio in Large Yellow Croaker Pseudosciaena crocea (Richardson)

ＴｈｅｌａｒｇｅｙｅｌｌｏｗｃｒｏａｋｅｒＰｓｅｕｄｏｓｃｉａｅｎａｃｒｏｃｅａ (Ｒｉｃｈａｒｄｓｏｎ)ｉｓｏｎｅｏｆｔｈｅｍｏｓｔｉｍｐｏｒｔａｎｔｃａｇｅｆａｒｍ ｉｎｇｓｐｅｃｉｅｓｉｎｓｏｕｔｈｅｒｎＣｈｉｎａ.Ｉｎｒｅｃｅｎｔｙｅａｒｓ,ｔｈｅｒａｐｉｄｄｅｖｅｌｏｐｍｅｎｔｏｆｙｅｌｌｏｗｃｒｏａｋｅｒｃｕｌｔｕｒｅｈａｓｂｅｅｎｔｒｏｕｂｌｅｄｂｙｖｉｂｒｉｏｉｎｆｅｃｔｉｏｎｒｅｑｕｉｒｉｎｇｒｅｓｏｒｔｔｏｄｒｕｇｓ,ｔｈｅａｂｕｓｅｏｆｗｈｉｃｈｄｅｔｅｒｉｏｒａｔｅｓｔｈｅｍｉ…