猪轮状病毒GD-01-2015的全基因序列分析及双夹心ELISA方法的建立

发布时间：2018-07-10 13:08

本文选题：轮状病毒 + 全基因组分析　；参考：《扬州大学》2017年硕士论文

【摘要】：轮状病毒(Rotavirus,RV)是人畜共患腹泻的重要病原之一,其中猪轮状病毒(Porcine Rotavirus,PoRV)是仔猪腹泻的重要病因之一。该病流行范围广,发病率较高,若与猪流行性腹泻病毒(PEDV)和猪传染性胃肠炎病毒(TGEV)混合感染会使病情加重,导致高死亡率,从而给畜牧业造成严重的经济损失。本论文就PoRV-GD-01-2015的全基因测序、抗PoRV的单克隆抗体的制备以及检测PoRV的ELISA方法的建立三个方面展开研究,对PoRV疫苗的研发和致病机理的研究具有重要意义。1.PoRV-GD-01-2015的全基因序列分析本实验将一份经RT-PCR检测猪轮状病毒病原阳性的临床腹泻粪样感染Vero细胞,成功分离到一株猪轮状病毒GD-01-2015株;参照已发表的A组轮状病毒CC0912-1设计合成11对引物,对GD-01-2015进行全基因克隆测序,并对其11个基因片段进行RotaC软件分析、遗传变异分析和生物信息学分析。结果显示,GD-01-2015完整基因型为G5-P[7]-15-R1-C1-M1-A1-N1-T1-E1-H1;GD-01-2015 与韩国毒株 K5 核酸同源性高达 99%;生物信息学分析预测发现各蛋白都具有良好的抗原性,其中VP1、VP3、VP4、VP7、NSP1、NSP2和NSP4属于稳定蛋白,VP7和NSP4分别含有2个和1个跨膜区结构,且VP7属于疏水性蛋白。2.抗PoRV的单克隆抗体的制备将研究一中的PoRV-GD-01-2015感染Vero细胞,并将浓缩的含有PoRV-GD-01-2015的细胞液作为免疫原免疫小鼠,经过两次融合,以间接免疫荧光(IFA)方法为筛选方法,成功获得两株抗PoRV的单克隆抗体,并命名为PoRV-2F 10和PoRV-6F5。经Western-Blot验证所得单抗只识别病毒抗原。经商品化试剂盒鉴定,所得两株单克隆抗体的类型均为IgG,Kappa链。将两株单抗制备腹水后,利用Protein G柱获得了纯化的抗体,为后续建立检测方法奠定了基础。3.双夹心ELISA方法的建立通过相加ELISA方法确定PoRV-2F10和PoRV-6F5为针对不同抗原位点的两株单克隆抗体。将两株单克隆抗体进行交叉配对实验,结果确定以PoRV-6F5为捕捉抗体,PoRV-2F10为酶标抗体建立了检测PoRV的双夹心ELISA方法;通过实验条件的优化,确定了捕捉抗体和酶标抗体的最佳工作浓度分别为3.5μg/mL和0.25μg/mL,待检抗原和酶标抗体的最佳孵育时间均为60min,最佳显色时间为20min;阳性临界值的确定实验结果显示,当OD4500.165时判为阳性;利用本实验建立好的双夹心ELISA方法检测PoRV,检测到的最低抗原量为1.3X105个TCID5o;交叉反应性实验结果显示本实验建立的双夹心ELISA方法只与PoRV有阳性反应,与TGEV和PEDV无交叉反应,与阴性Vero细胞也无交叉反应,表明该方法具有较好的特异性;通过批间批内重复实验,发现该方法的批内和批间的变异系数均小于10%,说明该方法可行、重复性好。
[Abstract]:Rotavirus rotavirus (RV) is one of the most important pathogens of zoonotic diarrhea, among which porcine rotavirus (PoRV) is one of the important causes of piglet diarrhea. The disease has a wide epidemic range and a high incidence. If mixed with porcine epidemic diarrhea virus (PEDV) and porcine infectious gastroenteritis virus (TGEV), the disease will be aggravated, resulting in a high mortality rate, thus causing serious economic losses to animal husbandry. In this paper, the whole gene sequencing of PoRV-GD-01-2015, the preparation of monoclonal antibody against PoRV and the establishment of Elisa for detecting PoRV were studied. The research and development of PoRV vaccine and the study of its pathogenic mechanism are important. 1. The whole gene sequence analysis of PoRV-GD-01-2015. In this experiment, a porcine rotavirus GD-01-2015 strain was isolated from Vero cells infected with fecal samples of clinical diarrhea positive for porcine rotavirus by RT-PCR. According to the published group A rotavirus CC0912-1, 11 pairs of primers were designed and synthesized. The GD-01-2015 gene was cloned and sequenced, and its 11 gene fragments were analyzed by RotaC software, genetic variation analysis and bioinformatics analysis. The results showed that the complete genotype of GD-01-2015 was G5-P [7] -15-R1-C1-M1-A1-T1-E1-H1-2015 and the nucleic acid homology of GD-01-2015 was 99% with Korean strain K5. Bioinformatics analysis predicted that all proteins had good antigenicity. VP1, VP3, VP7, NSP2 and NSP4 belong to stable protein, VP7 and NSP4, respectively. VP7 and NSP4 have two and one transmembrane structure respectively, and VP7 belongs to hydrophobic protein. The preparation of monoclonal antibody against PoRV will be used to infect Vero cells with PoRV-GD-01-2015, and the concentrated cell fluid containing PoRV-GD-01-2015 will be used as immunogen to immunize mice. After twice fusion, indirect immunofluorescence (IFA) method will be used as the screening method. Two monoclonal antibodies against PoRV were successfully obtained and named PoRV-2F10 and PoRV-6F5. The McAbs identified by Western-Blot only recognize viral antigens. The two McAbs were identified as IgG Kappa chain by commercial kit. After two McAbs were used to prepare ascites, the purified antibody was obtained by protein G column, which laid a foundation for the further establishment of detection method. 3. Establishment of double Sandwich Elisa method PoRV-2F10 and PoRV-6F5 were identified as two monoclonal antibodies against different antigen sites by additive Elisa. Two monoclonal antibodies were cross-matched. The results showed that a double sandwich Elisa method was established to detect PoRV by using PoRV-6F5 as capture antibody and PoRV-2F10 as enzyme labeled antibody. The optimal working concentration of capture antibody and enzyme labeled antibody were 3.5 渭 g / mL and 0.25 渭 g / mL, respectively. The optimal incubation time of antigen and enzyme labeled antibody were both 60 min and 20 min, and the positive critical value was determined when OD4500.165 was positive. The results of cross-reactivity test showed that the double sandwich Elisa method was only positive with PoRV, but not with TGEV and PEDV, and the results of cross-reactivity test showed that the double sandwich Elisa method had only positive reaction with PoRV, and had no cross reaction with TGEV and PEDV, and the results of cross-reactivity test showed that the double sandwich Elisa method was only positive for PoRV, and had no cross reaction with TGEV and PEDV. There was no cross reaction with negative Vero cells, which showed that the method had good specificity, and the coefficient of variation was less than 10% in both batches and batches, which indicated that the method was feasible and reproducible.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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