【作者】 吴加强；

【导师】 姜平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2001， 硕士

【摘要】 猪繁殖与呼吸综合征病毒（PRRSV）resp株是北美国家最常用的疫苗株之一，在Marc-145细胞上可产生典型的细胞病变（CPE），目前该毒株已广泛用于弱毒疫苗与灭活疫苗的生产与制备。但PRRSV-resp)株在Mare-145细胞上的增殖受到诸多因素的影响，滴度很不稳定，长期以来严重制约着疫苗的生产制备与推广应用。为此，木文对PRRSV-resp株在Marc-145细胞上的增殖条件进行了深入探讨与优化，为猪繁殖与呼吸综合征疫苗的大规模生产与推广应用打下了良好基础。试验表明，以PRRSV-resp株在Mare-145细胞上的滴度高低为标准，适合PRRSV增殖的各种最佳条件为：细胞密度10万-I5万/ml，培养基MEM，血清浓度8％，PH范围7.0-7.4，接毒量200TCID₅₀/ml，病毒吸附时间37℃，30分钟，接种后72-96小时收毒，收毒后于-20℃冻融3次。 环磷酰预处理技术常用于制备复杂组织中未知抗原或稀有抗原的抗体，但环磷酰胺的注射剂量和免疫程序很难把握。本文妥善解决了这一难题，并采用环磷酰胺预处理法成功制备了高纯度的兔抗PRRSV-resp株高免血清。在本试验中，以正常Mare-145细胞成分和环磷酰胺预处理试验兔，再以PRRSV-resp株经灭活、乳化制成油佐剂抗原多次免疫试验兔，获得了中和效价达1：32，ELISA效价达1：640的兔抗PRRSV高免血清，为下一步PRRSV的纯化与诊断奠定了基础。 初步建立了间接酶联免疫吸附试验（ELISA）和间接荧光抗体试验（IFA），用于检测杂交瘤细胞上清中的单克隆抗体。利用在猪肺泡巨噬细胞（PAM）上增殖的PRRSV-resp株，采用长程免疫法免疫BALB/C小鼠，用PEG-1500进行细胞融合，融合率为70.6％。初检阳性率为6.3％。经2次亚克隆获得了2株能稳定分泌PRRSV单克隆抗体的杂交瘤细胞株。免疫转印试验显示，这两株单抗分别可以与PRRSV 19KD蛋白（M蛋白）和45-50KD蛋白（GP₃）特异性结合，具有一定的诊断价值和进一步开发研究的前景。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome virus (PRRSV) resp-strain which is liable to result in typical cytopathic effect (CPE) is one of the most widely used vaccine strains, and it has been widespread applied to produce attenuated or inactivated vaccine of PRRS at present. But the propagation of PRRSV-resp strain on Marc-145 cell is affected by many factors and its titer hasn抰 always been stable for a long time, which is an obstacle to the production and application of PRRS vaccines. This thesis thoroughly studied and optimized the factors of the propagation of PRRSV-resp strain on Marc-145 cell, which had laid a good basis for large-scale production and application of PRRS vaccines. The experiment showed that taking the PRRSV-resp strain titer as the standard, the optimum fit for PRRSV propagation was as follows: cell concentration was 100,000-150,000 per ml; the medium was MEM plus 8% of fetal bovine serum; the PH of the medium ranged from 7.0 to 7.4; the inoculation dose of PRRSV was 200TC1D50 per nil; the absorbtion time was 30 minutes at 370C; the harvest time was 72-96 hours after virus inoculation; the virus needed to be freeze-thawwed 3 times at -20 0C after harvesting.Cyclophosphamide pretreatment technique is often used to prepare for the antibodies of unknown or rare antigens in complex tissues, but it is hard to master2the injection dose and immune procedure of cyclophosphamide. This puzzle was solved in the article, and hyperserum with high purity against PRRSV-resp strain by rabbit has been successfully produced with cyclophosphainide pretreatment technique. hi this experiment, the tested rabbits were pretreated with normal Marc-145 cell components and cyclophosphasnide, then vaccinated with inactivated and oil-emulsified porcine reproductive and respiratory syndrome virus. The neutralization value of the hyperserum against PRRSV obtained by rabbit was 1:32 and its ELISA value was 1:640. The hyperseruni set up a basis for purifying and detecting PRRSVIndirect enzyme-linked immunosorbent assay (I-ELISA) and indirect fluorescent antibody (IFA) were developed to detect monoclonal antibodies (McAb) secreted by hybridoma cell lines. The BALBIC mice were immunized several times with PR.RSV-resp strain replicating on porcine alveolar macrophage (PAM), then the spleen cells of the hyperimmunized mouse and SP2IO cells were fused with PEG-1500. The cell fusion rate of 70.6% and positive rate of 6.3% were obtained and two hybridoma cell lines secreting monoclonal antibodies were developed after two subclones. It indicated that the two monoclonal antibodies reacting specifically with 19KD (M) and 45-5OKD (GP3) proteins ofPRRSV by Western-blotting would certainly be useful for research and diagnostic purposes.更多还原