猪肺炎支原体P97蛋白单克隆抗体的制备

发布时间：2018-02-25 18:09

本文关键词： 猪肺炎支原体单克隆抗体 P97　出处：《中国农业科学院》2008年硕士论文　论文类型：学位论文

【摘要】： 猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)是引起猪气喘病(MycoplasmaPneumoniae of swine,MPS)的病原。MPS广泛分布于世界各地,以接触性、高度传染性、慢性、高发病率和低死亡率为特点,由于Mhp能够破坏呼吸道黏膜-纤毛屏障,从而继发其他致病菌的感染,给养猪业造成巨大的经济损失。该病早期检测比较困难以及缺乏特效的防治药物,因此在疾病控制方面至今未能取得令人满意的效果。伴侣蛋白Dnak是猪肺炎支原体的特异性外膜蛋白,伴侣蛋白Dnak的C端是其主要的抗原决定簇。P97蛋白是目前研究最为深入的Mhp表面的黏附因子,P97蛋白的C端也是其主要的抗原决定簇。本研究依据GenBank中公布的232株基因序列(AE017332),以Mhp中国分离株Yin-1为模板利用合成的特异性引物,扩增了DnaK基因的C末端,得到500bp的目的片段。扩增了P97基因的C末端,得到670bp的目的片段。并将PCR产物分别克隆到pET-30a载体上,经PCR、酶切方法鉴定正确后,并送测序。测序结果与GenBank公布的标准株yin-1株的DNA同源性为99%以上。鉴定阳性的重组质粒转化到大肠杆菌(DE3)中进行原核表达。获得的重组蛋白分别命名为30a-DnakC-P97C和30a-P97C。经超声裂解,SDS-PAGE分析,重组蛋白均主要以可溶形式存在于上清液中,分别在57kD和36kD处分别获得特异性条带。经Western-blot抗原性分析,均具有较好的免疫原性。利用Histag亲和层析柱,对重组蛋白30a-DnakC-P97C和30a-P97C进行分离纯化,纯化产物经SDS-PAGE电泳显示为单一蛋白条带,纯化效果较好。以30a-DnakC-P97C作为免疫抗原分别与弗氏完全佐剂和弗氏不完全佐剂乳化,免疫8周龄的雌性BALB/c小鼠,每只每次50μg,间隔两周免疫一次,最后一次加强免疫不加佐剂,3d后,取免疫小鼠脾细胞和骨髓瘤细胞SP2/0进行融合。以纯化30a-P97C作为包被抗原,用间接ELISA平行筛选阳性克隆,经有限稀释法三次亚克隆后,获得两株针对P97的能稳定分泌特定性抗体的杂交瘤细胞株,分别命名为A3C9和B4D5。生物学特性鉴定表明,腹水单抗A3C9和B4D5的间接ELISA效价分别为1∶10~5和1∶10~6,亚型鉴定分别为IgG2b和IgG2a亚类,且轻链均为k链。相加ELISA试验表明,针对P97的两株单抗识别的不是同一抗原表位。经Western-blot分析表明,两株单抗特异性地与猪肺支原体Yin-1株97KD左右的抗原成分发生反应。而与其他相关支原体:丝状支原体丝状亚种SC型标准株(PGI)、丝状支原体丝状亚种LC型标准株(Y-goat)、山羊支原体山羊肺炎亚种(Mccp)、猪鼻支原体(Mh)无交叉反应,表明单抗为针对猪肺炎支原体的特异性单抗。本试验获得的单抗为更深入的分析Mhp的结构、功能及生物学诊断提供有力工具。
[Abstract]:Mycoplasma hyopneumoniae (Mhp) is the pathogen of Mycoplasma-pneumoniae of swinesia (MPSs), which is widely distributed all over the world. It is characterized by contact, highly infectious, chronic, high morbidity and low mortality, because Mhp can destroy mucosal and ciliated respiratory barrier. As a result, the infection of other pathogenic bacteria has caused huge economic losses to pig industry. It is difficult to detect the disease early and lacks special control drugs, so it has not achieved satisfactory results in disease control up to now. Chaperone Dnak is a specific outer membrane protein of Mycoplasma pneumoniae. The C-terminal of chaperone protein Dnak is its main antigen determinant. P97 protein is the most deeply studied adhesion factor P97 protein C-terminal is also its main antigen determinant. This study is based on the published 232 strains of GenBank. As a result of the sequence AE017332, the Mhp Chinese isolate Yin-1 was used as template and the synthesized specific primers were used. The C-terminal of DnaK gene was amplified and the target fragment of 500bp was obtained. The C-terminal fragment of P97 gene was amplified and 670bp fragment was obtained. The PCR product was cloned into pET-30a vector and identified correctly by PCR and restriction endonuclease digestion. The DNA homology of yin-1 strain published by GenBank was more than 99%. The identified positive recombinant plasmid was transformed into E. coli to express prokaryotic expression. The obtained recombinant protein was named 30a-DnakC-P97C and 30a-P97C, respectively, and the recombinant protein was identified as 30a-DnakC-P97C and 30a-P97Crespectively. SDS-PAGE analysis of ultrasonic cleavage, The recombinant proteins were mainly soluble in the supernatant, and the specific bands were obtained at 57kD and 36kD, respectively. The Western-blot antigenicity analysis showed that the recombinant proteins had good immunogenicity. The recombinant proteins 30a-DnakC-P97C and 30a-P97C were separated and purified by Histag affinity chromatography. The purified products were identified as a single protein band by SDS-PAGE electrophoresis. Using 30a-DnakC-P97C as immune antigen and emulsifying with Freund's complete adjuvant and Freund's incomplete adjuvant respectively, female BALB/c mice of 8 weeks old were immunized with 50 渭 g of each mouse once every two weeks. Spleen cells of immunized mice and myeloma cells (SP2/0) were fused to purify 30a-P97C as coating antigen and indirect ELISA was used to screen the positive clones. Two hybridoma cell lines, named A3C9 and B4D5, which can stably secrete specific antibodies against P97, were obtained. The indirect ELISA titers of A3C9 and B4D5 were 1: 10 5 and 1: 10 6, respectively, and the subtypes were identified as IgG2b and IgG2a, respectively. The addition ELISA test showed that the two monoclonal antibodies against P97 did not recognize the same antigen epitope. Western-blot analysis showed that, The two McAbs reacted specifically with the antigen components of mycoplasma porcine Yin-1 strain 97KD or so, and with other related mycoplasma mycoplasma mycoplasma SC subspecies SC standard strain, mycoplasma mycoplasma mycoplasma serotype LC type standard strain, There was no cross reaction between Mycoplasma sheep and Mycoplasma suis. The results showed that the McAb was a specific monoclonal antibody against Mycoplasma pneumoniae. The McAbs obtained in this study provide a powerful tool for further analysis of the structure, function and biological diagnosis of Mhp.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【引证文献】