本文關(guān)鍵詞： 豬肺炎支原體 單克隆抗體 P97　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2008年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 豬肺炎支原體(Mycoplasma hyopneumoniae,Mhp)是引起豬氣喘病(MycoplasmaPneumoniae of swine,MPS)的病原。MPS廣泛分布于世界各地,以接觸性、高度傳染性、慢性、高發(fā)病率和低死亡率為特點(diǎn),由于Mhp能夠破壞呼吸道黏膜-纖毛屏障,從而繼發(fā)其他致病菌的感染,給養(yǎng)豬業(yè)造成巨大的經(jīng)濟(jì)損失。該病早期檢測(cè)比較困難以及缺乏特效的防治藥物,因此在疾病控制方面至今未能取得令人滿(mǎn)意的效果。 伴侶蛋白Dnak是豬肺炎支原體的特異性外膜蛋白,伴侶蛋白Dnak的C端是其主要的抗原決定簇。P97蛋白是目前研究最為深入的Mhp表面的黏附因子,P97蛋白的C端也是其主要的抗原決定簇。本研究依據(jù)GenBank中公布的232株基因序列(AE017332),以Mhp中國(guó)分離株Yin-1為模板利用合成的特異性引物,擴(kuò)增了DnaK基因的C末端,得到500bp的目的片段。擴(kuò)增了P97基因的C末端,得到670bp的目的片段。并將PCR產(chǎn)物分別克隆到pET-30a載體上,經(jīng)PCR、酶切方法鑒定正確后,并送測(cè)序。測(cè)序結(jié)果與GenBank公布的標(biāo)準(zhǔn)株yin-1株的DNA同源性為99%以上。鑒定陽(yáng)性的重組質(zhì)粒轉(zhuǎn)化到大腸桿菌(DE3)中進(jìn)行原核表達(dá)。獲得的重組蛋白分別命名為30a-DnakC-P97C和30a-P97C。經(jīng)超聲裂解,SDS-PAGE分析,重組蛋白均主要以可溶形式存在于上清液中,分別在57kD和36kD處分別獲得特異性條帶。經(jīng)Western-blot抗原性分析,均具有較好的免疫原性。 利用Histag親和層析柱,對(duì)重組蛋白30a-DnakC-P97C和30a-P97C進(jìn)行分離純化,純化產(chǎn)物經(jīng)SDS-PAGE電泳顯示為單一蛋白條帶,純化效果較好。以30a-DnakC-P97C作為免疫抗原分別與弗氏完全佐劑和弗氏不完全佐劑乳化,免疫8周齡的雌性BALB/c小鼠,每只每次50μg,間隔兩周免疫一次,最后一次加強(qiáng)免疫不加佐劑,3d后,取免疫小鼠脾細(xì)胞和骨髓瘤細(xì)胞SP2/0進(jìn)行融合。以純化30a-P97C作為包被抗原,用間接ELISA平行篩選陽(yáng)性克隆,經(jīng)有限稀釋法三次亞克隆后,獲得兩株針對(duì)P97的能穩(wěn)定分泌特定性抗體的雜交瘤細(xì)胞株,分別命名為A3C9和B4D5。生物學(xué)特性鑒定表明,腹水單抗A3C9和B4D5的間接ELISA效價(jià)分別為1∶10~5和1∶10~6,亞型鑒定分別為IgG2b和IgG2a亞類(lèi),且輕鏈均為k鏈。相加ELISA試驗(yàn)表明,針對(duì)P97的兩株單抗識(shí)別的不是同一抗原表位。經(jīng)Western-blot分析表明,兩株單抗特異性地與豬肺支原體Yin-1株97KD左右的抗原成分發(fā)生反應(yīng)。而與其他相關(guān)支原體:絲狀支原體絲狀亞種SC型標(biāo)準(zhǔn)株(PGI)、絲狀支原體絲狀亞種LC型標(biāo)準(zhǔn)株(Y-goat)、山羊支原體山羊肺炎亞種(Mccp)、豬鼻支原體(Mh)無(wú)交叉反應(yīng),表明單抗為針對(duì)豬肺炎支原體的特異性單抗。本試驗(yàn)獲得的單抗為更深入的分析Mhp的結(jié)構(gòu)、功能及生物學(xué)診斷提供有力工具。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 張美晶;豬肺炎支原體P97 C末端蛋白抗原表位分析及抗原捕捉ELISA方法初步研究[D];中國(guó)農(nóng)業(yè)科學(xué)院;2010年

2 祝永琴;豬肺炎支原體主要抗原基因在畢赤酵母中的分泌表達(dá)及間接ELISA檢測(cè)方法的建立[D];南京農(nóng)業(yè)大學(xué);2009年

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本文編號(hào)：1534605