【摘要】：豬細小病毒(Porcine Parvovirus,PPV)是引起豬繁殖障礙的主要病原之一。PPV的流行范圍非常廣泛,在世界各地均有感染,給養(yǎng)豬業(yè)和畜牧業(yè)造成了巨大的經(jīng)濟損失。VP2蛋白是PPV的結構蛋白之一,也是構成該病毒的主要衣殼蛋白,其攜帶了主要的抗原決定簇,決定著病毒的組織嗜性、抗原性、致病性等多種主要的生物學特性。本研究將實驗室保存的重組桿狀病毒Ac NPV-VP2同步接種于昆蟲細胞Sf9,36 h后收集病毒,并進行SDS-PAGE電泳,隨后利用Western blot對其結果進行分析,結果顯示蛋白成功的獲得了高效表達。通過Ni柱樹脂純化,以及切膠純化等方法,將表達的VP2蛋白進行純化,并利用抗His標簽蛋白單抗作為一抗,對純化后的蛋白進行Western blot分析,結果表明,純化效果良好,蛋白可用于后續(xù)的研究。將純化后得到的VP2蛋白作為免疫原,采用皮下注射的方式免疫6周齡的BALB/c小鼠,經(jīng)過三次基礎免疫和一次加強免疫后,利用細胞融合技術,將脾細胞與雜交瘤細胞進行融合。之后用間接ELISA方法進行篩選,經(jīng)過3次以上細胞亞克隆,最終成功獲得了1B3、2E5、5F8三株能夠穩(wěn)定分泌的抗VP2蛋白抗體的雜交瘤細胞。經(jīng)鑒定,3株雜交瘤細胞的亞型均為IgG1型;雜交瘤細胞培養(yǎng)上清效價為1:800-1 000,將雜交瘤細胞按適當?shù)牧扛骨蛔⑸湟呀?jīng)注射石蠟一周的BALB/c小鼠,檢測獲得的腹水效價為1:32 000-64 000。為進一步鑒定3株單克隆抗體的免疫反應性,收集同步接種豬細小病毒后的ST細胞,并以此為檢測用免疫原,檢測單克隆抗體的免疫原性。對其及進行了Western blot檢測,結果表明3株單抗均可以對PPV全病毒以及重組VP2蛋白特異性識別;間接免疫熒光結果表明,3株單抗均產(chǎn)生熒光,可以與PPV全病毒發(fā)生反應;特異性試驗表明,3株單抗均不與TEGV和PEDV等病毒發(fā)生反應,僅特異性與PPV發(fā)生反應;將在體外連續(xù)傳代3個月和凍存后再復蘇的3株雜交瘤細胞做了穩(wěn)定性分析,結果表明,3株單抗的穩(wěn)定性較好。本研究成功篩選了3株具有較高的反應原性和特異性的豬細小病毒結構蛋白VP2的單克隆抗體,為后期建立有效的PPV特異性血清學檢測方法奠定了理論依據(jù),同時也為PPV受體及感染機制的研究提供了物質基礎。
[Abstract]:Porcine parvovirus (Porcine Parvovirus,PPV) is one of the major pathogens causing reproductive disorders in pigs. PPV is widely prevalent and has been infected all over the world. VP2 protein is one of the structural proteins of PPV, and is also the main capsid protein of the virus. It carries the main antigenic determinant and determines the tissue and antigenicity of the virus. Pathogenicity and other major biological characteristics. In this study, recombinant baculovirus (Ac NPV-VP2) stored in laboratory was inoculated into insect cell Sf9,36 h and then collected by SDS-PAGE electrophoresis. The results were analyzed by Western blot. The results showed that the protein was highly expressed successfully. The expressed VP2 protein was purified by Ni column resin purification, and the monoclonal antibody against His label protein was used as the first antibody, and the purified protein was analyzed by Western blot. The results showed that the purification effect was good. Proteins can be used in subsequent studies. The purified VP2 protein was used as immunogen to immunize 6-week-old BALB/c mice by subcutaneous injection. After three basic immunization and one enhanced immunization, cell fusion technique was used. Spleen cells were fused with hybridoma cells. After being screened by indirect ELISA method, three hybridoma cells, 1B3O2E5O5F8, which could stably secrete antibodies against VP2 protein, were successfully obtained after more than 3 cell subclones. The subtypes of three hybridoma cells were identified as IgG1 type. The supernatant titer of hybridoma cell culture was 1: 800-1 000. The ascites titer of BALB/c mice which had been injected paraffin for one week was intraperitoneally injected with the appropriate amount of hybridoma cells. The ascites titer was between 1:32 and 64 000. In order to further identify the immunoreactivity of three strains of monoclonal antibodies, the ST cells inoculated with porcine parvovirus were collected and used as immunogen to detect the immunogenicity of monoclonal antibodies. The results of Western blot detection showed that all the three McAbs could specifically recognize the whole PPV virus and the recombinant VP2 protein, and the indirect immunofluorescence showed that all the McAbs produced fluorescence and reacted with the whole PPV virus. The specific test showed that the three McAbs did not react with TEGV and PEDV, but only with PPV. The stability of the three hybridoma cells which were subcultured in vitro for 3 months and resuscitated after cryopreservation were analyzed. The results showed that the stability of the three McAbs was better. In this study, three monoclonal antibodies against porcine parvovirus structural protein (VP2) with high reactivity and specificity were successfully screened, which laid a theoretical foundation for the establishment of an effective method for the detection of PPV specific serology. It also provides a material basis for the study of PPV receptor and infection mechanism.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.651

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