發(fā)布時間：2018-02-11 20:40

  本文關(guān)鍵詞： 非洲豬瘟病毒 抗體 免疫金 免疫層析 試紙條　出處：《揚(yáng)州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：非洲豬瘟(African swine fever, ASF)是由非洲豬瘟病毒(African swine fever virus, ASFV)引起的豬的一種急性、烈性、高度接觸性傳染性疾病。其臨床特征主要為全身出血、呼吸障礙和神經(jīng)癥狀,發(fā)病率和死亡率高,對養(yǎng)豬業(yè)的危害巨大。且目前尚無有效的疫苗用于該病的預(yù)防,主要通過檢疫后撲殺來控制其爆發(fā)和流行。因此,建立科學(xué)有效的檢測診斷方法對于非洲豬瘟的防范具有重要意義。為獲得ASFV P72,融合蛋白,本研究利用前期研究中構(gòu)建的重組菌株pET-30a-P72(BL21)接種到LB液體培養(yǎng)基,以IPTG進(jìn)行誘導(dǎo)后,收集菌體并以超聲波進(jìn)行裂解,裂解物經(jīng)SDS-PAGE分析,證明了該P(yáng)72融合蛋白可以在大腸桿菌BL21中大量表達(dá)。通過免疫印記技術(shù)分析可知,該融合蛋白可以與鼠抗His標(biāo)簽單抗特異性反應(yīng),將表達(dá)的P72重組蛋白經(jīng)His標(biāo)簽蛋白純化試劑盒純化后免疫新西蘭大白兔制備多克隆抗體。復(fù)蘇本實(shí)驗(yàn)室保存的抗ASFV的雜交瘤細(xì)胞株2D3,擴(kuò)大培養(yǎng)后接種于10周齡以上經(jīng)產(chǎn)BALB/c小鼠,采用體內(nèi)腹水誘生法制備了含有抗ASFV單抗的腹水。將收集的ASFV抗體分別經(jīng)飽和硫酸銨法、辛酸一硫酸銨聯(lián)合沉淀法、protein G柱親和層析法進(jìn)行純化,對純化后的抗體經(jīng)SDS-PAGE比較分析,最終選擇純化效果較好的protein G柱親和層析法。純化后的抗體經(jīng)紫外分光光度儀測定濃度,并用已建立的間接ELISA法測定其效價,單抗為1：160000,多抗為1：51200。為制備ASFV膠體金免疫層析試紙條,研究中采用直徑為30nm的膠體金顆粒標(biāo)記純化后的單抗2D3作為金標(biāo)抗體,以純化后的多抗固定于NC膜上作為檢測線,其最佳濃度為1.47 mg/mL;將葡萄球菌A蛋白(SPA)固定于NC膜上作為質(zhì)控線,其最佳濃度為1.0 mg/mL,組裝成用于檢測ASFV的膠體金免疫層析試紙條。以pET-30a和PGEX-6P-1為載體表達(dá)的ASFV P72蛋白(簡稱P3P蛋白和PXP蛋白)作為陽性樣品,PBS緩沖液、生理鹽水、蒸餾水等作為陰性樣品來檢測試紙條。結(jié)果顯示陽性樣品有明顯的兩個條帶,而陰性樣品只顯示一個條帶,且試紙條能檢測到的P3P蛋白和PXP蛋白的最低濃度分別為45ng/mL和60ng/mL。
[Abstract]:African swine fever (swine) is an acute, severe and highly contagious disease in pigs caused by African swine fever virus (swine). Its clinical characteristics are mainly systemic hemorrhage, respiratory disorders and neurological symptoms, high morbidity and mortality. At present, there is no effective vaccine for the prevention of the disease, mainly by culling after quarantine to control its outbreak and epidemic. In order to obtain ASFV P72, fusion protein, the recombinant strain pET-30a-P72 BL21 was inoculated into LB liquid medium and induced by IPTG. The cell was collected and cracked by ultrasonic wave. SDS-PAGE analysis showed that the P72 fusion protein could be expressed in a large number of Escherichia coli BL21, and the results of immunological imprinting analysis showed that the P72 fusion protein could be expressed in a large amount in Escherichia coli BL21. The fusion protein can react specifically with mouse anti His tag monoclonal antibody. The expressed recombinant protein P72 was purified by His tag protein purification kit and then immunized with polyclonal antibodies from New Zealand white rabbits. The hybridoma cell line 2D3, which was preserved in our laboratory, was resuscitated and inoculated into BALB/c mice over 10 weeks of age. Ascites containing anti ASFV monoclonal antibody were prepared by in vivo ascites induction method. The collected ASFV antibodies were purified by saturated ammonium sulfate method, combined with octanoic acid-ammonium sulfate precipitation method and protein G column affinity chromatography. The purified antibodies were compared and analyzed by SDS-PAGE. The purified antibody was determined by UV spectrophotometry, and the titer of the purified antibody was determined by indirect ELISA method. In order to prepare ASFV colloidal gold immunochromatographic strip, the purified monoclonal antibody 2D3, labeled with colloidal gold particles of 30 nm in diameter, was used as the gold standard antibody, and the purified polyantibody was fixed on the NC membrane as the detection line, the monoclonal antibody was 1: 160000 and the polyantibody was 1: 51200.To prepare the ASFV colloidal gold immunochromatographic strip, the purified McAb 2D3 was used as the gold standard antibody. The optimum concentration is 1.47 mg / mL. Staphylococcal protein A SPAs are immobilized on the NC membrane as the quality control line. The best concentration was 1.0 mg / mL, and the colloidal gold immunochromatographic strip was assembled to detect ASFV. The ASFV P72 protein (P3P protein and PXP protein) expressed on pET-30a and PGEX-6P-1 was used as positive sample buffer solution and normal saline. The results showed that there were two obvious bands in the positive sample, but only one band in the negative sample. The lowest concentrations of P3P protein and PXP protein were 45 ng / mL and 60 ng / mL, respectively.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65

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