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1、中國農(nóng)業(yè)大學(xué)碩士學(xué)位論文豬圓環(huán)病毒2型抗體間接ELISA檢測試劑盒的研制和應(yīng)用姓名:李寧申請學(xué)位級別:碩士專業(yè):預(yù)防獸醫(yī)學(xué)指導(dǎo)教師:楊漢春;郭鑫20040601中文摘要利用已構(gòu)建的基因工程重組菌BL21(DE3)表達(dá)豬圓環(huán)病毒2型(PCV2)ORF2編碼的結(jié)構(gòu)蛋白.并成功摸索出一種快速純化該蛋白的方法。用純化的蛋白作為包被抗原,建立了檢測豬血清中PCV2抗體的問接ELISA方法;以該方法為基礎(chǔ)初步組裝出試荊盒,并進(jìn)行了初步應(yīng)蹦。對ELISA的反應(yīng)條件進(jìn)行了探索,確定了最適工作條件。結(jié)果表明,重組蛋白抗原的昂適包被濃度為O.199/mL,最適包被條件為37"C2h加4"C過夜;待檢血清的
2、稀釋度為1:40;HRP.兔抗豬IgG的工作濃度為1:800;待檢血清和酶標(biāo)二抗的反應(yīng)條件和反應(yīng)時間均為37"C30min:底物室溫顯色10-15min:陰陽性臨界值為0.4。經(jīng)阻斷試驗(yàn)、交叉反應(yīng)試驗(yàn)和重復(fù)性試驗(yàn),表明建立的間接ELISA方法是特異的,且重復(fù)性很好;與間接免疫熒光試驗(yàn)(IFA)進(jìn)行比較,其敏感性和特異性分別是94.3%和90%,說明兩種檢測方法間無顯著性差異:進(jìn)行了試劑盒的組裝及其保存期試驗(yàn),結(jié)果表明試劑盒在4℃下至少可保存三個月。用試劑盒檢測人工感染PCV2組織毒的SPF小香豬血清,并繪制血清中抗體變化的動態(tài)曲線。結(jié)果顯示,攻毒后14,--2l天血清抗體出現(xiàn)陽轉(zhuǎn)化,抗
3、體水平的高峰期出現(xiàn)在感染后28---35天,其后,抗體水平開始下降。用試劑盒對我國13個地區(qū)70個規(guī)?;B(yǎng)豬場的2007份豬血清進(jìn)行了PCV2感染的普查,結(jié)果表明自1997年以來PCV2感染已在我國普遍存在。關(guān)鍵詞:PCV2:重組蛋白:ELISA;試劑盒;應(yīng)用AbstractInthisstudy,thestructuralproteinencodedbyORF2ofPCV2wasexpressedinrecombinantE.coliBL21(DE3)andavalidmethodofpuri5,ingthePCV2-ORF2recombinantfusionproteinwasde
4、veloped.Usingthepurifiedrecombinantproteinascoatingantigen,anindirectELlSAwasestablishedfordetectionofantibodyinsernmtoPCV2.ELISAkitwasprimarilypreparedandappliedfordetectionofantibodiesinserunlsamplesfrompigletsinfectedexperimentallyandswinefarms.Thevario/isfactorsandconditionsforELISAwereexplo
5、red.a(chǎn)ndtheoptimalreactionconditionsweredetermined.ItwasshownthattheoptimalconcentrationofPCV2-ORF2mcombinantproteinforcoatingofplatewas0.1gg/mL,theoptirealcoatingconditionofmcombinantproteinwas3TCfor2hoursand4"Covemight,thedilutionofserumsamplewasl:40.theworkingconcentrationofHRP-labeledrabbitan
6、ti-proteinIgGwas1:800,sevtllnsampleforde'reefingandHRP-labeledrabbitanti·porcineIgGshouldbemcubatedat37"Cfor30minbeforeterminatedwiththestoppingsolution.ThethresholdforELISAwas0.4.TheblockingtestforthereactionbetweenthepositiveserulnofPCV2andtherecombinantproteinwascarriedoutbyusingPCV2harvested
7、incellculture.TheresultsindicatedthatPCV2couldblockthereactioncompletely.Itwasalsoconfirmedthattherewasnocrossreactionbythecrossingtest.Meanwhile,theELISAhadidealrepeatabilitywhileitwasdonebothinthesamedayandthedifferentdays