本文選題：傳染性支氣管炎 + N蛋白　； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：傳染性支氣管炎病毒(infectious bronchitis virus,IBV)可引起雞的一種急性、高度接觸傳染性的呼吸道和泌尿生殖道疾病——雞傳染性支氣管炎(infectious bronchitis,IB)。自1931年被首次報(bào)道后,IB在世界各地均有發(fā)生,IBV現(xiàn)已衍生出多種血清型。IBV可與其它病原體混合感染,引起嚴(yán)重的并發(fā)癥,給養(yǎng)禽業(yè)帶來巨大損失。IBV抗原性復(fù)雜,易發(fā)生基因重組和點(diǎn)突變,而且不同血清型間的交叉保護(hù)效力低,使IB的防控極具挑戰(zhàn)性,因此,開發(fā)簡便、快速的診斷方法,對(duì)IB的防控非常重要。核衣殼蛋白(N蛋白)是IBV的主要結(jié)構(gòu)蛋白,相對(duì)較保守,本研究針對(duì)N蛋白制備了兔多克隆抗體(PcAb)和鼠單克隆抗體(MAb),建立了檢測IBV的雙抗體夾心ELISA檢測方法,為IBV的診斷提供了一種行之有效的手段。1.針對(duì)IBVN蛋白的兔多克隆抗體和鼠單克隆抗體的制備本研究以IBVQXL毒株為基礎(chǔ),對(duì)其N蛋白的序列進(jìn)行抗原性分析,用PCR方法擴(kuò)增抗原性較強(qiáng)區(qū)域?qū)?yīng)的N基因片段,然后將產(chǎn)物連至克隆載體上,經(jīng)基因測序鑒定正確后,再以pET-32a(+)為載體,構(gòu)建表達(dá)N蛋白片段的重組原核表達(dá)質(zhì)粒,轉(zhuǎn)化宿主菌BL21(DE3)后誘導(dǎo)表達(dá)并純化,獲得的N蛋白片段用作免疫原,分別免疫成年兔和6-8周齡的BALB/C小鼠。當(dāng)免疫兔的血清效價(jià)達(dá)到1:104時(shí),心臟采血制備抗N蛋白的PcAb血清;當(dāng)免疫鼠的血清效價(jià)達(dá)到1:104時(shí),取脾臟與SP2/0細(xì)胞融合,利用間接ELISA鑒定、篩選雜交瘤細(xì)胞,最終獲得四株能穩(wěn)定分泌抗N蛋白MAb的細(xì)胞株6F4、7D8、3G2、5C9,制備腹水并鑒定、純化后,進(jìn)一步用Western blot鑒定,結(jié)果表明獲得的單抗可與N蛋白特異結(jié)合。2.檢測IBV的雙抗體夾心ELISA方法的建立本研究以制備的PcAb為包被抗體、MAb為檢測抗體,建立檢測IBV的雙抗體夾心ELISA方法。優(yōu)化后的反應(yīng)條件為:包被抗體最佳稀釋度為1:64000,檢測抗體最佳稀釋度為1:4000,封閉液為1%BSA-PBST,酶標(biāo)抗體稀釋度為1:10000,顯色底物作用時(shí)間為15 min。特異性實(shí)驗(yàn)表明,該方法對(duì)禽流感病毒(AIV)、新城疫病毒(NDV)及禽腺病毒(FAdV)無交叉反應(yīng)。重復(fù)性實(shí)驗(yàn)表明,該方法的批內(nèi)、批間變異系數(shù)均小于8%。該方法與檢測IBV的PCR方法的陽性符合率為95.9%,陰性符合率為85.2%�？傊�,本研究制備的抗體可以和IBV的N蛋白特異性反應(yīng),以此為基礎(chǔ)建立的雙抗體夾心ELISA檢測IBV方法的特異性好、可重復(fù),且檢測結(jié)果可靠,為IB防控提供了良好的診斷方法。
[Abstract]:Infectious bronchitis virus (IBV) can cause acute and highly contagious respiratory and urogenital diseases in chickens.Since it was first reported in 1931, IBV has been found in various parts of the world. IBV can be mixed with other pathogens, causing serious complications and causing huge losses to poultry industry. IBV antigenicity is complex.Gene recombination and point mutation are easy to occur, and the cross-protection efficacy between different serotypes is low, which makes the prevention and control of IB extremely challenging. Therefore, it is very important to develop a simple and rapid diagnostic method for the prevention and control of IB.Nucleocapsid protein (N) is the main structural protein of IBV, which is relatively conserved. In this study, rabbit polyclonal antibody (PcAb) and mouse monoclonal antibody (McAb) were prepared for N protein, and a double antibody sandwich ELISA method was established for the detection of IBV.It provides an effective method for the diagnosis of IBV.The preparation of rabbit polyclonal antibody and mouse monoclonal antibody against IBVN protein. Based on the IBVQXL strain, the sequence of N protein was analyzed antigenically, and the N gene fragment corresponding to the region with strong antigenicity was amplified by PCR.The recombinant prokaryotic expression plasmid expressing N protein fragment was constructed by using pET-32a () as vector. The recombinant plasmid was transformed into host strain BL21DE3) and then induced and purified.The obtained N protein fragments were used as immunogen to immunize adult rabbits and 6-8 week old BALB/C mice respectively.When the titer of serum of immunized rabbits reached 1: 104, the PcAb serum of anti-N protein was obtained from the heart, and when the titer of serum of immunized mice reached 1: 104, the spleen was fused with SP2/0 cells, and the hybridoma cells were screened by indirect ELISA identification.Finally, four cell lines that stably secreted anti-N protein MAb were obtained. Ascites were prepared and identified by Western blot. The results showed that the obtained McAbs could specifically bind to N protein.Establishment of a double antibody sandwich ELISA method for detection of IBV A double antibody sandwich ELISA method was established for the detection of IBV using the prepared PcAb as the coated antibody to detect the antibody.The optimized reaction conditions were as follows: the best dilution of coated antibody was 1: 64000, the best dilution of antibody was 1: 4000, the blocking solution was 1BSA-PBST, the dilution of enzyme-labeled antibody was 1: 10000and the reaction time of chromogenic substrate was 15 min.The specificity test showed that the method had no cross reaction against avian influenza virus (AIV), Newcastle disease virus (NDV) and avian adenovirus (FAdV).The repeatability experiment showed that the coefficient of variation was less than 8%.The positive coincidence rate and negative coincidence rate between this method and PCR method were 95.9 and 85.2, respectively.In a word, the antibody prepared in this study can react with the N protein of IBV, and the double antibody sandwich ELISA based on this method has good specificity, repeatability and reliable detection results, which provides a good diagnostic method for IB prevention and control.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65

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