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  本文關(guān)鍵詞： 鴨圓環(huán)病毒 ORF3基因 克隆表達(dá) 單克隆抗體 間接競爭ELISA　出處：《四川農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：鴨圓環(huán)病毒被歸類于圓環(huán)病毒科圓環(huán)病毒屬,該病毒ORF3所編碼的蛋白功能目前尚未闡釋。為了制備針對該蛋白的單克隆抗體,給進(jìn)一步探索該蛋白功能提供素材,論文對本實驗室分離鑒定的DuCV ORF3基因開展了以下實驗：DuCV ORF3基因的生物信息學(xué)分析、ORF3基因的克隆表達(dá)及純化、單克隆抗體的制備、間接競爭ELISA方法的建立和應(yīng)用,結(jié)果如下：1. DuCV ORF3的生物信息學(xué)分析GH01株DuCV全長1988 by,ORF3基因A、T、G、C含量分別為21.89%、25.25%、24.58%、28.28%,共297bp,位于全基因組第399至第103位堿基,編碼98個氨基酸,分子量約11.6 KD。該蛋白二級結(jié)構(gòu)中無規(guī)則卷曲占大部分,含量約為62.3%,α-螺旋和p-折疊結(jié)構(gòu)分別占31.6%和6.1%；沒有保守區(qū)、信號肽、跨膜區(qū),提示該蛋白可能是該病毒進(jìn)化的關(guān)鍵蛋白,且既不是分泌性蛋白也不是跨膜蛋白；有三個潛在的磷酸化位點,沒有糖基化位點；.抗原表位主要集中在第2～12、第35-40、第49～53、第62-65、第72～75、第77～84位氨基酸,免疫原性良好。2. DuCV基因的克隆表達(dá)及純化PCR特異性擴(kuò)增ORF3基因后進(jìn)行T-A克隆,再用EcoR Ⅰ和Xhol這兩種內(nèi)切酶消化后連接到表達(dá)載體上,成功構(gòu)建了重組原核表達(dá)載體：pET-32a-ORF3和pGEX-6p-ORF3.重組載體分別轉(zhuǎn)化進(jìn)Rosetta (DE3) E.coli菌后用IPTG成功誘導(dǎo)表達(dá)了30 KD和35 KD的ORF3融合蛋白,與預(yù)期大小一致,對pET-32a-ORF3的最佳IPTG誘導(dǎo)濃度為0.8 mM,而對pGEX-6p-ORF3是0.2 mM,這兩種融合蛋白都主要是以包涵體的形式存在。利用Ni2+親和層析及包涵體洗滌和SDS-PAGE切膠回收相結(jié)合的方法分別純化pET-32a-ORF3和pGEX-6p-ORF3重組蛋白,得到了高純度的蛋白。Western Blot檢測顯示這兩種蛋白均能夠與鴨圓環(huán)病毒陽性血清特異性結(jié)合,表明它們都具有免疫學(xué)活性。3. DuCV ORF3單克隆抗體的制備純化的pET-32a-ORF3蛋白經(jīng)梯度復(fù)性后以100μg/只腹腔注射Balb/c雌性小鼠進(jìn)行免疫,效價達(dá)到104后,將骨髓瘤細(xì)胞與其脾細(xì)胞按1：4的比例混勻,在PEG4000作用下進(jìn)行融合反應(yīng)。用建立的間接ELISA法(最佳抗原包被濃度1μg/孔)篩選陽性細(xì)胞,融合率為25%。經(jīng)過3次有限稀釋,最終達(dá)到100%的陽性,獲得4株穩(wěn)定分泌抗ORF3抗體的雜交瘤細(xì)胞(依次命名為1、2、3、4號)。利用ORF3陽性雜交瘤細(xì)胞制備腹水型抗體,抗體的效價在1：25600～1：102400之間,均為IgM亞類,并能夠與ORF3蛋白特異性結(jié)合。4.間接競爭ELISA方法的建立與應(yīng)用利用純化的pET-32a-ORF3融合蛋白作為包被抗原,建立了穩(wěn)定可靠的DuCV ORF3抗體的間接競爭ELISA免疫學(xué)檢測方法。利用棋盤滴定法優(yōu)化包被抗原和腹水單抗的最佳梯度,再確定出最優(yōu)血清稀釋度、競爭作用時間和cut-off判定值。結(jié)果表明該方法的最佳條件為pET-32a-ORF3純化蛋白以1μg/包被,5%BSA封閉后,1：800的DuCV陽性血清37℃放置30 min,直接加入1：4000的腹水型單克隆抗體,混勻后37℃競爭反應(yīng)40 min,HRP-羊抗鴨Ig G二抗孵育再TMB顯色。該檢測方法cut-off判定值為10.9%,只特異性的與DuCV陽性血清反應(yīng),靈敏度為血清稀釋1：1600倍。將該方法與PCR檢測方法對45份未知樣品進(jìn)行檢測并比較,結(jié)果PCR檢測出37個陽性,而本實驗方法檢測出25個陽性,且這25個樣品在PCR檢測中也均為陽性,符合率為100%。
[Abstract]:Duck circovirus is classified in the circoviridae circovirus genus, the virus ORF3 encoding protein function has not yet been explained. In order to prepare monoclonal antibody against this protein, to further explore the function of this protein to provide material, the DuCV ORF3 gene on the isolation and identification of the laboratory to carry out the following experiments: the bioinformatics analysis of DuCV ORF3 gene, ORF3 gene cloning, expression and purification of monoclonal antibody preparation, establishment and application of indirect competitive ELISA method, the results are as follows: analysis of GH01 strain was 1988 by DuCV 1. DuCV ORF3 biological information, ORF3 gene A, T, G, C contents were 21.89%, 25.25%, 24.58%, 28.28% 297bp, located in the whole genome, 399th to 103rd nucleotides, encoding 98 amino acids, random coil accounted for most of the molecular weight of about 11.6 KD. of the two protein secondary structure, content is about 62.3%, alpha helix and p- folding structure Accounted for 31.6% and 6.1%; no conserved signal peptide, transmembrane region, suggesting that the protein may be a key protein in the virus evolution, and neither secreted protein nor transmembrane protein; there are three potential phosphorylation sites, no glycosylation sites.; epitope mainly concentrated in second 12, article 35-40, article 62-65, forty-ninth ~ 53, seventy-second ~ 75, seventy-seventh ~ 84 amino acid, cloning and expression of immunogenicity.2. DuCV gene and purification of PCR specific ORF3 gene was amplified by T-A and cloned, EcoR I and Xhol two is connected to the expression vector after digestion, success to construct a Recombinant Prokaryotic expression vector pET-32a-ORF3 and recombinant vector pGEX-6p-ORF3. was transformed into Rosetta (DE3) E.coli strain by IPTG after successfully induced expression of 30 KD and 35 KD ORF3 fusion protein, consistent with the expected size, the best IPTG on pET-32a-ORF3 induced concentration For 0.8 mM, which is 0.2 mM for pGEX-6p-ORF3, the two kinds of fusion protein mainly existed in the form of inclusion body. By using the method of Ni2+ gel extraction combined with affinity chromatography and inclusion body washing and SDS-PAGE respectively. The pET-32a-ORF3 and pGEX-6p-ORF3 recombinant protein, the protein of high purity.Western Blot detection showed that the two proteins were capable of binding and duck circovirus positive serum specific, showed that they all have the immunological activity of purified.3. DuCV ORF3 monoclonal antibody against pET-32a-ORF3 protein was refolded by gradient with 100 g/ intraperitoneal injection of Balb/c female mice was up to 104 after the myeloma cells and spleen cells by 1:4 the mixing, the fusion reaction in the presence of PEG4000. With the developed ELISA method (the optimal antigen concentration of 1 g/ hole) screening positive cells, the fusion rate was 25%. after 3 times Limited dilution, eventually reaching 100% positive, obtained 4 hybridoma cell strains secreting anti ORF3 antibodies (designated as 1,2,3,4). The preparation of ascites antibody by ORF3 positive hybridoma cells, antibody titer was 1:25600 ~ 1:102400, were subtype IgM, and can be combined with ORF3 protein the establishment and application of.4. indirect competitive ELISA method using the purified pET-32a-ORF3 fusion protein as antigen to establish an indirect competitive DuCV method for immunological detection of ELISA antibody of ORF3 is stable and reliable. The chessboard titration optimization package was the best gradient antigen and monoclonal antibodies, and then determine the optimal serum dilution, competition time and cut-off to determine the value. The results showed that the best conditions of the method for purification of pET-32a-ORF3 protein in 1 g/ package, 5%BSA closed, 1:800 DuCV positive serum of 37 DEG C for 30 min, directly adding 1 Type 4000: ascites monoclonal antibody, mixed competition reaction 37 C 40 min, HRP- Ig G two anti Goat anti duck incubation TMB color. The detection method to determine cut-off value of 10.9%, a specificity and sensitivity of DuCV positive sera, sera were diluted 1:1600 times. The method with PCR the detection method for the detection of 45 samples and compared the results of 37 positive PCR was detected, and the experimental method to detect 25 positive, and the 25 samples in PCR assay were also positive, the coincidence rate was 100%.

【學(xué)位授予單位】：四川農(nóng)業(yè)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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