【摘要】：豬流行性腹瀉(PED)是一種急性、感染性高的腸道病毒性疾病,對(duì)新生豬仔造成很高的死亡率,豬流行性腹瀉病毒(PEDV)為其致病原。PEDV屬于尼多病毒目、冠狀病毒科,α冠狀病毒亞科(或?qū)?,基因組為單股正義RNA病毒,基因組大小約28 kb。PEDV變異較大,其臨床癥狀、病理變化以及流行特點(diǎn)都與傳染性胃腸炎和輪狀病毒病的十分相似。目前,PED在世界范圍內(nèi)廣泛流行,但還沒有很好的預(yù)防和診斷方法。為此本研究選擇PEDV S基因的保守序列進(jìn)行原核表達(dá),制備相應(yīng)抗體,并初步建立一套檢測(cè)PEDV抗體的間接ELISA方法。具體研究?jī)?nèi)容如下:1 PEDV部分S基因的合成及重組質(zhì)粒的構(gòu)建通過DNAstar軟件對(duì)豬流行性腹瀉病毒流行毒株S基因進(jìn)行序列比對(duì)與分析,選取位于502-641氨基酸位置處的S1蛋白保守序列,利用大腸桿菌密碼子偏嗜性進(jìn)行密碼子優(yōu)化,Overlap PCR合成,獲得417 bp基因片段,連接到克隆載體pJET/1.2-blunt上,并構(gòu)建重組表達(dá)質(zhì)粒pET32a-S417和pXXGST-S417。2重組質(zhì)粒的表達(dá)與純化將獲得的重組表達(dá)質(zhì)粒轉(zhuǎn)化到BL21感受態(tài)細(xì)胞進(jìn)行誘導(dǎo)表達(dá),并通過割膠純化,獲得重組蛋白GST-S417,分子量31 kD左右,濃度0.3 mg/mL左右,純度達(dá)95%以上;通過過柱純化,獲得重組蛋白His-S417,分子量30 kD左右,濃度0.1 mg/mL左右,純度達(dá)90%以上。3多克隆抗體的制備以純化的GST-S417重組蛋白作為免疫原,免疫新西蘭大白兔制備多克隆抗體,免疫量500μg/只。以純化的His-S417重組蛋白作為包被抗原,檢測(cè)免疫血清中抗體的效價(jià),ELISA檢測(cè)結(jié)果表明血清效價(jià)達(dá)1:10000以上,而且制備的多克隆抗體能特異性識(shí)別目的蛋白。該研究表明所選的S蛋白區(qū)域具有一定的抗原性。4間接ELISA檢測(cè)方法的初步建立包被純化的GST-S417蛋白,摸索最佳間接ELISA條件,結(jié)果顯示:包被量0.32μg/孔,血清最佳稀釋比1:800,HRP標(biāo)記的抗豬抗體稀釋比為1:3000時(shí),檢測(cè)效果最好,陰性臨界值為0.340,有良好的重復(fù)性和特異性,與市場(chǎng)銷售的診斷試劑盒符合率較高,達(dá)72.7%,為進(jìn)一步開發(fā)診斷試劑盒提供了理論和技術(shù)基礎(chǔ)。
[Abstract]:Porcine epidemic diarrhea (PED) is an acute and highly infectious enterovirus disease, which causes a high mortality to newborn piglets. Porcine epidemic diarrhea virus (PEDV) is the causative agent. PEDV belongs to the family Nidoviridae and coronavirus. The genome of 偽 coronavirus subfamily (or genus) is a single-stranded sense RNA virus, and the genome size is about 28 kb.PEDV. The clinical symptoms, pathological changes and epidemic characteristics of 偽 coronavirus are very similar to those of transmissible gastroenteritis and rotavirus disease. At present, PED is popular all over the world, but there is no good method of prevention and diagnosis. In this study, the conserved sequence of PEDV S gene was selected for prokaryotic expression, the corresponding antibody was prepared, and a set of indirect ELISA method for detecting PEDV antibody was established. The main contents are as follows: 1 the synthesis of S gene of PEDV and the construction of recombinant plasmid were used to sequence alignment and analysis of S gene of porcine epidemic diarrhea virus epidemic strain by DNAstar software. The S1 protein conserved sequence located at the 502-641 amino acid position was selected. The codon bias of Escherichia coli codon was used to optimize the, Overlap PCR synthesis. The 417 bp gene fragment was obtained and ligated to the clone vector pJET/1.2-blunt. The expression and purification of the recombinant expression plasmid pET32a-S417 and pXXGST-S417.2 were constructed. The recombinant expression plasmid was transformed into BL21 receptive cells for induction and expression, and the recombinant protein GST-S417, was obtained by gel purification. The molecular weight is about 31 kD, the concentration is about 0.3 mg/mL, and the purity is over 95%. The molecular weight of recombinant protein His-S417, was about 30 kD, the concentration was about 0.1 mg/mL, and the purity of polyclonal antibody was over 90%. The purified recombinant GST-S417 protein was used as immunogen. New Zealand white rabbits were immunized with polyclonal antibodies (500 渭 g / mouse). The purified His-S417 recombinant protein was used as the coating antigen to detect the titer of the antibody in the immune serum. The results of ELISA showed that the titer of the serum was over 1: 10000, and the polyclonal antibody could specifically recognize the target protein. This study shows that the selected S protein region has a certain antigenicity. 4 the preliminary establishment of indirect ELISA detection method for the purification of GST-S417 protein, explore the best indirect ELISA conditions, the results showed that the coating amount of 0.32 渭 g / pore, When the dilution ratio of anti-porcine antibody labeled with 1: 800 or HRP was 1: 3000, the detection effect was the best, the negative critical value was 0.340, which had good repeatability and specificity, and was in good agreement with the diagnostic kit sold on the market. It provides a theoretical and technical basis for the further development of diagnostic kit.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

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