本文選題：豬圓環(huán)病毒型 + ORF��； 參考：《中國獸醫(yī)科學(xué)》2017年04期

【摘要】：ORF3蛋白是豬圓環(huán)病毒2型(porcine circovirus type,2PCV2)的一個(gè)重要的非結(jié)構(gòu)蛋白。根據(jù)GenBank數(shù)據(jù)庫中PCV2的ORF3基因序列設(shè)計(jì)引物,以PCV2基因組為模板進(jìn)行PCR擴(kuò)增,并構(gòu)建重組表達(dá)質(zhì)粒。將重組質(zhì)粒轉(zhuǎn)染至BL21感受態(tài)細(xì)胞,進(jìn)行最佳誘導(dǎo)條件的選擇,采用Ni-NTA親和層析柱純化表達(dá)產(chǎn)物。以純化后的重組ORF3蛋白作為酶標(biāo)包被抗原,優(yōu)化反應(yīng)條件,建立PCV2間接ELISA(indirect ELISA,iELISA)診斷方法。結(jié)果顯示,重組質(zhì)粒經(jīng)PCR鑒定、酶切鑒定和測序鑒定構(gòu)建成功。重組蛋白表達(dá)的最佳誘導(dǎo)條件為37℃、8 h、1 mmol/LIPTG。Western-blot結(jié)果表明,該重組蛋白具有較好的免疫原性。Ni-NTA親和層析純化結(jié)果表明,280 mmol/L咪唑?yàn)樽罴严疵摑舛�。iELISA的最佳包被抗原質(zhì)量濃度為0.03 mg/m L,待檢血清最佳稀釋度為1∶100,酶標(biāo)抗體最佳工作濃度為1∶4 000。批內(nèi)和批間試驗(yàn)結(jié)果表明,該方法具有較好的重復(fù)性。此iELISA與PCR檢測方法具有良好的一致性。結(jié)果表明,基于ORF3蛋白建立的PCV2間接ELISA診斷方法,可以為臨床上rCap蛋白基因工程疫苗免疫豬場進(jìn)行鑒別診斷。
[Abstract]:ORF3 protein is an important nonstructural protein of porcine circovirus type 2 (porcine circovirus type 2 PCV2. According to the sequence of ORF3 gene of PCV2 in GenBank database, primers were designed and PCR amplification was carried out using PCV2 genome as template, and the recombinant expression plasmid was constructed. The recombinant plasmid was transfected into BL21 cells and the optimal induction conditions were selected. The expression product was purified by Ni-NTA affinity chromatography. The purified recombinant ORF3 protein was used as the coated antigen, and the reaction conditions were optimized. An indirect Elisa method for the diagnosis of PCV2 was established. The results showed that the recombinant plasmid was successfully constructed by PCR, enzyme digestion and sequencing. The optimal inducing conditions for the expression of recombinant protein were as follows: 1 mmol / L LIPT G. Western-blot at 37 鈩，

本文編號(hào)：2070992