鴨坦布蘇病毒JM株E蛋白的截斷表達(dá)及間接ELISA方法的建立
發(fā)布時間：2018-10-24 21:18
【摘要】：本研究利用RT-PCR擴(kuò)增鴨坦布蘇病毒(Duck Tembusu virus,DTMUV)JM株E基因截斷片段(822 bp),并將其克隆至原核表達(dá)載體p ET32a(+),成功構(gòu)建了重組質(zhì)粒p ET32a-E。重組質(zhì)粒轉(zhuǎn)化大腸桿菌Rosseta,經(jīng)IPTG誘導(dǎo)得到了高效表達(dá)。Western blot分析表明,重組蛋白能與DTMUV陽性血清發(fā)生特異性反應(yīng)。將純化好的DTMUV E蛋白作為包被抗原,建立了檢測DTMUV血清抗體的間接ELISA方法。經(jīng)過條件優(yōu)化,確定了抗原最適包被濃度為0.093μg/孔,血清的最佳稀釋度為1∶100。批內(nèi)和批間重復(fù)試驗的最大變異系數(shù)分別為3.53%和9.73%。用建立的ELISA方法對免疫鴨坦布蘇病毒滅活疫苗的鴨血清和對照鴨血清進(jìn)行抗體檢測,同時與攻毒保護(hù)試驗進(jìn)行比較,兩者的陽性符合率為86.67%,陰性符合率為100%。
[Abstract]:In this study, the truncated fragment of E gene (822 bp),) of (Duck Tembusu virus,DTMUV JM strain was amplified by RT-PCR and cloned into prokaryotic expression vector p ET32a (),. The recombinant plasmid p ET32a-E. was successfully constructed. The recombinant plasmid transformed into Escherichia coli Rosseta, was induced by IPTG to obtain the highly expressed. Western blot. The results showed that the recombinant protein could react specifically with the DTMUV positive serum. The purified DTMUV E protein was used as the coated antigen, and an indirect ELISA method for detection of DTMUV serum antibody was established. The optimum coating concentration of antigen was 0.093 渭 g / pore and the optimal dilution of serum was 1: 100. The maximum coefficients of variation were 3.53% and 9.73%, respectively. The ELISA method was used to detect the antibody of duck serum and control duck serum of the inactivated vaccine. The positive coincidence rate was 86.67 and the negative coincidence rate was 100.
【作者單位】： 廣東省農(nóng)業(yè)科學(xué)院動物衛(wèi)生研究所
【基金】：廣東省科技計劃項目(2013B020307001、2014A040401049、2014A020208052、2015B020203003、2015B070701015、2016A020210049) 廣州市科技計劃項目(201510010250) 廣東省農(nóng)業(yè)科學(xué)院院長基金項目(201436、201412)
【分類號】：S852.65
[Abstract]:In this study, the truncated fragment of E gene (822 bp),) of (Duck Tembusu virus,DTMUV JM strain was amplified by RT-PCR and cloned into prokaryotic expression vector p ET32a (),. The recombinant plasmid p ET32a-E. was successfully constructed. The recombinant plasmid transformed into Escherichia coli Rosseta, was induced by IPTG to obtain the highly expressed. Western blot. The results showed that the recombinant protein could react specifically with the DTMUV positive serum. The purified DTMUV E protein was used as the coated antigen, and an indirect ELISA method for detection of DTMUV serum antibody was established. The optimum coating concentration of antigen was 0.093 渭 g / pore and the optimal dilution of serum was 1: 100. The maximum coefficients of variation were 3.53% and 9.73%, respectively. The ELISA method was used to detect the antibody of duck serum and control duck serum of the inactivated vaccine. The positive coincidence rate was 86.67 and the negative coincidence rate was 100.
【作者單位】： 廣東省農(nóng)業(yè)科學(xué)院動物衛(wèi)生研究所
【基金】：廣東省科技計劃項目(2013B020307001、2014A040401049、2014A020208052、2015B020203003、2015B070701015、2016A020210049) 廣州市科技計劃項目(201510010250) 廣東省農(nóng)業(yè)科學(xué)院院長基金項目(201436、201412)
【分類號】：S852.65
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