Veterinary Faculty of Hunan Agricultural University, Changsha, Hunan Province, People's Republic of China.
Res Vet Sci. 2012 Oct;93(2):710-5. doi: 10.1016/j.rvsc.2011.12.010. Epub 2012 Jan 9.
Fragment of 759 bp DNA spanning the Matrix 1 (M1) gene of Avian Influenza Virus (AIV) was inserted into an expression vector pET28c to construct a recombinant plasmid pET28c-M1. The pET28c-M1 plasmid was transformed into the Escherichia coli BL21 (DE3) competent cell to produce a recombinant strain E. coli 21 (DE3). After being induced by Isopropyl-b-D-galactopyranoside (IPTG), E. coli 21 (DE3) expressed a 28-kDa fusion protein at a high level. This protein can bind anti-AIV (H5N1) positive serum by Western-blot analysis. After being denatured, renatured, and purified by Ni(2+)-column, the fusion protein was used as an antigen to develop Matrix 1 Enzyme-Linked Immunosorbent Assay (M1-ELISA) for detecting antibodies against AIV from chicken serum. We found that this indirect M1-ELISA was sensitive for differentiating antisera against AIV and antisera against other six kinds of avian viruses apart from AIV and this method is more sensitive than Hemagglutination Inhibition (HI) test. When compared with HI test and ELISA (IDEXX) in evaluating 581 serum samples from field vaccinated chickens, this assay showed 93.3% agreement ratio with the HI test, as well as 96.0% agreement ratio with ELISA (IDEXX). In a preliminary application, the assay successfully detected 19 AIVs from 51 nonvaccinated chicken lungs. It concludes that an indirect ELISA was successfully developed for detecting AIV. The assay is specific and sensitive. The application will greatly contribute to the long-term prevention and control of avian influenza in China.
一段长 759bp 的 DNA 片段跨越了禽流感病毒（AIV）的基质 1 （M1）基因，被插入表达载体 pET28c 中，构建了一个重组质粒 pET28c-M1。pET28c-M1 质粒被转化到大肠杆菌 BL21（DE3）感受态细胞中，产生了一个重组大肠杆菌 21（DE3）菌株。在异丙基-β-D-硫代半乳糖苷（IPTG）的诱导下，大肠杆菌 21（DE3）高水平表达了一个 28kDa 的融合蛋白。该蛋白可以通过 Western-blot 分析与抗 AIV（H5N1）阳性血清结合。经过变性、复性和 Ni（2+）柱纯化后，融合蛋白被用作抗原，开发了基质 1 酶联免疫吸附试验（M1-ELISA），用于检测鸡血清中的抗 AIV 抗体。我们发现，这种间接 M1-ELISA 能够敏感地区分抗 AIV 血清和抗除 AIV 以外的六种禽病毒血清，并且该方法比血凝抑制（HI）试验更敏感。当与 HI 试验和 ELISA（IDEXX）在评估 581 份来自田间接种鸡的血清样本时，该试验与 HI 试验的一致性为 93.3%，与 ELISA（IDEXX）的一致性为 96.0%。在初步应用中，该试验成功地从 51 份未接种鸡肺中检测到了 19 种 AIV。综上所述，成功开发了一种用于检测 AIV 的间接 ELISA。该试验具有特异性和敏感性。该应用将极大地有助于中国长期预防和控制禽流感。
