Centre for Biotechnology, Jawaharlal Nehru Technological University, Hyderabad, India.
J Virol Methods. 2013 Jan;187(1):20-5. doi: 10.1016/j.jviromet.2012.07.026. Epub 2012 Jul 31.
Recombinant avian influenza vaccines offer several advantages over the conventional vaccines. In this study, the haemagglutinin (HA) gene of highly pathogenic avian influenza H5N1 was cloned and expressed as His tagged protein in methylotropic yeast Pichia pastoris. The expression of recombinant HA (rHA) protein was confirmed by SDS-PAGE and western blot analysis. The rHA protein was purified using Ni-NTA affinity chromatography under denaturing conditions and the functions of the protein was assessed by the haemagglutinin assay after refolding. The immunogenicity of the rHA was evaluated by immunizing four groups of mice with different payloads (2.5, 5.0, 10 and 25μg) of purified rHA and the production of rHA specific antibodies were analysed by haemagglutinin inhibition assay (HI) and enzyme-linked immunosorbent assay (ELISA). An antigen specific immune response was observed against rHA indicating that the rHA antigen could be used as a vaccine candidate against avian influenza. These results suggest that this strategy would pave the way for the development of rapid and cost effective method for the production of an avian influenza vaccine.
重组禽流感疫苗相对于传统疫苗具有多项优势。在本研究中，我们克隆了高致病性禽流感 H5N1 的血凝素（HA）基因，并在甲醇营养型酵母毕赤酵母中表达为 His 标签蛋白。通过 SDS-PAGE 和 Western blot 分析证实了重组 HA（rHA）蛋白的表达。使用 Ni-NTA 亲和层析在变性条件下纯化 rHA 蛋白，并在复性后通过血凝试验评估蛋白的功能。通过用不同载量（2.5、5.0、10 和 25μg）的纯化 rHA 免疫四组小鼠来评估 rHA 的免疫原性，并通过血凝抑制试验（HI）和酶联免疫吸附试验（ELISA）分析 rHA 特异性抗体的产生。针对 rHA 观察到了抗原特异性免疫反应，表明 rHA 抗原可用作抗禽流感疫苗的候选物。这些结果表明，这种策略将为开发快速且具有成本效益的禽流感疫苗生产方法铺平道路。
