H5N1亚型AIVHA抗原表位的高效表达及其抗原活性分析

血凝素(hemagglutinin,HA)蛋白是禽流感病毒(avian influenza virus,AIV)的一个重要表面抗原性蛋白,在疾病诊断和防治上有重要意义。本研究为了探讨一种更为简便有效的HA重组蛋白表达途径,利用生物信息学软件,对H5N1亚型AIVHA基因编码的氨基酸序列进行分析,在分析其在大肠杆菌中的密码子偏好性、稀有密码子分布情况及有关蛋白的抗原性等重要特性后,构建了HA抗原表位重组表达质粒pET-32a(+)-HA。经测试,该重组质粒在1mmol/LIPTG诱导剂作用下诱导过夜,能在大肠杆菌Rosetta-gami B(DE3)中高效表达,并得到48.1kD大小的目的重组表达蛋白。重组蛋白用6×His-tagged protein纯化试剂盒纯化后,与福氏佐剂等量混合制备成抗原,以200μg/鸡的剂量皮下注射2月龄SPF鸡3次,采血分离血清。Western-Blot试验结果表明,该重组表达蛋白能分别与所制备的高免鸡血清及H5N1亚型AIV阳性血清发生特异性反应,在硝酸纤维素膜上出现特异性杂交带。说明本试验研究的HA抗原重组表达蛋白具有良好的免疫原性和反应原性,保留了HA蛋白的抗原活性,提示该重组蛋白在H5亚型AIV的防治技术研究中具有重要的实际应用价值。

High Level Expression and Antigenic Analysis of H5N1 AIV Hemagglutinin Epitopes

Hemagglutinin(HA) is an important surface antigen protein of avian influenza virus(AIV),and plays an important role in diagnosis,prevention and treatment of AIV.The purpose of this study is to develop an easier and more efficient way to express HA recombinant protein.The open reading frame(ORF) of H5N1 AIV HA gene was analyzed using bioinformatics software.Its codon bias to Escherichia coli,rare codon’s contributions in the sequence and the antigenicity of HA protein were used to construct a recombinant expression plasmids of HA epitopes,pET-32a(+)-HA.When these recombinant plasmids were induced overnight with 1 mmol/L IPTG solution,they could be highly expressed in E.coli Rosetta-gami B(DE3),and the desired recombinant proteins of 48.1 kD could be got.After purified by 6×His-tagged protein purification kit,the recombinant proteins were mixed with equal amount of Freund’s Adjuvant Complete/Incomplete to make the antigen.Then every seven days this antigen was hypodermically injected into two-months-old SPF chickens with a dose of 200 μg per chicken,totally three times.Serums were collected from chickens after last injection.Western-Blot results showed that the recombinant protein could hybridize with both the serum from the highly-immunized chicken and the serum from H5N1 AIV positive chicken.Specific bands were present on the nitrocellulose membrane.This indicates that the recombinant protein expressed by HA antigen gene segment has great immunogenicity and antigenicity and keeps the antigen activity of HA protein.This study suggests that the recombinant protein has practical value in the diagnosis,prevention and treatment of H5N1 AIV.