禽流感病毒HA蛋白的原核表达与鉴定
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摘要
为了实现禽流感病毒血凝素(HA)蛋白在原核系统中高效表达,根据Gen Bank发表的HA(ID=DQ023145. 1)基因序列,在不改变HA蛋白氨基酸序列的情况下,根据大肠杆菌密码子偏好性优化基因序列,化学合成HA全基因。将其克隆到pET28a(+)载体中,构建重组质粒pET28a(+)-HA,转化至BL21工程菌进行诱导表达,利用His-tag镍柱纯化HA重组蛋白,并对纯化后的重组蛋白进行SDS-PAGE分析及Western-blot鉴定。结果表明:当重组菌株p ET28a(+)-HA/BL21(DE3)诱导条件为30℃、IPTG浓度为1 mmol/L诱导8 h时,HA蛋白表达量最高; SDS-PAGE电泳显示经镍柱纯化后HA蛋白纯度较高,Western blot实验发现在预期的70 kD处有明显的蛋白印迹条带,说明已成功实现了在大肠杆菌中高效表达禽流感病毒HA蛋白,为后续开展禽流感检测方法及疫苗的研究奠定了基础。
In order to achieve high expression of avian influenza virus hemagglutinin( HA) protein in prokaryotic expression system,based on the HA( ID = DQ023145. 1) gene sequence published by GenBank,Without changing the amino acid sequence of the HA protein,according to the large intestine bacillus codon preference optimized gene sequence,the whole gene was synthesized into HA gene,and cloned into pET28 a( +) vector to construct recombinant plasmid p ET28 a( +)-HA,which was transformed into BL21 engineering strain for induction expression,using His-tag The recombinant protein of HA was purified by nickel column,and the purified recombinant protein was identified by SDS-PAGE and Western-blot. The results showed that when the recombinant strain p ET28 a( +)-HA/BL21( DE3) was induced at 30 ℃ and IPTG concentration was 1 mmol/L for 8 h,the expression of HA protein was the highest. SDS-PAGE electrophoresis showed that HA protein was purified by nickel column. The purity was higher,and Western blot showed a significant Western blot at 70 KD,which was consistent with expectations. It shows that the HA protein of avian influenza virus is highly expressed in E. coli,which lays a foundation for the subsequent research on avian influenza detection methods and vaccines.
In order to achieve high expression of avian influenza virus hemagglutinin( HA) protein in prokaryotic expression system,based on the HA( ID = DQ023145. 1) gene sequence published by GenBank,Without changing the amino acid sequence of the HA protein,according to the large intestine bacillus codon preference optimized gene sequence,the whole gene was synthesized into HA gene,and cloned into pET28 a( +) vector to construct recombinant plasmid p ET28 a( +)-HA,which was transformed into BL21 engineering strain for induction expression,using His-tag The recombinant protein of HA was purified by nickel column,and the purified recombinant protein was identified by SDS-PAGE and Western-blot. The results showed that when the recombinant strain p ET28 a( +)-HA/BL21( DE3) was induced at 30 ℃ and IPTG concentration was 1 mmol/L for 8 h,the expression of HA protein was the highest. SDS-PAGE electrophoresis showed that HA protein was purified by nickel column. The purity was higher,and Western blot showed a significant Western blot at 70 KD,which was consistent with expectations. It shows that the HA protein of avian influenza virus is highly expressed in E. coli,which lays a foundation for the subsequent research on avian influenza detection methods and vaccines.
引文
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[2] SMRT S T,DRANEY A W,LORIEAU J L. The influenza hemagglutinin fusion domain is an amphipathic helical hairpin that functions by inducing membrane curvature.[J]. Journal of Biological Chemistry(J Biol Chem),2015,290(1):228-38.
[3] GAO H,CUI H,CUI X,et al. Expression of HA of HPAI H5N1 Virus at US2 Gene Insertion Site of Turkey Herpesvirus Induced Better Protection than That at US10Gene Insertion Site[J]. Plos One(PLo S One),2011,6(7):e22549.
[4] MATSUOKA Y,SWAYNE D E,THOMAS C,et al.Neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of influenza H5N1 viruses for mice[J]. Journal of Virology(J Virol),2009,83(9):4704.
[5]杨昭君. H5N1禽流感病毒HA基因在原核系统的表达及其活性检测[D].广州:南方医科大学,2014.
[6] NICHOLAS R A J,AYLING R D. Mycoplasma bovis:disease,diagnosis,and control[J]. Veterinary Science(Vet Sci),2003,74:105-112.
[7] OCTAVIANI C P,OZAWA M,YAMADA S,et al. High level of genetic compatibility between swine[J]. Journal of Virology(J Virol),2010,84(20):10918-10922.
[8]王玲玲,吴胜昔,曾政,等.新城疫病毒F蛋白的原核表达及鉴定[J].黑龙江畜牧兽医,2018(7):36-39.
[9]张文帅,温恬,迟莹,等.禽流感病毒(H5N1)血凝素蛋白真核表达载体的构建与表达[J].细胞与分子免疫学杂志,2012,28(1):91-93.
[10]余华,蔡家利.禽流感病毒检测方法研究进展[J].动物医学进展,2009,30(12):78-81.
[11] OCTAVIANI C P,OZAWA M,YAMADA S,et al. High Level of Genetic Compatibility between ine-Origin H1N1and Highly Pathogenic Avian H5N1 Influenza Viruses[J]. Journal of Virology(J Virol),2010,84(20):10918-10922.
[12] JIANG Y,ZHANG H,LI C,et al. Increased expression and immunogenicity of optimized H5 subtype avian influenza HA gene containing chicken bisa codons[J]. Immunological Journal,2005.
[13]吴艳菊,张治业,倪小舒,等. H5N1亚型禽流感病毒NA和NS1蛋白原核表达及多克隆抗体制备[J].黑龙江畜牧兽医,2017(11):1-4.
[14]邹淑梅,于在江,张烨,等.禽流感病毒H5N1血凝素基因和神经氨酸酶基因在真核酵母菌中的表达[J].实验动物科学,2011,28(3):1-5.