禽白血病病毒p27基因的原核表达及生物信息学分析

Prokaryotic expression and bioinformatics analysis of Avian leukosis virus p27 gene

为了进一步研究禽白血病病毒(Avian leukosis virus,ALV)p27蛋白的结构与功能,并获得具有反应原性的重组蛋白p27,试验通过PCR技术克隆p27基因,构建克隆载体pMD19-T-p27,并应用生物信息学软件对获得的p27基因序列进行分析,构建重组表达载体pET-28a-p27,并将其转化至大肠杆菌BL21(DE3)感受态细胞中诱导表达,通过SDS-PAGE和Western-blot对获得的重组蛋白p27进行分析。结果表明:通过PCR技术克隆获得ALV p27基因,并成功构建了克隆载体pMD19-T-p27;生物信息学分析发现,p27蛋白由239个氨基酸组成,分子式为C(1140)H(1853)N(323)O(339)S6,理论等电点为6.23,无信号肽和跨膜区,含有8个丝氨酸磷酸化位点和13个苏氨酸磷酸化位点,无糖基化位点,共有9个抗原表位,二级结构和三级结构中以α-螺旋为主;成功构建了重组表达载体pET-28a-p27,并在大肠杆菌BL21(DE3)感受态细胞中高效表达;诱导6小时时蛋白质表达量最大,分子质量约为27 ku,且以可溶性为主;重组蛋白p27具有较好的反应原性和特异性。说明试验获得了具有反应原性的重组蛋白p27,为建立禽白血病快速诊断方法和新型疫苗的研发提供了理论基础。

In order to study the structure and function of Avian leukosis virus(ALV) p27 protein,and to obtain a recombinant p27 protein with good immunogenicity,PCR method was used in this experiment for cloning p27 gene,and the recombinant clonging vector pMD19-T-p27 was constructed. Bioinformatics softwares were applied to analyze the obtained p27 gene sequence,and the recombinant expression vector pET-28 a-p27 was constructed,which was transformed into E. coli BL21(DE3) competent cells and inducible expression was carried out. SDS-PAGE and Western-blot methods were used for the analysis of the obtained recombinant p27 protein. The results showed that ALV p27 gene was obtained by PCR cloning,and the recombinant clonging vector pMD19-T-p27 was successfully constructed. Bioinformatics analysis found that p27 protein consisted of 239 amino acids,and the molecular formula was C(1140)H(1853)N(323)O(339)S6. The theoretical isoelectric point was 6.23 and there was no signal peptide or transmembrane region. There were 8 serine phosphorylation sites and 13 threonine phosphorylation sites,with no glycosylation sites. There were 9 antigenic epitopes,and α-helix was dominant in the secondary structure and tertiary structure. The recombinant expression vector pET-28 a-p27 was successfully constructed,and was highly expressed in E. coli BL21(DE3) competent cells. SDS-PAGE result showed that protein expression was the highest at 6 h induction,with a molecular mass of about 27 ku and mainly soluble. Western-blot result showed that the recombinant p27 protein had good immunogenicity and specificity. The results suggested that the recombinant p27 protein with immunogenicity was obtained in this experiment,which provided a theoretical basis for the establishment of a rapid diagnostic method and a new vaccine for avian leukosis.