禽白血病病毒p27基因的原核表达及生物信息学分析
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摘要
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)S_6,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)S_6. 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.
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)S_6. 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.
引文
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[13] 马超英.禽呼肠孤病毒与禽白血病病毒双重RT-PCR检测方法的建立及应用[J].中国畜牧兽医,2013,40(2):53-56.
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[15] 胡海,钱琨,张丹阳,等.禽白血病病毒衣壳蛋白p27基因绝对定量PCR方法的建立及应用[J].中国家禽,2015,37(20):16-20.
[2] 孙舒,胡海,程晓薇,等.禽白血病病毒衣壳蛋白表达载体的构建及应用[J].中国家禽,2017,39 (18):18-22.
[3] 赵子君,饶明章,陈建,等.K亚群禽白血病病毒5′LTR序列及启动活性分析[J].畜牧兽医学报,2018,49(4):754-760.
[4] CHEN M,GRANGER A J,VANBROCKLIN M W,et al.Inhibition of Avian leukosis virus replication by vector-based RNA interference[J].Virology,2007,365(2):464-472.
[5] SILVA R F,FADLY A M,TAYLOR S P.Development of a polymerase chain reaction to differentiate Avian leukosis virus (ALV) subgroups:detection of an ALV contaminant in commercial Marek’s disease vaccines[J].Avian Dis,2007,51(3):663-667.
[6] WANG Y,XU S,LI S,et al.Lamivudine inhibits the replication of ALV-J associated acutely transforming virus and its helper virus and tumor growth in vitro and in vivo[J].Front Microbiol,2015,6:1306.
[7] 常超越,闫艳娟,李蕴玉,等.蛋种鸡禽白血病与大肠杆菌病混合感染的诊治[J].黑龙江畜牧兽医,2018(14):130-132.
[8] 毛娅卿.禽白血病病毒抗原/抗体ELISA检测方法的建立及准种分析[D].北京:中国农业大学,2014.
[9] 谢华丽.禽白血病抗原和抗体ELISA检测方法的建立[D].武汉:华中农业大学,2013.
[10] 王广龙,王皓然,王勇,等.ELISA检测方法在禽白血病净化中的应用[J].中兽医学杂志,2017(1):67-68.
[11] 仇钰,秦爱建,钱琨,等.间接ELISA检测抗禽白血病病毒抗体方法的建立[J].中国家禽,2010,32(22):16-20.
[12] YUN B,LI D,ZHU H,et al.Development of an antigen-capture ELISA for the detection of Avian leukosis virus p27 antigen[J].J Virol Methods,2013,187(2):278-283.
[13] 马超英.禽呼肠孤病毒与禽白血病病毒双重RT-PCR检测方法的建立及应用[J].中国畜牧兽医,2013,40(2):53-56.
[14] 邢秀皎.禽白血病病毒逆转录环介导等温扩增检测方法的研究[D].北京:中国兽医药品监察所,2011.
[15] 胡海,钱琨,张丹阳,等.禽白血病病毒衣壳蛋白p27基因绝对定量PCR方法的建立及应用[J].中国家禽,2015,37(20):16-20.