摘要
目的原核表达小反刍兽疫病毒(peste-des-petits ruminants virus,PPRV)N基因,评价其抗原性,并进行生物信息学分析。方法参照GenBank中登录的PPRV(Nigeria/75/1)N基因序列,人工合成该基因,克隆至原核表达载体pET-30a(+),IPTG诱导N基因原核表达,表达产物进行SDS-PAGE及Western blot分析。运用Expasy Protparam和SOPMA软件对PPRV N基因编码的氨基酸序列进行生物信息学分析。结果成功表达了PPRV N基因,其融合蛋白相对分子质量约64 400,主要以包涵体形式存在,并可被PPRV羊免疫血清特异性识别。PPRV N蛋白含524个氨基酸,相对分子质量约57 900,亲水性氨基酸占57.63%,疏水性氨基酸占42.17%,碱性氨基酸占12.21%,酸性氨基酸占14.12%,等电点理论值为5.11;亲水性平均系数为-0.296,不稳定系数为48.20;二级结构存在242个α螺旋(占46.18%),42个β转角(占8.02%),164个无规则卷曲(占31.3%),76个延伸链(占14.50%)。结论原核表达的PPRV N蛋白具有良好的特异性和抗原性,为单克隆抗体的制备及快速诊断方法的建立提供了参考。
Objective To express the N gene of peste-des-petits ruminants virus（PPRV） in prokaryotic cells,evaluate its antigenicity and analyze its bioinformatics. Methods According to the published sequence of N gene of PPRV Nigeria75/1 strain in Gen Bank,the gene was synthesized and cloned into prokaryotic expression vector pET-30a（＋）. Fusion protein N was expressed under induction of IPTG and analyzed by SDS-PAGE and Western blot. Expasy Protparam and SOPMA software were used to analyze the sequence of amino acid encoded by N gene of PPRV. Results PPRV N fusion protein with a relative molecular mass of about 64 400 was successfully expressed,which mainly existed in a form of inclusion body and was recognized specifically with serum of sheep immunized with PPRV. Bioinformatics analysis showed that the N protein,with a relative molecular mass of 57 900,contained 524 amino acid residues,in whic h hydrophilic and hydrophobic amino acids accounted for 57. 63% and 42. 17%,while alkaline and acidic amino acids accounted for 12. 21% and 14. 12%,respectively. The theoretical isoelectric point was 5. 11,while the mean hydrophilicity coefficient was-0. 296,and the coefficient of instability was 48. 20. The secondary structure of the protein consisted of242 alpha helices（46. 18%）,42 beta sheet（8. 02%）,164 irregular coil（31. 3%）and 76 extended strands（14. 50%）.Conclusion The N protein of PPRV expressed in prokaryotic cells showed good specificity and antigenicity,which provided a reference for the preparation of monoclonal antibody and development of rapid diagnostic methods.
出处
《中国生物制品学杂志》
CAS
CSCD
2017年第8期806-810,共5页
Chinese Journal of Biologicals
基金
国家科技支撑计划项目(2013BAD12B04)
关键词
小反刍兽疫病毒
N蛋白
原核表达
生物信息学分析
Peste des petits ruminants virus（PPRV）
N protein
Prokaryotic expression
Bioinformatics analysis
