传染性法氏囊病病毒C4毒株VP2基因的原核表达及生物信息学分析  

Prokaryotic Expression and Bioinformatics Analysis of VP2 Gene of Infectious Bursal Disease Virus C4 Strain

作　　者：王洁琼[1] 赵玉杰[1] 周云飞[1] 黄宗梅[1] 陈盼盼[1] 周薇帆 刘琳[1] 李新生[1] WANG Jie-qiong;ZHAO Yu-jie;ZHOU Yun-fei;HUANG Zong-mei;CHEN Pan-pan;ZHOU Wei-fan;LIU Lin;LI Xin-sheng(College of Animal Science and Veterinary Medicine, Henan Agricultural University,Zhengzhou 450002, China)

机构地区：[1]河南农业大学牧医工程学院,郑州450002

出　　处：《中国畜牧兽医》2017年第12期3585-3591,共7页China Animal Husbandry & Veterinary Medicine

基　　金：国家十三五科技专项(2017YFD0500701)

摘　　要：试验旨在研究一株传染性法氏囊病病毒(IBDV)河南分离株的毒力特征及其与VP2氨基酸序列特征的关系。通过提取IBDV C4株RNA,利用RT-PCR扩增其VP2基因,与其他不同毒力IBDV毒株进行核苷酸及推导的氨基酸序列比对分析,同时使用pET-32a(+)原核表达载体表达VP2基因,用SDS-PAGE和Western blotting检测重组VP2蛋白的表达。结果显示,扩增的IBDV C4株的VP2基因序列在进化关系上属于超强毒力IBDV(vvIBDV)分类,与选取的vvIBDV毒株代表毒株核苷酸序列同源性在98.1%~98.7%之间,其七肽区为S-W-S-AS-G-S(第326—332位氨基酸)符合超强毒株特征,且222(A)、256(I)、294(I)和299(S)位氨基酸与超强毒力毒株的4个特征性氨基酸一致;但IBDV C4毒株的VP2蛋白氨基酸序列与超强毒力毒株代表毒株UK661相比,201(D/G)、281(G/R)、313(V/A)位氨基酸不同,其中281位氨基酸的改变处于279—290的小亲水区内,与病毒抗原性有关;构建的pET-32a(+)-VP2原核表达载体在大肠杆菌BL21感受态细胞上表达出分子质量约67ku的重组VP2蛋白,为进一步比较201(G)、281(R)、313(A)位氨基酸差异导致的抗原特性改变提供了研究基础。本试验结果表明,IBDV C4株VP2基因与vvIBDV毒株VP2基因的主要特性一致,但也有3处氨基酸与代表毒株UK661存在差异,这些改变可能与中国IBDV毒株毒力的进化有关。This study was aimed to investigate the relationship between the virulence characteristics of infectious bursal disease virus(IBDV)C4strain and its VP2amino acid sequence.The RNA of IBDV C4strain was extracted,and its VP2gene was amplified by RT-PCR.VP2nucleotide sequences and deduced amino acids of different virulent IBDV strains were compared.At the same time,prokaryotic expression vector pET-32a(+)was used to express the VP2gene.The expression of recombinant VP2protein was detected by SDS-PAGE and Western blotting.The results showed that the VP2gene of IBDV C4strain belonged to the very virulent infectious bursal disease virus(vvIBDV)in evolutionary relationship,the VP2nucleotides homology between IBDV C4strain and other vvIBDV strains were98.1%to98.7%,and there were no mutations in S-W-S-A-S-G-S(326-332amino acids)and222(A),256(I),294(I)and299(S).The VP2amino acid sequence of IBDV C4strain was consistent with the characteristics of other vvIBDV strains.However,there were three differences amino acids sites at201(D/G),281(G/R)and313(V/A)between the amino acids of the C4strain and the very virulent strain UK661.And the change of281(R)was in the small hydrophilic region of279to290,which was related to the antigenicity of the virus;The recombinant VP2protein molecular weight expressed in Escherichia coli BL21was about67ku.This study provided a basis for further research on antigenic changes resulting from amino acid variation of201(G),281(R)and313(A).These results indicated that the VP2gene of the IBDV C4strain was consistent with the major characteristics of the vvIBDV strain VP2gene.The difference of three amino acid sites in the vvIBDV strain C4might be related to the evolution of virulence of IBDV strain in China.

关 键 词：传染性法氏囊病病毒(IBDV) VP2 分子特征 原核表达 

分 类 号：S852.659.4[农业科学—基础兽医学]