Expression of the nucleoprotein of rabies virus inEscherichia coli and mapping of antigenic sites
Expression of the nucleoprotein of rabies virus inEscherichia coli and mapping of antigenic sites
复制标题
狂犬病病毒核蛋白在大肠杆菌中的表达及抗原位点定位
作者：
Hideo Goto;N. Minamoto;Hiroshi Ito;Ting Rong Luo;M. Sugiyama;T. Kinjo;A. Kawai
狂犬病病毒核蛋白(N)在狂犬病病毒和狂犬病相关病毒中具有很好的保守性，因此对狂犬病病毒核蛋白(N)的抗原性进行了研究。将RC-HL株的N基因克隆到表达载体pET3a中，构建成重组表达载体pET3a。ColiBL21(DE3)进行转化。在异丙基-1-硫代-D-半乳糖苷的诱导下，转化子产生了一种大小(56k-Da)的蛋白，其大小与真正的N蛋白几乎相同。该蛋白还与我们的一组N蛋白特异性单抗(N-MAb)反应，其中包括针对抗原位点I和IV的抗体。通过使用该cDNA，产生了各种缺失突变体并在其中表达。用Western印迹法检测突变蛋白与N-单抗的反应性。C端67个氨基酸残基的缺失并不意味着它们与针对I和IV位点的任何N-MAb的反应性消失。然而，当C末端缺失91个或更多残基时，蛋白质失去了反应性，这表明抗原I和IV被定位到一个最多包含24个氨基酸残基的小区域，从360位到383位。将该24个氨基酸序列与其他几个Lyssav株N蛋白的相应区域进行比较，发现抗原点I被定位在360到369位。
SummaryInvestigations were performed to delineate the antigenic sites I and IV of rabies virus nucleoprotein (N), the former of which is well conserved among the rabies and rabies-related viruses. The N cDNA of the RC-HL strain was inserted into an expression vector pET3a, with which theE. coli BL21(DE3) was transformed. Upon induction with isopropyl-1-thio-ß-D-galactoside, the transformants produced a protein with a size (56k-Da) almost identical to that of the authentic N protein. The protein also reacted with a panel of our N protein-specific monoclonal antibodies (N-MAbs) including the antibodies against the antigenic sites I and IV. By using the cDNA, various deletion mutants were generated and expressed inE. coli to examine the reactivity of mutant proteins with N-MAbs by Western blot analysis. Deletion of the C-terminal 67 amino acid residues did not abolish their reactivity with any of the N-MAbs specific for the sites I and IV. When 91 residues or more were deleted from the C-terminus, however, the protein lost the reactivity, indicating that the antigenic sites I and IV are mapped to a small region which is comprised of at most 24 amino acid residues from positions 360 to 383. Comparison of the 24-amino acid sequence with the corresponding region of N protein of several other Lyssavirus strains suggests that the antigenic site I is mapped to positions 360 to 369.
DOI：
10.1073/pnas.88.5.2001
发表时间：
1991-03-01
影响因子：
11.1
作者：
FU, ZF;DIETZSCHOLD, B;KOPROWSKI, H
通讯作者：
KOPROWSKI, H
ERA 狂犬病病毒核蛋白和非结构蛋白上的抗原位点。
DOI：
10.1099/0022-1317-66-10-2125
发表时间：
1985
期刊：
The Journal of general virology
影响因子：
--
作者：
Lafon,M;Wiktor,TJ
通讯作者：
Wiktor,TJ