The study of the antigenic and sialic acid binding sites on the hemagglutinin molecule of influenza virus by recombinant DNA technique.

将B-1-23病毒Hal区190位氨基酸发生改变的变异株A/USSR/90/77(A/USSR/77)(H1N1)的HA基因全长克隆到表达载体SV40中。重组病毒感染的CV-1细胞上表达的HA蛋白与感染B-1-23病毒的细胞上表达的HA蛋白的吸血或融合活性无明显差异。用5株抗A/USSR/77病毒HA的单抗检测表达的HA蛋白的抗原性。除mAb264外，其余单抗均可与HA蛋白反应。为了确定表位264的区域，我们制备了7个经定点突变修饰的HA cDNA，并将其克隆到SV40表达载体(SVHA)中。Raymond等人的核苷酸序列分析表明，A/BASIS/11/78的变异氨基酸残基为219(Lys)和227(Glu)，A/Lackland/3/78的189(Lys)和225(Asp)可能包含在表位265区域。我们的结果表明，在这些氨基酸中，有190和219个残基，可能还有189个残基，涉及到表位264，但不包括残基225和227。

Full-length cDNA of HA gene derived from B-1-23 virus, a variant of A/USSR/90/77 (A/USSR/77) (HlNl) which had been selected with monoclonal antibody (mAb) 264 and contained an amino acid change at position 190 of the HAl region, was cloned into the SV40 expression vector. Hemabsorbing or fusion activities of the HA protein expressed on CV-1 cells infected with the recombinant virus were indistinguishable from those of the authentic HA protein on B-1-23 virus infected cells. The antigenicity of the expressed HA protein was examined with five monoclonal antibodies to the HA of A/USSR/77 virus. The HA protein reacted with all mAbs except for mAb264. To define the area of epitope 264, we prepared seven HA cDNAs modified by site specific mutagenesis, and cloned them into the SV40 expression vector (SVHA). Raymond et al showed that changed amino acid residues 219 (lys) and 227 (Glu) in A/Brazil/11/78, and 189 (Lys) and 225 (Asp) in A/Lackland/3/78 were presumaly included in the area of epitope 265 by the nucleotide sequence analysis. Of those amino acids, our results shows it is the residues 190 and 219 and possibly residue 189 that are involved in the epitope 264, but neither the residue 225 nor 227.