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为了探讨风疹病毒包膜糖蛋白E1中二硫键对风疹病毒细胞融合活性的影响,在构建重组载体pBSK-SPE2E1的基础上,利用PCR定点突变与体内同源重组相结合的方法,构建了11个突变体,分别将E1外功能区的11个半胱氨酸残基突变为其它氨基酸残基,从而去除一个二硫键,利用Giemsa染色法定性检测由此引起的细胞融合情况,流式细胞术检测导入的外源DNA在细胞表面的表达效率,血吸附检测重组表达的突变体蛋白的受体识别活性。结果表明E1外功能区的10个二硫键对RV的细胞融合活性都有重要影响,任何一个二硫键的去除均导致E1的细胞融合活性丧失;其中第5和第8个半胱氨酸残基所形成的二硫键与E2和E1的相互作用有关,第3、第4和第13个半胱氨酸残基所形成的二硫键可能直接影响E1的细胞融合功能。
In order to investigate the effect of disulfide bond on the rubella virus cell fusion activity of rubella virus envelope glycoprotein E1, a recombinant vector pBSK-SPE2E1 was constructed based on the combination of site-directed mutagenesis with homologous recombination in vivo to construct 11 Mutants, 11 cysteine residues in the E1 outer domain were mutated to other amino acid residues, respectively, to remove one disulfide bond, and the resulting cell fusion was detected qualitatively by Giemsa staining. Flow cytometry The efficiency of expression of the introduced foreign DNA on the cell surface was measured by the blood adsorption assay, and the receptor recognition activity of the recombinantly expressed mutant protein was examined. The results showed that 10 disulfide bonds outside the E1 functional domain have an important influence on the cell fusion activity of RV, and any disulfide bond removal leads to the loss of the cell fusion activity of E1. The fifth and the eighth cysteines The disulfide bonds formed by the residues are related to the interaction of E2 and E1. The disulfide bonds formed by the 3rd, 4th and 13th cysteine residues may directly affect the cell fusion function of E1.