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目的明确M122蛋白与宿主因子Myst4相互作用的位点。方法以诱饵载体pGBKT7-M122为模板,采用PCR方法扩增不同长度大小的M122基因片段,并将其分别插入至pMD-T simple载体,构建含有不同长度M122基因片段的重组质粒。将构建成功的各个重组质粒分别用限制性内切酶EcoRI和SalI进行双酶切鉴定,并送测序。将测序正确的重组质粒采用同样的内切酶进行双酶切,凝胶回收试剂盒回收不同长度大小的M122基因片段,并将其分别亚克隆至pGBKT7-BD载体构建含有不同长度M122基因片段的诱饵质粒。将构建成功的各个诱饵质粒分别用限制性内切酶EcoRI和SalI进行双酶切鉴定,并送测序。将测序正确的各个诱饵质粒分别与pGADT7-Myst4质粒共同转化至酵母菌株AH109感受态细胞,转化后的酵母细胞分别涂板于营养缺陷培养基SD/-Trp/-Leu和SD/-Trp/-Leu/-His/-Ade/X-α-Gal平板。M122与宿主因子Myst4相互作用的位点通过营养缺陷筛选确定。结果成功扩增了编码不同M122蛋白突变体的基因片段,并将其正确插入诱饵载体,构建了含有不同长度大小M122基因片段的诱饵质粒;通过酵母双杂交筛选明确了M122蛋白与宿主因子Myst4相互作用的结合位点位于M122蛋白的1～148氨基酸。结论 M122蛋白的1～148氨基酸是其与宿主因子Myst4相互作用所必需的,为研究M122蛋白在MCMV致神经系统损伤的分子机制中的作用提供了实验基础。
Objective To clarify the interaction between M122 protein and host factor Myst4. Methods The bait vector pGBKT7-M122 was used as a template to amplify M122 gene fragments of different length by PCR and inserted into pMD-T simple vector respectively to construct a recombinant plasmid containing different lengths of M122 gene fragment. The constructed recombinant plasmids were double digested with restriction endonucleases EcoRI and SalI respectively and sequenced. The recombinant plasmid with the correct sequencing was double-digested by the same endonuclease. The M122 gene fragment of different length was recovered by the gel recovery kit and subcloned into the pGBKT7-BD vector to construct the M122 gene fragment with different length Bait plasmid. Each constructed bait plasmid was double-digested with restriction endonucleases EcoRI and SalI respectively and sequenced. The bait plasmids with the correct sequencing were co-transformed into the competent cells of yeast strain AH109 respectively with the pGADT7-Myst4 plasmid. The transformed yeast cells were plated on SD / -Trp / -Leu and SD / -Trp / Leu / -His / -Ade / X-α-Gal plate. The site of M122 interaction with the host factor Myst4 was determined by selection of auxotrophy. Results The gene fragment encoding different M122 protein mutant was successfully amplified and inserted into the bait vector successfully. The bait plasmid containing M122 gene fragment of different length was constructed. By yeast two-hybrid screening, M122 protein was identified with the host factor Myst4 The binding site of action is between 1 and 148 amino acids of the M122 protein. Conclusion The 1-181 amino acids of M122 protein are necessary for its interaction with host factor Myst4 and provide the experimental basis for studying the role of M122 protein in the molecular mechanism of nervous system damage induced by MCMV.