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目的为建立人巨细胞病毒(HCMV)活动性感染检测方法,表达HCMV早期复制的标志物pp65蛋白,以制备相应的抗体。方法将pp65全基因和其亲水性强、有抗原代表性的片段(pp65hp)分别克隆到表达载体pET22b(+)中诱导表达,并以免疫印迹考察重组蛋白的抗原性。结果重组rpp65全蛋白表达量低于全菌体蛋白的5%,而rp65hp蛋白获得高效表达,约为全菌体蛋白的40%,并与HCMVIgG抗体特异性结合。结论亲水性pp65hp片段有较好的抗原性,可以制备相应的抗体,来检测HCMV活动性感染。
Objective To establish a detection method of human cytomegalovirus (HCMV) active infection and express the pp65 protein, which is an early marker of HCMV replication, to prepare the corresponding antibody. Methods The full-length pp65 gene and its highly hydrophilic, antigen-presenting fragment (pp65hp) were cloned into the expression vector pET22b (+) to induce expression. The antigenicity of the recombinant protein was examined by Western blotting. Results The expression of recombinant rpp65 protein was lower than 5% of the total bacterial protein, while the protein of rp65hp was highly expressed, about 40% of the total bacterial protein and specifically bound to HCMVIgG antibody. Conclusion Hydrophilic pp65hp fragment has good antigenicity, and the corresponding antibody can be prepared to detect HCMV active infection.