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目的构建甲型流感病毒血凝素(HA)信号肽(SP)与HA唾液酸受体结合部位(RB)所含T、B表位的基因片段(RBT、RBB)真核表达质粒,研究其在真核细胞中的表达与免疫特性。方法通过重叠延伸PCR法(overlap-PCR)将HA的SP分别与RB、RBT、RBB通过一段多肽接头Gly4Ser融合为SP-RB、SP-RBT和SP-RBT,将其分别插入pcD-NA3.1(+)载体,构建质粒,鉴定正确后转染MDCK细胞,RT-PCR、免疫荧光、MTT检测该质粒的表达和分泌。结果融合基因真核表达质粒构建成功,在MDCK细胞中成功表达,转染细胞培养上清能刺激淋巴细胞增殖。结论SP-RB、SP-RBT、SP-RBB真核表达载体成功构建,表达产物能刺激淋巴细胞增值,为流感病毒唾液酸受体结合部位的T、B细胞表位免苗研究提供初步依据,也为流感核酸疫苗的研制奠定了基础。
Objective To construct eukaryotic expression plasmids of RBT and RBB contained in influenza virus (HA) signal peptide (SP) and HA sialic acid receptor binding site (RB) In eukaryotes expression and immune characteristics. Methods SP-RB, SP-RBT and SP-RBT were fused with RB, RBT and RBB to SP-RB, SP-RBT and SP-RBT respectively by overlapping peptide linker Gly4Ser by overlap-PCR. (+) Vector was constructed and transfected into MDCK cells. The expression and secretion of this plasmid were detected by RT-PCR, immunofluorescence and MTT. Results The fusion gene eukaryotic expression plasmid was successfully constructed and successfully expressed in MDCK cells. Transfection of cell culture supernatants stimulated lymphocyte proliferation. Conclusion The eukaryotic expression vector SP-RB, SP-RBT and SP-RBB were constructed successfully. The expressed product stimulated the proliferation of lymphocytes and provided a preliminary basis for the study on the immunoprecipitation of T and B cell epitopes in the binding site of influenza virus sialic acid receptor. Also laid the foundation for the development of influenza nucleic acid vaccine.