流感病毒受体结合表位真核载体的构建及表达

Construction of eukaryotic expression plasmid for receptor binding of influenza virus and their expression

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 vectors for the signal peptide（SP） of hemagglutinin（HA） of influenza A virus and its acylneuraminate receptor binding（RB） sites incorporating gene segments of T and B epitopes（RBT and RBB,respectively） and to observe the expression of target protein by and immunological characteristics of eukaryotic cells.Methods A polypeptide linker Gly4Ser was used to splice SP with RB,RBT,and RBB by overlap-PCR to respectively construct SP-RB,SP-RBT,and SP-RBT fusion genes.These genes were inserted into eukaryotic expression plasmids pcDNA3.1（＋）.After identification,pcDNA3.1（＋）/SP-RB,pcDNA3.1（＋）/SP-RBT,and pcDNA3.1（＋）/SP-RBB were transfected into MDCK cells.The expression of these genes was analyzed with RT-PCR,immunofluorescence assay,and lymphocyte proliferation test with MTT.Results pcDNA3.1（＋） expression vectors incorporating SP-RB,SP-RBT,and SP-RBT fusion genes were successfully constructed.The fusion protein was detected in MDCK transfected with pcDNA3.1（＋）/ SP-RB,pcDNA3.1（＋）/SP-RBT,or pcDNA3.1（＋）/SP-RBB and stimulated lymphocyte proliferation.Conclusion Eukaryotic expression plasmids for SP-RB,SP-RBT and SP-RBT fusion genes were successfully constructed and the expression products stimulated lymphocyte proliferation,establishing a solid foundation for further study of receptor binding,immunological study of T and B cell epitopes of influenza virus,and study of DNA vaccines.