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目的构建表达甲型H5N1流行性感冒(流感)病毒基质蛋白(Matrix Protein,M)2(M2)与1(M1)基因的脱氧核糖核酸(Deoxyribonucleic Acid,DNA)疫苗与腺病毒(Adenovirus,Ad)载体疫苗,将其联合免疫小鼠,对免疫效果进行评价。方法以中国分离的首株人H5N1亚型禽流感病毒(Avian Influenza Virus,AIV)[A(甲型)/Anhui(安徽)/1/2005]作为研究对象,聚合酶链反应(Polymerase Chain Reaction,PCR)扩增全长M2和M1基因,构建共表达H5N1亚型AIVM2和M1基因的重组pStar-M2/M1。利用Ad-Easy载体系统,在293细胞中包装出表达M2基因的重组Ad-M2和表达M1基因的重组Ad-M1。按初次免疫-加强免疫程序,分别用重组pStar-M2/M1和重组Ad-M2+Ad-M1免疫BALB/c小鼠,共免疫4次,每次间隔14d。第1、3次用DNA疫苗,第2、4次用重组Ad疫苗,每次免疫前及末次免疫后13d采集血清,用于检测体液免疫应答。末次免疫后14d,以H1N1亚型流感病毒A/PuertoRico(PR,波多黎各)/8/34/株进行攻击。病毒攻击后14d内,逐日观察小鼠的存活及体重变化情况。结果用间接免疫荧光(Indirect Immunofluorescence Assay,IFA)方法检测到了重组pStar-M2/M1、Ad-M2、Ad-M1载体上相应插入基因的表达,将其联合免疫小鼠后,酶联免疫吸附试验(Enzyme-linked Immunosorbent Assay,ELISA)检测到小鼠血清中的抗H5N1亚型流感病毒M2、M1的IgG抗体。病毒攻击实验结果表明,免疫后小鼠对流感病毒A/PR/8/34(H1N1)株的攻击有一定的交叉保护作用。结论成功构建了表达甲型H5N1流感病毒M2与M1基因的DNA疫苗与重组Ad疫苗,联合免疫小鼠后刺激产生了特异性的体液免疫应答,并对异源毒株具有一定的交叉保护作用。
Objective To construct Deoxyribonucleic Acid (DNA) vaccine and adenovirus (Adenovirus) that express Matrix Protein (M) 2 (M2) and M1 gene of Influenza A (H5N1) Carrier vaccine, its combined immunization of mice, the immune effect was evaluated. Methods The first isolate of H5N1 Avian Influenza Virus (AIV) [A (type A) / Anhui (Anhui) / 1/2005] isolated from China was used as research object. Polymerase Chain Reaction PCR) to amplify full-length M2 and M1 genes and construct a recombinant pStar-M2 / M1 co-expressing the H5N1 subtypes AIVM2 and M1 genes. Using Ad-Easy vector system, recombinant Ad-M2 expressing M2 gene and recombinant Ad-M1 expressing M1 gene were packaged in 293 cells. BALB / c mice were immunized with recombinant pStar-M2 / M1 and recombinant Ad-M2 + Ad-M1, respectively, for 4 immunizations with an interval of 14 days. The first and the third DNA vaccine, the first 2,4 times with recombinant Ad vaccine, before each immunization and 13d after the last immunization serum was collected for the detection of humoral immune response. 14 days after the last immunization, the H1N1 subtype influenza A / Puerto Rico (PR, Puerto Rico) / 8/34 / strain was challenged. Within 14 days after the virus attack, the mice were observed daily for changes in survival and weight. Results The expression of the corresponding inserted gene in recombinant pStar-M2 / M1, Ad-M2 and Ad-M1 vectors was detected by indirect immunofluorescence assay (IFA) IgG antibodies against H5N1 subtype influenza virus M2 and M1 in mouse sera were detected by Enzyme-linked Immunosorbent Assay (ELISA). The result of virus attack experiment showed that the mice had some cross-protection against the challenge of influenza A / PR / 8/34 (H1N1) strain after immunization. Conclusion DNA vaccine and recombinant Ad vaccine expressing M2 and M1 genes of Influenza A H5N1 influenza virus were constructed successfully. The combination of DNA vaccines and recombinant Ad vaccines resulted in specific humoral immune responses stimulated by the combined immunized mice and cross-protection against heterologous strains.