甲型H_5N_1流行性感冒病毒基质蛋白2与基质蛋白1双基因载体疫苗的构建及其免疫学评价

Construction and Immunological Evaluation of Mammalian Cell Vector Co-expressing Matrix Protein 2 and Matrix Protein 1 Genes of H_5N_1 Influenza Virus

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 recombinant Deoxyribonucleic Acid （DNA）and adenoviral vaccines expressing matrix protein 2 （M2）and Ml genes of H5N1 influenza virus and evaluate the effect of combined immunization in mice. Method Based on the human H5N1 avian influenza virus strain A/Anhui/1/2005 isolated in China, M2 and M） genes were amplified by Palymerase Chain Reaction（PCR）and then subcloned into pStar vector to construct two genes co-expressing recombinant DNA vaccine pStar-M2/ Ml. On the other hand, using Ad-Easy adenovirus vector system, two recombinant adenoviruses, namely Ad-M2 and Ad-M1 were constructed. Then the recombinant DNA vaccine and adenoviral vaccines were combined in immunization of BALB/e mice with a prime-boost regime. On day 0 and day 28, the mice / were immunized with DNA vaccine, and on day 14 and day 42, with recombinant adenovirus vaccines. Blood samples were collected before administered and 13 days after the final injection for Eazyme-Linked Immunosorbent Assay（ELISA）. 14 days after the final injection, the mice were attacked with H1N1 influenza virus A/Puerto Rieo/8/34/（A/ PR/8/34）. Survival rate and weight change of the miee were observed for 14 days. Results After transfeetion of 293 cells with the pStar-M2/M, Ad-M2 ~nd Ad-M1, indireet immunofluoreseenee assay（IFA）confirmed that M2 and M1 genes on the recombinant vectors expressed successfully. After combined immunization, ELISA showed that the vaccines successfully induced specific IgG antibody in serum. Experiment of influenza virus challenge showed that cross-protection against H1N1 influenza virus A/PR/8/34 was achieved. Conclusions Recombinant DNA and adenoviral vaccines expressing M2 and M1 genes of influenza H5N1 virus were constructed successfully. Specific immune responses were induced and cross-protection against influenza virus was achieved after combined immunization in mice.