【摘要】 目的引入6种不同的Kozak类似序列制备口蹄疫DNA疫苗,比较分析Kozak类似序列中不同碱基组成对口蹄疫DNA疫苗表达水平及免疫效果的影响作用。方法在口蹄疫DNA疫苗抗原编码基因VP1起始位点引入Kozak通用序列及所设计的5种Kozak类似序列(KZ1-KZ6),插入真核表达载体pVAX1,制备口蹄疫DNA疫苗,对6～8周龄BALB/c小鼠进行免疫,用间接ELISA分析实验动物外周血VP1的抗体滴度；同时插入带有绿色荧光蛋白的融合表达载体pEGFP-N1构建6种体外表达质粒,转染HeLa细胞,进行瞬时表达并分析体外表达水平。结果引入含三联密码AAA的Kozak类似序列KZ6的疫苗质粒pKV6接种动物后产生的免疫抗体滴度高于其他组；相应的体外表达质粒pKG6转染HeLa细胞后体外表达水平亦高于其他组。结论含三联密码AAA的Kozak类似序列KZ6的口蹄疫DNA疫苗,能特异性地增强口蹄疫DNA疫苗的表达水平及免疫效果。更多还原

【Abstract】 Objective To construct the Foot-and-Mouth disease DNA vaccine with six different Kozak similar sequences,and to compare and analyze the effect of different nucleotides of Kozak.Sequences on expres- sion and immunizing efficiency of Foot-and-Mouth disease DNA vaccine.Methods Six different Kozak similar se- quences(KZ1-KZ6)were inserted into the initiation site of the antigenic gene coding VP1 within the FMD DNA vaccine and were connected with the vector pVAX1,FMD DNA vaccine were constructed to immunize female BALB/c mice aged 6-8 weeks.The anti-VP1 antibodies values in peripheral blood of the experimental animals were analyzed using indirect enzyme linked immunusorbent assay.After the antigenic gene coding VP1 had been rebuilt. Then the sequences were inserted into the fusion expression vector pEGFP-N1 of green fluorescent protein.The se- quences were inserted into the fusion expression vector pEGFP-N1,the in vitro expression plasmids containing GFP were constructed and transfected HeLa cells for transient expression.Results There was a significant difference of expression efficiency by HeLa cells between the pKG6 and other groups(P＜0.05).The mice produced signifi- cantly higher anti-VP1 antibody level after DNA immunization with pKV6 than that of other recombinant plasmids (P＜0.05).Conclusion FMD DNA vaccine inserted Kozak similar sequences of Triple codon AAA can specifi- cally increase the vaccine protein expression.更多还原