融合基因DNA疫苗STxB-VP1和MDC-VP1对CVB3的免疫效果研究

Construction of STxB-VP1 and MDC-VP1 fusion DNA vaccine and study on its immunological effects on mice

目的构建融合基因DNA疫苗pcDNA3/STxB-VP1和pcDNA3/MDC-VP1,并免疫小鼠,观察其免疫效果。方法从痢疾志贺菌PCR扩增志贺毒素B亚单位(STxB)基因片段,用RT-PCR法从小鼠脾细胞扩增巨噬细胞来源的趋化因子(MDC)基因片段。将这两个基因片段分别与柯萨奇病毒B组3型(CVB3)的衣壳蛋白VP1通过一个编码15肽的柔性接头(Linker)相连接,构建真核表达质粒pcDNA3/STxB-VP1和pcDNA3/MDC-VP1。120只BALB/c小鼠随机均分为6组,分别在胫骨前肌注射pcDNA3、pcD-NA3/STxB、pcDNA3/MDC、pcDNA3/VP1、pcDNA3/STxB-VP1和pcDNA3/MDC-VP1,每3周1次,共3次。每次免疫后20d检测血清中中和抗体水平,最后1次免疫后20d用7LD50的CVB3进行致死性攻击,观察小鼠生存率。结果成功构建了融合基因疫苗pcD-NA3/STxB-VP1和pcDNA3/MDC-VP1。各组小鼠的生存率分别是10%、10%、15%、40%、20%和75%,中和抗体水平及血中病毒滴度均与生存率一致。结论与pcDNA3/VP1相比,pcDNA3/MDC-VP1可诱导产生高水平的中和抗体,提高小鼠生存率,而pcDNA3/STxB-VP1仅诱导出低水平的中和抗体,小鼠的生存率降低。

Objective To Construct fusion gene DNA vaccine pcDNA3/STxB-VP1 and fusion gene DNA vaccine pcDNA3/MDC- VP1, then the two vaccines were inoculated to mice and stuy their immunological effects. Methods B subunit of Shiga Toxin （STxB） gene fragment were amplified by PCR from the DNA of Shigella dysenteriae and Macmphage-derived chemokine （MDC） gene fragment was amplified by RT-PCR from the total RNA of a mouse spleen cells. The DNA fragments of STxB and MDC waere respectively linked to coxsackievirus 133 （CVB3） VP1 by a DNA sequence which encode a flexible polypeptide （15 amino acids） to construct eukaryotic expression plasmids pcDNA3/STxB-VP1 and pcDNA3/MDC-VP1. BALB/c mice were randomized to 6 groups which were respectively immunized pcDNA3, pcDNA3/STxB, pcDNA3/MDC, pcDNA3/VP1, pcDNA3/STxB-VP1 and pcDNA3/MDC-VP1 for 3 times at 3-week intervals, intramuscularly （i. m. ） in tibialis anterior muscle. The levels of the serum neutralizing antibodies were detected 20d after each inoculation. The mice were challenged with 7 LD60 CVB3 3 weeks after the last immunization. Results The fusion gene vaccines pcDNA3/STxB-VP1 and pcDNA3/MDC-VP1 were constructed successfully. The survival rates of each group were 10%, 10%, 15%, 40%, 20% and 75%, respectively, and the levels of neutralizing antibody titers, virused titers, were all consistent with those survive rates in each group. Conclu- sion Comparing with pcDNA3/VP1, pcDNA3/MDC-VP1 vaccine can induce a high level of neutralizing antibody titers and result in a higher survival rate in mice, while pcDNA3/STxB-VP1 induce a low level of neutralizing antibody titers and result in a lower survival rate in mice .