小鼠β防御素2与柯萨奇病毒B3 VP1融合基因疫苗的构建和免疫效果研究

Construction of the mouse beta-defensin-2 and coxsackievirus B3 VP1 fusion DNA vaccine and study on its immunological effects in mice

目的:构建小鼠β-防御素2(Mouse beta-defensin-2,mBD2)与柯萨奇病毒B组3型(Coxsackievirus B3,CVB3)VP1融合基因疫苗,并观察其对小鼠的免疫效果。方法:克隆mBD2基因,构建重组质粒pcDNA3/mBD2和pcDNA3/mBD2-L-VP1。将4～6周龄BALB/c雄性小鼠随机分为5组,分别于股四头肌注射PBS(A组)、pcDNA3(B组)、pcDNA3/mBD2(C组)、pcDNA3/VP1(D组)、pcDNA3/mBD2-L-VP1(E组),每次接种剂量100μg/只,3周免疫1次,共3次。每次免疫后第14天眼眶静脉取血,用微量中和试验滴定血清中和抗体效价。第三次免疫后第21天,每组随机取3只小鼠,制备脾淋巴细胞悬液,用CCK-8细胞计数法检测特异性CTL的杀伤活性;每组取3只小鼠以3LD50CVB3病毒攻击,第7天取血处死,检测血清病毒滴度。结果:成功克隆了mBD2基因,构建了pcDNA3/mBD2和pcDNA3/mBD2-L-VP1两种重组质粒;D组和E组血清中和抗体滴度随免疫次数增加而提高(P<0.01);E组血清中和抗体滴度和脾细胞特异性CTL杀伤活性显著高于其他组(P<0.01),血中CVB3病毒滴度显著低于其他组(P<0.01)。结论:pcDNA3/mBD2-L-VP1能诱导小鼠对CVB3产生较强的体液免疫和细胞免疫,能有效抑制病毒在体内增殖。

Objectlve：To construct recombinant DNA vaccine expressing the fusion protein of mouse beta-defensin-2（mBD2） and CVB3VP1 and to evaluate the specific immune response induced. Methods：The mBD2 gene was cloned by RT-PCR and two recombinanted eukaryotic expression plasmids pcDNA3/mBD2 and pcDNA3/mBD2-L-VP1 were constructed. BALB/c mice were divided into 5 groups randomly and inoculated in quadriceps at 3-week interval for 3 times with PBS, pcDNA3, pcDNA3/mBD2, pcDNA3/VP1 and pcDNA3/mBD2-L-VP1.14 days after every inoculation,the sera were collected to titer of neutralizing antibody. Three weeks after the last immunization,specific CTL activities were tested with the spleen cells and the viral titers in the blood were evaluated for 3 mice in every group that had been challenged with 3LD50 CVB3 and killed 7 days post the infection. Results：The mBD2 gene were cloned and two recombinant plasmids of pcDNA3/mBD2 and pcDNA3/mBD2-L-VP1 were constructed successfully. After three times of immunization, the recombined DNA vaccine pcDNA/mBD2-L-VP1 induced higher level of neutralizing antibody and the virus titers were lower in this group （ P 〈 0.01 ）. In addition, the specific CTL cytotoxity activity in pcDNA31 mBD2-L-VPI group was significantly enhanced （ P 〈 0.01 ）. Conclusion： pcDNA3/mBD2-L-VPI vaccine can induce stronger humoral and cellular immune responses against CVB3 and decrease virus titers in blood effectively.