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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。
OBJECTIVE: To construct the VP1 fusion gene vaccine against mouse beta-defensin-2 (mBD2) and Coxsackievirus B3 (CVB3) and to observe its immunogenicity in mice. Methods: The mBD2 gene was cloned and the recombinant plasmids pcDNA3 / mBD2 and pcDNA3 / mBD2-L-VP1 were constructed. Male BALB / c mice aged 4 to 6 weeks were randomly divided into 5 groups: PBS (group A), pcDNA3 (group B), pcDNA3 / mBD2 (group C) and pcDNA3 / VP1 Group) and pcDNA3 / mBD2-L-VP1 (group E). The inoculation dose was 100μg / dose and immunized once every 3 weeks for 3 times. On the 14th day after each immunization, blood was collected from the orbital vein and serum neutralizing antibody titers were titrated with a micro-neutralization test. On the 21st day after the third immunization, three mice in each group were randomly selected to prepare splenic lymphocyte suspension. Specific cytotoxic activity of CTL was detected by CCK-8 cell counting method. Three mice in each group were immunized with 3LD50CVB3 virus Attack, blood sacrificed on day 7, serum titer was tested. Results: The mBD2 gene was successfully cloned and two recombinant plasmids, pcDNA3 / mBD2 and pcDNA3 / mBD2-L-VP1, were constructed. The titer of serum neutralizing antibody in groups D and E increased with the increase of immunization times (P <0.01) The serum neutralizing antibody titer and splenocyte specific cytotoxicity of CTL were significantly higher than other groups (P <0.01). The CVB3 virus titer in blood was significantly lower than that of other groups (P <0.01). Conclusion: pcDNA3 / mBD2-L-VP1 can induce murine CVB3 produce strong humoral and cellular immunity, which can effectively inhibit the virus in vivo proliferation.