水痘-带状疱疹病毒糖蛋白H/L重组真核表达质粒的构建及其免疫效果评价

Construction and immune effect of recombinant eukaryotic expression plasmid for varicella zoster virus glycoprotein H/L

目的构建水痘-带状疱疹病毒(varicella zoster virus,VZV)糖蛋白H(glycoprotein H,gH)和糖蛋白L(glyco-p rotein L,gL)重组真核表达质粒,用质粒免疫小鼠,评价其免疫效果。方法以提取的VZV基因组DNA为模板,PCR扩增gH胞外区基因(g H795)和gL全长基因,与pCI-neo载体连接,构建重组真核表达质粒pCI-neo-VZV-gH795-His和p CI-neo-VZV-gL-His;将质粒单独或与CpG1826佐剂共同免疫小鼠,免疫周期为0、3、5周,采血周期为3、5、7周,检测血清抗体效价、中和效价和膜免疫荧光抗体(fluorescent antibody to membrane antigen,FAMA)效价。结果重组真核表达质粒pCI-neo-VZV-g H795-His和pCI-neo-VZV-gL-His经双酶切(XbaⅠ/EcoRⅠ)和测序证明构建正确。单质粒免疫小鼠,血清抗体效价较低,双质粒免疫小鼠,血清抗体效价微弱升高,双质粒与佐剂共同免疫小鼠,血清抗体效价达1∶6000以上,CpG1826对于抗体产生具有较明显的促进作用。仅双质粒与佐剂共免疫组小鼠血清显示出中和活性,免疫后7周,血清中和效价达1∶62。双质粒与佐剂共免疫组免疫后7周小鼠血清FAMA效价为1∶8~1∶16,其余各组均为阴性。结论gH(或gHgL复合物)具有一定的免疫原性,本实验为其进一步的免疫原性研究奠定了基础。

Objective To construct the eukaryotic expression plasmid for glycoprotein H(gH)and glycoprotein L(gL)of varicella zoster virus(VZV)and evaluate its immune effect in mice.Methods The gH extracellular domain(gH795)gene and full-length of g L gene were amplified by PCR using extracted genomic DNA of VZV as template and inserted into vector pCI-neo to construct eukaryotic expression plasmids pCI-neo-VZV-gH795-His and pCI-neo-VZV-gL-His respectively.Mice were immunized with the plasmids and/or CpG1826 adjuvant at weeks 0,3 and 5,of which the serum samples were collected at weeks 3,5 and 7 and determined for the titers of serum antibody,neutralizing antibody and fluorescent antibody to membrane antigen(FAMA).Results Both restriction analysis(XbaⅠ/EcoRⅠ)and sequencing proved that recombinant eukaryotic expression plasmids pCI-neo-VZV-g H795-His and pCI-neo-VZV-gL-His were constructed correctly.The serum antibody titer of mice immunized with either of the plasmids alone was relatively low,while that with the two plasmids increased slightly,and that with the two plasmids plus CpG1826 adjuvant was more than 1∶6000,indicating that CpG1826 significantly promoted the production of antibodies.However,the neutralizing antibody titer was detected only in the sera of mice immunized with the two plasmids plus CpG1826 adjuvant,which reached 1∶62 seven weeks after immunization.The serum FAMA titer of mice 7 weeks after immunization with the two plasmids plus CpG1826adjuvant was 1∶8~1∶16,while those in other groups were undetected.Conclusion The gH(or gHgL complex)shows a certain immunogenicity,which lays a foundation of further study on immunogenicity.