单纯疱疹病毒I型糖蛋白D胞外区的真核表达及生物学活性分析

Eucaryotic expression and bioactivity analysis of the recombinant HSV-gD1

摘要:

单纯疱疹病毒 (Herpes simplex virus，HSV) 包膜糖蛋白D (glycoprotein D，gD) 是HSV的结构蛋白之一，具有重要抗原表位，是目前疫苗研究的热点。为了分离纯化HSV gD1糖蛋白胞外区片段并对其生物学活性进行分析，本研究将化学合成的gD1胞外区基因片段克隆至真核表达载体pCEP4，重组质粒转染HEK293细胞进行瞬时表达，产物经Western blotting检测后用亲和层析法进行分离纯化，ELISA检测其抗原性。以纯化的重组蛋白作为抗原免疫小鼠，ELISA测血清特异性抗体效价以评价其免疫原性。构建的重组质粒经测序显示基因序列完全正确。表达产物的Western blotting分析发现，在相对分子量约46 kDa处有外源蛋白表达，与预期蛋白带一致。用Ni柱得到了纯化的重组gD1蛋白，ELISA检测显示其具有良好的抗原性，免疫小鼠7周后血清中抗体效价达到5×103。重组gD1蛋白的抗原性及免疫原性分析为HSV检测试剂和基因重组亚单位疫苗的研制提供了实验依据。

Abstract:

Envelope proteins of herpes simplex virus (HSV) plays a vital role not only in the infection process of adsorption and invasion but also in the stimulation to the organism that gives rise to immune response. Among the envelope proteins, glycoprotein D (gD), which can induce specific immune response, are the primary targets of humoral and cellular immunity of the host. In order to analyze the antigenicity and immunogenicity of HSV-gD1, we chemically synthesized the extracellular domain fragment gene of gD1, cloned it into eucaryotic expression vector pCEP4, and transfected the HEK293 cells with the recombinant vector. Then we identified the recombinant protein by Western blotting, and detected antigenicity of the protein by ELISA. Finally, we used the purified gD1 protein to immunize Kunming mice in 1, 3, 5 weeks, and collected antiserum in 3, 5 and 7 weeks. We titrated the sera for the detection of anti gD1 using an ELISA assay. Gene sequencing analysis demonstrated that the recombinant plasmid pCEP4-gD1 was constructed successfully. Western blotting analysis indicated one major protein band, which molecular weights is approximate 46 kDa corresponding to the truncated forms of gD1 protein, was observed. ELISA assay showed that the expressed recombinant protein gD1 had good antigenicity. After the third immunization, antibody titer of the mouse anti-gD1 was at least 5×103. The successful expression of the recombinant protein gD1, which can induce humoral immune response, lays a foundation for serological diagnosis and vaccine study of HSV.