单纯疱疹病毒1型糖蛋白gD的表达纯化及多克隆抗体的制备

Expression and purification of herpes simplex virus type 1 glycoprotein D and preparation of polyclonal antibodies

目的表达及纯化单纯疱疹病毒1型(Herpes simplex virus type 1,HSV-1)糖蛋白D(glycoprotein D,gD)并制备多克隆抗体。方法双酶切pcDNA 3.1-HSV1-gD和pFastBacTM I质粒,gD基因克隆到pFastBacTM I载体,连接产物转化E.coli DH10Bac感受态细胞并鉴定重组杆状病毒质粒Bacmid-gD。将构建正确的重组杆粒转染Sf9细胞包装杆状病毒。杆状病毒经传代扩增后,诱导重组gD蛋白的表达,使用Ni-NTA亲和层析及凝胶过滤层析纯化重组gD蛋白,进行15%SDS-PAGE电泳鉴定并采用肽指纹图谱分析纯化的gD蛋白。将纯化的gD蛋白进行热稳定性试验检测其在不同缓冲液条件下的稳定性。用重组gD蛋白免疫小鼠,利用ELISA法检测血清抗体滴度。结果成功构建重组质粒pFastBacTM I-gD,转化E.coli DH10Bac感受态细胞后,经蓝白斑筛选鉴定重组杆状病毒质粒Bacmid-gD,鉴定正确的重组质粒感染Sf9细胞,包装出重组杆状病毒,获得滴度为8×108 pfu/ml的P3代杆状病毒。重组杆状病毒再次感染Sf9细胞能够表达高纯度可溶性的重组gD蛋白。不同缓冲液条件下重组gD蛋白稳定性良好。用gD蛋白免疫小鼠,获得ELISA效价为1×105的多克隆抗体。结论成功构建重组杆状病毒质粒Bacmid-gD,表达的HSV-1 gD蛋白稳定及免疫原性良好,制备的抗gD蛋白多克隆抗体滴度高,为HSV-1的亚单位疫苗的制备鉴定了基础。

Objectives To express and purify herpes simplex virus type 1 glycoprotein D and to prepare polyclonal antibodies against it.Methods The plasmids pcDNA 3.1-HSV1-gD and pFastBacTM I were double digested.The gD gene fragment was cloned into the pFastBacTM I vector to construct the recombinant plasmid pFastBacTM I-gD.After the recombinant plasmid pFastBacTM I-gD was transformed into E.coli DH10 Bac competent cells,the recombinant baculovirus plasmid Bacmid-gD was identified using blue-white screening and analyzed using PCR.The identified plasmid Bacmid-gD was transfected into Sf9 cells,and the recombinant baculovirus was obtained.It was then used to infect serum-free Sf9 cells to express the gD protein in the supernatant.The gD protein was purified using affinity chromatography and gel filtration chromatography.The purified gD protein was detected using 15%SDS-PAGE and analyzed using MALDI-TOF.Then,the gD protein was subjected to a Thermofluor assay to examine the stability of the gD protein under different buffer conditions.BALB/c mice were immunized with the purified gD protein combined with Freund’s adjuvant via subcutaneous injections,anti-gD serum was separated,and the antibody titer was measured using indirect ELISA.Results Blue-white screening and PCR analysis confirmed that the recombinant plasmid Bacmid-gD was successfully constructed.After infection of Sf9 cells,a P3 baculovirus was obtained at a titer of 8×108 pfu/mL.The soluble recombinant gD protein was expressed in Sf9 cells.Cells were infected with the P3 baculovirus and then the protein was purified using Ni-NTA affinity chromatography and gel filtration chromatography.The purified protein was identified using 15%SDS-PAGE.It had a molecular weight of approximately 56 ku,which is the same as the predicted value.Mass spectrometry indicated that the protein was recombinant gD protein.The Thermofluor assay indicated that the recombinant gD protein was stable under different buffer conditions.A polyclonal antibody at a titer of 1×105 according to ELISA was obtained by immunizing BALB/c mice with the recombinant gD protein.Conclusion The recombinant plasmid Bacmid-gD was successfully constructed.The HSV-1 gD protein was successfully expressed and displayed good stability and immunogenicity.High titers of anti-gD polyclonal antibodies can provide a basis for vaccine preparation.