弓形虫复合抗原ROP2-SAG1优势表位与人乳头瘤病毒16型晚期结构蛋白L1融合体的构建及其在真核细胞中的表达

Construction of the RSepitope-HPV16L1 fusion protein using the dominant epitope of ROP2-SAG1 antigen from Toxoplasma gondii and late structural protein 1 of HPV type 16 and its expression in eukaryotic cells

目的构建弓形虫复合抗原ROP2-SAG1免疫优势表位(RSepitope)与人乳头瘤病毒16型晚期结构蛋白L1(HPV16L1)的融合体(RSepitope-HPV16L1),验证其在真核细胞中的表达。方法优化RSepitope基因序列,构建pc DNA3.1/RSepitope重组真核表达质粒。从T-HPV16L1质粒中扩增HPV16L1基因片段,构建pc DNA3.1/RSepitope-HPV16L1重组质粒。双酶切、PCR、测序鉴定正确后,将2个重组质粒分别转染至非洲绿猴肾细胞COS-7细胞,培养48 h后收集细胞,提取RNA,RT-PCR扩增相应的目的基因。Western blotting和间接免疫荧光实验检测重组蛋白RSepitope和RSepitope-HPV16L1在细胞中的表达。结果构建的重组质粒pc DNA3.1/RSepitope和pc DNA3.1/RSepitope-HPV16L1经PCR扩增和双酶切分别获得282 bp和1 500 bp片段,与预期结果一致,测序结果正确。RT-PCR结果显示,以2个重组质粒转染细胞的c DNA为模板,分别扩增到282 bp和1 500 bp的目的条带。Western blotting分析结果显示,以RSepitope免疫血清为一抗,重组蛋白RSepitope和RSepitope-HPV16L1分别在相对分子质量(Mr)约为10 000和65 000处出现特异性条带;以HPV16L1单克隆抗体为一抗,重组蛋白RSepitope-HPV16L1在Mr65 000处出现特异性条带。间接免疫荧光实验结果显示,以RSepitope免疫血清为一抗,重组质粒pc DNA3.1/RSepitope和pc DNA3.1/RSepitope-HPV16L1转染细胞内均观察到绿色荧光;以HPV16L1单克隆抗体为一抗,重组质粒pc DNA3.1/RSepitope-HPV16L1转染细胞内观察到特异性绿色荧光。结论弓形虫优势表位RSepitope与HPV16L1融合体构建成功,且可在真核细胞中表达。

Objective To construct the RSepitope-HPV16 L1 fusion protein using the dominant epitope of ROP2-SAG1 antigen from Toxoplasma gondii（RSepitope） and late structural protein 1 of HPV type 16（HPV16 L1）,and verify its expression in eukaryotic cells. Methods The recombinant plasmid pc DNA3.1/RSepitope was constructed after optimizing the RSepitope gene sequence. The gene fragment of HPV16 L1 was amplified from the plasmid T-HPV16 L1 preserved in our lab, and the recombinant plasmid pc DNA3.1/RSepitope-HPV16 L1 was constructed. The recombinant plasmid was verified by enzymatic digestion, PCR amplification and sequencing, and transfected into the COS-7 cells. After 48 h of culture, cells were collected to extract RNA. RT-PCR was performed to amplify target genes. Meanwhile, Western blotting and indirect immunofluorescence test were performed to detect the expression of target proteins RSepitope and RSepitope-HPV16 L1 in cells. Results After amplification and enzymatic digestion, a 282-bp fragment was obtained from pc DNA3.1/RSepitope, and a 1 500 bp fragment was obtained from pc DNA3.1/RSepitope-HPV16 L1. RT-PCR using c DNA of the recombinant plasmid-transfected cells as the template produced specific bands of 282 bp and 1 500 bp, respectively. Western blotting analysis showed that when the RSepitope anti-serum was used as the primary antibody, specific bands with relative Mrof 10 000 and 65 000 were seen for RSepitope and RSepitope-HPV16 L1, respectively. When anti-HPV16 L1 monoclonal antibody was used as the primary antibody, a specific band with relative Mrof 65 000 was seen for RSepitope-HPV16 L1. Indirect immunofluorescence analysis using RSepitope anti-serum as the primary antibody produced green fluorescence in cells transfected with pc DNA3.1/RSepitope and pc DNA3.1/RSepitope-HPV16 L1. When the HPV16 L1 monoclonal antibody was used as the primary antibody, green fluorescence was seen only in cells transfected with pc DNA3.1/RSepitope-HPV16 L1. Conclusion The RSepitope-HPV16 L1 fusion is constructed using RSepitope and HPV16 L1 and could be expressed in eukaryotic cells.