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目的制备基于Eppin优势中和B细胞表位的新型DNA避孕疫苗。方法采用PCR技术扩增mIL-4和Eppin B细胞表位基因,将其克隆到真核表达载体pVITR02-mcs,构建双盒表达载体,并将双盒表达质粒转染CHO细胞,检测其体外表达;将双盒表达质粒DNA与具有穿膜活性的HIV-1 TAT49-57阳离子肽,通过正负电荷吸引原理,制备成模拟病毒颗粒样的疫苗,并以DNA阻滞试验、DNaseⅠ保护试验和扫描透射电镜对该疫苗进行鉴定。结果 RT-PCR和免疫印迹结果表明转染细胞中均有目的分子的存在,在电荷比为4的条件下,该疫苗能形成类似病毒样的颗粒。结论成功制备了基于Eppin优势中和B细胞表位的模拟病毒颗粒样疫苗,为进一步研究避孕效果奠定了基础。
Objective To prepare a novel DNA contraceptive vaccine based on the Eppin dominant neutralizing B cell epitopes. Methods The mIL-4 and Eppin B cell epitope genes were amplified by PCR and cloned into the eukaryotic expression vector pVITR02-mcs to construct a two-box expression vector. The two-box expression plasmid was transfected into CHO cells to detect the expression of mIL-4 and Eppin B cells in vitro . The double-box expression plasmid DNA and the transmembrane HIV-1 TAT49-57 cationic peptide were prepared into a mimic virus particle-like vaccine by positive and negative charge attraction. The DNA vaccine, DNAase I protection test and scanning The vaccine was identified by transmission electron microscopy. Results The results of RT-PCR and Western blotting showed that all the transfected cells had the target molecule. Under the charge ratio of 4, the vaccine could form virus-like particles. Conclusion MVD vaccine based on Eppin ’s dominant neutralizing B cell epitopes was successfully prepared, which laid the foundation for further study of contraceptive effects.