【作者】 李轶杰； 张富春； 王焱冰； 金华利； 王宾；

【Author】 LI Yi-Jie ZHANG Fu-Chun WANG Yan-Bing JIN Hua-Li WANG Bin( Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China; State Key Laboratory for Agro-Biotechnology, College of Biology, China AgricultureUniversity, Beijing 100094, China)

【机构】 新疆大学生命科学与技术学院分子生物学重点实验室； 中国农业大学生物学院农业生物技术国家重点实验室；

【摘要】 构建含高表达信号肽、提高抗原溶解性片断及新型佐剂C3d的草原兔尾鼠透明带3真核表达载体,探讨提高草原兔尾鼠透明带3DNA疫苗免疫原性的策略。通过基因重组依次在去信号肽和跨膜区的草原兔尾鼠透明带3核心序列两端分别融合人α胰岛素抑制剂(αk-antitrypsin,AAT)的信号肽、大鼠寡聚软骨基质蛋白(rat cartilage oligomerization matrix protein,COMP)片段及3分子的C3d序列形成LZP3嵌合体。分别插入真核细胞表达质粒pcDNA3的CMV启动子下游形成重组质粒pcD-NA3-AAT-COMP-LZP3 (pacD-ACL)和pcDNA3-AAT-COMP-LZP3-C3d3(融合有3个拷贝C3片段的pcD-ACL;pcD-ACLC)。免疫BALB/c后取血清用酶联免疫法检测LZP3抗体水平的变化。结果表明,免疫第4周时pcD-ACLC与pcDNA3-LZP3组产生的LZP3特异性抗体水平大致相当,且高于pcD-ACL组产生的抗体水平。第8周时与LZP3全长核酸疫苗(pcDNA3-LZP3)相比pcD-ACL和peD-ACLC均能产生较强的免疫效果,其中后者产生的抗体水平明显高于pcD-ACL。通过融合高表达分泌蛋白信号肽、增加抗原溶解度的序列和分子佐剂C3d3能显著提高LZP3的免疫原性。更多还原

【Abstract】 The zona pellucida (ZP) protein have been proposed as candidate immunogens for development of a contraceptive vaccine. DNA vaccines expressing ZP protein have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in murine models. This paper reported using a DNA vaccine expressing a fusion of the Lagurus zona pellucida 3 (LZP3) protein and various elements, such as a secretion leader sequence from the highly expressed human gene encoding α 1 -antitrypsin (AAT), a highly soluble and stably folded domain from the rat cartilage oligomerization matrix protein (COMP), and three copies of the complement component, C3d3, to enhance the titers of neutralizing LZP3-specific antibody. Analysis of titers of the antibody raised in vaccinated mice at different time points indicated that immunizations with the DNA expressing pcDNA3-AAT-COMP-LZP3-3C3d had similar titers of anti-LZP3 antibodies compared to that with DNA expressing pcDNA3-LZP3 and higher than that of pcD-NA3-AAT-COMP-LZP3 at weeks 4. However, the level of LZP3-specific antibodies produced at week 8 following immunization with pcDNA3-AAT-COMP-LZP3-C3d3 is higher than that of pcDNA3-LZP3 and pcDNA3-AAT-COMP-LZP3, and the latter is observably higher than pcDNA3-LZP3. These results suggested that mediate efficient antigen secretion in vivo, a fused piece of sequence improving antigens solubility, as well as C3d3, genetic adjuvant, could enhance the antibody responses level using a plasmid vector containing a strong heterologous signal sequence. This approach might be an efficient way to improve the antibody level of LZP3 DNA vaccination.更多还原