猴B病毒包膜蛋白gD与细胞受体分子nectin-1的相互作用机制研究

B virus (Cercopithecine herpesvirus1) is an α-herpesvirus that naturally infects conventional populations of macaques, and in any typical population over 60% of adult animals are infected. Serious disease due to BV is rare in macaques, but when transmitted to humans, BV has a propensity to invade the central nervous system and has a fatality rate greater than 70%..Our previous studies shows that only antibodies to envelope glycoprotein D (gD) inhibited BV infection in Vero cell, but not antibodies to gB or gC. Antibodies to nectin-1, a memberof the nectin family of adhesion molecules belonging to the immunoglobulin superfamily, blocked completely BV infection in CHO-K1 expressing nectin-1 but only part of in Vero cell. These data indicated that interactions of gD and nectin-1 was important for BV infection. In the present study, functional domain(s) on gD and nectin-1 would be identified by gene-fragment phage display libraries and predicted by bioinformatics or base on results of related-alpha herpesviruses (especially herpes simple virus type 1, HSV-1). Then, mutations in functional domains would be constructed and used to product recombinant protein and transfect to cell line. To these ends, mixed recombinant mutants with target cell expressing wild-gD (Vero-W-gD) or wild-nectin-1 (CHO-K1-W-nectin), mixed Vero cell expressing mutant gD and viral gB, gH and gL with CHO-K1-W-nectin or CHO-K1 expressing mutant nectin-1 with Vero cell expressing four viral glycoproteins (gD, gB, gH and gL). The reactions of recombinant mutants with target cell were detected by cell enzyme-linked immunosorbent assay (CELISA), cell fusion assay was used to assess functional activity of the gD and nectin-1 mutants,and founctions of domains were validated by BV infection. Our study aims to identify functional domains of BV gD and cellular receptor nectin-1 so as to elucidate the mechanisms of BV infection.

B病毒在自然宿主（猴群）中的感染率>60%，感染动物无明显临床症状并终生带毒，人感染后的死亡率>70%，是重要的动物源性传染病。.我们前期发现，只有针对B病毒gD的抗体能阻止病毒感染；抗nectin-1抗体部分影响Vero细胞感染，完全阻断表达nectin-1的CHO-K1细胞感染，表明gD与nectin-1相互作用是B病毒感染的关键环节。鉴于B病毒与单疱病毒高度同源，推测其入侵机制相似，即病毒与受体互作包括膜结合、分子变构、膜融合等过程。本项目以噬菌体展示技术为基础，辅以生物信息学和同源病毒研究结果，筛选gD和nectin-1互作的功能域；构建功能域突变体，制备突变体及突变体转染细胞；最后，重组突变体及转染细胞与表达野生型互作蛋白的靶细胞作用，通过ELISA、细胞融合、病毒感染等实验，揭示两个互作分子的功能域及各功能域的作用。本课题旨在阐明B病毒感染的分子机制，为防治措施选择提供新靶点。

B病毒是以猴为自然宿主且对人高度致死的疱疹病毒属成员，在猴群中的感染率约为60%，人感染后的死亡率超过75%。当前，由于缺乏特异性治疗手段，净化实验猴群、探索B病毒感染机制是疾病防控的主要研究方向。本课题构建了稳定表达B病毒膜蛋白gD、gB和gB+gD的工程细胞，血清学评价显示，细胞表达产物的敏感性显著高于大肠表达产物，联合使用gD和gB重组蛋白的准确性高于单个分子。进而，通过ELISA参数优化，成功研制了B病毒ELISA抗体检测试剂盒；与国外金标准试剂盒相比，新试剂盒的准确性为98.28%~98.95%，且具有安全、低廉、反差明显等优点。此外，克隆表达了猴nectin-1基因，构建了稳定表达猴nectin-1的CHO-K1工程细胞，感染实验表明，HSV-1、HSV-2和PRV在nectin-CHO-K1工程细胞上实现了病毒增殖，说明nectin-1是这些疱疹病毒的特异性受体，但其功能不是增加病毒结合，而是参与病毒侵入过程。细胞融合实验发现，发生细胞融合至少需要4种B病毒膜蛋白（gB、gD、gH、gL）以及细胞受体nectin-1共同参与，gD分子上的53TDPPDVRRVY62、239VDGIGMLPRFIPENQRIVAVY259和nectin-1分子的59SVKITQVTWQ69、78KQNVAIYNPSMGVSVL93、127EFATFPTGN136都是影响膜融合的重要功能区，这些部位缺失将导致细胞融合消失；但gD重组蛋白不与nectin-CHO-K1细胞结合，nectin-1的三个功能区肽段也不与gD重组蛋白结合，说明二者之间并不存在强烈的相互作用。. 我国实验灵长类动物的生产和出口占全球50%以上，本课题研制的猴B病毒血清抗体ELISA检测试剂盒如果申报成功（正在申报），不仅能显著降低实验灵长类动物产业界的生产成本，也为高品质实验灵长类动物（如SPF）培育提供了技术支撑。