适应婴儿肠道的双歧杆菌菌株高通量筛选体系建立

The colonization of Bifidobacterium in infant gut is important to hosts' health and development. To develop the probiotics for infant, screening bifidobacteria strains which could colonize in infant gut is the primary step. The culture method screening is time and labor consuming, and the results are not reliable due to the different environment between vitro and gut. Therefore, a new high-throughput screening method should be developed for these probiotics. In this proposal, LnbB and LnpA gens, the key enzymes in HMOs metabolism pathway, will be cloned and sequenced from 10 species of Bifidobacterium (more than 500 strains). The genes will be classified based on alignment, similarity analysis and clustering of sequences. The LnbB and LnpA genes in different classifications were combined and transformed into Bifidobacterium (LnbB-; LnpA-) with vectors. The enzymatic activity of LnbB and LnpA, as well as degradation of LNT (core structure of tpye I HMOs) were determined for recombinants in vitro. Based on the results, the relationship between gene classification and HMOs utilization will be confirmed, and genotype of LnbB and LnpA contribute to high activity of HMOs utilization will be revealed. In vivo study, different bifidobacteria recombinants with LNT will be fed to mice, and the number of recombinants in colon will be determined. Ultimately, we will set up a new high-throughput method for screening bifidobacteria strains, based on the classification of LnbB and LnpA genes. This study could contribute to the development of infant probiotics.

双歧杆菌的肠道定植对婴儿健康及生长发育起关键作用。筛选能够在婴儿肠道中定植的双歧杆菌菌株是开发适合婴儿的益生菌产品必须环节。传统培养法筛选菌株费时费力，且可靠性差，亟需建立新型菌株高通量筛选体系。本项目拟对10个种（亚种）500株以上双歧杆菌的母乳寡糖代谢关键酶LnbB和LnpA基因进行测序；利用大规模序列比对、相似度分析、多算法聚类等生物信息学手段，对基因进行分类；将不同类别的LnbB和LnpA基因组合，克隆转化于双歧杆菌宿主，体外评价不同重组子LnbB和LnpA酶活性以及对I类母乳寡糖的降解能力，明确基因类别与母乳寡糖代谢活性的关系，揭示高活性重组子LnbB和LnpA基因序列特征；通过动物实验，在母乳寡糖存在条件下，验证不同重组双歧杆菌的定植能力。通过以上研究，建立基于LnbB和LnpA基因分类的新型双歧杆菌菌株高通量筛选体系，为开发适应婴儿肠道的益生菌菌株提供保障。

双歧杆菌的肠道定植对婴儿健康及生长发育起关键作用。筛选能够在婴儿肠道中定植的双歧杆菌菌株是开发适合婴儿的益生菌产品必须环节。传统培养法筛选菌株费事费力，且可靠性差，亟需建立新型菌株高通量筛选体系。本项目对10个种（亚种）263株双歧杆菌的母乳寡糖代谢关键酶LnbB和LnpA基因进行测序；利用序列比对等手段，将LnpA基因分为3类，LnbB分为5类；将不同类别的LnbB和LnpA基因组合，克隆转化于双歧杆菌NCC2705宿主，体外评价不同重组对I类母乳寡糖的降解能力，证实I类LnpA和III类LnbB具有较强的母乳寡糖代谢活性，且代谢LNT、2-FL活性较强。本研究为开发适应婴儿肠道的益生菌菌株提供理论基础。