基因重组PACAP27衍生多肽RP2制备及促角膜上皮细胞增殖研究  

Preparation of Recombinant PACAP 27-derived Peptide RP2 and Its Effects on Promoting Corneal Epithelial Cell Proliferation

作　　者：王东波[1,2] 马义[1,2] 王孝丽[1,2] 赵绍军[1,2] 韩磊[1,2] 洪岸[1,2] 

机构地区：[1]暨南大学细胞生物学系 [2]暨南大学生物医药研究院 广东省生物工程药物重点实验室 基因工程药物国家工程研究中心,广州510632

出　　处：《中国生物工程杂志》2015年第3期61-65,共5页China Biotechnology

基　　金：国家自然科学基金面上项目(81373314);广东省自然科学基金(S2012010008756);高等学校博士学科点专项科研基金(20124401120012);广东省高等学校科技创新项目(2012KJCX0015);广东省教育部产学研结合项目(2010B090400544,2013B090500105);广州市科技计划项目(2011J4300112)资助项目

摘　　要：利用基因工程技术高效制备具有促进角膜上皮细胞增殖功能的垂体腺苷酸环化酶激活肽(PACAP 27)衍生多肽RP2,在细胞水平初步研究其生物学作用,为其用于角膜损伤治疗提供实验基础。采用基因重组技术表达重组肽RP2,经Chitin-Beads和HPLC纯化、SDS-PAGE和质谱鉴定后,研究其对小鼠角膜上皮细胞增殖的影响。实验结果表明:利用基因重组技术制备的PACAP 27衍生多肽RP2的分子量为3.3 k Da,纯度达96%;分别用重组肽RP2、PACAP 27及PBS作用于小鼠角膜上皮细胞,处理24 h及48 h时,RP2处理组角膜上皮细胞增殖率分别为(49.6±3.1)%、(93.0±1.7)%,PACAP 27处理组细胞增殖率分别为(29.0±2.4)%、(78.8±2.6)%,PBS对照组细胞增殖率为(20.2±1.1)%,(40.9±3.3)%。利用建立的重组多肽制备技术条件,可实现PACAP衍生多肽RP2高效制备,制备的RP2可有效促进角膜上皮细胞的增殖,从而有望成为一种新型角膜损伤治疗候选药物。PACAP 27-derived peptide RP2 which can promote promote the epithelial cell proliferation is efficiently producted by genetic engineering and be used to study its effects on corneal epithelial cells,which can provide an experimental basis for the treatment for corneal injury. Recombinant peptide RP2 was expressed by genetic recombinant technology after the prokaryotic expression, purification by Chitin-Beads column, and producted,analyzed、identification by HPLC, SDS-PAGE and mass spectrometry, then its effects on mouse corneal proliferation were studied. Experimental results showed that the molecular weight of RP2 is 3.3 kDa. and the purity is 96%. Mouse corneal epithelial cells were respectively treated with recombinant peptide RP2、 PACAP 27 and PBS for 24 h and 48 h,The proliferation rate of mouse corneal epithelial cells treated with recombinant peptide RP2,is （49.6±3.1）% and（93.0±1.7）%, the PACAP 27 group is （29.0±2.4）% and （78.8±2.6）%,while the PBS group is （20.2±1.1）% and （40.9±3.3）%. Therefore, the producted recombinant peptide RP2 can significantly stimulate corneal epithelial cell proliferation, so it will likely to become a new candidate drug for corneal injury.

关 键 词：基因重组 垂体腺苷酸环化酶激活肽 重组肽 角膜上皮细胞 

分 类 号：Q819[生物学—生物工程]