表达HIV单链抗体的重组乳酸杆菌的构建及表型分析  

Construction and Phenotypic Analyses of Recombinant Lactobacillus Expressing Single-Chain Antibody of HIV

作　　者：安明晖[1,2,3] 田文 韩晓旭[1,2,3] 尚红 AN Ming-hui;TIAN Wen;HAN Xiao-xu;SHANG Hong(NHC Key Laboratory of AIDS Immunology(China Medical University),The First Affiliated Hospital of China Medical University,Shenyang 110001,China;Key Laboratory of AIDS Immunology of Liaoning Province,The First Affiliated Hospital of China Medical University,Shenyang 110001,China;Key Laboratory of AIDS Immunology,Chinese Academy of Medical Sciences,Shenyang 110001,China)

机构地区：[1]中国医科大学附属第一医院国家卫生健康委员会艾滋病免疫学重点实验室,沈阳110001 [2]中国医科大学附属第一医院辽宁省艾滋病免疫学重点实验室,沈阳110001 [3]中国医学科学院艾滋病免疫学重点实验室,沈阳110001

出　　处：《中国生物工程杂志》2019年第10期1-8,共8页China Biotechnology

基　　金：国家自然科学基金青年项目(81401655)资助项目

摘　　要：近年来,基于抗体药物的被动免疫策略在人类免疫缺陷病毒(HIV)预防和治疗中获得一定的成效,但仍需要定期补充抗体,才能维持体内长期有效的药物浓度,如何延长抗体药物的作用时间是亟待解决的问题。根据乳酸杆菌的食品安全性和黏膜定植稳定性,把HIV单链抗体连接到具有宿主同源启动子和信号肽的乳酸杆菌表达质粒pldhl0373中,电转化乳酸杆菌WCFS1感受态细胞,通过红霉素筛选出阳性单菌落,发酵培养后菌液经Western blot检测证明了单链抗体得到表达,并具有抗病毒能力。与野生型WCFS1菌株相比,重组菌体外生长能力未发生改变,发酵24h后菌液的CFU均可达到109/ml;单链抗体基因的插入不会明显改变重组菌黏膜细胞定植能力,每平方厘米单层上皮细胞可稳定定植105CFU的菌落。以宿主黏膜共生菌为媒介,在黏膜细胞表面实现抗病毒蛋白的长期稳定表达为预防HIV黏膜传播提供了一种新的思路。Recently,although the passive immunization strategy based on antibody drugs has achieved certain results in the prevention and treatment of HIV,supplement of antibodies is necessary to maintain its long-term effective concentration.The key question is how to prolong the active time of antibodies.Due to the food safety and mucosal colonization stability of lactobacillus,the anti-HIV single-chain antibody fragments were inserted in its expression plasmid with homologous promotor and signal peptide,and plasmid was transformed into competent cells of WCFS1 strains by electroporation.Positive single colonies were screened out by erythromycin,and after culture,the anti-HIV single-chain antibodies were detected to be expressed in the bacterial supernatant and the surpernatant can inhibit the HIV pseudovirus.Compared with the wild-type WCFS1 strain,the growth capacity of the recombinant strain in vitro did not changed and the CFU of the bacterial solution could reach to109/ml after 24 hours’fermentation;the colonization ability of the recombinant strain also did not change obviously,and the each square centimeter of monolayer epithelial cells could bind 105CFU colonies.Expressing anti-viral proteins via a live recombinant lactobacillus as host rectal mucosal colonizers gave a new proposal to prevent HIV homosexual transmission.

关 键 词：HIV单链抗体 植物乳酸杆菌 启动子 信号肽 黏膜上皮细胞 

分 类 号：Q815[生物学—生物工程]