Highly efficient expression, purification of recombinant LTB protein and its activity against mucosal immunoadjuvant by nasal immunization  

作　　者：王静 李琳琳 郑瑾 于军 杨筱凤 耿宜平 来宝长 王一理 司履生 

机构地区：[1]Institute of Cancer Research, School of Life Science and Technology, Medical Sciences of Xi’an Jiaotong University, Xi’an 710061, China [2]Institute

出　　处：《Chinese Medical Journal》2003年第7期1115-1117,共3页中华医学杂志（英文版）

基　　金：agrantfromthe"863"Program (No .2 0 0 1AA2 15 2 2 1)andtheNationalNaturalScienceFoundationofChina(No 3 0 0 70 848)

摘　　要：Objective To develop an efficient expression, purification system of recombinant Escherichia coli heat labile enterotoxin B subunit (rLTB) and study its activity against mucosal immunoadjuvant by nasal immunization Methods A recombinant, pMMB68 LTB was generated by cloning the LTB cDNA fragment into an expression vector (pMMB68) and transformed it into the host strain marine vibrio VSP60 The relevant target protein was identified using SDS polyacrylamide gel electrophoresis (SDS PAGE) and Western blot Sephacryl S 100 gel filtration chromatography was carried out for purification of rLTB in engineering bacteria VSP60 BALB/c mice received hen egg lysozyme (HEL) alone or combined with rLTB by nasal administration After three times immunization, IgG and IgA antibody levels in serum or small intestine wash samples were determined using ELISA Results rLTB protein was highly expressed in VSP60 After gel filtration with Sephacryl S 100, the purity of rLTB reached 98 1%, the yield rate was about 52% After immunization, IgG and IgA antibody responses specific to HEL in system and mucosa of HEL+rLTB groups were significantly increased, compared with the HEL alone group ( P <0 001) Conclusions A set of protocols for large scale rLTB preparation has been established, which is simple, efficient and applicable The rLTB protein we prepared was proved to be a powerful mucocal adjuvant, which could greatly enhance systemic and mucosal immune responses to nasally co administered antigenObjective To develop an efficient expression, purification system of recombinant Escherichia coli heat labile enterotoxin B subunit (rLTB) and study its activity against mucosal immunoadjuvant by nasal immunization Methods A recombinant, pMMB68 LTB was generated by cloning the LTB cDNA fragment into an expression vector (pMMB68) and transformed it into the host strain marine vibrio VSP60 The relevant target protein was identified using SDS polyacrylamide gel electrophoresis (SDS PAGE) and Western blot Sephacryl S 100 gel filtration chromatography was carried out for purification of rLTB in engineering bacteria VSP60 BALB/c mice received hen egg lysozyme (HEL) alone or combined with rLTB by nasal administration After three times immunization, IgG and IgA antibody levels in serum or small intestine wash samples were determined using ELISA Results rLTB protein was highly expressed in VSP60 After gel filtration with Sephacryl S 100, the purity of rLTB reached 98 1%, the yield rate was about 52% After immunization, IgG and IgA antibody responses specific to HEL in system and mucosa of HEL+rLTB groups were significantly increased, compared with the HEL alone group ( P <0 001) Conclusions A set of protocols for large scale rLTB preparation has been established, which is simple, efficient and applicable The rLTB protein we prepared was proved to be a powerful mucocal adjuvant, which could greatly enhance systemic and mucosal immune responses to nasally co administered antigen

关 键 词：Escherichia coli heat  labile enterotoxin immunoadjuvant mucosal 

分 类 号：R392[医药卫生—免疫学]