Highly efficient expression, purification of recombinant LTB protein and its activity against mucosal immunoadjuvant by nasal immunization.
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 antigen.
Wang
Jing
J
Institute of Cancer Research, School of Life Science and Technology, Medical Sciences of Xi'an Jiaotong University, Xi'an 710061, China.
Li
Linlin
L
Zheng
Jin
J
Yu
Jun
J
Yang
Xiaofeng
X
Geng
Yiping
Y
Lai
Baochang
B
Wang
Yili
Y
Si
Lusheng
L
D000276:Adjuvants, Immunologic / Q000008:administration & dosage / Q000494:pharmacology*; D000281:Administration, Intranasal; D000818:Animals; D001427:Bacterial Toxins / Q000008:administration & dosage / Q000302:isolation & purification* / Q000494:pharmacology; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000008:administration & dosage / Q000302:isolation & purification* / Q000494:pharmacology; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D009297:Nasal Mucosa / Q000276:immunology*; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000302:isolation & purification* / Q000494:pharmacology
D000276:Adjuvants, Immunologic; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli