RESUMEN
To reconstruct the heat-labile enterotoxin subunit B (LTB) gene of Escherichia coil in order to increase the outputs of the prokaryotic expression on recombinant LTB (rLTB), and to determine its immune adjuvant activity on mucosa,the nucleotide sequence of the whole length of LTB gene was synthesized according to the preferred codons of E. coli, and the prokaryotic expression plasmid pET32a-rLTB and its expression system in E. coli BL21DE3 were reconstructed. The recombinant plasmid was extracted and the inserted sequence of rLTB gene was determined. Meanwhile, the expression quantity of the reconstructed rLTB was identified by SDS-PAGE and BioRad agarose image analyzing system, and compared with that of the un-reconstructed rLTB. The abilities of the reconstructed rLTB and the un-reconstructed rLTB to bind with bovine GM1 were determined by means of GM1-ELISA assay. By using the recombinant urease subunit B as antigen, the effects of the reconstructed and the un-reconstructed rLTB on the improvement of immune protection of BALB/c mice infected with Helicobacter pylori strain SS1 and the induction of S-IgAs in infected mice were assayed. The experimental results showed that the expression quantity of the reconstructed rLTB approached upto 35.4% of the total bacterial proteins after induction with 1 mmol/L IPTG for pET32a-rLTB-E. coliBL21DE3 and to be 12.6 times higher than that of the un-reconstructed rLTB (2.8 %). In addition, both the abilities of the recombinant reconstructed LTB and the un-reconstructed rLTB to bind with bovine GM1 could be demonstrated by GM1-ELISA. The immune protection rate of the recombinant urease subunit B in the infected mice was 66.7%; and it could reach up to 91.7% with a significantincrease of the specific S-IgA level, when it was immunized with the reconstructed or the un-reconstructed rLTB. It is concluded that the reconstructed LTB gene in the present study shows a remarkable increased outputs of expression of this gene with a strong immune adjuvant activity on mucosa.
RESUMEN
Objective To observe the changes of LoVo cell growth by galectin-1.Methods Eukaryotic expression vector of galectin-1 was constructed and transfected into LoVo cells using lipofectamineRM 2000.Immunochemistry was employed to detect galectin-1 expresson.LoVo cell proliferation and apoptosis were observed.Results Galectin-1 eukaryotic expression vector pEGFP-C1/GAL1 was successfully constructed.Three cell clones,p-GAL1-LoVo and p-LoVo,transfected with pEGFP-C1/GAL1 and pEGFP-C1 correspondingly,and LoVo were cultured successfully.Galectin-1 expression downregulated Bcl-2 level only occurring in p-GAL1-LoVo cells.p-GAL1-LoVo cell proliferation was similar to p-LoVo and LoVo cells,but p-GAL1-LoVo cell apoptosis was increased(9.61?0.56)%,as compared with p-LoVo and LoVo cells,[(3.56?0.53)% and(3.46?0.46)% respectively](P