Role of MUC2 gene in the regulation of rat intestinal barrier function by probiotics / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate MUC2 expression in rat colons induced by probiotics and its effects on the inhibition of E.coli K1 (E44) penetration of the intestinal barrier by probiotics.</p><p><b>METHODS</b>SD rats were subjected to intragastric administration of probiotics, E44, or probiotics +E44 on a daily basis for 7 days, and MUC2 expression in the colons was determined by RT-PCR. MUC2-targeted shRNA (shRNA MUC2) and scrambled shRNA plasmids (shRNA NC) were respectively transfected into Lovo cells, and the efficiency of MUC2 knockdown was determined using qRT-PCR. Competitive exclusion assay was used to evaluate the effects of the probiotics against E44 adhesion and invasion.</p><p><b>RESULTS</b>Intestinal MUC2 mRNA expression was up-regulated in the rats after intragastric administration of probiotics, while E44 administration caused significantly lowered MUC2 expression. MUC2 expression was down-regulated (by 66.7%) by transfection with shRNA MUC2 in Lovo cells as compared with the negative control and mock control cells. The inhibition of E44 adherence and invasion by probiotics was significantly attenuated in transfected Lovo cell culture (in which the relative adhesion and invasion rates of E44 were 56.64% and 66.64%, respectively) as compared with those in the control group.</p><p><b>CONCLUSION</b>The up-regulation of MUC2 in rat colons can be one of the mechanisms of the probiotics in antagonizing the translocation of the pathogenic bacteria. Silencing MUC2 expression causes attenuated inhibitory effect of the probiotics on E. coli K1 penetration across human intestinal epithelial cells.</p>

Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique.</p><p><b>METHODS</b>Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry.</p><p><b>RESULTS</b>Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)].</p><p><b>CONCLUSION</b>Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).</p>