Regulation of matrix metalloproteinase production and tumor cell invasion by four monoclonal antibodies against different epitopes of HAb18G/CD147 extracellular domain.

HAb18G/CD147, a membrane spanning molecule and highly expressed in hepatocellular carcinoma (HCC) cells, was shown to stimulate the production of matrix metalloproteinases (MMPs) in the interaction of tumor cells and fibroblasts. Studies on the EMMPRIN/CD147 showed that CD147 extracellular domain is involved in the induction of MMPs. To study the biological molecular function of HAb18G/CD147 extracellular domain (HAb18G/CD147-ED) on production of MMPs following mediated tumor cell invasion, we isolated four novel monoclonal anibodies (MAbs)-1B3, 3B3, HAb18Gedomab1, and HAb18Gedomab2-against HAb18G/CD147-ED by immunization of BALB/c mice with purified HAb18G/CD147-ED fragments, which were efficiently expressed in Escherichia coli. Gelatin zymography and Boyden chamber assays were used to identify the production of MMPs in the co-cultured human fibroblast and HCC cells, and to quantify the migrated cells in the presence of the generated MAbs. The results showed that two MAbs (1B3 and 3B3) inhibited [corrected] the secretion of MMP-2 and [corrected] the HCC cell invasion, whereas the other two MAbs (HAb18Gedomab1 and HAb18Gedomab2) had reverse function [corrected] FCM additive assay showed that four MAbs recognized different epitopes of HAb18G/CD147-ED. Taken together, the results suggest that various regions of HAb18G/CD147-ED participated in the regulation of MMP secretion.

[Construction and identification of the E.coli-BCG shuttle vector expressing lipoprotein Der p2 on cell wall of mycobacterium vaccae].

AIM: To construct the E.coli-BCG shuttle vector carrying and expressing dust mite antigen gene Der p2 on cell wall of mycobacterium vaccae. METHODS: The gene fragment encoding 19 kDa antigen and the upstream control element (19-ss) was amplified by PCR from the mycobacteria tuberculosis H37Rv.Subsequently, the 19-ss gene was cloned into the E.coli-BCG shuttle vector pOLYG, which can schlepped and expressed exogenous antigen gene on cell wall of mycobacteria and containing the Der p2 gene. The expression of Der p2 gene in mycobacterium vaccae determined by indirect immunofluorescence staining. RESULTS: Sequencing proved that the cloned sequence of 19-ss gene was correct. The constructed E.coli-BCG shuttle vector (pCW) containing 19-ss gene had function of shuttle between E.coli and mycobacteria, and mediated the expression of antibiotic resistance gene. Indirect immunofluorescence staining indicated that the Der p2 gene was expressed in the form of lipoprotein on surface of the mycobacterium vaccae. CONCLUSION: E.coli-BCG shuttle vector has been constructed successfully, which can express exogenous antigen gene as a chimeric protein on cell wall.

[Protective efficacy of DNA vaccines encoding mycobacterium tuberculosis Ag85B protein].

AIM: To explore protective efficacy of pTB30m and pTB30s encoding Ag85B protein against infection with M. tuberculosis H (37)R (v). METHODS: BALB/c mice were infected intravenously with 5x10(5) CFU of M. tuberculosisH (37)R (v) six weeks after the last vaccination of pTB30m and pTB30s. At the same time, normal BALB/c mice were injected intravenously with 5x10(6) T cells separated from immunized BALB/c mouse spleen through nylon wool column, and challenges were injected with 10(5) CFU of M. tuberculosis immediately intravenously. After 4 weeks, the CFU in spleens of infected mice were counted respectively. RESULTS: Vaccination with pTB30m and pTB30s produced significant protection against M. tuberculosis proliferation in the mouse spleens following challenge. As compared with the saline-injected mice, CFU in spleens of the mice vaccinated with pTB30m or pTB30s were reduced significantly, being 0.645(log(10) CFU, P<0.01) and 0.839(log(10) CFU, P<0.001), respectively. In contrast, bacterial load in the mice spleens vaccinated with empty plasmid only had little reduction. After adoptive immunization by T cells from the mice vaccinated with pTB30m and pTB30s, the mice might induce partial protection against M. tuberculosis proliferation in the mouse spleens following challenge. CONCLUSION: Protective efficacy of the mice immunized with pTB30s against challenge with M. tuberculosis virulent strain was better than that immunized with pTB30m.Thus, pTB30s is a promising DNA vaccine with respect to the prevention and treatment of tuberculosis.