[Construction of a dual-promoter DNA expression plasmid pCN-SSIE harboring gene encoding SBR of Streptococcus mutans and verification of its immunogenicity as DNA vaccine].

PURPOSE: To construct a dual-promoter expression plasmid that harbors the target gene encoding SBR of Streptococcus mutans and can be applied as DNA vaccine especially suitable for using attenuated Salmonella as delivery vector to elicit effective mucosal immune responses because of its advantage of possessing dual-promoter. METHODS: Genes encoding SBR and green fluorescence protein gene (EGFP) were amplified by PCR and inserted to the proper sites of vector pCMVnir. Then IRES sequence was inserted between the genes coding for SBR and EGFP. Furthermore, a DNA fragment encoding tissue-type plasminogen activator (tPA) signal peptide was fused to the 5' end of target gene. Thereby, construction of the dual-promoter expression plasmid pCN-SSIE was completed and then the plasmid was analyzed with DNA sequencing and endonuclearase digestion mapping. The expressions of SBR protein by attenuated Salmonella SL3261 and CHO cell transformed or transfected by the plasmid were tested respectively. Finally, BALB/c mice were immunized through injecting intramuscularly with plasmid pCN-SSIE and anti-SBR specific IgG in serum was tested. RESULTS: Both DNA sequencing and endonuclearase digestion mapping showed that the construction of pCN-SSIE was successful with its open reading frame being correct. The expressions of SBR protein in transformed attenuated Salmonella SL3261 and transfected CHO were detected, and anti-SBR specific IgG levels in serum of immunized mice were markedly higher than the control. CONCLUSION: The construction of the dual-promoter expression plasmid pCN-SSIE was successful and the plasmid can express in prokaryocyte and eukaryocyte and elicit dramatic immune response when applied as DNA vaccine in experimental animal.

[Inhibition of multidrug resistance related P-gp expression in human neuroblastoma by antisense peptide nucleic acid].

OBJECTIVE: To investigate the efficiency of a peptide nucleic acid (PNA) delivery system by using liposome via PNA-DNA hybrids and to test the inhibitive action of antisense PNA on expression of multidrug resistance (MDR) related P-glycoprotein (P-gp) in human neuroblastoma cell line SK-N-SH. METHODS: Two antisense PNAs were designed targeting at MDR-1 mRNA and then combined with partially complement DNAs respectively. The hybrids were delivered into cells using cationic liposome. The transfection efficiency, expression of P-gp and MDR-1 mRNA, intracellular adarimycin (ADM) were measured by flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), and high performance liquid chromatography (HPLC). RESULTS: Transfection of PNA increased the cell average fluorescence intensity significantly and the extent of increase was dependent on the concentration of PNA. After being transfected by both PNAs, P-gp expression of SK-N-SH cells decreased significantly and the intracellular ADM level was increased by about 3 times. The level of MDR-1 mRNA expression slightly decreased after transfection, but no statistical significance was observed. CONCLUSIONS: PNA can be delivered into tumor cells in form of PNA-DNA hybrids by cationic liposome. Properly designed antisense PNA can inhibit MDR related P-gp expression of SK-N-SH cells efficiently and specifically.

Ornithine decarboxylase gene is overexpressed in colorectal carcinoma.

AIM: To investigate the ornithine decarboxylase (ODC) gene expression in colorectal carcinoma, ODC mRNA was assayed by RT-PCR and ODC protein was detected by a monoclonal antibody against fusion of human colon ODC prepared by hybridoma technology. METHODS: Total RNA was extracted from human colorectal cancer tissues and their normal counterpart tissues. ODC mRNA levels were examined by RT-PCR. ODC genes amplified from RT-PCR were cloned into a prokaryotic vector pQE-30. The expressed proteins were purified by chromatography. Anti-ODC mAb was prepared with classical hybridoma techniques and used to determine the ODC expression in colon cancer tissues by immunohistochemical and Western blotting assay. RESULTS: A cell line, which could steadily secrete anti-ODC mAb, was selected through subcloning four times. Western blotting reconfirmed the mAb and ELISA showed that its subtype was IgG2a. RT-PCR showed that the ODC mRNA level increased greatly in colon cancer tissues (P<0.01). Immunohistochemical staining showed that colorectal carcinoma cells expressed a significantly higher level of ODC than normal colorectal mucosa (98.6+/-1.03% vs 5.26+/-5%, P<0.01). CONCLUSION: ODC gene overexpression is significantly related to human colorectal carcinoma. ODC gene expression may be a marker for the gene diagnosis and therapy of colorectal carcinoma.

[The construction of SCID-Hu IC mice model and application in rAd5HPV16L1-E7 vaccine].

OBJECTIVE: To construct human-SCID chimeric mice through implantation of mononuclear cells from human cord blood and study the immunoreaction of SCID-Hu IC mice immunized with rAd5HPV16L1-E7 vaccine. METHODS: (1) Experiment groups were injected with the suspension of mononuclear cells from human cord blood through a tail vein; the control ones were injected with non serum RPMI 1640 medium. Eight weeks after implantation, blood was collected and human serum IgG level in the mice were tested, and human CD45, CD3 and CD19 were determined. (2) SCID-Hu IC mice were divided into two groups: in group A the mice were immunized intraperitoneally with rAd5HPV16L1-E7 virus and in group B the mice were immunized through nasal drip with rAd5HPV16L1-E7 virus. At the end of fourth week, the serum specific IgG antibody to rAd5HPV16L1-E7 virus, IFN-gamma in culture medium of spleen lymphocyte and T-lymphocyte propagation were tested. RESULTS: (1) In the experiment groups, the number of mice positive for human IgG was 10/15, the average values of CD45, CD3 and CD19 were (9.39+/-4.21), (3.25+/-3.99) and (1.69+/-0.75), respectively. In the control ones, the human IgG, CD45, CD3 and CD19 were negative. (2) The results in the experiment groups showed that the IFN-gamma and T-lymphocyte stimulated by HPV16 protein were higher than those in the non-stimulated group (P less than 0.05). CONCLUSION: (1) The results indicated that the construction of human-SCID chimaera through the implantation of mononuclear cells from human cord blood into SCID mice was successful. They also indicated that the reconstructed SCID-Hu IC mice has the ability to produce immune response against rAd5HPV16L1-E7 recombinant virus.

Cloning and expression of ornithine decarboxylase gene from human colorectal carcinoma.

AIM: To construct and express ODC recombinant gene for further exploring its potential use in early diagnosis of colorectal carcinoma. METHODS: Total RNA was extracted from colon cancer tissues and amplified by reverse-transcription PCR with two primers, which span the whole coding region of ODC. The synthesized ODC cDNA was cloned into vector pQE-30 at restriction sites BamH I and Sal I which constituted recombinant expression plasmid pQE30-ODC. The sequence of inserted fragment was confirmed by DNA sequencing, the fusion protein including 6His-tag was facilitated for purification by Ni-NTA chromatographic column. RESULTS: ODC expression vector was constructed and confirmed with restriction enzyme digestion and subsequent DNA sequencing. The DNA sequence matching on NCBI Blast showed 99 % affinity. The vector was transformed into E. coli M15 and expressed. The expressed ODC protein was verified with Western blotting. CONCLUSION: The ODC prokaryote expression vector is constructed and thus greatly facilitates to study the role of ODC in colorectal carcinoma.