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Human beta defensin 2 (HBD-2) may play an important role in human defense against infection. Its antimicrobial capacity has been fully documented in in vitro study. In order to evaluae its in vivo effects, we developed an HBD-2 transgenic mouse model. The HBD-2 minigene containing CMV promoter, full length of HBD-2 cDNA and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57 X ICR hybridized mouse by microinjection, and offspring were produced. DNA was isolated from the tails of the mouse pups, and the HBD-2 minigene incorporation was analyzed by PCR using HBD-2 specific primers. The HBD-2 gene expression in the multi-tissues of transgenic mice was determined at mRNA level by RT-PCR and at peptide level by immunohistological staining with the use of HBD-2 monoclonal antibody. The results showed that among 17 F0 transgenic mice, HBD-2 positive signal was determined by PCR in 4 mice, suggesting that HBD-2 minigene has been incorporated into the offspring mice. Meanwhile, a widespread expression of HBD-2 mRNA and peptide was detected in the F1 transgenic mice's multi-tissues such as trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium and brain.

This study sought to clarify the distribution of HMGN2 in HeLa cells. The recombinant eukaryotic expression vectors pcDNA3. 1-myc-his-HMGN2 and pEGFP-N1-HMGN2 were constructed, and then were transfected into HeLa cells. immunocytochemistry staining indicated that HMGN2 were present not only in HeLa nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant by ELISA with rabbit anti-serum against HMGN2 and mouse anti-His6 monoclonal antibodies. The confocal microscope observation showed the same subcellular localization as that of immunocytochemistry staining. There results suggested that HMGN2 could be present in the nucleus and cytoplasm of HeLa cell as well as in the extracellular environment.

For the purpose of detecting the HBD-2 expression at protein level, the recombinant prokaryotic expression vector pGEX-1lambdaT-HBD-2 was constructed and the E. coli-based product of GST-HBD-2 fusion protein was prepared. When rabbit was immunized with the fusion protein, the anti-serum against HBD-2 was produced. After caprylic acid and ammonium sulfate precipitation, high titer of specific polyclonal antibody against HBD-2, which was detected by ELISA and Western blot, was obtained. This result suggests that recombinant peptide fusion protein could be used instead of the conjugate of peptide-albumin or peptide-thyroid globulin to produce antibody. The obtained antibodies could be used for revealing the tissue distribution of HBD-2 and the regulation of its gene expression.

The recombinant PSMA DNA vaccine for active immunotherapy of prostate cancer was investigated. Two DNA vaccine recombinant plasmids, pcDNA3.1/PSMA and pcDNA3.1/hBD-2-PSMA, were constructed by inserting the hBD-2 gene and PSMA gene into an eukarytoic expression vector pcDNA3.1. Expression of the two recombinants was detected in transfected COS-7 cells and inoculated mouse muscular cells by RT-PCR and immunohistochemical method. When immunized with pcDNA3.1/PSMA and pcDNA3.1/hBD-2-PSMA, the immunized BALB/c mice acquired specific antibody and T cell response to PSMA. The quantity of the spleen lymphocytes and their CTL activity against PSMA gene transfected-BALB/3T3 cells significantly increased in the immunized mice, and the CTL activity of lymphocytes from pcDNA3.1/hBD-2-PSMA immunized mice was significantly higher than that of pcDNA3.1/PSMA immunized mice. This result suggests that pcDNA3.1/hBD-2-PSMA would probably be developed as a DNA vaccine for the immunotherapy of prostate cancer.

experimental pancreatitis and explore the mechanism of bacterial translocation at the molecular level in mice with severe acute pancreatitis.Thirty-six ICR mice were randomly divided into 6 groups with 6 mice in each group. The animals in the experimental groups (from A to E) received seven intraperitoneal injections of caerulein (50 microg/kg body weight) at hourly intervals over 6 hours, and were killed at 9, 18, 24, 48 and 72 hours after the first injection. The control group (F) received intraperitoneal injection of the same volume of saline, and the animals were killed at the 18th hour after the first injection. Blood and pancreatic tissue samples were obtained after the animals were killed. Amylase and pancreatic pathological alterations were observed. The ileum sequential segments were removed from each mouse. The amplification products of RT-PCR were electrophoresed and the images were analyzed by UVI software.Acute necrotizing pancreatitis in ICR mice were induced by the intraperitoneal injection of caerulein at large doses. Markedly pathological lesions were observed at the 18th hour after the first injection of caerulein. And, intestinal cryptdin-4 mRNA expression was down regulated slightly at the 9th hour and most markedly at the 18th hour (P0.05). After 24 hours, the cryptdin-4 mRNA expression recovered to the normal level gradually.Acquired cryptdin-4 deficiency may play an important role in the pathogenesis of bacterial tanslocation in acute necrotizing pancreatits.

beta-defensins possess a broad spectrum of antimicrobial activity. In this study its in vivo antibacterial effect was evaluated by using gene transfer. Rat beta-defensin-2 (rBD2) recombinant pBK-CMV-rBD2 and pCD-NA-3, 1-Myc-His(+)-rBD2 were constructed. Then, by use of liposome agent, the recombinants were delivered into rat airway via tracheal injection. The rBD-2 mRNA expression was detected in the trachea by RT-PCR and its protein expression was determined in the lungs by the tag His immunostaining, 24 hours after inoculation via trachea, the count of P. areuginosa in the lung of rat transfected with pBK-CMV-rBD2 markedly decreased, compared with the control (n = 8, P = 0.003). The data presented in this study provide evidence that airway beta-defensin-2-gene transfer can protect the rat against bacterial infection in vivo, suggesting the beta-defensins as part of the innate host defense system can be of potential applicability.

The aim of this study is to explore the possibility and technical itinerary of establishing an mammal engineering cell line in which hBD-2 can be effectively expressed, secreted, detected, separated and purified. The full hBD2 cDNA was inserted into the multi clone site of a eukaryotic expressive plasmid pcDNA3.1/Myc-His(+) and located closely at the upstream of two tag gene (myc and 6 Poly-histidines) so as to construct another recombinant eukaryotic expressive vector of hBD-2 gene: rpcDNA3.1/Myc-His/hBD-2. By the use of RT-PCR with special primers, a band of 240 bp was amplified from COS-7 cells transfected by this recombinant plasmid, which matched full length of cDNA coding hBD2 plus myc epitope and 6 poly-histidines tags. Western blot analysis with specific anti-histidines antibody revealed that the lysate of COS-7 cells transfected by rpcDNA3.1/Myc-His/hBD-2 had a strong band with molecular weight of about 10 Kd that was approximate to the size of chiasmic peptide. Antibacterial activity assay showed that obvious bacterial inhibition occurred in both lysate and supernatant of COS-7 cells transfected by rpcDNA3.1/Myc-His/hBD-2.