Correction to: Adenovirus-mediated LIGHT gene modification in murine B-cell lymphoma elicits a potent antitumor effect.

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Genomic comparisons between paired bacterial strains with strong and weak GC skews.

A majority of known eubacterial genomes are characteristic of GC skew, i.e., the leading strand has exceeding number of G over C. The cause of this compositional bias is still not very clear. In this study, we chose five pairs of genomes from distantly related bacterial genera, i.e., Buchnera, Haemophilus, Mycoplasma, Mycobacterium, and Synechococcus, each containing one with strong GC skew and the other with weak GC skew. Through comparison of the orthologous genes in these genera, we found that neither chromosomal rearrangement nor CDS skew has direct relationship with GC skew.

Downregulation of microRNA 99a in oral squamous cell carcinomas contributes to the growth and survival of oral cancer cells.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression by either translational inhibition or mRNA degradation. miRNAs play pivotal roles in physiological functioning and pathological progression. The function of microRNA-99a (miR-99a) in oral squamous cell carcinoma (OSCC) remains unclear. In the present study, we investigated the roles of miR-99a in OSCC development and the underlying mechanisms in 25 cases of primary OSCC tissues and Tca-8113 cells. The cells were analyzed using FACS analysis and western blotting. Results showed that the expression levels of miR-99a were markedly decreased in OSCC tissues compared with the adjacent non-tumor tissues (n=25). The results of in vitro experiments showed that miR-99a mimics significantly inhibited the proliferation of Tca-8113 cells, a tongue squamous carcinoma cell line, and that miR-99a mimics markedly induced the apoptosis of Tca-8113 cells. Furthermore, we demonstrated that mammalian target of rapamycin (mTOR) was directly targeted by miR-99a, as the overexpression of miR-99a in Tca-8113 cells downregulated the protein expression level of mTOR. Thus, our findings suggest that the downregulation of miR-99a in OSCC tissues is associated with tumor development. miR-99a regulates the growth and survival of OSCC cells and may be exploited as a biomarker and therapeutic target for patients with OSCC.

Piperine suppresses tumor growth and metastasis in vitro and in vivo in a 4T1 murine breast cancer model.

AIM: To investigate the effects of piperine, a major pungent alkaloid present in Piper nigrum and Piper longum, on the tumor growth and metastasis of mouse 4T1 mammary carcinoma in vitro and in vivo, and elucidate the underlying mechanisms. METHODS: Growth of 4T1 cells was assessed using MTT assay. Apoptosis and cell cycle of 4T1 cells were evaluated with flow cytometry, and the related proteins were examined using Western blotting. Real-time quantitative PCR was applied to detect the expression of matrix metalloproteinases (MMPs). A highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model was used to evaluate the in vivo antitumor activity. Piperine was injected into tumors every 3 d for 3 times. RESULTS: Piperine (35-280 µmol/L) inhibited the growth of 4T1 cells in time- and dose-dependent manners (the IC(50) values were 105 ± 1.08 and 78.52 ± 1.06 µmol/L, respectively, at 48 and 72 h). Treatment of 4T1 cells with piperine (70-280 µmol/L) dose-dependently induced apoptosis of 4T1 cells, accompanying activation of caspase 3. The cells treated with piperine (140 and 280 µmol/L) significantly increased the percentage of cells in G(2)/M phase with a reduction in the expression of cyclin B1. Piperine (140 and 280 µmol/L) significantly decreased the expression of MMP-9 and MMP-13, and inhibited 4T1 cell migration in vitro. Injection of piperine (2.5 and 5 mg/kg) dose-dependently suppressed the primary 4T1 tumor growth and injection of piperine (5 mg/kg) significantly inhibited the lung metastasis. CONCLUSION: These results demonstrated that piperine is an effective antitumor compound in vitro and in vivo, and has the potential to be developed as a new anticancer drug.

IFN-γ producing T cells contribute to the increase of myeloid derived suppressor cells in tumor-bearing mice after cyclophosphamide treatment.

It has been reported that treatment with cyclophosphamide (CTX) as a chemotherapeutic drug in cancer patients or tumor-bearing mice can result in an increase in the proportion of myeloid derived suppressor cells (MDSCs) in blood and lymphoid organs. Here we sought to clarify the possible mechanism of this unwanted increase in proportion of MDSCs in tumor-bearing mice after CTX treatment. We found that both CD4(+) T cells and CD8(+) T cells underwent an expansion and activation before the increase of MDSCs in the early period of CTX treatment in 4T1 breast tumor-bearing mice. The proportion of MDSCs in nude mice lacking T cells after CTX therapy was comparable to that in nude mice without CTX treatment. T cell transfer to 4T1-bearing nude mice enhanced the proportion of MDSCs in tumor-bearing mice after CTX therapy. The co-culture of MDSCs and T cells in vitro also showed that CD4(+) T cells and CD8(+) T cells could facilitate the expansion and survival of MDSCs, and this effect was mediated by IFN-γ released by T cells. These results gave an explanation of the unwanted consequence resulted from CTX treatment in tumor-bearing mice. It also provided some insights into the strategies for eliminating the bad side of CTX treatment and to make it more effective in cancer therapy.

Reduced tumorigenesis of EG7 after interleukin-10 gene transfer and enhanced efficacy in combination with intratumorally injection of adenovirus-mediated lymphotactin and the underlying mechanism.

Although interleukin-10 (IL-10) is commonly regarded as an immunosuppressive cytokine, a wealth of evidence is accumulating that IL-10 also possesses some immunostimulating antitumor properties. Previous studies demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. In this study, we explored the tumorigenesis of EG7 cells transduced with IL-10 gene. In vivo, IL-10 gene transfer reduced tumorigenic capacity of EG7 cells and prolonged survival of the EG7 tumor-bearing mice. It was found that the cytotoxicities of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) were enhanced. Assessment of the immune status of the animals showed prevalence of a systemic and tumor-specific Th2 response (high levels of IL-4 and IL-10). To improve the therapeutic efficacy, we combined with intratumoral injection of adenovirus-mediated lymphotactin (Ad-Lptn) into the overestablished EG7 tumor model. More significant inhibition of tumor growth were observed in EG7 tumor-bearing mice that received combined treatment with IL-10 and Lptn gene than those of mice treated with IL-10 or Lptn gene alone. The highest NK cells and CTL activity was induced in the combined therapy group, increasing the production of IL-2 and interferon-γ (IFN-γ) significantly but decreasing the expression of immune suppressive cells (CD4(+)Foxp3(+) Treg cells and Gr1(+)CD11b(+) MDSCs). The necrosis of tumor cells was markedly observed in the tumor tissues, accompanying with strongest expression of Mig (monokine induced by interferon-gamma) and IP-10 (interferon-inducible protein 10), weakest expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2). In vivo, depletion analysis demonstrated that CD8(+) T cells and NK cells were the predominant effector cell subset responsible for the antitumor effect of IL-10 or Lptn gene. These findings may provide a potential strategy to improve the antitumor efficacy of IL-10 and Lptn.

[Establishment of loop-mediated isothermal amplification method for detection of Legionella pneumophila].

OBJECTIVE: To establish a simple and rapid molecular detection for Legionella pneumophila. METHODS: The loop-mediated isothermal amplification (LAMP) was applied for detection of Legionella pneumophila. A set of primers were designed to identify six special areas in mip gene of Legionella pneumophila. Genomic DNAs from 13 bacterial strains,including 8 Legionella pneumophila strains and 5 other bacterial strains were amplified by LAMP and general PCR method to evaluate the specificity and sensibility of LAMP. RESULT: All positive tubes produced visible white precipitation, and no precipitation was observed in others. By adding smart green fluorescent dye, all Legionella pneumophila positive tubes presented a strong green fluorescence, while others showed weak fluorescence. The detection rate of LAMP was higher than that of general PCR. The detection limits were 576fg with genomic DNA of Legionella pneumophila,and 8 cfu/mL with positive water samples. CONCLUSION: LAMP detection of Legionella pneumophila is an effective and low-cost method with high specificity and sensitivity requiring no special equipment.

Adenovirus-mediated LIGHT gene modification in murine B-cell lymphoma elicits a potent antitumor effect.

Here, we investigated the antitumor effect of adenovirus-mediated gene transfer of LIGHT, the tumor-necrosis factor (TNF) superfamily member also known as TNFSF14, in the murine A20 B-cell lymphoma. LIGHT gene modification resulted in upregulated expression of Fas and the accessory molecule--intercellular adhesion molecule-1 (ICAM-1) on A20 cells and led to enhanced A20 cell apoptosis. LIGHT-modified A20 cells effectively stimulated the proliferation of T lymphocytes and interferon (IFN)-gamma production in vitro. Immunization of BALB/c mice with a LIGHT-modified A20 cell vaccine efficiently elicited protective immunity against challenge with the parental tumor cell line. Adenovirus-mediated gene transfer of LIGHT by intratumoral injection exerted a very potent antitumor effect against pre-existing A20 cell lymphoma in BALB/c mice. This adenovirus-mediated LIGHT therapy induced substantial splenic natural killer (NK) and cytotoxic T lymphocyte (CTL) activity, enhanced tumor infiltration by inflammatory cells and increased chemokine expression of CC chemokine ligand 21 (CCL21), IFN-inducible protein-10 (IP-10) and monokine induced by IFN-gamma (Mig) from tumor tissues. Thus, adenovirus-mediated LIGHT therapy might have potential utility for the prevention and treatment of B-cell lymphoma.

[Preparation and identification of polyclonal antibody against cysteinyl leukotriene receptor 2].

OBJECTIVE: To prepare and identify a polyclonal antibody against cysteinyl leukotriene receptor (CysLT(2)receptor). METHODS: Rabbits were immunized with KLH-coupled CysLT(2) receptor peptide to prepare the polyclonal antibody (pAb). The titer of the pAb in rabbit plasma was detected by indirect ELISA, and the specificity of the pAb was tested by antigen blockade. The tissue distribution of CysLT(2) receptor was detected by Western blot and immunohistochemistry with the prepared pAb. RESULT: The pAb showed a titer higher than 1/1047296, and was specific to CysLT(2) receptor, without cross-reaction with the antigens of CysLT(1) receptor and GPR17. A higher expression of CysLT(2) receptor in kidney, brain and lung of rats and mice was detected by Western blot analysis using the prepared pAb. The molecular weight of CysLT(2) receptor protein was about 40 kD. Immunohistochemical examination showed that CysLT(2) receptor was expressed mainly in the neuron, and partly in astrocytes in rat brain. CONCLUSION: The prepared CysLT(2) receptor pAb has high sensitivity and specificity, and can be used in Western blot and immunohistochemistry.

[Method for screening cysteinyl leukotriene receptor 2 antagonists and preliminary screening of compounds].

OBJECTIVE: To establish a method for screening cysteinyl leukotriene receptor 2 (CysLT(2)) antagonists and to preliminarily screen a series of synthetic compounds. METHODS: Rat glioma cell line (C6 cells) highly expressing CysLT(2) receptor was used. Intracellular calcium concentration was measured after stimulation with the agonist LTD(4),which was used to screen compounds with antagonist activity for CysLT(2) receptor. Bay u9773, a CysLT1/CysLT(2) receptor non-selective antagonist, and AP-100984, a CysLT(2) receptor antagonist, were used as control. RESULT: PT-PCR showed a higher expression of CysLT(2) receptor in C6 cells. LTD(4) at 1 mumol/L significantly increased intracellular calcium in C6 cells; the maximal effect was about 37.5% of ATP, a positive stimulus.LTD(4)-induced increase of intracellular calcium was blocked by CysLT(2) receptor antagonists, but not by CysLT(1) receptor antagonists. Among the synthetic compounds, D(XW-)1,2,13,23,29 and 30 inhibited LTD(4)-induced increase of intracellular calcium. CONCLUSION: LTD(4)-induced change in intracellular calcium in C6 cells can be used as a screening method for CysLT(2) receptor antagonists. The compounds, D(XW-)1,2,13,23,29 and 30, possess antagonist activity for CysLT(2) receptor.

[Preparation and identification of a polyclonal antibody against novel cysteinyl leukotriene receptor GPR17].

OBJECTIVE: To prepare and identify a polyclonal antibody (pAb) against GPR17, a novel cysteinyl leukotriene receptor. METHODS: Rabbits were immunized with KLH-coupled GPR17 peptide to prepare the pAb. The titer of the pAb in rabbit plasma was detected by indirect ELISA, and the specificity of the pAb was tested by antigen blockade. GPR17 tissue distribution was detected by Western blot with the pAb. RESULTS: The pAb showed a titer as high as 1:16 364,and was not cross-reacted with the antigens of CysLT(1) and CysLT(2) receptors. A higher expression of GPR17 in the rat brain and heart was detected using the newly prepared pAb. The molecular weigh of GPR17 protein was about 43 kD. CONCLUSION: The prepared GPR17 pAb has high sensitivity and specificity,and can be used in Western blot for detecting GPR17.

EBAG9 inducing hyporesponsiveness of T cells promotes tumor growth and metastasis in 4T1 murine mammary carcinoma.

The estrogen receptor-binding fragment-associated gene 9 (EBAG9) has been identified as an estrogen-responsive gene and was recently identified as a tumor-promoting and prognostic factor for renal cell carcinoma. We investigated whether EBAG9 expression was correlated with primary tumor growth and distant tumor metastasis in a murine breast carcinoma model. Knockdown expression of EBAG9 by small interfering RNA significantly suppressed tumor growth and metastasis in vivo in a highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model. 4T1 cells stably overexpressing EBAG9 developed larger and faster tumor growth and lung metastasis compared with parental 4T1 or 4T1 expressing vector alone. Strong specific cytotoxic T lymphocyte activity and enhanced gamma-interferon and interleukin-2 productions were induced in mice that received EBAG9 small interfering RNA therapy. Gene silencing of EBAG9 prolonged the survival of tumor-bearing mice and induced more intensive infiltration of CD8+ T cells in tumor mass. EBAG9 induced apoptosis of T cells, enhanced glycogen synthase kinase 3beta phosphorylation and inhibited gamma-interferon production of T cells when T lymphocytes were cocultured with 4T1 cells overexpressing EBAG9. Furthermore, overexpression of EBAG9 in 4T1 cells was accompanied with enhanced expression of chemokine (C-X-C motif) receptor 4, which might be involved in tumor metastasis. Taken together, our results suggested that EBAG9 promoted primary 4T1 mammary carcinoma growth and distant metastasis, and EBAG9 small interfering RNA exerted overt regression of tumor growth and metastasis. These findings might provide insights into the mechanism through which tumors evade immunosurveillance and provide a strategy for therapeutic intervention of cancer metastases.

Significance of SHP-1 and SHP-2 expression in human papillomavirus infected Condyloma acuminatum and cervical cancer.

Human papillomaviruses (HPVs) are a group of DNA viruses that infect the skin and mucous membranes. Type HPV6/11 is closely related to Condyloma acuminatum, while HPV16/18 is the principal cause of cervical cancer. In this study, we examined the expression of protein tyrosine phosphatases SHP-1 and SHP-2 in Condyloma acuminatum, cervical cancer and the relationship between SHP-1/SHP2 expression and HPV infection. Forty Condyloma acuminatum cases, 20 cervical cancer cases and 20 normal human foreskins were examined for HPV infection by in situ hybridization and the expression of SHP-1 and SHP-2 were examined by immunohistochemistry. Results demonstrated that positive expression rates of HPV6/11, HPV16/18, and HPV31/33 were 98%, 10%, and 7.5% in Condyloma acuminatum, 10%, 85%, and 25% in cervical cancer. Only one normal foreskin demonstrated positive staining for HPV16/18. Positive expression rates of SHP-1 and SHP-2 were 80% and 85% in Condyloma acuminatum, 85% and 90% in cervical cancer. The SHP-1 and SHP-2 expressions were mainly distributed in the prickle layer of Condyloma acuminatum and were diffusely distributed in cervical cancer cells. Only 35% and 30% of foreskins demonstrated weak staining in the basal layer cells. There were statistically significant correlations among the infection of HPV and the expression of SHP-1 and SHP-2 in both Condyloma acuminatum and cervical cancer (P < 0.05). SHP-1 expression has a positive correlation with SHP-2 expression. Our results demonstrate putative roles of SHP-1 and SHP-2 in the progression of both Condyloma acuminatum and cervical cancer after HPV infection.

[Expression of tyrosine phosphatase containing C-src homology SH-2 in benign prostate hyperplasia].

OBJECTIVE: To explore the expression of tyrosine phosphatase containing C-src homology SH-2 (SHP-1 and SHP-2) in benign prostate hyperplasia. METHODS: With En Vision two-step method, the expression of SHP-1 and SHP-2 was detected in 10 cases of normal prostate tissue, 30 cases of BPH, 20 cases of PIN, 20 cases of high differential Pca and 20 cases of low differential Pca. RESULT: The expression of SHP-2 in normal group was mainly distributed in the cytoplasm of secretive cells and basal cells, and a little part in the nucleu. In BPH it was distributed equally in the plasm and nucleu. In PIN, high differential Pca and low differential Pca, SHP-2 expressed mainly in nucleu. The average dyeing index of SHP-2 in each group is 0.4, 1.7, 2.1, 2.2 and 2.6. SHP-1 positive expression in normal prostate, BPH, PIN and high differential Pca showed differentiating layer staining in the cytoplasm of secretive cells and basal cells, while not in low differential Pca. The average dyeing index of SHP-1 in each group is 1.8, 1.8, 1.5, 1.2 and 0.4. CONCLUSION: There are transformation in signal transduction relation with SHP-1 and SHP-2 in the progress of prostate cell proliferation, differentiation and malignant. The abnormal activation and distribution of SHP-2 might induce prostate reconstruction and hyperplasia, even carcinoma.

[Adhesional inhibiton of polyclonal anti-porin I antibody].

OBJECTIVE: To investigate the blockness effects of purified polyclonal anti-porin I antibody on N. gonorrhoeae adherence to genitourinary tract epithelia of BALB/c mouse. METHODS: Polyclonal anti GST-PI antibody was generated by immunizing rabbit with GST-PI fusion protein which was constructed and expressed by ourselves. The purified immunoglobulin G was obtained by ammonium sulphate deposition and DEAE cellulose chromatography. Mice model of gonorrhea was established. In order to evaluate the effects of PI-IgG on gonococcus adhesion to vagina mucus, the macroscopic and pathological assessing as well as gonococcus culture was employed after gonococcus challenge on PI-IgG immunized mice. RESULT: No pus and pathological inflammation were observed on mice vagina mucus treated with 1 mg/ml PI-IgG 3 hours before gonococcus challenge. Gonococcus could not be detected in the smears and washing solutions from vagina. Pathological inflammation was found in mice treated with anti PI-IgG, in which the concentrations were lower than 1 mg/ml or the treated time was longer than 3 hours prior to gonococcus challenge. CONCLUSION: The purified anti PI-IgG can effectively inhibit the adherence and infection of gonococci to genitourinary tract epithelia of BALB/c mice. In addition, the blocking duration of anti PI-IgG is associated with antibody concentration.

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells.

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4+, CD8+, CD19+ cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34+ stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.

[Preparation of monoclonal antibodies against human mesenchymal stem cells].

OBJECTIVE: To prepare monoclonal antibodies (McAbs) against human mesenchymal stem cells (hMSCs) and to study their biological characteristics. METHODS: BALB/C mice were immunized with pooled hMSCs. McAbs were prepared by hybridoma technique and their biological characteristics were analyzed by indirect immunofluorescence, immunohistochemistry and flow cytometry. RESULT: Five hybridoma cell lines were successfully established, which secret McAbs specifically against hMSCs. Investigations showed that all these McAb reacted only to hMSCs and had no cross-reaction to other human cells, the relative affinities of 5 McAbs were 1x10(6) (ZUB1), 1x10(5) (ZUB4), 1x10(6) (ZUC3), ZUE12 (1x10(5)) and 1x10(5) (ZUF10), respectively. Isotype analysis showed that ZUB1, ZUE12, ZUF10 against the same isotype, while ZUC3, ZUB4 against other two different isotypes alone. Flow cytometric analysis showed that the positive expression rate of cultured hMSCs was 87.39% (ZUB1), 88.07% (ZUB4), 88.12% (ZUC3), 69.89% (ZUE12) and 83.67% (ZUF10). CONCLUSION: The prepared five McAbs can specifically react against hMSCs, which can be used for selection and study of hMCSs.

[Experiment treatment of collagen-induced arthritis in rats with recombinant plasmid containing vasoactive intestinal peptide gene].

OBJECTIVE: To investigate the therapeutical effect of recombinant plasmid containing vasoactive intestinal peptide gene (pcDNA3.1+/VIP) on collagen-induced arthritis (CIA) in rats. METHODS: The experimental arthritis was induced by intradermal injection of bovine type II collagen emulsified in Freund's adjuvants in male SD rats. The rats then were given intra-articular injection with recombinant plasmid (pcDNA3.1+/VIP). The levels of serum TNF-alpha, IL-4 and IL-2 were detected by Avidin-Biotin Peroxdase Complex-enzyme-linked immunosorbent assay (ABC-ELISA) and the pathological changes in the joint of rats were observed. RESULT: Histological examination showed massive inflammatory infiltration in the joint with destruction of bone and cartilage, while the severity of pathological changes in synovia of VIP-treated rats was markedly reduced. Compared with normal group, the serum TNF-alpha, IL-2 levels of CIA rats were significantly increased (P <0.05) and IL-4 level was decreased (P<0.05). Compared with control and pcDNA3.1+ -treated CIA rats, serum TNF-alpha and IL-2 levels of pcDNA3.1+/VIP-treated rats were decreased and IL-4 level was increased (P<0.05). CONCLUSION: Recombinant plasmid containing vasoactive intestinal peptide gene (pcDNA3.1+/VIP) can reduce the clinical and histological severity of established CIA and it might be a promising candidate for treatment of rheumatoid arthritis.

[Study on lysosomes degradation of ricin A chain].

OBJECTIVE: To study lysosomes involvement in the degradation of ricin A chain. METHODS: A lysosome-targeted singal KFERQ was added to the C terminus of rRTA by DNA recombinant technology. A pKK223.3 expression system in E. coli was used to produce recombinant ricine A chain (rRTA) and rRTA-KFERQ. Recombinant proteins were purified by affinity chromatography using Blue-Sepharose 6B. The cytotoxicity of recombinant proteins was measured by the MTT method. RESULTS: Recombinant RTA-KFERQ was 49.87%, 54.18% and 88.68% less cytotoxic than RTA itself on the three cell lines HEPG2, Hela and A549, respectively. CONCLUSION: Lysosomes can degrade, but not completely inactivate RTA in different cells, suggesting cells may have other degradation pathways for RTA.

[Construction of recombinant plasmid containing mouse vasoactive intestinal peptide gene and its expression in COS-7 cells].

OBJECTIVE: To construct the eukaryotic expression plasmid containing mouse vasoactive intestinal peptide(VIP) gene with biological activities. METHODS: VIP cDNA including the sequences of signal peptide was cloned from mouse thymus by RT-PCR, and then inserted into the mammalian expression vector pcDNA3.1 between Hind III and EcoR I restriction sites. COS-7 cells were transfected with pcDNA3. 1-VIP using liposome, the expression of VIP was identified by Western blot and ELISA. Supernatant of transfected cell culture was added to LPS-stimulated macrophages and the TNF-alpha production in cell medium was observed by ELISA. RESULTS: The cloned VIP cDNA was confirmed by enzyme digestion and DNA sequencing. The expression of VIP was detected in the pcDNA3. 1-VIP transfected COS-7 cells by Western blot and ELISA. The VIP in culture supernatant potently inhibited TNF-alpha production by LPS-induced Macrophages in vitro. CONCLUSION: The eukaryotic expression plasmid that expresses biological active murine VIP has been constructed successfully.