| BackgroundThymosin alpha-1(Tal), a biologically active peptide consisted of28amino acid residues and firstly separated from calf thymus extract. Many clinical trials and studies on animals have demonstrated that (1) Tαl increased the efficiency of T cell maturation, stimulated precursor stem cell differentiation into CD4+/CD8+T cells and balanced CD3/CD4+/CD8+T cells of peripheral blood. By stimulating NK cells and CTL, Tal could directly kill virally infected cells. Tal also exert its immunoregulatory function by increasing IL-2and decreasing Th2cytokines such as IL-4and IL-10, by upregulating specific cytokine receptors such as high-affinity IL-2cytokine receptors.(2) Tαl could decrease tumor cell growth both in vitro and vivo and shown therapeutic usefulness in several types of cancer. Tal exhibit inhibitory effects on the growth of tumor, reducing tumor progression, show enhancing effects of chemotherapy agents.(3) Tal had protective effects against oxidative damage. Tal had a positive influence on liver superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity and thereby limited free radical damages to hepatic tissue. It was reported that Tαl could improve pancreatic functions by reducing malondialdehyde (MDA), increasing GSH level and enhancing the activities of both SOD and catalase (CAT).(4) When it was combined with chemotherapeutics in treating cancers, Tα1could prevent patients from chemotherapy-induced neurotoxicities. Moreover, Tα1has potent effects in promoting endothelial cell migration, angiogenesis as well as wound healing.Tα1has been used in clinic widly for the diagnosis and treatment of many diseases, including viral infections, such as chronic hepatitis B and C, primary immunodeficiency diseases (AIDS) and acute respiratory syndrome (SARS). Additionally, Tα1shows remarkable effects in the treatment of other diseases such as severe sepsis, gastrointestinal and systemic infectious disorders, and spontaneous peritonitis in individuals with cirrhosis. However, Tα1peptide used in clinic is mainly obtained by artificial synthesis. Injection is still the main administration route for peptide drugs such as artificially synthetic Tα1. It is inconvenient and even causes many side effects including allergy and infection. Tα1can also be purified from fresh calf thymus tissue, genetic engineering hosts including Escherichia coli, Pichia pastoris and plants. However, it is difficult to isolate and purify Tα1peptide from complex liquor or tissues in chemical synthesis and genetic engineering process. Insufficient purity and high expenses limit its availability for clinical applications.Bifidobacterium are the dominant Gram-positive and anaerobic bacteria which are ubiquitous and endosymbiotic inhabitants of the gastrointestinal of human and other warm-blooded animals and exert a range of beneficial health effects including improving intestinal microbial balance, preventing infection, reducing lactose intolerance, protecting against colon cancer. Recently, researchers successfully developed a new oral delivery system for many peptide drugs using engineered bifidobacterium as the carrier. The human oxyntomodulin (OXM), interleukin10(IL-10) and interferon-α2b (IFN-α2b)-transformed bifidobacteria were produced using the oral delivery system and applied in experimental obesity, colitis and myocarditis treatment in mice.In this study, a human Tal gene transformed B.longum was selected and its effects on cellular immunity in mice by oral administration were evaluated. This may provide a new manufacture and delivery approach for peptides such as Tal. ObjectiveIn order to improve administration convenience, a transformed-Bifidobacteri um.longum with human thymosin alpha-1(Tal) gene (BL-Tal) was produced a nd its effects on mice immunity by oral administration were investigated. MethodsThe human Tal expression vector pBBADs-Tal was constructed by inserting the human Tal coding sequence at the Bpi I and Xba I sites of plasmid pBBADs-GFP to replace GFP DNA fragment. The empty control vector (pBBADs-0) was constructed by cutting the GFP DNA fragment of plasmid pBBADs-GFP and ligating with DNA ligase.B.longum was transformed with pBBADs-Tal and pBBADs-0by electroporation respectively and selected by selective plates supplemented with Ampicillin. After gram stain was performed to identify the morphology of transformed B.longum, genome DNA were extracted and16s rDNA fragment were identified by PCR, plasmids were extracted to sequence.Transformed bacteria were cultured in MRS broth containing60μg ampicillin ml-1(final concentration) until the absorption of bacterium suspension at OD695nm reached～0.8,0.2%L-arabinose (final concentration)was added to induce the expression of target gene. Culture supernatants and pallets were collected and stored at-70℃after continuous induction for0,12,24,36h, respectively. The peptide in the culture supernatants and pallets of the transformed B.longum was detected by ELISA and Western blot. Six-week-old male Balb/c mice weighting18-20g were randomly divided into three groups and treated as follows:the BL-Tal group (n=8) was administrated0.1ml (6×109cells per ml) pBBADs-Tal transformed B.longum (BL-Tal) induced with0.2%L-arabinose for24h by intragastric administration every another day; the BL-0group (n=8) used as the negative control was administrated0.1ml (6×109cells per ml) pBBADs-0transformed B.longum (BL-0); the NS group (n=8) used as blank control was administrated0.1ml NS (0.9%saline). The intestinal contents of ileocecum, peripheral blood; spleens and thymuses were harvested. Lymphocytes were separated from spleens and thymuses respectively by Lymphocyte Separation Medium. Tal content in serum and intestinal content was also detected by ELISA kit. Several typical cytokines in serum (IFN-γ, TNF-α, IL-4, IL-12), CD3+CD4+, CD3+CD8+and CD4+CD8+T cell subsets in blood, spleen and thymus were detected by Flow CytoMeter.To investigate the effect of administration of BL-Tal for a long period on lymph nodes and thymus, another24mice were divided into3groups and orally administrated0.1ml (6×109cells per ml) BL-Tal induced with0.2%L-arabinose for24h every another day for3months. The pBBADs-0transformed B.longum (BL-0) and the NS were used as negative control and blank control respectively. The thymus, spleen and lymph nodes were surveyed after animals were sacrificed and removed for histological examination.Results were presented as mean±SD and analyzed with SPSS13.0statistical software (IBM, USA). The one-way ANOVA was performed for multiple comparisons, followed by the Bonferroni test method. When the equal variance test failed to produce results, the Dunnett T3test was selected. A probability value of less than0.05was considered statistically significant. ResultsThe plasmids containing Tal gene or without Tal gene were constructed and transformed into B.longum respectively. Gram staining, amplifying16s rDNA by PCR, and sequencing of plasmids extracted from transformed B.longum identified transformed B.longum successfully.Tal can be detected in both culture supernatant and pallet lysates after induced by0.2%L-arabinose. Western blot shows that bands (～3.3kDa) were observed in both culture supernatant and pallet lysates after induced by0.2%L-arabinose while no band was observed in BL-0, untransformed bacteria and preinduced BL-Tal. Furthermore, the level of Tal in supernatant reached maximum at24h of induction.Oral administration of bifidobacterium for2weeks shown that (1) The Tal concentration in intestinal contents was46.45±3.50pg/ml in BL-Tal group,13.64±2.92pg/ml in BL-0group and15.13±2.60pg/ml in NS group. The Tal level of BL-Tal group was increased significantly than that of BL-0group and NS group (P<0.001). No difference of Tal level in intestinal contents was found between BL-0group and NS group (P=0.997).(2) The Tal concentration in serum was15.61±1.65pg/ml in BL-Tal group,14.14±1.72pg/ml in BL-0group and15.07±2.17pg/ml in NS group. No statistical differences presented between BL-Tal group and the negative or blank control group respectively (P>0.05).(3) The average concentration (pg/ml) of IFN-γ, IL-12、TNF-α and IL-4were47.32±3.96、65.86±3.71、9.57±1.42、13.76±2.85in BL-Tal group,36.77±4.43、54.79±5.73、9.37±2.07、14.08±2.32in BL-0group,31.26±4.68、50.91±5.16、9.08±1.74、15.0O±3.31in NS group. The concentration of IFN-y was higher than that of BL-0group and NS group (P=0.001and P<0.001respectively), the concentration of IL-12also higher than that of BL-0group and NS group (P=0.002and P<0.001respectively). No differences were found in concentration of IFN-y and IL-12between BL-0group and NS group (P=0.089and P=0.475respectively), no differences of TNF-a and IL-4levels were found between BL-Tal group and BL-0group or NS groups (P>0.05).(4)In BL-Tal group, the percentage of CD3+CD4+cells was59.66±3.05in blood,22.05±2.26in spleen, and16.07±1.57in thymus; The percentage of CD3+CD8+cells was6.40±0.72in blood,6.80±0.62in spleen, and7.12±0.79in thymus; The percentage of CD4+CD8+cells was0.21±0.06in blood,3.49±0.64in spleen, and10.08±1.37in thymus. In BL-0group, the percentage of CD3+CD4+cells was47.64±2.49in blood,13.83±1.04in spleen, and12.46±1.68in thymus; The percentage of CD3+CD8+cells was3.09±0.60in blood,0.60±0.14in spleen, and4.08±0.65in thymus; The percentage of CD4+CD8+cells was0.21±0.08in blood,0.85±0.14in spleen, and6.08±1.07in thymus. In NS group, the percentage of CD3+CD4+cells was44.45±2.65in blood,10.54±1.19in spleen, and12.08±1.61in thymus; The percentage of CD3+CD8+cells was2.84±0.56in blood,0.45±0.11in spleen, and3.71±0.61in thymus; The percentage of CD4+CD8+cells was0.24±0.07in blood,0.44±0.07in spleen, and6.03±0.96in thymus. The proportion of CD3+CD4+and CD3+CD8+cells of BL-Tal group were increased significantly in blood, spleen and thymus, while CD4+CD8+in BL-Tal group increased markedly in thymus and spleen than that of BL-0group and NS group respectively (P=0.001). Furthermore, the proportion of CD3+CD4+and CD4+CD8+cells in spleen of BL-0group were up-regulated than that of NS group (P<0.001). No differences of CD4+CD8+in blood and spleen were found between BL-Tα1group and BL-0group and NS group(P>0.05), no differences of CD3+CD8+in spleen, CD3+CD4+, CD3+CD8+and CD4+CD8+in thymus and blood of BL-0group were found compared with those of NS group (P>0.05).The enlargement of thymus, spleen and lymph nodes were observed markedly in5mice among the total8mice of the BL-Tal group after oral administration treatment for three months, no thymic hyperplasia and lymphadenectasis were observed in BL-0group and NS group.ConclusionThe human Tα1gene transformed B.longum bifidobacterium was constructed, and Tα1expression and secretion could be detected in the Tal-transformed bifidobacterium both in vitro and vivo. Tal-transformed B.longum does have the effects on promoting T cell proliferation and differentiation, increasing the percentage of CTL and Thl cell in blood, spleen and thymus, stimulating immuneorgan growth and improve the cellular immunity through Thl pathway by oral administration. In our study, bifidobacterium was used as a delivery for human Tα1. The Tα1-transformed B.longum with bioactive Tal expression can be taken by oral administration, and exert its function directly in gut without purification. It is very convenient for production and administration, and has extremely price advantage than Tα1peptide. This may provide a new manufacture and delivery approach for peptides such as Tα1. |
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