Development of thermostable dextranase from Streptococcus mutans (SmdexTM) through in silico design employing B-factor and Cartesian-ΔΔG.

The thermal stability of enzymes dramatically limits their application in the industrial field. Based on the crystal structure, we conducted a semi-rational design according to the B-factor and free energy values to improve the stability of dextranase from Streptococcus mutans (SmdexTM). The B-factor values of Asn102, Asn503, Asp501 and Asp500 were the highest predicted by B-FITTER. Then Rosetta was used to simulate the saturation mutations of Asn102, Asn503, Asp501 and Asp500. The mutated amino acid was designed according to the change of acG. The results showed that the thermal stability of N102P, N102C, D500G, and D500T was improved, and the half-lives of N102P/D500G and N102P/D500T at 45 °C were increased to 3.14 times and 2.44 times, respectively. Analyzing the interaction of amino acids by using Discovery Studio 4.5, it was observed that the thermal stability of dextranase was improved due to the increase in hydrophobicity and the number of hydrogen bonds of the mutant enzyme. The catalytic efficiency of N102P/D500T was increased. Compared with the hydrolyzed products of SmdexTM, the mutant enzymes do not change the specificity of hydrolysates.

Acute and Sub-chronic Toxicity Study of Recombinant Bovine Interferon Alpha in Rodents.

INTRODUCTION: Recombinant bovine interferon alpha (rBoIFN-α) has been demonstrated to have antiviral activity. However, no conduct of acute or chronic toxicity tests has been reported. MATERIAL AND METHODS: Specific pathogen-free Sprague Dawley rats were administered doses at different concentrations through intraperitoneal or intravenous injection. After the administration (single for an acute toxicity test over 14 days or daily for a sub-chronic toxicity test over 30 days), the rats' behaviour and other indicators and the degree of toxic reaction were continuously monitored. Blood was collected for haematological and serum biochemical examinations. At the end of the experiments, the rats were sacrificed for necropsy and histopathological tissue analysis. RESULTS: The external performance, behaviour characteristics, and changes in body temperature and body weight of the rats in each subgroup were comparable to the normal control subgroup. Except for a few cases, there were no lesions in the viscera's pathological structures, and the blood parameters and biochemical indicators were not noticeably different from those of the control subgroup. CONCLUSION: This study suggests that rBoIFN-α seems to be safe for rats, and its use may foster the development of the cattle industry in China by protecting livestock health.

Expression, Purification, and Bioactivity of a Soluble Recombinant Ovine Interferon-tau in Escherichia Coli.

INTRODUCTION: Ovine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein. MATERIAL AND METHODS: First, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography. RESULTS: The protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays. CONCLUSION: Purified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

Inhibitory effects of recombinant porcine interferon-α on porcine transmissible gastroenteritis virus infections in TGEV-seronegative piglets.

Our previous research obtained purified recombinant porcine interferon-α (rPoIFN-α) containing thioredoxin (Trx) fusion tag in E. coli Rosetta (DE3). Here, we evaluate the efficacy of this rPoIFN-α to prevent piglets from the infection of the transmissible gastroenteritis virus (TGEV) attack. In this experiment, twenty-five TGEV-seronegative piglets were randomly divided into five groups. Group 1 was positive control and only challenged with TGEV; Pigs in groups 2-4 were pretreated with 2 × 10(7)IU/pig, 2 × 10(6)IU/pig, and 2 × 10(5)IU/pig rPoIFN-α before TGEV challenge. The fifth group is a negative control group. The animals of this group are pretreated only with Trx protein-containing PBS solution without TGEV challenge. After 48 h of rPoIFN-α pretreatment, the pigs in groups 1-4 were challenged by TGEV, and the pigs in group 5 were administered with PBS. The surveillance results show that Pigs pre-treated with 2 × 10 (7) IU/pig rPoIFN-α are fully aligned with the violent TGEV attack. Pigs pretreated with 2 × 10 (6) IU/pig rPoIFN-α are partially aligned with the violent TGEV attack. Though piglets pretreated with 2 × 10(6) IU/pig or 2 × 10(5)IU/pig rPoIFN-α cannot be adapted to the challenge of TGEV. However, the use of this dose of rPoIFN-α could put off the clinical signs of pigs than the positive control group of the above. These results indicate that rPoIFN-α can protect pigs from the infection of potential TGEV or delay the appearance of clinical symptoms, and its effect is dose-dependent.

Recombinant Porcine Interferon Alpha Enhances Immune Responses to Killed Porcine Reproductive and Respiratory Syndrome Virus Vaccine in Pigs.

In this study, the immunoadjuvant effects of recombinant porcine interferon alpha (rPoIFNα) on the killed virus vaccine (KV) of porcine reproductive and respiratory syndrome virus (PRRSV) in pigs were investigated. The experimental pigs were divided into six groups, including normal control group, rPoIFNα control group, PRRSV KV control group, KV+40,000 U rPoIFNα immunization group, KV+400,000 U rPoIFNα immunization group, and KV+4,000,000 U rPoIFNα immunization group. The experimental pigs were boosted immunized on the 28th day after the initial immunization, and the heparinized blood and serum samples were collected at different time points of these two immunizations to detect and evaluate the immune responses of pigs after immunization by ELISA assay, neutralization assay, flow cytometry, and so on. The results showed that the proportion of the levels of PRRSV-specific antibodies, neutralizing antibodies, stimulation index, IL-4, IFN-γ, and lymphocytes within the groups immunized with KV+rPoIFNα were significantly higher than that group immunized with KV alone. The humoral and cellular immune responses in pigs were markedly enhanced by rPoIFNα after the coadministration with KV vaccine. Therefore, we tentatively think that rPoIFNα is a potential immune promoter with prospects for future applications in the pig industry.

Enhancing immunogenicity and protective efficacy of inactivated avian influenza H9N2vaccine with recombinant chicken IFN-α in chicken.

Control of currently circulating re-assorted low-pathogenicity avian influenza (LPAI) H9N2 is a major concern for both animal and human health. Thus, an improved LPAI H9N2 vaccination strategy is needed to induce complete immunity in chickens against LPAI H9N2 virus strains. Cytokines play a crucial role in mounting both the type and extent of an immune response generated following infection with a pathogen or after vaccination. To improve the efficacy of inactivated LPAI H9N2 vaccine, prokaryotic expression recombination chicken interferon-α (rchIFN-α) was used as vaccine adjuvant.In this study chIFN-α was used as adjuvant in inactivated AI H9N2 vaccine, modulated the immune response of chickens against the vaccine antigen through enhanced humoral and Th1-biased cell-mediated immunity, compared to chickens that received single AI H9N2 vaccine. To further test the protective efficacy of this improved vaccination regimen, immunized chickens were challenged with a high dose of LPAI H9N2 virus. Combined administration rchIFN-α showed markedly enhanced protection compared to single administration of the vaccine, as determined by mortality, clinical severity, and feed and water intake. This enhancement of protective immunity was further confirmed by reduced rectal shedding and replication of AIV H9N2 in challenged chickens. Our results indicate the value of combined administration of rchIFN-α to generate an effective immunization strategy in chickens against LPAI H9N2.

Cloning and characterization of a phosphate transporter gene in Dunaliella salina.

The full-length cDNA of a Na(+) -dependent Pi transport gene (DsSPT1) in Dunaliella salina was cloned by 3' and 5' Rapid Amplification of cDNA Ends (RACE), with an open reading frame (ORF) encoding 716 predicted amino acids, which exhibited 60.5% identity to that of Na(+) -dependent Pi transport 1 (DvSPT1) from Dunaliella viridis. Hydrophobicity and secondary structure prediction revealed 11 conserved transmembrane domains similar to those found in DvSPT1 from D. viridis and PHO89 from Saccharomyces cerevisiae. The result of real-time quantitative PCR showed that expression level of DsSPT1 was enhanced at first and reached its peak at 90 min after salt stress; however, D. salina cells rapidly absorbed extracellular inorganic phosphorus which was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) during the first 5 min under salt stress. It suggested that D. salina on the absorption of inorganic phosphorus was regulated at DsSPTI posttranslational level.