Abstract:Abstract：Biological active resources are various and abundant in the ocean. With this realization, active proteins and peptides especially marine bacteria active proteins and peptides have attracted much attention recently. The achievements in the study of bioactivities of marine bacterial proteins and peptides were reviewed in this paper. Acquisition and potential applications of these marine bacteria active products were then proposed. Additionally, we focused on the prospective outline on the study of this field.

Abstract:Abstract: Nisin, a lantibiotic produced by some species of Lactococcus lactis, has broad antibacterial spectrum against Gram-positive bacteria species, especially those with close phylogenetic relationship to the nisin producing strain. The broad use of nisin did not lead to widespread resistence. However, some non-nisin producing bacteria could develope certain mechanisms of resistance against nisin when growing under laboratory or nature selection pressure. Nisin resistance mainly involved two strategies, namely, the non-specific physiological isolation mechanism (by the change of cell wall or membrane structure and composition) and the specific protease-mediated mechanism. This review introduced the advances in the study of nisin resistance mechanism.

Abstract:Abstract: [Objective] We studied the physiological, biochemical properties and metabolism of Clostridium lituseburense P4-1 from soil in Namucuo. [Methods] We adopted Hungate anaerobic technique to get Strain P4-1 from soil in Namucuo. Through physiological, biochemical and phylogenetic analysis, we identified the strain P4-1. [Results] Cells were Gram-positive and spore-forming. It grew between 13 and 40℃ (optimum at 37℃), between pH value 5.0 and 10.0 (optimum at 7.5－8.0), and with the presence of NaCl between 0%－5%. Strain P4-1 could metabolize many carbon sources including glucose, melibiose and mannitol. Metabolites of glucose were acetate, butyrate, propionate, CO2, and little H2. Based on 16S rDNA studies, strain P4-1 was most close to Clostridium lituseburense DSM 797(M59107) with 98.7% similarity. Strain P4-1 could degrade p-toluene sulfonate. [Conclusion] Strain P4-1 tolerated low temperature, salt and could degrade p-toluene sulfonate. Its metabolites produced by fermentation of glucose could improve the soil micro-environment. It was significant for strain P4-1 to be utilized in the wastewater treatment.

Abstract:Abstract: [Objective] To analyze the biodiversity and distribution of the yeast species of Qula in Tibet and milk cake in Yunnan, and to provide essential data for the utilization of yeasts in the traditional dairy products of China. [Methods] Forty-one yeast strains were isolated from 5 samples of Qula in Tibet and 8 samples of milk cake in Yunnan. The isolates were identified by the large-subunit (26S) rDNA gene D1/D2 domain sequences analysis. [Results] The population of yeast in Qula varied from 106 cfu/g to 107 cfu/g. The content of yeast ranged from 102 cfu/g to 106 cfu/g in milk cake. The average population of yeast in Qula was higher than milk cake for 34 folds. These strains were grouped in 12 species belonging to 10 genera. The dominant species in Qula were Pichia fermentans and Saccharomyces cerevisiae, but Candida zeylanoides and Pichia cactophila were major population in milk cake. The results showed that Pichia was the dominant genera both in Qula and milk cake. [Conclusion] There existed that yeasts of great biodiversity both of Qula in Tibet and milk cake in Yunnan, but quite different from each other.

Abstract:Abstract: [Objective] To enrich the epidemiologic data of the waterfowl origin avian influenza virus (AIV). [Methods] The full genome of A/Muscovy Duck/Fujian/FZ01/2008(H6N6), which was first isolated from domestic Muscovy duck in Fujian Province in southern China at 2008, was cloned and sequenced by reverse transcription polymerase chain reaction (RT-PCR)．[Results]The motif of hemagglutinin (HA) cleavage site was PSMKVIV↓GL, which is a molecular characteristic of low pathogenic AIV, and the intravenoys pathogenicity index (IVPI) was 0.15. The HA gene, NP gene, M gene and PB2 gene nucleotide sequences had similarity highest with A/duck/Kingmen/E322/04(H6N2) at 94.2%, 95.7%, 97.2% and 95.6%, shared the same phylogenetic lineage. The neuraminidase (NA) gene had similarity highest with A/duck/Eastern China/01/2007(H4N6) at 97.1 per cent, and also had 11 continuous amino acids (TNSTTTIINNN) deletion in the NA gene stalk region, which was first reported had deletion 11 amino acids in N6 subtype AIV NA genes’ ORF. The NA gene had familiar phylogenetic relationship with H4N6 subtype AIV. The NS gene、PB1 gene and the PA gene had genetically close relationships with the H5N1 high pathogenic AIV strains isolated in Hong Kong at 1997. The eight genes also showed any immediate ancestor with other H6N6 subtype avian influenza viruses isolated in North America. [Conclusion] These data showed that A/Muscovy Duck/Fujian/FZ01/2008 (H6N6) was possibly a reassortant virus derived from H6N2 subtype、H4N6 subtype and H5N1 subtype AIV.

Abstract:Abstract: [Objective] Xanthomonas oryzae pv. oryzae (Xoo) possesses a type III secretion system (T3S), encoded by a hpa-hrp-hrc cluster, including hrpD6, to inject T3S effectors into plant cells to trigger hypersensitive response (HR) in nonhost tobacco and pathogenicity in susceptible host rice. However, it is unclear what roles of Xoo hrpD6 gene plays in HR in tobacco and in pathogenicity in rice. [Methods] In this study, we constructed a deletion mutant of hrpD6 gene by using marker-exchange method. PCR and Southern blot analysis demonstrated that the hrpD6 gene was knocked out successfully. [Results] in planta assays indicated the hrpD6 mutant, ?PhrpD6, lost the ability to induce HR in tobacco, to trigger water-soaked symptoms in seedlings rice and to cause bacterial blight in adult rice. Importantly, the bacterial growth in rice tissues was tremendously reduced. Complementation assays confirmed that hrpD6 gene could restore HR induction in tobacco, pathogenicity and bacterial growth in rice to the mutant ?PhrpD6. Reverse transcriptional polymerase chain reaction (RT-PCR) revealed that the expression of hrpD6 was not only induced by rice cells, but also controlled by hrpG and hrpX. Intriguyingly, the expression of hpa1, encoding a harpin protein, was found to be dependent on hrpD6, implying that hrpD6 regulates the expression of hpa1. Immunobloting assay confirmed that the mutation of hrpD6 affect the secretion of Hpa1 through T3S. [Conclusion] The mutant lost the ability of triggering hypersensitive response in nonhost tobacco and pathogenicity in host rice is due to that hrpD6 regulates the expression of hpa1 gene and the mutation in hrpD6 affects the secretion of T3S effectors, like Hpa1, through T3SS. Our results provide molecular clues to understand whether hrpD6 is involved in the formation of T3S apparatus and in regulation of other hpa-hrp-hrc gene expression or not for HR induction in tobacco and pathogenicity in rice.

Abstract:Abstract: [Objective] An aerobic denitrifying bacterium, strain 2-8, was isolated from a biological aerated filter in a recirculating marine aquaculture system, phylogeny and characteristics of the strain was further studied. [Methods] Sequence of the 16S rRNA gene was analyzed and the factors affect the denitrifying ability of strain 2-8 were investigated, including carbon source, C/N ratio, initial pH, NaCl concentration, temperature and shaker speed. [Results] It was identified as Pseudomonas sp. 2-8 based on the analysis of its 16S rRNA gene sequence which showed highest similarity (99.9%) to Pseudomonas segetis FR1439T (AY770691). The results indicated that carbon source and C/N ratio exhibited significant influences on aerobic denitrifying capacity of strain 2-8. Strain 2-8 could grow a little on acetate, succinate and citrate as sole carbon source, and the removal rates of NO3--N at 140 mg/L were higher than 65% despite of the accumulation of NO2--N around 35 mg/L. It grew quite well on glucose as sole carbon source, however, the removal rate of nitrate was not so high as on other carbon source. The optimum C/N ratio was 15, as lower C/N ratios may lead to nitrite accumulation. The optimum temperature and pH for its aerobic denitrification were 30 oC and 7.5, respectively. Strain 2-8 could grow and exhibit aerobic denitrifying ability at a wide range of NaCl concentrations (0-30 g/L). The highest nitrogen removal appeared under the condition of 160 r/min shaking culture. [Conclusion] When cultured in the conditions of NO3--N at 140 mg/L, sodium citrate as sole carbon source, C/N ratio at 15, pH 7.5, NaCl at 30g/L, 30 oC and 160 r/min of the shaker, strain 2-8 removed up to 92% of the nitrogen within 48 hours, and no nitrite accumulation.

Abstract:Abstract:【Objective】The aim of our work is to investigate the effects of overexpression of two acetyl-CoA synthase genes, ACS1 and ACS2, on the physiological functions of Saccharomyces cerevisiae. 【Methods】 We overexpressed ACS1 and ACS2 in S. cerevisiae CEN. PK2 with shuttle vector pY26-TEF-GPD. We determined and compared the physiological parameters of the parent strain to the ACS1/2 overexpressed strains, including the intracellular acetyl-CoA content, ATP content, mevalonate pathway, and the tolerance to ethanol stress. 【Results】Compared to the parent strain, the overexpression of ACS1 and ACS2 led to: (1) The intracellular acetyl-CoA content increased by 2.19-fold (ACS1) and 5.02-fold (ACS2), respectively; (2) The intracellular ATP content increased by 3.92-fold (ACS1) and 2.05-fold (ACS2), respectively; (3) The transcription levels of the seven key genes in mevalonate pathway were upregulated, therefore, more carbon flux was channeled into the mevalonate pathway, which could provide precursor for terpenes synthesis; (4) The tolerance to high content of ethanol was enhanced, especially for the ACS1 overexpression strain. 【Conclusion】 The results presented here demonstrated that the overexpression of acetyl-CoA synthase can enhance the carbon flux into mevalonate pathway and improve the tolerance of S. cerevisiae to high content of ethanol, which is the main byproduct of the fermentation process with the yeast.

Abstract:Abstract: [Objective] To investigate the antibacterial activity and mechanism of luteolin on Staphylococcus aureus. [Methods] The antibacterial activity and mechanism experiments were carried out by using 2, 3, 5-triphenyl tetrazolium chloride (TTC), determining membrane penetrability, the change of SDS-PAGE protein spectra and 4'6-diamidino-2-phenylindole (DAPI) staining assay. [Results] Luteolin could affect the membrane permeability of Staphylococcus aureus, but not destroy the membrane integrity directly. After treated with luteolin for 16 hours, the total content of proteins decreased to 64.54%, the quantity of both DNA and RNA reduced to 48.44% and 39.35% respectively. The activity of DNA topoisomerase I and II was inhibited completely by 1.6 mg/mL luteolin. [Conclusion] Luteolin showed obvious antibacterial activity against Staphylococcus aureus. The antibacterial mechanism of luteolin is that it could inhibit the activity of DNA topoisomerase I and II, which resulted in some decrease in the nucleic acid and protein synthesis.

Abstract:bstract: [Objective] Bradysia hygida salivary glands antimicrobial peptide 1 (BhSGAMP-1) is one of the antimicrobial peptides involved in a preventive mechanism of defense of the fly Bradysia hygida. To know better about the molecular characterization of this antimicrobial peptide, we expressed and purified the modified BhSGAMP-1-S and investigated its antimicrobial activity. [Methods] We synthesized the gene of BhSGAMP-1-S designed with preferred codons of Escherichia coli and expressed it as a fusion protein in E.coli TB1 by using pMAL-c2X as vector. We purified the fusion protein using amylase resin affinity chromatography. In addition, we cleaved the fusion protein by enterokinase and the released recombinant BhSGAMP-1-S was separated by size exclusion chromatography, then followed by reversed-phase high performance liquid chromatography. We analyzed the antimicrobial activities of the purified recombinant BhSGAMP-1-S by bioassay. [Results] The fusion protein was mostly expressed in soluble form under the optimized conditions. The recombinant BhSGAMP-1-S was produced with a pure yield of 0.38 mg/100 mL culture medium. Antimicrobial assays demonstrated that the recombinant BhSGAMP-1-S was active against several Gram-positive and Gram-negative bacteria and fungi. [Conclusion] It appears to be first successful production of the recombinant BhSGAMP-1-S from fly Bradysia hygida. Data presented here confirm that the recombinant BhSGAMP-1-S is now ready for further studying and characterize their antimicrobial properties.

Abstract:Abstract: [Objective] To achieve fast, safe and stable expression of Burkholderia cepacia lipase in Pichia pastoris. [Methods] We first amplified B. cepacia lipase gene, and then analyzed the codon usage of B. cepacia and Pichia, lipase gene signal peptide with bioinformatics methods. On this basis, we applied the overlap PCR to modify the lipase gene and finally got the optimized gene with Pichia codon usage and lower G + C content. Subsequently, we cloned the optimized and wild lipase gene into vector pGAPZα and pPIC9K, respectively. As a result, constitutive expression vector pGAPlipW, pGAPlipO and inducible expression vector pPIClipW, pPIClipO were obtained. Finally, we electroporated these expression vectors into GS115, and therefore, got a series of engineering strains. After fermentation and NTA resin purification, the enzymatic properties of lipase were studied. [Results] The lipase activities of pPIClipW, pPIClipO, pGAPlipW and pGAPlipO were 37.8 U/mL, 129.5 U/mL, 40.2 U/mL, and 184.3 U/mL, respectively. The optimized lipase activity increased 4.6-fold. Enzymatic properties study showed that the optimal temperature and pH was 60 ℃ and 9.0, respectively. The lipase was rather stable at 40℃-65℃ and pH 6.0-pH10.0. [Conclusion]After overlap PCR modification, the lipase expression efficiency in Pichia was significantly increased，which indicates that the overlap PCR modification is a potential strategy for lipase overexpression. The GAP promoter is more appropriate than the AOX1 promoter for the B. cepacia lipase expression. Additionally, the recombinant lipase whose enzymatic properties were identical to the wild type satisfies the needs of industrial application.

Abstract:Abstract: [Objective] We identified a new isolated naringinase-producing yeast strain named as Jmudeb008, and analyzed its naringinase-producing ability cultured with different composition and concentration of carbon sources. [Methods] The strain was identified based on conventional phenotypic methods and sequences of the D1/D2 region of 26S rDNA and 5.8S-ITS. Media with different composition and concentration of carbon sources were used in shaking culture of Jmudeb008. The activity of naringinase was evaluated by analyzing the concentration of naringin, naringenin and glucose during 48 h culture. [Results] The Jmudeb008’s sequences of the D1/D2 region of 26S rDNA and 5.8S-ITS were 99% identical with Cryptococcus laurentii. Further glucose fermentation test, urease test, DBB (diazotization based blue）test and nitrate reduction test were coincided with results of DNA sequencing. Therefore, Jmudeb008 was identified as Cryptococcus laurentii. When Jmudeb008 was cultured in the medium with naringin as the only carbon source, it could synthesize naringinase. However, when glucose was available, the synthesis of naringinase was repressed. [Conclusion] The new isolated naringinase-producing yeast strain Jmudeb008 was identified as Cryptococcus laurentii. The glucose in medium repressed naringinase expression.

Abstract:Abstract: [Objective] Anthracnose caused by Colletotrichum gloeosporioides (Penz.) Sacc. is a main disease in citrus production. To develop an effective biocontrol measure against citrus postharvest anthracnose, we screened antagonistic microbes and obtained a bacterial strain 1404 from the rhizospheric soil of chili plants in Nanning city, Guangxi, China. The objectives of the present study were to: (1) identify and characterize the antagonistic bacterium; (2) to evaluate the efficacy of the antagonistic strain in controlling citrus postharvest anthracnose disease. [Methods] Strain 1404 was identified by comparing its 16S rDNA sequence with related bacteria from GenBank database, as well as analyzing its morphological, physiological and biochemical characters. The antagonistic stability of the strain 1404 was determined by continuously transferring it on artificial media. The effect of the strain on suppressing citrus anthracnose at postharvest stage was tested by stab inoculation method. [Results] The 16S rDNA of strain 1404 was amplified with primers PF1 (5′-AGAGTTTGATCATGGCTCAG-3′) and PR1 (5′-TACGGTTACCTTGTTACGACTT-3′) and its sequence submitted to GenBank (accession number: GU361113). Strain 1404 clustered with the GenBank-derived Brevibacillus brevis strains in the 16S-rDNA-sequence-based phylogenetic tree at 100 % bootstrap level. The morphological traits, physiological and biochemical characters of strain 1404 agreed with that of Brevibacillus brevis. Less change in the suppressive ability of antagonist against growth of Colletotrichum gloeosporioides was observed during four continuous transfers on artificial media. The average control efficacy of the strain was 64.9 % against the disease 20 days after the antagonist application. [Conclusion] Strain 1404 was identified as Brevibacillus brevis based on its morphological traits, phyiological and biochemical characters as well as 16S rDNA sequence analysis. The antagonist was approved to be a promising biocontrol agent. This is the first report of Brevibacillus brevis as an effective antagonist against citrus postharvest anthracnose disease.

Abstract:Abstract: [Objective] In order to investigate the diversity of oil-degrading bacteria in the surface seawater across the India Ocean, and to obtain new oil-degrading bacteria. [Methods] Potential oil-degrading bacteria were selected out via 1:1 mixture of diesel and crude oil as sole carbon source. Meanwhile, the community structure of 13 enrichments was analyzed by polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE). [Results] We obtained 51 unique strains of 29 genera after screening via morphological, physiological, biochemical and 16S rRNA analyses. They mainly belonged to α and γ- Proteobacteria. The four genera Alcanivorax (accounting for 18%), Novosphingobium (10%), Marinobacter (6%) and Thalassospira (6%) were the most predominant bacteria. Ecological analyses showed that the bacteria had high diversity with Shannon-Winner index (H) of 4.57968, and distributed even with Evenness index (E) as 0.8664771. Then Further experiments revealed oil-degrading capability of 49 strains. In addition, our investigation revealed oil-degrading ability of genera Sinomonas, Knoellia and Mesoflavibacter for the first time. DGGE fingerprint patterns indicated that the genus Alcanivorax was an important oil-degrading bacteria in the surface seawater across the India Ocean. [Conclusion] This study demonstrated a high diversity of the oil-degradation bacteria in the surface seawater of Indian Ocean, these bacteria are of potential in bioremediation of marine oil pollution.

Abstract:Abstract: [Objective] S-layer proteins of Lactobacillus acidophilus were extracted and purified, then the effect of Lactobacillus acidophilus and its S-layer proteins against the adhesion and invasion of salmonella typhimurium to caco-2 cells were investigated. [Methods] S-layer proteins were purified by anion-exchange column {diethylaminoethyl (DEAE) DE52}, and the inhibition of Lactobacillus acidophilus and its S-layer proteins were studied against the adhesion and invasion of salmonella typhimurium on Caco-2 cells. [Results] S-layer proteins exhibited strongly inhibitory effects of adhesive and invading properties of Salmonella typhimurium. In the adhesive experiments (competitive, exclusive and displacement), Salmonella typhimurium adhesion was reduced by S-layer proteins and the ability of adherence to Caco-2 cells were 1.17 %±5.97 %, 8.71 %±1.36 % and 10.56 %±0.92 % , respectively (p＜0.01). The influence to inhibit the competitive adhesion of Salmonella typhimurium was optimal. Furthermore, the S-layer proteins showed a stronger effect than Lactobacillus acidophilus to inhibit Salmonella typhimurium adhesion on Caco-2 monolayers (p＜0.01). Moreover, invasion of Salmonella typhimurium to Caco-2 monolayers was inhibited by S-layer proteins. [Conclusion] S-layer proteins inhibited adherence and invasion of Salmonella typhimurium. The result can merit a highlight for preventive or probiotic therapy in human or animals with disease caused by Salmonella typhimurium.

Abstract:Abstract: [Objective] We studied the colonization ability and the distribution of the recombinant Lactobacillus casei in mouse intestine. [Methods] We used Green fluorescent protein (GFP) gene as reporter in constructing the recombinant plasmid pLA-GFP, which was electrotransformed into the host cells L. casei. Six-week-old female SPF BALB/c mice were orally fed with the recombinant L. casei of approximately 109. Groups of at least three mice per condition were killed at 1.5 h, 3 h, 12 h, 1 d, 3 d, 5 d, 6 d, 7 d, and its duodenum, jejunum, ileum, caecum intestinal tract rinse solution was sampled separately. The recombinant bacteria in intestinal tracts were examined by plate culture count. [Results] The molecular weight of the recombinant protein was about 69 kDa in the result of western blot. The GFP fusion protein on the cell surface was confirmed by fluorescence microscopy and flow cytometric analysis. A portion of the recombinant L. casei was able to adhere and colonize in different regions of murine intestinal tract, and the planting peak was appeared on day 6 postinoculation. The ratio of the sixth day to the first day of the recombinant L. casei adhered to the intestinal mucosa in the duodenum, jejunum, ileum, and caecum was 16.49%, 25.08%, 47.71%, and 41.03%, respectively. [Conclusion] The recombinant L. casei stably expressing GFP could colonize mouse intestine. The field planting rule was ileum > caecum > jejunum > duodenum. Our findings indicated that L. casei used as a deliver vector in oral vaccine is feasible, but the impact on intestinal immune mechanism in mice is needed more research.

Abstract:Abstract: [Objective] In this study the immunogenicity and protective efficacy of 5 pertactin recombinants against Bordetella bronchiseptica (Bb) challenge is shown. [Methods and results] The complete coding sequence (2 040 bp) of the prn gene (PRN) and its fragments, 5’-terminal 1 173 bp fragment (PN), 3’-terminal 867 bp fragment (PC), two copies of region I (654 bp; PRI) in PN, and two copies of region II (678 bp; PRII) in PC, were separately cloned into the prokaryotic expression vector pGEX-KG, and expressed in the Eschierichia coli BL21 (DE3) using induction by IPTG. The recombinant proteins were named GST-PRN, GST-PN, GST-PC, GST-2PRI and GST-2PRII. All 5 recombinant proteins showed immunological reactivity in the Western-blot analysis. Mice, immunized subcutaneously with two doses of the purified proteins mixed with an equal volume of Freund’s adjuvant, produced robust PRN-specific IgG antibody levels. When challenged, 6 of 9 mice in GST-2PRI group and all 9 mice in the other groups survived intranasal challenge with three times the 50% lethal dose (LD50) of virulent Bb HH0809. After challenge with 10 LD50 7/9, 3/9, 6/9, 1/10 and 6/10 of the mice survived. Furthermore, complete protection against intraperitoneal (i.p.) challenge with 10 LD50 of HH0809 was observed in mice that were injected i.p. with 0.5 ml rabbit anti-GST-PRN, GST-PN, GST-PC or GST-2PRII serum. Only 1 of 10 mice survived in the group of mice that received anti-GST-2PRI, and no survivors were noted in the group of mice that received PRN-absorbed rabbit antiserum (0/5). [Conclusion] In this study, we showed the immunogenicity and protective efficacy of 5 pertactin recombinants against Bb challenge, with GST-PC>GST-PN, and GST-2PRII>GST-2PRI. These findings built a good foundation for the further research into highly efficient vaccine against bordetellosis.

Abstract:Abstract: [Objective] To identify the exotoxin-specific motifs/domains in bacterial exotoxin sequences, and to expand understanding of bacterial exotoxins pathogenic mechanisms. [Methods] We constructed a non-pathogenic bacterial proteins database and collected 89 bacterial exotoxin sequences from Virulence factor database(VFDB), then we analyzed these protein sequences by motif /domain search using InterProScan(www.ebi.ac.uk/Tools/InterProScan/). [Results] We identified 39 exotoxin-specific motifs/domains in 89 bacterial exotoxin sequences. [Conclusion] The identified exotoxin-specific motifs/domains were closely related to the functions of the exotoxins and could be used as template to search for new exotoxins by mining pathogenic bacterial geomes. The analysis of the acquired Gene Ontology (GO) items was to further expend our understanding of bacterial exotoxin pathogenic mechanisms.

Abstract:Abstract: ［Objective］ To construct an E. coli-streptomyces shuttle vector pMF that can integrate into the genome of streptomyces by site-specific integration.［Methods］We inserted the integrase gene ΦC31 int and attP site into pLSB2, a suicidal streptomyces plasmid. The resulting conjugably transferable vector which contains the activator promoter system actⅡ-ORF4/PactⅠfrom Strepromyces coelicolor A3(2) could be integrated into the genome of streptomyces by site-specific integration.［Results］The plasmid pMF was conjugably transferred with high frequency into S. coelicolor M145, S. lividans TK24 and Saccharopolyspora erythraea 2338 from E. coli. Southern blotting results showed that pMF was able to integrate into the genome of streptomyces. We also confirmed functional protein expression by cloning a putative S-adenosylmethionine synthetase (SAM-s) gene from Sacc. spinosa S08-4 into pMF and conjugated into S. coelicolor M145. Protein expression were confirmed using Western blotting.［Conclution］pMF can be used as an effective tool for site-specific integration expression of foreign gene in streptomyces.

Abstract:Abstract：[Objective] We expressed mature chicken interleukin-18 (mChIL-18) in Pichia pastoris. [Methods] The mChIL-18 gen was reconstructed by using site-specific mutagenesis based on the Pichia pastoris-preferred codons. The recombinant plasmid pPIC9K/mChIL-18 was constructed and transformed to Pichia pastoris GS115 by electroperation. Multi-copy recombinant strains were screened by Geneticin (G418). The expression of mChIL-18 protein was induced by methanol. SDS-PAGE and Western-blot were used to analyze the expressed products. The bioactivity of mChIL-18 was measured by methyl thiazolyl tetrazolium assays and chicken embryo fibroblasts-vesicular stomatitis virus (CEF-VSV) system. [Results] The protein of mChIL-18 could be secreted by GS115. The optimum expression conditions, a rate of 480 mg/L, were obtained as follows: temperature 28 ℃, pH 6.5, methanol concentration 2% and expression time 120 h. The obtained mChIL-18 protein could stimulate T lymphocytes proliferation. IFN-γ induced by mChIL-18 could directly inhibit the growth of VSV in CEF, and its antiviral activity was about 1.7×104 U/mL which was produced by 400 ng/mL of mChIL-18. [Conclusion] The high expression of bioactive recombinant mature chicken interleukin-18(mChIL-18) in Pichia pastoris had been achieved.

Abstract:Abstract: [Objective] To make clear the molecular characterization of subgroup J Avian Leukosis Viruses (ALV-J) isolated from chickens with clinical hemangioma, as well as to get more information for controlling the spread of ALV-J in layer chickens flocks. [Method] We amplified the full-length viral cDNA sequences of three layers isolates associated with simple hemangioma or coexisting of hemangioma and myeloid leukosis (ML) by PCR. We also obtained some partial sequences of 3 layer isolates related to hemangioma and 1 layer isolate from ML case. Then we analyzed and compared the sequences by using DNAstar software. [Results] The phylogentic analysis showed the significant differences in the complete sequences between isolates from layer hemangioma cases and from broilers, which were grouped to two branches in the phylogenetic tree. We noted a special 19bp insertion mutation in Primer Binding Site (PBS)-Leader sequences in both JS09GY3 and JS09GY6 isolates from layer chickens with hemangioma and ML, which sequence was same to Rous Associated Virus type 1 (RAV-1), Rous Associated Virus type 2 (RAV-2) and Rous sarcoma virus (strain Schmidt-Ruppin B) (RSV-SRB). In addition, different continuous sequences deletions were found in the U3 regions of NHH and JS09GY5. By motif analysis, we found some distinct motifs including c-Est-1, TCF11 and C\EBP only in the isolates from layers with hemangioma. The five isolates associated with layer hemangioma exhibited intact E element sequences but almost identical substantial deletion was found in all Chinese broiler isolates. An 11bp continuous nucleotide insertion in the E element of JS09GY3 was found. [Conclusion] Isolates from layer showing hemangioma and broilers exhibited evident difference. We found some special mutation sites in U3, DR1 and the E element showed some potential relationship with the host breeds and the tumor phenotype, which function needs to be investigated in future. The isolates from layer cases with coexisting of hemangioma and ML were the recommbinants of ALV-J and other retroviruses.