In present study,bovine Lactoferricin was first secretly expressed in Pichia pastors yeast expression system.The synthesized LfcinB gene fragment was cloned into expression vector pPIC9K,and then obtained recombinant plasmid,designated as pPIC9K-LfcinB,was linearized and transformed into Pichia pastors strains SMD1168 by electroporation.The transformants were screened with Geneticin and multiply-copy colonies were harvested,in which LfcinB gene was verified to inserted into yeast chromosome stably.The positive recombinant Pichia strains were induced with methanol to express LfcinB in culture supernatant.It's expressive products has high activity of killing bacteria.We concluded that LfcinB gene was cloned and integrated into yeast chromosomes,and obtained expression peptide was tested to have high antibacterial activity.

A fragment containing amino acid residues 561～578 of HSV-2 glycoprotein G(gG2) was obtained by PCR assembling technique,and doubly cloned into vector pET-KDO.The recombinant plasmid was transformed to BL21(DE3)plysS.Fusion protein,of molecular weight about 39kDa was highly expressed by induction of IPTG.Western blot result showed the fusion protein had good antigenicity.After putification and digestion,the purity reached 95%.The digested purified protein was analysed by ELISA and showed good sensitivity and specificity.The recombinant protein should be useful for type-specific serodiagnosis of HSV-2.

Objective:To establish a human lung squamous carcinoma cell line and to study its biological characteristics. Methods:Lung squamous carcinoma specimens were freshly resected during operation;the tissues were incubated in vitro and the cell line was named CHLH-1.The biological characteristics of the cells were studied by light microscopy,electron microsco- py,chromosome analysis and transplantation experiment.Results:Cells from the specimens of the primary tumor,the CHLC- 1 cell line and the cells from transplanted tumor possessed the characteristics of malignant squamous epithelium under light and electron microscope.The cell growth curve,doubling time and mitotic index were also observed in vitro.Nuclear chromosome analysis revealed that the tumor was a subtriploid with a mode of 60-68 per cell.Tumor nodes were observed under the skin of nude mice by heterogenic transplantation.Conclusion:The characteristics of the established cell line suggest that it is a newly established human squamous carcinoma cell line.

Objective To investigate the children's body environmental Se and T-2 toxin level in their staple food in Kaschin-Beck disease(KBD)relative active regions in Aba state of Sichuan province in 2008.Methods We took X-ray photograph of the right hand on children aged 7-13 years in 48 villages from 11 counties in Aba state.The relative active regions of KBD were chosen according to the X-ray result and historical status of KBD.The children's urine and hair,drinking water and their staple food werr sampled.Selenium contents in urine,hair,water and food samples were determined by naphthalene fluorescence,and T-2 toxin in staple food samples were detected by ELISA kits.Results In 2145 X-ray films,66 films were positive,and the children's KBD positive rate was 3.08%(66/2145).The KBD positive rate was respectively 10.98%(29/264)and 8.52%(19/223)in Maerkang county,Jinchuan county and it was 0.75%(3/400)in Rangtang county,historically serious endemic area.The selenium content in urine of children aged 7-13 years in Maerkang county,Jinchuan county and Rangtang county was (10.41±4.67), (10.11±3.65), (8.42±2.68)μg/g Cr, respectively, there was no statistical difference among three counties(F=0.901, P>0.05). The selenium content in hair of children aged 7-13 years in Maerkang county[(0.18±0.04)mg/kg] was lower than that in Jinchuan county[(0.21±0.04)mg/kg, P<0.05].The selenium content in water in Jinchuan county [(0.225±0.124 )μg/L ] was lower than that in Maerkang county and Rangtang county[(0.320±0.092), (0.339±0.105)μg/L, all P<0.05]. The selenium content in staple food in Jinchuan county(0.0033 mg/kg) was lower than that in Maerkang county and Rangtang county(0.0258,0.0137mg/kg, Z=-6.146,-3.042, all P<0.017). The T-2 toxin level in flour in three counties was 19.60,17.95,26.25 ng/g,respectively,there was no statistical difference among three counties(X2=5.623, P>0.05).The T-2 toxin level in grain Maerkang county (10.72 ng/g) was higher than that in Jinchuan county and Rangtang county (3.74,3.30 ng/g, Z=-6.315,-4.407,all P<0.017). T-2 toxin contamination in flour was more severe than that in grain (Z=-6.690,-5.493,-3.676, all P<0.05). Conclusions In 3 relative active KBD regions of Aba state,the children's selenium nutritional status and the T-2 toxin contamination level in their staple food is consistent with the distribution of KBD.

AIM:To investigate the inhibitory effect of sinapine thiocyanate(ST)on hyperglycemia,hyper-lipemia,atherosclerosis and hepatocellular steatosis of ApoE-/-mice with insulin resistance(IR)and the possible mecha-nisms.METHODS:ApoE-/-male mice(n=60)were assigned randomly into control group ,saline group,rosiglitazone group and ST treatment groups(at low,middle and high doses )with 10 mice in each group.The mice in control group were fed with fundamental diet ,while the mice in other groups were fed with high-fat diet for 12 weeks.The mice in ST groups were given gavage with different doses of ST(10,30 and 90 mg· kg-1· d-1)simultaneously,while the mice in rosiglitazone group received gavage with rosigltazone(1.33 mg· kg-1 · d-1 ).In the last 3 weeks,the mice in control group received daily intrape-ritoneal injection of physiological saline ,and IR was induced in other groups by daily intrape-ritoneal injection of dexamethasone(0.8 mg/kg).The blood sample was collected and fasting plasma glucose was tested weekly through tail vein.After all animals fasted for 12 h at the end of the 12th week,they were sacrificed and the levels of fasting insulin,tumor necrosis factor-α(TNF-α),triglyceride,total cholesterol and liver lipids were measured.The li-ver tissue and aortic immobilized sections were detected by HE staining.The expression of the proteins related to liver lipid metabolism and skeletal muscle MAPK signaling pathway was determined by Western blot.RESULTS:ST showed dose-dependently reduced serum lipids ,plasma glucose and TNF-α(P<0.05),delayed hepatocellular steatosis and atheroscle-rosis,and dose-dependently regulated hepatic lipid metabolism signaling molecules(HMGR and SREBP-2)and MAPK signaling molecules(ERK and p38)(P<0.05).CONCLUSION:ST has the biological potential of reducing blood li-pids and relieving IR.The mechanism may be related to the regulation of liver lipid metabolism and skeletal muscle MAPK signaling pathway.

OBJECTIVETo understand the rate of viral carrying status among rodents as well as genotypes and distribution of Hantaviruses (HV) isolated in Hunan province.

METHODSWith DFA, the HV antigen in lung tissues of rodents was detected. The total viral RNA was extracted from the lung tissues of the HV infected rats and amplified with reverse transcrition-polymerase chain reaction (RT-PCR), using the HV genotype specific primers. The amplified genes were then sequenced and subjected to genotyping and homologic analysis.

RESULTSThe average density of rodents was 3.15% and the virus carrying rate among rodents was 1.31%. Data from genotype analysis showed that the HV isolated from seven lung specimens taken from Rattus norvgicus, Apodemus agraius, Mus musculus, Rattus flavipectus among indoor rodents in Shaodong and Liuyang belonged to HV type II (SEOV), and one isolated from Apodemus agraius in Shaungfen belonged to HV type I (HTNV) among outdoor rodents. Six strains were sequenced successfully and the homology between six srains was 88.3%-100%. The homology of HN1, HN2, HN4, HN6 came from Liuyang and the HN7 and HN8 from Shaodong were both 100% while the homology between L99 and the strains from Liuyang and Shaodong were 94.4% and 88.3% respectively.

CONCLUSIONHV type II (SEOV) and the HV type I (HTNV) were all existed in Hunan province while SEOV was the main genotype.

Animals ; Genotype ; Hantavirus ; classification ; genetics ; Hemorrhagic Fever with Renal Syndrome ; virology ; Phylogeny ; RNA, Viral ; genetics ; Rats ; Reverse Transcriptase Polymerase Chain Reaction

Adult ; Age Factors ; Athletic Injuries ; epidemiology ; Blood Group Antigens ; Body Mass Index ; Humans ; Male ; Military Personnel ; education ; statistics & numerical data ; Physical Education and Training

PBD-1 is an antibacterial peptide that plays an important role in defence system of porcine. To produce PBD-1 with bioactivity in Pichia pastoris, according to published amino acid sequence of porcine beta-defensin 1(PBD-1) and the partiality codon of yeast, the PBD-1 gene was synthesized by PCR and cloned into pPIC9K to construct the recombinant expression vector pPIC9K-PBD-1, the obtained recombinant plasmid was linearized by Sal I, and then transformed into SMD1168 by electroporation. Under the control of the promoter AOX1, an approximately 4.5 kD PBD-1 peptide was expressed. Antibacterial activity assay shows that the PBD-1 has the antibacterial activity on Staphylococcus aureus. This is the first secreted expression of porcine beta-defensin 1 gene in Pichia pastoris.
Animals ; Anti-Bacterial Agents ; biosynthesis ; isolation & purification ; pharmacology ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; genetics ; Pichia ; genetics ; Protein Engineering ; Staphylococcus aureus ; drug effects ; Swine ; genetics ; beta-Defensins ; biosynthesis ; genetics ; isolation & purification ; pharmacology

Objective To study the effect of Itk down regulation on Jurkat cell proliferation and inflammatory cytokines production, and provide useful data for Itk as an attractive target for potential drugs.Methods Three shRNAs against different region of Itk were constructed and cotransfected with pEGFP-C1-hItk. The shRNA, which can knock down Itk, was selected and packed into lentivirus. After Jurkat cells were transfected with shRNA lentivirus, the change of Itk protein expression, cell proliferation and cytokines production was observed. Results Itk mRNA was reduced about 55% in Jurkat cells transfected with ItkshRNA1, compared with that in control cells shRNAnon (P ＜ 0. 05 ). Knocking down Itk expression had a profound inhibitory effect on Jurkat cell proliferation. In addition, there was a substantial decrease in level of cytokines, such as IL-2, IL-5, IL-10 and IFN-γ, produced by cell transfected with Itk-shRNA1.Conclusion Knocking down Itk expression can inhibit Jurkat cell proliferation and inflammatory cytokines production.

Objective To investigate the Mrna and protein expressions of 11β-Hydroxysteroid dehydrogenase type 2(11βHSD2)in patients with atrial fibrillation.Methods Right and left atrial lateral wall tissue samples were obtained during mitral/aortic valve replacement operation from 25 patients with rheumatic heart valve disease(12 in sinus rhythm and 13 in chronic atrial fibrillation).Realtime quantitative PCR and Western blot were used to determine the Mrna and protein expressions of 11βHSD2 in atria specimens.The distribution of 11βHSD2 in human atrial tissue Was analyzed by specific immunohistochemical staining.Echocardiography examination was performed before operation.Results The left atrial diameters were significantly higher in the atrial fibrillation group as compared to sinus rhythm group (P<0.01).Similarly,Mrna expression of 11βHSD2(0.86±0.14 vs 0.33±0.12 in right atria,0.95± 0.15 vs 0.37±0.10 in left atria,all P<0.01)and protein expression of 11βHSD2(1.18±0.64 vs 0.71±0.21 in right atria,P<0.01:and 1.36±0.58 vs 0.85±0.15 in left atria,P<0.05)were also significantly upregulated in atrial fibrillation groups than those in sinus rhythm groups.The mRNA and protein expressions of 11βHSD2 were similar between left atria and right atria both in fibrillation and sinus groups (all P>0.05).The special immunohistochemical staining demonstrated that 11βHSD2 Was abundant in the human atrial myocardium and located mainly in the cytoplasm.Conclusion These findings suggested that upregulated 11βHSD2 might be associated to the development and persistence of atrial fibrillation.