Objective To construct a eukaryotic expression vector in Pichia pastoris containing human tissue factor( hTF) gene,in order to achieve high level secretory expression in extracellular.Methods Expression plasmid, pGAPZaA-hTF, was constructed by inserting the synthesized sequence encoding human extracellular tissue factor into yeast expression vector pGAPZaA and transformed into Pichia pastoris SMD1168H with electroporation.Having been selected by Zeocin, transformants containing hTF cDNA were expressed in YPD, extracellular proteins were detected by SDS-PAGE and confirmed by Western bolt.Results Successfully constructed the recombinant pGAPZaA-hTF expression system in Pichia pastoris.SDS-PAGE showed that the molecular weight of the expression product was about 37 -40 kDa.Western-blot indicated that it was human extracellular tissue factor.The crude yield of total protein in medium was up to 1 g/L, more than 80% of which was hTF.Conclusion Truncated rTF gene is expressed in Pichia pastoris and the active products are secreted into the medium which have the same activation as the native TF.

Pullulan is a linear glucosic polysaccharide produced by the polymorphic fungus Aureobasidium Pullulans, which has long been applied for various applications in medical and food industry due to its security, stability and low adhesive ability.At present, the two problems in restricting pullulan industrial production are the low polysaccharide production and melanin secreted which is hard to erase completely, giving the following process some problem.As a starting point, this review article collects and analyzes the progress on the breeding of pullulan high-yield strain without melanin in recent years, in order to find more efficient strains breeding methods, laying a foundation for further breeding of pullulan high-yield strain without melanin.

Nisin, produced by several strains in the growth process of Lactococcus lactis, is a natural antimicrobial polypeptide.Now, Nisin has served as an effective and safe food additive extensively used in food industry in many countries and regions because of its excellent antimicrobial activity.However, the current production of Nisin is largely fermented by lactobacillus and its industrialized production still can not meet enormous market needs, therefore establishing reasonably high-yield Nisin strains is of great significance.This review mainly summarizes the development pathway of molecule based on the functional expression of Nisin biosynthetic genes and regulation of gene expression, and also the study status on high Nisin-producing strains which provides practical foundation for further study on expected strains as well as some useful guidance for large-scale industrialized production of Nisin.

We enrolled 60 patients with American Association of Anesthesiologists grade Ⅰ - Ⅱ undergoing unilateral total knee arthroplasty. All patients received combined epidural and spinal anesthesia,and a nerve stimulator was used to guide placement of a femoral nerve catheter. Patients were divided into three groups according to the catheter location on X-ray : psoas muscle group ( n = 18 ), iliacus muscle group (n = 19) and local group (n =23). Visual analog scale (VAS) pain scores were recorded at rest and with movement at 4, 24 and 48 h postoperatively and sensory blockade of the femoral, obturator and lateral femoral cutaneous nerves was recorded at 24 h.There were no significant differences in femoral nerve blockade among the three groups. Obturator nerve blockade was significantly better in the psoas muscle group than in the iliacus muscle and local groups, and was also better in the local group than in the iliacus muscle group. There was no significant difference in lateral femoral cutaneous nerve blockade between the psoas muscle and iliacus muscle groups, but there was better blockade in both these groups than in the local group. At 4 h postoperatively, VAS pain scores at rest were significantly lower in the psoas muscle group than in the iliacus muscle and local groups, but there were no significant differences in VAS pain scores with movement among the three groups. At 24 and 48 h postoperatively, VAS scores at rest and with movement were significantly lower in the psoas muscle group than in the iliacus muscle and local groups.

Objective:To explore the role of T lymphocytes activation co-stimulation pathway in asthma pathogenesis and the ability of therapy asthma with Anti-CD86mAb.Methods:The blood samples were taken from 28 asthma children( including 18 male and 10 female, age 1 year-8.08 years) and 15 normal children( including 7 male and 8 female, age 3.25 years-10 years).ELISA was used to detect the levels of IL-4、IL-13、IFN-? in culture supernatants of PBMC stimulated with PHA and treated with mouse anti human CD86mAb. Results:①When treated control PBMC with anti-CD86mAb, the level of IL-4 in control group(13.30?4.66 pg/ml) was lower than that of mouse IgG control group (15.20?5.22 pg/ml,P

Objective:To investigate the induction and differentiation potential of ADSCs by tissue culture method,and to preliminary study on the origin of ADSCs.Methods:Using adipose tissue culture method to culture human ADSCs.The third generation of ADSCs for the adipogenic and osteogenesis differentiation,and staining by oil red O and alizarin red S.HE staining was performed after the seventh day culture of adipose tissue.Results:The primary human ADSCs were successfully cultured with adipose tissue culture method.ADSCs cultured to the eighth generation,still maintained a good proliferation ability and cell morphology.ADSCs can be successfully induced into adipose cells and bone cells.ADSCs were mainly distributed around the mesenchymal vascular and connective tissue,by HE staining of adipose tissue after seven days of culture.Conclusion:The cells that were cultured with adipose tissue have the potential to adipogenic and osteogenesis differentiation.The ADSCs were mainly distributed around the mesenchymal vascular and connective tissue.

BACKGROUND:Tissue engineering provides new ideas and approaches for repair of cartilage defects.
OBJECTIVE:To develop a complete set of solutions for construction of tissue engineered cartilagein vitro, with chondrocytes as seed cels and cross-linked sodium hyaluronate as scaffold materials.
METHODS: New Zealand rabbit articular chondrocytes were isolated, counted, and then cultured and passaged to prepare cellsuspension. Toluidine blue staining, RT-PCR and immunocytochemistry were exerted to evaluate the cultured cels. Chondrocytes were seeded and co-cultured with cross-linked sodium hyaluronate scaffold for 21 days. Then, RNA was isolated for RT-PCR, and frozen sections were prepared for morphological observation and immunohistochemistry study.
RESULTS AND CONCLUSION:The chondrocytes could adhere to the cross-linked sodium hyaluronate scaffold and aggregate, growing between fibers or adhering to the scaffold in a monolayer manner. The transcripts of cartilage specific aggrecan gene and colagen type II alpha 1 gene and cartilage specific protein colagen type II were expressed in cel-scaffold complexes to maintain the phenotype of chondrocytes. Cel-scaffold complexes co-cultured in vitro can form cartilage extracelular matrix, by which tissue engineered cartilage is expected to be obtained.

Objective To retrospectively study the high risk factors for biliary complication (BC) and the application of the Clavien system to classify BC in a large cohorts of subjects undergoing liver transplantations (LT).Methods The clinical data of 181 patients who received LT from Jan.2004 to Dec.2008 were studied.BC was classified using the Clavien system.The risk factors of biliary complication were evaluated by using a binary forward stepwise logistic regression analysis.Results 14.4％ (26/181) recipients developed BC (BC group).In 84.6％ (22/26) patients the BC was above the Clavien Ⅲ b.Regression analysis of BC revealed that the placement of a T tube (P =0.0090,OR=31.177),RIld (P=0.0094,OR＜0.001).RI1w (P=0.0013,OR＞999.999) were significantly associated with the development of BC.Regression analysis of BC above Clavien Ⅲ b revealed that RIld (P=0.0065,OR＜0.001,RI1w (P=0.0022,OR＞999.999) were significantly associated with the development of BC above Clavien Ⅲ b.Conclusions The Clavien classification system was useful to classify BC.The placement of a T tube was an independent risk factor to predict BC,it was not a factor for BC above Clavien Ⅲ b.Hepatic arterial insufficiency (HAI) was an independent risk factor for BC and BC above Clavien Ⅲ b.

Objective To construct a eukaryotic expression vector in Pichia pastoris containing human fibrinogen gene, in order to achieve high level secretory expression in extracellular.Methods Expression plasmid,pGAPZαA-FGB-FGG-FGA-AOX1,was constructed by inserting the synthesized sequence encoding human fibrinogen(FGA, FGB,FGG) and then introduced into Pichia pastoris SMD1168H by electroporation.Transformants were availably screened by Zeocin resistance,the expression of recombinant protein was identified by SDS-PAGE and Western blot analysis, the protein yield was tested by ELISA assay.After ultrafiltration and purification, the biological activity of protein was detected.Results The crude yield of human fibrinogen in Pichia pastoris supernatant reached 15 mg/L in flask and the biological aggregation activity was determined.Conclusion The human fibrinogen gene was obtained and successfully expressed in Pichia pastoris and the active products were secreted into the medium.

Objective To construct a prokaryotic expression vector in BL21 to secretorily expressα-Cyclodextrin Glycosyltransferase(α-CGTase). Methods α-CGT gene was amplified from Bacillus macerens genome by PCR.pET26b and α-CGT gene were connected after digested with Nco I, Xho I respectivly, and then transformed into Escherichia coli BL21 strain.α-CGTase was expressed in fermentation culture medium and AA-2G was prepared by using α-CGTase, VC and starch.Results α-CGTase was expressed secretorily and the enzyme activity was up to 120 U/mL.AA-2G was prepared by the biotransformation of VC and starch using α-CGTase which proved to be correct by HPLC.Conclusion AA-2G was prepared by using self-madeα-CGTase, after optimized the preparation conditions the yield of AA-2G was 17.46 g/L, and the conversion rate reached 58.2%(mg/mg).