Objective To prepare recombinant human prothrombin-2 expressed in Pichia pastoris, and assay the enzymatic and clotting activities of prothrombin-2 activated by prothrombin activator ecarin.Methods Human prothrombin-2 gene and Echis carinatus ecarin gene were synthesized separately on the basis of the cDNA sequences published in GenBank.The gene of prothrombin-2 was cloned into the expression vector pPICZαA.The expression vector pPICZαA/prothrombin-2 was transformed into glycoengineered P.pastoris, and then prothrombin-2 engineered P.pastoris was screened.The expression products were induced by methanol, purified by two-step chromatography and identified by diges-tion by PNGase F and analysis of pepetide fingerprint.The ecarin gene was cloned into the expression vector pcDNA3.1. The expression vector pcDNA3.1/Ecarin was transformed into HEK 293T cells and the culture supernatant of HEK 293T/Ecarin was collected.The reaction product of HEK 293T/Ecarin cell culture supernatant and purified prothrombin-2 was analyzed by S-2238,which was the chromogenic substrate for thrombin.Fibrinogen was used to measure blood clotting time. Results The purified protein of P.pastoris expressed prothrombin-2 culture supernatant was 37 ×103 .The relative molecular mass(Mr) of the purified protein was reduced to 35 ×103, which was consistent with the theoretical Mr of prothrombin-2 molecular weight.The purified protein was proved to be prothrombin-2 by peptide fingerprint identification. The purified prothrombin-2 processed by HEK 293T/Ecarin culture supernatant could hydrolyze S-2238 to produce yellow pNA, and D405 of pNA increased with the volume of the processed prothrombin-2 that could promote the plasma coagulation.The blood clotting time was close to that of the thrombin kit.Conclusion Prothrombin-2 is prepared by P.pastoris and activated toα-thrombin by ecarin.This technique may replace the method of extraction of prothrombin from plasma and can be used for the treatment of war wounds or for future clinical research.

Objective To develop an interleukin-4(IL-4) antagonist named M5-IgG1Fc protein constructed by genetic engineering of antibody Fc fragment-cytokine mutein fusion protein which has a long half-life time in plasma.M5-IgG1 Fc protein binds to IL-4 receptor but cannot activate downstream signalling pathway , which provides a basis for drug develop-ment for allergic diseases .Methods The synthesized interleukin-4 mutant gene ( named M5 ) was cloned into the expres-sion vector pBV220 and transformed into E.coli DH5α.Chimeric gene M5-IgG1Fc obtained by overlap extension (SOE) method was transformed into glycoengineered Pichia pastoris GJK01 through expression vector pPICZαA .Then M5-IgGFc fusion protein was obtained by protein purification after being induced by methanol in 72 hours.The anti-IL-4 biologicial ac-tivity assay of M5 and M5-IgG1 Fc was performed with CTLL-2/IL-4R cells and detected with MTT colormetry .Finally,the half-life time of M5 and M5-IgG1 Fc protein in mice was compared by detecting the remaining amount in plasma with ELISA kit.Results The M5 protein expressed in E.coli and M5-IgG1 Fc fusion protein expressed in P.pastoris GJK01 both had IL-4 antagonistic bioactivity .The EC50 of both, which inhibited 5.6 ×10 -2 nmol/ml of IL-4, were 0.31 ±0.05 and 0.77 ± 0.03 nmol/ml,respectively.The maximum of M5 in plasma at 0.5 h was 5.8 ×10 -2 nmol/ml but the remaining amount was 2.8%of the maximum at 2 h.M5 protein could not be detected after administration at 8 h because of the detection line . The maximum of M5-IgG1 Fc fusion protein was 4.7 ×10 -2 nmol/ml,while fusion protein M5-IgG1 Fc decreased to 4.3%of its maximum at 120 h and could not be detected at 168 h.Conclusion M5 protein has IL-4 antagonistic bioactivity .M5-IgG1 Fc fusion protein expressed in glycoengineered P.pastoris GJK01 has IL-4 antagonistic bioactivity and long retention time in mice,which can be potentially used for treatment of allergic diseases .

In order to solve the problems of erratic drug absorption and low bioavailability after oral administration for poorly-water soluble drugs due to low solubility, a series of novel pharmaceutical dosage forms as solid dispersion, liposome, microemulsion, vesicle, cyclodextrin inclusion complexes and drug nanocrystal have been developed in recent years. Among which drug nanocrystal attracts more attentions for its simpler preparation method, higher drug loading and easier manufacturing technology in the design of dosage forms suitable for different administration routes. In this paper, the nanocrystals of the poorly-water soluble drugs prepared based on bottom-up and top-down technologies were introduced. The characteristics and applications of the nanocrystal-based dosage forms as suspension, tablet and capsule were also introduced and carefully evaluated with the focus on their pharmacokinetics, pharmacodynamics and tissue targeted drug distribution after delivery by oral administration, intravenous injection and pulmonary inhalation. The advantages of drug nanocrystals in their therapeutics effects over the bulk drugs were discussed together with the inherent mechanism. Finally, the problems existing in basic research and scaled-up manufacture of drug nanocrystal as well as the possible ways of solution were listed out so as to make the nanocrystal-based preparations exert their maximum therapeutic effect after clinical application.

Objective To express and purify mammalian glycosylation modified trimeric Ebola virus trimeric glycoprotein (EBOV-GP) using novel glycoengineered Pichia pastoris.Methods The EBOV-GP and EBOV-GPΔMLDΔTM genes were cloned into the pPICZ-αA vector,electrochemically converted to glycoengineered Pichia pastoris,and compared with EBOV-GP expressed in HEK-293T cells.Glycosylation was analyzed by PNGaseF and EndoH digestion,while the target protein was purified by affinity chromatography and ion exchange chromatography.N-terminal sequencing was used to determine whether the signal peptide was correctly cleaved during protein translation and gel column analysis was used to find out whether the trimeric structure was formed.Results The results of PNGaseF showed that EBOV-GP expressed by glycoengineered Pichia pastoris and HEK-293T cells had the same relative molecular mass and N-glycosylation degree.EndoH digestion showed that the N-glycosylation modification of EBOV-GPΔMLDΔTM was in a non-high mannose form.N-terminal sequencing showed that the signal peptide of the GP protein itself was correctly excised.Gel column analysis showed that the purified protein was in a trimeric form.Conclusion An EBOV-GP is obtained with complex glycosylation modification based on Glycoengineered Pichia pastoris.

Objective To analyze the methylation status of MUC2 gene in pancreatic carcinoma cell lines and peripheral blood of pancreatic carcinoma patients,and to explore the role of MUC2 methylation in the early diagnosis of pancreatic carcinoma.Methods Human pancreatic cancer cell lines of SW1990,ASPC,PANC1,BxPC3,PaTu8988 and CFPAC1,and 40 peripheral blood samples of pancreatic carcinoma patients,15 cases of chronic pancreatitis,25 cases of normal controls were collected,and the methylation status of MUC2 gene was detected by methylation sensitive restriction endonuclease PCR.Results MUC2 methylation was not detected in PANC1,BxPC3,PaTu8988,but was detected in ASPC,CFPAC1,SW1990.Among the peripheral blood samples,the rate of methylation in pancreatic cancer was 40.O%(n=16),in chronic pancreatitis was 0%,in normal controls was 4.0%(n=1),and the difference among the three groups was statistically significant(P＜0.01).The methylation of MUC2 gene CpG islands for the diagnosis of pancreatic carcinoma had a sensitivity of 40%,specificity of 97.5%,accuracy of 68.8%,positive predictive value of 94.1%and negative predictive value of 61.9%.Conclusions The detection of MUC2 hypermethylation in peripheral blood samples may be an potential marker for early diagnosis of pancreatic carcinoma.

Objective To express the hemagglutinin of H7N9 in Pichia pastoris and analyze its immunogenicity. Methods The HA [1 -525 amino acids(aa)] of H7N9 [A/Hangzhou/1/2013(H7N9)] lacking the C-terminal transmembrane anchor-coding sequence was amplified by PCR and cloned into the expression vector pPICZαA.The plasmid pPICZ-HA7-S-was transformed into P.pastoris and the HA1-525 was detected by ELISA.Then the HA1-525 was precipitated by PEG20000.After immunization with the HA1-525 , the anti-HA7 antibody in mouse serum was detected by ELISA and hemagglutinin inhibition ( HI) test.Results The HA1-525 was expressed in P.pastoris after being induced with methanol. Western blotting confirmed that there were specific dispersion bands and the molecular weight of HA1-525 decreased to 58 × 103 after being digested by endo H.Anti-HA7 antibody was found in serum of HA1-525 immunized mice and the hemaggluti-nation-inhibition titers reached 1∶700 after the third doses.Conclusion This study shows the HA1-525 expressed in P.pastoris can induce the neutralizing antibody in mice.

Adenosine receptors (AR) play an important role in the regulation processes for body temperature and vigilance states. During our previous studies, we noticed that aminophylline (a non-selective, blood-brain-barrier penetrably AR antagonist) could attenuate the effects of YZG-330 [(2R,3S,4R,5R)-2-(hydroxymethyl-5-(6-(((R)-1-phenylpropyl)amino)-9H-purin-9-yl)tetrahydrofuran-3, 4-diol] on lowering the body temperature. Hereby, we focused ourselves on the character of thermal regulation effect of YZG-330 in mice and tried to specify the receptor subtype via giving typical adenosine receptor antagonists. The results showed that both of the magnitude and lasting time of the effect that YZG-330 played on decreasing body temperature are in a dose-dependent manner: within the next 3 hour after intragastric administration (ig) of 0.25, 1 or 4 mg . kg-1 YZG-330, the extreme values on body temperature decreasing were (1.2 ± 0.3) °C, (3.6 ± 0.4) °C (P<0.001) and (7.4±0.5) °C (P<0.001), separately; whereas the duration that body temperature below 34 °C were 0, (10±5) and (153±4) min, separately. Adenosine A1 receptor (A1R) antagonist (DPCPX) could effectively reverse YZG-330's effect on decreasing body temperature, with intraperitoneal administration of DPCPX (5 mg . kg-1) 20 min prior than YZG-330 (4 mg.kg-1, ig), the extreme value on body temperature decreasing was (3.5 ± 0.7) °C (P<0.001), the duration that body temperature below 34 °C was (8±6) min (P<0.001). However, adenosine A2a receptor antagonist, SCH-58261, did not show any influence on the effects of YZG-330 at all. Combined with the fact that 8-SPT (a non-selective, blood-brain-barrier impenetrably AR antagonist) did not reverse the effect of YZG-330, we come to the conclusion that central-adenosine A, receptor plays a significant role on the thermal regulation effect of YZG-330.
Adenosine ; analogs & derivatives ; pharmacology ; Adenosine A1 Receptor Antagonists ; pharmacology ; Animals ; Body Temperature Regulation ; drug effects ; Mice ; Pyrimidines ; pharmacology ; Receptor, Adenosine A1 ; physiology ; Triazoles ; pharmacology ; Xanthines ; pharmacology

Objective To prepare endo-beta-N-acetylglucosaminidase H (Endo-H) expressed in Pichia pastoris, and apply it to N-glycosylation analysis .Methods One complete gene was synthesized on the basis of the cDNA sequence encoding Streptomyces plicatus reported in GenBank .The gene was cloned into the expression vector pPIC 9.The expression vector pPIC9-Endo-H was transformed into P.pastoris(JC308).The expression products were induced by methanol , puri-fied by two-step chromatography , used to analyze the glycan structures of RNaseB by the DNA sequencer assisted fluoro-phore-assisted carbohydrate electrophoresis (DSA-FACE)methods, and finally compared with peptide-N-asparagine amidase F(PNGase F).Results This enzyme expressed in P.pastoris(JC308) had the ability to hydrolyze natural or denatured high-mannose type of oligosaccharide linked by β-1,4-glycosidic bonds , but not complex-type oligosaccharide .The result of DSA-FACE showed that carbohydrate chains of Man 5 GlcNAc-Man9 GlcNAc could be obtained when RNaseB was hydrolyzed by Endo-H, and that Man5 GlcNAc2-Man9 GlcNAc2 chains became available when RNaseB was hydrolyzed by PNGase F . Conclusion Endo-H expressed in P.pastoris has bioactivity which can be used to analyze N-glycosylation with the method of DSA-FACE.

Berberine is a naturally occurring benzylisoquinoline alkaloid with a wide range of pharmacological activities, such as antibacterial, anticancer, hypolipidemic, antidiabetic and antidiarrheal. Although berberine has a wide range of curative effects, the extremely low bioavailability (< 1%) limits its clinical application. Pure berberine preparations have not yet been approved for any specific disease. The low oral bioavailability of berberine is mainly due to poor solubility caused by self-aggregation under acidic conditions, low permeability, P-glycoprotein (P-gp)-mediated efflux, and liver and intestine metabolism. To improve the oral bioavailability of berberine, researchers have adopted a variety of strategies, including the application of various nano-delivery systems, penetration enhancers and P-gp inhibitors, structural modifications, and development of berberine derivatives. Improving the oral bioavailability of berberine can improve the pharmacological activity of berberine, reduce the dosage, and then reduce the toxic and side effects. This review summarized the various pharmacological activities, metabolism progress and pharmacokinetic characteristics of berberine, the newly discovered berberine target intestinal microbiota and focused on the strategies to improve the oral bioavailability of berberine by improving solubility and permeability, inhibiting P-gp efflux, and structural modification. The research on berberine was prospected, which provided guidance for the in-depth study of berberine.

Objective To obtain a strain of glycoengineering yeast with higher N-glycosylation efficiency by overexpressing N-glycosyltransferase.Methods Through the selecting marker URA3 gene, a new glycoengineering yeast strain named 4-32-STT3D was constructed, which could overexpress the Leishmania major N-glycosyltransferase staurosporine and temperature sensitivity3 D subunit(STT3D) under the control of an inducible alcohol oxidase 1(AOX1) promoter.We analyzed the N-glycosylation status of anti-human epidermal growth factor receptor 2 ( HER2 ) antibody and granulocyte macrophage colony stimulating factor (GM-CSF) expressed in 4-32-STT3D using SDS-PAGE,Western blotting and peptide-N-asparigineamidase F(PNGase F).Finally the effect of STT3D on the growth rate of glycoengineering yeast was detected.Results SDS-PAGE showed that anti-HER2 antibody expressed in 4-32-HL had two components:the first one with a relative molecular mass 55 ×103 was glycosylated,while the second one with 50 ×103 was non-glycosylated,but anti-HER2 antibody expressed in 4-32-HL-STT3D had the component of 55 ×103 only without any non-glycosylated 50 ×103 .The above components became 50 ×103 with the digestion of PNGaseF.All of them proved to be antibodies by Western blotting.As a report protein,GM-CSF expressed in 4-32-GM-CSF had two components: 22 ×103 and 20 ×103, while in 4-32-GM-CSF-STT3D there was only one with 22 ×103 .All these components became 18 ×103 with the digestion of PNGase F.Statistical analysis showed that without induction,STT3D had no effect on the growth rate of glycoengineering yeast, while great effect was observed when STT3D was induced.Conclusion Glycoengineering yeast with the overexpression of N-glycosyltransferase has higher N-glycosylation efficiency.