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 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 .

Objective:Toprepare poly-DL-lactide-poly (PELA) microspheres encapsulating recombinant tissue inhibitors of metalloproteinase-1 (TIMP-1) adenovirus, and to investigate their effects on the proliferation of hepatocellular carcinoma HepG2 cells. Methods: The microsphere was constructed by encapsulating recombinant adenovirus containing TIMP-1 in biodegradable PELA. The diameter of the microsphere, quantity of virus encapsulated, loading rate, and releasing kinetics were measured. HepG2 cells were infected with the microspheres; the infection efficiency was examined by fluorescent microscope; and the ultrastructure was observed by TEM. The expression of TIMP-1 mRNA in HepG2 cells was examined by semi-quantitative RT-PCR, and the proliferation of HepG2 cells was detected by MTT assay. Results: The microsphere encapsulating recombinant TIMP-1 adenovirus was successfully constructed, with its diameter, entrapment efficiency, and virus loading rate being 1.965, 60.0%, and 10.5×10~8/mg, respectively. About 60% of the viruses were released within 120 h, and the total releasing time was longer than 240 h. Infection with rAdTIMP-1 PELA microsphere efficiently induced TIMP-1 expression in HepG2 cells, and significantly inhibited the proliferation of HepG2 cells, with the inhibitory rate being 47%. Conclusion: PELA microsphere encapsulating recombinant TIMP-1 adenovirus can markedly inhibit the proliferation of HepG2 cells, which provides an experimental basis for the combining macromolecular chemistry and gene therapy for treatment of hepatocellular carcinoma.

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 investigate the value of optimizing the thoracic CT scanning dose on reducing radiation dose.Methods 50 patients were undergone CT scanning using CARE Dose 4D technique,the mAs of each slices,CT dose index of volume(CTDIvol) and images qualities were evaluated respectively.And the results were compared with traditional thoracic CT scanning(200mAs,15.31mGy).Results Compared with routine sequence,the exposure dose of singe-slice was decreased by 48.2% when the CARE Dose 4D technique was used(maximal decreasing 84%),CTDIvol was reduced about 32.98%(maximal reduction of 56.5%),there was statistically significant difference(P

Objective To evaluate the correlations between CT features and histopathologic subtypes of lung adenocarcinomas presenting as pure ground-glass nodules (pGGN) of 1 cm or less in maximal diameter. Methods CT appearances, pathology and clinical data of 95 patients (97 lesions) who underwent curative resection of lung adenocarcinomas presenting as pGGN≤1 cm in diameter from March 2011 to February 2015 were retrospectively analyzed. Of the 97 lung adenocarcinomas, there were 19 atypical adenomatous hyperplasia (AAH) (19.6%), 31 adenocarcinoma in situ (AIS) (31.9%), 19 minimally invasive adenocarcinoma (MIA) (19.6%) and 28 invasive pulmonary adenocarcinoma (IPA) (28.9%). Fifty (51.5%) were preinvasive (AAH+AIS) and 47 (48.5%) were invasive (MIA+IPA). Lesion size and density were compared among pathologic subtypes using analysis of variance (ANOVA). Lesion size were compared between preinvasive and invasive lesions using 2?independent samples t?test. Lesion location, presence of bubble?like sign, air bronchogram, vessel changes, margin, and tumor?lung interface were compared among histopathologic subtypes using chi?square test. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the cut-off point of size in discriminating preinvasive lesions from invasive lesions. Results Of the 97 lesions, there were no statistically significant differences among histopathologic subtypes in terms of lesion density, presence of bubble?like sign, air?bronchogram, and margin (P>0.05). Mean size of AAH, AIS, MIA and IPA was (0.72 ± 0.19), (0.82 ± 0.14), (0.84 ± 0.11) and (0.85 ± 0.16) cm respectively. There were statistically significant differences among histopathologic subtypes in terms of lesion size (F=3.16, P=0.028). The vessel changes occurred in 2 of AAH, 11 of AIS, 10 of MIA and 17 of IPA. There were statistically significant differences among histopathologic subtypes in terms of vessel changes (χ2=13.22, P=0.004). Lesions with clear tumor?lung interface were in 10 of AAH, 24 of AIS, 17 of MIA, and 26 of IPA. There were statistically significant differences among histopathologic subtypes in terms of tumor?lung interface (χ2=12.67, P=0.005). The optimal cutoff value of lesion size for differentiating preinvasive lesions from invasive lesions was 0.82 cm (sensitivity, 61.7%;specificity, 62.0%). Conclusion Lesion size, vessel changes, and lung?tumor interface may indicate the invasiveness of lung adenocarcinoma presenting as pGGNs of≤1 cm in diameter.

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.

PurposeTo investigate the multi-slice spiral CT features of lung primary invasive mucinous adenocarcinoma, a rare malignancy with few relevant literature. Materials and Methods Forty-one patients with pathology proven primary invasive mucinous adenocarcinoma of the lungs underwent MSCT examination, of which 27 patients received intravenous contrast. Imaging features were analyzed, and the patients were divided into groups according to morphology, density and enhancement.Results For 41 patients, 40 cases had one lesion, and one case had more than one lesion; the lesions were in the right upper lobe in 5 cases, right middle lobe in one case, right lower lobe in 17 cases, left upper lobe in 5 cases, and left lower lobe in 12 cases. There was solitary lesion in 40 cases. The other patient had multiple lesions. 29 cases showed nodules/masses, while non-mass lesions in 12 cases. Ground glass opacity was seen in 4 cases, single consolidation in 11 cases, and mixed consolidation in 26 cases. There was significant contrast enhancement in nodules/masses compared to non-mass lesions (t=5.00, 6.51 and 14.06,P<0.05).Conclusion MSCT shows a diversity of imaging features of primary invasive mucinous adenocarcinom, including single round lesion, lower lobe in both side; the density is lower than muscle on CT scan, and presents mild to moderate enhancement on enhancement scan round lesions has high density and enhancement range than irregular-shaped lesions.

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.

Objective To determine the correlation between coronary artery calcium score (CACS) and carotid calcification, and their risk factors. Methods 162 cases underwent coronary and carotid pre-contrast CT scan with dual-source CT scanner within 2 weeks. The reconstructed parameters were the same. The calcifications of coronary and carotid arteriae were quantified by calculating the Agatston score. The main risk factors such as age, sex, hypertension, total cholesterol, low-density-lipoprotein cholesterol (LDL), high-density-lipoprotein cholesterol (HDL), diabetes, smoking and coronary heart disease history were recorded. The CACS and carotid calcium scores were comparatively analyzed using Spearman's correlative analysis. The relativity between the risk factors and CACS scores, carotid calcium scores. Results In 162 subjects, there was positive relativity between CACS and carotid cal-cium score(r=0.690, P<0.01). Logistic regression analysis showed that CACS were of relativity with age, diabetes, total cholesterol and LDL cholesterol,otherwise, carotid calcium scores only with age and diabetes. Conclusion There is significant correlation be-tween CACS and carotid calcium score, but their risk factors are not same.