AIM:The homologous recombination takes place in E.coli BJ5183 between shuttle vector and adenoviral backbone vector.Recombinants are selected for kanamycin resistance,and the linearized recombinant plasmid is transfected into 293 cells.In this study,AdEasy system was used to generate recombinant adenovirus vector carrying rat interleukin-10(IL-10) gene.METHODS:The experiment was conducted in Department of Microbiology,Qingdao University Medical College from August 2006 to May 2007.①The materials included five clean-grade SD rat,a shuttle vector pAdTrack-CMV,an adenoviral backbone plasmid pAdEasy-1,E.coli.BJ5183 and DH10B,and human embryo kidney 293 cells,which were given as a present by professor Luo.All animal treatment was accorded with the ethical standards.②Total RNA was extracted from spleen cells of rat stimulating by lipopolysaccharide.IL-10 cDNA was amplified by using RT-PCR.The gene of interest was firstly cloned into a shuttle vector pAdTrack-CMV.The resultant plasmid was linearized by digesting with restriction endonuclease Pme Ⅰ,and subsequently transformed into E.coli.BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1.Finally,the linearized recombinant plasmid was transfected into adenovirus packaging cell lines 293 cells.Recombinant adenovirus vAd-IL-10 was obtained.The expression of green fluorescent protein(GFP) was observed under fluorescent microscope.293 cells were cultured in 96-well culture plate with 1 ?104 cells per well.Condensed virus stock solution was added into the plate at different ratios.Recombinant adenoviruses titer was determined.Three days later,total RNA was extracted from 293 cells by TRIzol one-step method,and contaminant DNA was digested by DNaseⅠ.Electrophoresis detected the expression of IL-10 mRNA.RESULTS:①GFP expression was observed 16 hours after packing of the linearized recombinant adenoviruses in 293 cells.The amplification product of replicationdeficient Ad-IL-10 virus was transfected into adenovirus packaging cell lines 293 cells.When the fourth and fifth generations were seeded in virus for 24-48 hours,fluorescence was found in almost cells,and 5.5?108 pfu/mL titer of Ad-IL-10 was obtained.Titer of vAd-GFP was 9.0?108 pfu/mL.②Total RNA was extracted from transfected 293 cells.Electrophoresis showed that 580bp amplification product was expressed obviously and recombinant adenovirus was duplicated in 293 cells.CONCLUSION:The recombinant adenoviral vector carrying IL-10 gene is successfully constructed using AdEasy system.Moreover,vAd-IL-10 recombinant adenovirus with high titer is prepared and transcribed in 293 cells.

Systemic sclerosis is an autoimmune disease characterized by skin thickening and tightness.Digestive system is the most commonly damaged system secondary to skin,and as to unspecific clinical manifestations,this complication is often ignored in the early,the prognosis and quality of life of patients will be affected.This article overviews the recent research developments on pathology,clinical features,management of systemic sclerosis with digestive system involvement,to draw attention to early diagnosis and treatment of this condition for the improvement of the prognosis.

Objective To evaluate the quality of four domestic and three imported fourth-generation HIV diagnostic reagents.Methods The specificity and sensitivity of these assays were analyzed when testing HIV negative samples and HIV-1 RNA positive samples.The relative seroconversion sensitivity index was analyzed when testing BBI seroconversion panels.Results The sensitivity of seven 4th-generation assays were 100％ (95％ CI:99.86％-100％ ),and one sample at the window period of HIV-1 infection were detected as positive.Of the seven assays,one imported assay exhibited the relative largeδ + value (1.0892),and the small δ+ value were found on the remaining six assays (0.0836-0.3003 ).For the samples negative for HIV antibody,varying degrees of false positives were observed on the seven assays ( specificity:97.80％ -99.60％,δ- value:-1.3803 to -0.4778).When testing the BBI seroconversion panels,the relative seroconversion sensitivity index of domestic assays were -0.500-0,however,which of imported assays were -0.600 and -0.700.Conclusion The seven reagents exhibited high sensitivity and specificity.The 4th generation HIV assays can be used as blood screening reagents to find the samples at window period of HIV-1 infection,thus indicating the certain meaning in reducing the transmission risk of HIV-1 for fourth-generation HIV diagnostic reagents.However,the better efficiency to detect HIV-1 early infection was observed on the imported assays than on the domestic assays.

To explore the protective effect of adenovirus mediated IGF-Ⅰgene(Ad-IGF-Ⅰ)transfer on non-obese diabetic(NOD)mice. The results showed that the incidence of diabetes and degree of insulitis were significantly reduced in mice receiving Ad-IGF- Ⅰ . Treatment with Ad-IGF- Ⅰ significantly decreased apoptosis rate,expression of Fas and caspase-3, and increased expression of Bcl-xl. This study indicates the potential of IGF- Ⅰ gene therapy in protecting NOD mice from insulitis and apoptosis.

Objective To evaluate the differences between the third and the fourth generations of anti-HIV assays,and different kits within the same generation.Methods A total of 989 HIV-negative samples,185 samples positive for HIV-1 RNA.1st-generation international references of HIV antibodies and samples from 9 sets of BBI seroconversion panels were detected by 8 kits of the third generation and 4 kits of the fourth.Results The fourth generation kits can detect HIV infection earlier than the third generation kits.However,the detected days of HIV infection with different kits of the fourth generation were different whilst no significant difierences were found with difierent kits of the third generation.Furthermore,the capacity of detecting samples with different genotypes for different reagents was different,especially the capacity of domestic reagents on detecting HIV-1 O group and HIV-2 samples was relatively weak.Conclusion These data provided information to improve the quality of anti-HIV diagnostic reagents further.

Objective To investigate the correlation of previous hepatitis B virus (HBV) infection with the incidence risk of chronic pancreatitis (CP).Methods This was a case control study.Five hundred and seventy-one patients with CP admitted in the Department of Gastroenterology, Changhai Hospital, Second Military Medical University between January 2015 and October 2016 were enrolled, and 1216 sex and age matched health individuals were also enrolled as the control group.The 5 serum HBV markers(HBsAg,HBsAb, HBeAg, HBeAb and HBcAb) were detected and their correlation with CP incidence was analyzed.Results The positive rate of HBsAg in the CP group and the control group were 3.0% and 3.8%, respectively, and the difference was statistical significant.(OR=0.039, 95% CI 0.02~0.80, P<0.00), but in all the HBsAg positive models (HBVM) the difference of CP and control groups was not statistical significant.HBsAb positive rate in CP group and the control group were 51.8% and 75.0%, respectively, and the difference was statistical significant(P<0.000).HBeAg positive rate in CP group and the control group were 1.1% and 0.1%, the difference was statistical significant (P<0.05), but in all the HBeAg positive models, the CP group and the control group had no statistical difference (P>0.05).The positive rate of HBeAb in the CP group and the control group were 24.3% and 10.8%, respectively, and the difference was statistical significant(P<0.00).The positive rate of HBcAb in the CP group and the control group were 50.1% and 16.5%, respectively,and the difference was statistical significant(P<0.000).In the(HBsAb+, HBeAb+, HBcAb+), (HBsAb+, HBcAb+), (HBeAb+, HBcAb+), (HBcAb+) models, the positive rate in CP group was significantly higher than that of the control group(P<0.000).Multivariate regression analysis showed that the positivity of HBsAb and HBeAb were the protection factors for the occurrence of CP(P<0.05),and HBcAb positivity was the independent risk factor for CP (OR=6.931,P<0.000).Conclusions HBsAb and HBeAb poitivity were the protectors for CP, while HBcAb positivity could be considered as an independent risk factor for CP.

Objective To investigate the feasibility of low-tube-voltage in combination with the three-dimensional adaptive iterative dose reduction (AIDR-3D) algorithm in performing lower extremity computed tomography angiography (CTA).Methods A total of 60 patients suspicious of lower extremity arterial occlusion were randomized into control group (120 kV,a =30) and experimental group (100 kV,n =30).The CTA was undertaken with a 320-row scanner (Toshiba Aquilion ONE),and the images was reconstructed with filtered back projection (FBP) algorithm in control group and FBP as well as the AIDR-3D algorithm in experimental group.The subjective image quality,vascular density (VD),noise,signal-to-noise ratio (SNR),contrast-to-noise ratio (CNR),and dose length product (DLP) were compared between two groups.Results The DLP was significantly lower in experimental group than that in control group [(503.5± 104.7) vs.(1 099.4 ± 151.7) mGy·cm,t =15.7,P ＜0.05].The images in experimental group with 100 kV and FBP protocol had significantly increased VD and noise (t =-3.13,-3.61,P ＜ 0.05) than that in the control.The images in experimental group with AIDR-3D had significantly lower noise and higher SNR and CNR than that with FBP (t =13.59,2.14,P ＜ 0.05),also significantly lower noise and significantly higher VD,SNR,and CNR than that in the control (t =-3.75,-4.19,-4.15,P ＜ 0.05).Conclusions Low-tube-voltage (100 kV) combined with AIDR-3D reconstruction could significantly improve the image quality and reduce radiation dose in lower extremity CTA with a 320-row CT scanner.Trial registration Chinese clinical trial registry,ChiCTR-DPD-16008054.

Objective To pan and characterize anti-HIV-1 Fab by the phage antibody library technology. Methods Total RNA were extracted from lymphocytes which were isolated from peripheral blood collected from asymptomatic HIV-1 infected donors with high titer antibody against HIV-1. The genes of heavy chains Fd fragment and light chains of antibody were amplified by RT-PCR. The phagmids pComb3X cloned Fd and light chain genes were transformed into E. coli XL1-Blue by electroporation to construct phage Fab library. By three runs of "absorption-elution-neutralization-enrichment", the clones were induced by IPTG and characterized by ELISA. The positive clones were sequenced and analyzed the sequences. Subsequently, Fab antibodies of these positive clones were induced to expressed and purified, then the recombinant virus neutralization assay was performed. Results A phage Fab library was constructed with 8×106 members, and 11 positive clones were obtained by detecting IPTG-induced-expressing Fabs with ELISA. By analysis of the sequences, 10 light chain genes and 8 Fd genes were ensured to be obtained. Compared with the genes of anti-HIV-1 antibodies in HIV sequence database, the gene sequences we obtained were highly homologous to some patent genes of anti-HIV-1 gp120 antibodies in HIV sequence database( light chains with 60%-90% identity, Fd with 71%-85% identity); The CDRs of these positive clones were determined by comparing the positive clone genes with antibodies' genes in V base database, furthermore, CDRH3 of these positive clones has the length of 12-22 aa. Strand shift had little effect to improving affinity of our Fab clones. Fab antibodies were induced to express at the concentration of ＞ 10 mg/L. Three Fab antibodies neutralize HIV-1 virus to some extent. Conclusion The studies will provide the basis on further study on the anti-HIV-1 Fabs obtained successfully.

Objective To investigate the feasibility of low-c oncentration iso_osmolar contrast agent together with low tube voltage and iterative reconstruction algorithm in rabbit liver computed tonography (CT) perfusion imaging.Methods A total of 15 bealthy New Zealand rabbits were scanned twice of liver CT perfusion scans each with 24 hours interval.The first scan (routine group) was acquired at 100 kV and 100 mAs with ultravist (370 mg/ml),while the second (double low group) was acquired at 80 kV and 100 mAs with iodixanol (270 mg/ml) at 24 hours after the first scan.The obtained images were reconstructed with filtered back projection (FBP) and adaptive iterative dose reduction (AIDR-3D)algorithms in the controlled and experimental groups,respectively.The perfusion parameters including hepatic artery perfusion(HAP),portal vein perfasion(PVP),hepatic perfusion index(HPI),and total liver perfusion(TLP) and image quality as image quality score,average CT value of abdomen aorta,signalto-noise ratio(SNR),carrier-to-noise ratio(CNR),and figure of merit(FOM) were compared used pair ttest or Mann-Whitney U-test between the two groups wherever appropriate.The effective radiation dose and iodine intake were also recorded and compared.Results The image quality and perfusion parameters had no significantly different between the two groups except for FOM.The effective radiation dose and iodine intake were 38.79％ and 27.03％ lower in the double low group.Conclusions Low concentration iso _osmolar contrast agent (iodixanol,270 mg/ml) together with low tube voltage (80 kV) helps to reduce radiation dose and iodine intake without compromising perfusion parameters and image quality in liver CT perfusion imaging.

Objective To explore the feasibility of coronary computed tomographic angiography (CCTA) for obese patients with lower tube voltage (100 kV) and lower contrast media concentration (270 mgI/ml) using iterative reconstruction.Methods A total of 48 patients with body mass index greater than 30 kg/m2 were included and randomly divided into 2 groups according to random number table method.The images of the control group were obtained using iodine 370 mgI/ml, a tube voltage of 120 kV, and traditional filtered back projection (FBP) image reconstruction.Patients in the test group were injected with isotonic low concentration contrast media (270 mgI/ml), scanned with a lower tube voltage (100 kV), and adaptive iterative noise reduction image reconstruction algorithm (AIDR-3D) was used.Two experienced physicians scored the image quality in a double-blind way.Independent sample t-test was used to compare the effective dose (E), average CT values, signal to noise ratio (SNR), contrast to noise ratio (CNR), the figure of merit (FOM), image quality scores and the total iodine intake.Side effect was also evaluated.Results The subjective scores for control group and test group were not significantly different (P ＞ 0.05).The scores of two physicians were consistency (Kappa =0.88, P ＜ 0.05).The average CT values, SNR and CNR for the two groups were not significantly different (P ＞ 0.05), but the FOM of the test group was significantly higher than that of the control group (t =-9.250,-8.604,-9.158,-5.341, P ＜ 0.05).Effective dose in the test group was (1.61 ± 0.41) mSv, lower than that of the control group (t =8.373, P ＜ 0.01).The total iodine and iodine injection rate in the test group were both lower than in the control group (t =7.628, 8.480, P ＜ 0.01).The incidence of contrast mediarelated discomfort in the test group was lower than control group (x2 =18.70, 6.25, P ＜ 0.05).Conclusions For obese patients, isotonic low concentration of contrast media and low-dose CCTA could be feasible, which substantially reduce the radiation dose and iodine intake without sacrificing image quality.Trial registration Chinese clinical trial registry, ChiCTR-DPD-15007510.