Construction of recombinant adenovirus expressing capsid protein of serotype O foot-and-mouth disease virus and analysis of its immunogenicity / 生物工程学报

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×109.1 TCID50/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.

An experimental study of SCAD recombinant adenovirus treating heart failure following myocardial infarction in rats / 中国药理学通报

Aim To investigate the effects of overexpression of short-chain acyl-CoA dehydrogenase (SCAD) recombinant adenovirus on heart failure following myocardial infarction in rats, and to explore the relationship between SCAD and heart failure. Methods The rat model of heart failure following myocardial infarction was established by ligation of left anterior descending coronary artery (LAD). The SCAD recombinant adenovirus was injected into the apical wall. The experimental groups were divided into 6 groups; Sham + NS group, LAD + NS group, Sham + Ad-GFP group, Sham + Ad-SCAD group, LAD + Ad-GFP group and LAD + Ad-SCAD group. Systolic blood pressure (SBP) of tail artery was measured after surgery and the cardiac morphological changes were detected. The changes of SCAD mRNA expression, protein expression, enzyme activity, ATP content and free fatty acid content were examined. Results Compared with sham group, LAD + NS group showed heart failure significantly. Compared with LAD + NS group, SBP from LAD + Ad-SCAD group increased obviously after surgery, collagen deposition was apparently reduced and myocardial fibrosis was markedly improved. Compared with Sham + NS group, the SCAD mRNA, protein expression, enzyme activity and ATP content in LAD + NS group obviously decreased, and the contents of free fatty acids markedly increased. Compared with LAD + NS group, the above indicators of LAD + Ad-SCAD group were evidently reversed after treated with SCAD recombinant adenovirus. Conclusion Overexpression of SCAD recombinant adenovirus in heart can significantly improve heart failure caused by LAD.

Nucleoprotein vaccine induces cross-protective cytotoxic T lymphocytes against both lineages of influenza B virus

PURPOSE: The influenza B virus diverges into two antigenically distinct lineages: B/Yamagata and B/Victoria. Influenza B is the dominant circulating virus during some influenza seasons, and recent data demonstrated that influenza A and B infection similarly cause severe clinical symptoms in hospitalized patients. Nucleoprotein (NP) is a good target for a universal influenza vaccine. This study investigated whether NP epitope variation within two lineages affects the dominant cytotoxic T lymphocyte (CTL) responses induced by vaccination and the resultant protective immunity. MATERIALS AND METHODS: The NP of B/Yamagata/16/1988, the representative strain of the Yamagata lineage, includes a dominant CTL epitope, FSPIRITFL, while B/Shangdong/7/1997 from the Victoria lineage has one amino acid difference in this sequence, FSPIRVTFL. Two recombinant replication-deficient adenovirus (rAd)-vectored vaccines expressing either NP were prepared (rAd/B-NP(I) and rAd/B-NP(V), respectively) and administered to BALB/c mice intranasally. To examine the efficacy of vaccination, antibody responses, CTL responses, and morbidity/mortality after challenge were measured. RESULTS: Both vaccines induce similar antibody and CD8 T-cell responses cross-reacting to both epitopes, and also confer cross-protection against both lineages regardless of amino acid difference. CONCLUSION: The rAd-vectored vaccine expressing the NP could be developed as universal influenza B vaccine which provides broader protection.

Cytotoxic activity of mammaglobin A-specific CD8+ cytotoxic T lymphocytes and cytokine-induced killers against breast cancer cells in vitro / 西安交通大学学报(医学版)

Objective: To compare the cytotoxic activity of mammaglobin A-specific CD8+ cytotoxic T lymphocytes (CTLs) and cytokine-induced killers (CIKs) against breast cancer cells in vitro. Methods: PBMCs were isolated in vitro from the peripheral blood of healthy volunteers. Then DCs, CTLs and CIKs were isolated, induced and cultured from PBMCs in vitro. CD8+ CTLs were purified with immunomagnetic beads from CTLs. DCs were infected with recombinant adenovirus encoding mammaglobin A(Ad-MGBA). CTLs and CIKs were co-cultured with DCs being infected with Ad-MGBA. The cytotoxic activity of mammaglobin A-specific CD8+ CTLs and CIKs against breast cancer cells was compared by flow cytometry. Results: The apoptosis rate of breast cancer cell MDA-MB-415, which expressed MGBA was 63.07% by CD8+ CTL killing and 48.35% by CIK killing (P<0.05). The apoptosis rate of breast cancer cell MDA-MB-231, which could express MGBA was 14.62% by CD8+CTL killing and 29.29% by CIK killing (P<0.05). Conclusion: The cytotoxic activity of antigen-specific CD8+ CTLs for the same antigen-expressing oncology cells is higher than CIKs. However, for different antigen-expressing oncology cells, the cytotoxic activity of CD8+CTLs is lower than that of CIKs.

Clinical value of adenovirus-mediated down regulation of TIMP-1 on liver fibrosis in rats under ultrasound microbubble / 实用医学杂志

Objective Analysis of the effect and the mechanism of adenovirus with down regulation of matrix metalloproteinase inhibitor 1 (TIMP-1) achieved targeting by ultrasound microbubbles combined with ultrasound irradiation for liver fibrosis in rats. Methods Recombinant adenovirus-mediated with down regulation of TIMP-1 gene was constructed and a mixture of recombinant adenovirus and ultrasound contrast agent was prepared.The rat liver fibrosis model was established and divided into 5 groups : model group; adenovirus group (recombinant adenovirus); adenovirus microbubble group (mixture of recombinant adenovirus and ultrasound contrast agent); experimental group (ultrasound irradiation + mixture of recombinant adenovirus and ultrasound contrast agent); ultrasound adenovirus group (ultrasound irradiation + recombinant adenovirus). The rats were sacrificed after 24 hours and liver sections were prepared. The expression of EGFP in each group was observed and the transfection rate was analyzed. The liver slices were stained by Masson to judge the stage of liver fibrosis. ANOVA analysis was used to compare the levels of alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , bydroxyproline (Hyp) , hyaluronic acid (HA) , type IV collagen (CIV) and laminin (LN) in each group. The relative expression levels of TIMP-1 and matrix metalloproteinase 13 (MMP-13) in each group were detected by Western blot. Results The transfection rate of the experimental group was higher than that of the adenovirus group (q = 3.418) , the adenovirus microbubble group (q = 3.756) and the ultrasonic adenovirus group (q = 5.502) , and the difference was statistically significant (P < 0.05); Pathological examination showed that the degree of fibrosis in the experimental group and the grade of liver fibrosis were lower than the other groups (P < 0.01). The activities of ALT, AST, HA, LN, CIV and Hyp in the experimental group were lower than those in the other 4 groups.Western blot showed that the level of TIMP-1 protein expression was highest while the level of MMP-13 protein expression was lowest in the experimental group than those in the other groups. Conclusion Adenovirus with down regulation of TIMP-1 achieved targeting by ultrasound microbubbles combined with ultrasound irradiation can inhibit the activity of TIMP-1 and improve the degree of liver fibrosis. Gene therapy is an potential therapeutic method in the application of treating liver fibrosis.

Study on Inhibitory Effects of Recombinant Adenovirus Ad-GFP-C197 on 3 Kinds of Tumor Cells / 中国药房

OBJECTIVE:To study the inhibitory effects of recombinant adenovirus Ad-GFP-C197, which prepared by adenovirus vector system-loading human telomerase reverse transcriptase(hTERT)C fragment(C197),on the proliferation of 3 kinds of tumor cells in vitro. METHODS:Ad-GFP-C197 was amplified and purified with HEK293 cells. Human gastric cancer cells SGC7901,human breast cancer cells MCF7 and human colorectal cancer cells CaCO2 were infected by Ad-GFP-C197 respectively. Using blank adenovirus carrier (Ad-GFP) as reference,the protein expression of C197 in 3 kinds of tumor cells infected by Ad-GFP-C197 was detected by Western blot assay. The inhibitory effects of Ad-GFP-C197 on 3 kinds of tumor cells were detected by MTT assay. The cell proliferation curve was drawn and the proliferation inhibition rate was calculated. RESULTS:The protein expression of C197 was not detected in 3 kinds of tumor cells infected by Ad-GFP,while significant protein expression of C197 was found in above cells infected by Ad-GFP-C197. The proliferation curves of the 3 kinds of tumor cells infected by Ad-GFP-C197 were significantly inhibited with the time extended,and the proliferation inhibitory rate reached 37.31%-41.42%. CONCLUSIONS:Ad-GFP-C197 shows significant inhibitory effects on the proliferation of SGC7901,MCF7 and CaCO2 cells, which is rapid to make up for the slow effect of other telomerase inhibitors.

Effect of miR-125b on dermal papilla cells of goat secondary hair follicle

Background: MicroRNAs (miRNAs) are endogenous noncoding RNAs that regulate various biological processes. miR-125b is a miRNA that has been reported to be critical for hair follicle (HF) morphogenesis and development. We identified that the expression of miR-125b varies during an individual hair cycle (anagen, catagen, and telogen) in the skin of cashmere goats. We constructed a gain model (by overexpressing miR-125b) and a loss model (by inhibiting endogenous miR-125b) based on dermal papilla cells (DPCs) to further investigate the role of miR-125b in HF cycle. In addition, we used a dual-luciferase system to highlight the predicated target genes of miR-125b. Results: We found that miR-125b affects the expression of FGF5, IGF-1, SHH, TNF-α, MSX2, LEF-1, FGF7, NOGGIN, BMP2, BMP4, TGF-ß1, and ß-catenin. The dual-luciferase assay further validated a direct interaction between miR-125b and FGF5 and TNF-α. Conclusion: miR-125b affects the expression levels of genes related to hair cycle and may also play a critical role in regulating the periodic development of HF.

rAAV1 mediated SER CA2a gene transfer improves heart failure canine cardiac function by down-regulat-ing MMP-9 expression / 实用医学杂志

Objective To investigate the effect and mechanism of recombinant adenovirus associated vi-rus type 1 (rAAV1) mediated SERCA2a gene transfer on cardiac function in canine with heart failure (HF). Methods 20 healthy male beagle dogs were randomly divided into control group,HF group,HF+EGFP group and HF+SERCA2a group,5 dogs in each group. The canine cardiac function,apoptosis and MMP-9 expression in cardiac myocytes were detected by TUNEL and immunohistochemistry methods. Results The left ventricular dia-stolic diameter (LVDD),left ventricular systolic diameter (LVSD) and left ventricular posterior wall dimension (LVPWD)in the HF and HF+EGFP groups,were significantly higher than those of the control and HF+SERCA2a groups,and the ejection fraction(EF)in the former two groups was significantly lower than that of the control group and HF+SERCA2a group(P<0.05). The left ventricular systolic pressure(LVSP)and left ventricular maximal rate of pressure rise(+dp/dtmax)in the HF and HF+EGFP groups were both significantly lower than those of the control group and HF+SERCA2a group,while left ventricular end diastolic pressure(LVEDP)and left ventricular maximal rate of pressure decline(-dp/dtmax)significantly higher than those of the control and HF+SERCA2a groups(P<0.05). The apoptosis index of HF+SERCA2a group was significantly lower than that of group HF and group HF+EGFP(P<0.05). The MMP-9 light density of HF+SERCA2a group was significantly higher than that of the control group(P < 0.05). Conclusion RAAV1 mediated CA2a SER gene transfer can effectively improve the cardiac function of HF dog,probably by inhibiting the apoptosis of cardiac muscle cells and down-regulating the expression of MMP-9.

rAAV1 mediated SER CA2a gene transfer improves heart failure canine cardiac function by down-regulat-ing MMP-9 expression / 实用医学杂志

Objective To investigate the effect and mechanism of recombinant adenovirus associated vi-rus type 1 (rAAV1) mediated SERCA2a gene transfer on cardiac function in canine with heart failure (HF). Methods 20 healthy male beagle dogs were randomly divided into control group,HF group,HF+EGFP group and HF+SERCA2a group,5 dogs in each group. The canine cardiac function,apoptosis and MMP-9 expression in cardiac myocytes were detected by TUNEL and immunohistochemistry methods. Results The left ventricular dia-stolic diameter (LVDD),left ventricular systolic diameter (LVSD) and left ventricular posterior wall dimension (LVPWD)in the HF and HF+EGFP groups,were significantly higher than those of the control and HF+SERCA2a groups,and the ejection fraction(EF)in the former two groups was significantly lower than that of the control group and HF+SERCA2a group(P<0.05). The left ventricular systolic pressure(LVSP)and left ventricular maximal rate of pressure rise(+dp/dtmax)in the HF and HF+EGFP groups were both significantly lower than those of the control group and HF+SERCA2a group,while left ventricular end diastolic pressure(LVEDP)and left ventricular maximal rate of pressure decline(-dp/dtmax)significantly higher than those of the control and HF+SERCA2a groups(P<0.05). The apoptosis index of HF+SERCA2a group was significantly lower than that of group HF and group HF+EGFP(P<0.05). The MMP-9 light density of HF+SERCA2a group was significantly higher than that of the control group(P < 0.05). Conclusion RAAV1 mediated CA2a SER gene transfer can effectively improve the cardiac function of HF dog,probably by inhibiting the apoptosis of cardiac muscle cells and down-regulating the expression of MMP-9.

Clinical efficacy and safety of recombinant adenovirus-p53 combined with concurrent radiotherapy and hyperthermia in treatment of advanced soft tissue sarcoma:a study of 76 patients / 中华放射肿瘤学杂志

Objective To evaluate the efficacy and safety of recombinant adenovirus-p53(rAdp53) injection combined with radiotherapy and hyperthermia in the treatment of unresectable advanced soft tissue sarcoma.Methods In this retrospective study, we evaluated 76 patients with unresectable advanced primary or recurrent soft tissue sarcoma treated in our hospital from November 2005 to November 2012.These patients received radiotherapy and hyperthermia with rAdp53(p53 group, n=41) or without rAdp53(control group, n=35).rAdp53((1-2)×1012viral particles each time, once a week, 8 times on average) was injected into the tumor or infused into the pelvic cavity.Radiotherapy (2 Gy each time, 5 times a week) was performed for the planning target volume at 56.3±5.3 Gy in the p53 group and 58.1±4.2 Gy in the control group, with no significant difference between the two groups (P>0.05).Superficial or deep thermotherapy was employed 8 times on average (twice a week).Clinical features, response rate, time to progression (TTP), overall survival (OS), and adverse events were compared between the two groups (P>0.05).The Kaplan-Meier method was used to calculate OS;the log-rank test was used for survival difference analysis and univariate prognostic analysis;the chi-square test was used for comparison of categorical data.Results At 2 months after treatment, the p53 group had significantly increased response rate (partial response+ complete response+ stable disease)(85% vs.54%, P=0.003) and local control rate (49% vs.23%, P=0.020) as well as prolonged TTP (12 months vs.5 months, P=0.010) and OS (48 months vs.31 months, P=0.049), as compared with the control group.No adverse events caused by radiotherapy and hyperthermia except transient fever were seen in the two groups.Conclusions Concurrent radiotherapy and hyperthermia combined with rAdp53 injection is effective and safe for patients with advanced soft tissue sarcoma.

Immune responses induced by BCG prime and single dose of recombinant adenovirus Ad5-CEAB boosted strategy in mice / 中国人兽共患病学报

Tuberculosis (TB) remains an enormous health burden worldwide.To date,Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the unique anti-TB vaccine available for humans,which provides an important but limited protection from the Mycobacterium tuberculosis (Mtb) infection.It is therefore an urgent need to develop better vaccines and vaccination strategies to prevent the spread of Mtb infection.Heterologous prime-boost vaccination strategies using both BCG and novel anti-TB vaccines have been demonstrated to induce robust immune responses than BCG alone.We have previously demonstrated that a recombinant adenoviral vector Ad5-CEAB co-expressing CFP10,ESAT6,Ag85A and Ag85B of Mtb was able to induce robust antigen-specific immune responses in mice.In the present study,we examined immunological effects of Ad5-CEAB in the mice primed with BCG and boosted with a single dose of the virus via an intranasal route.Results demonstrated that this vaccination strategy could effectively induce strong antigen-specific mucosal and humoral immune responses.These immune responses were characterized with an increased productions of cytokines (IL-12 and IFN-γ),increased concentration of secretary IgA (sIgA) in bronchoalveolar lavage fluid (BALF) and serum IgG in mice in comparison with mice in BCG group.These data suggested that the regimen of BCG prime-single dose of Ad5-CEAB boosted strategy was novel for inducing antigen-specific immune responses in response to Mtb antigens in vivo,which warrants for further development of adenoviral-based vaccine against Mtb infection.

Construction of Recombinant Adenovirus Vector Expressing Mouse SPINK5 Gene and Its Curative Effect on Skin Lesions in Atopic Dermatitis Mice / 中国医科大学学报

Objective To construct a recombinant adenovirus vector expressing mouse SPINK5 gene,and observe its curative effect on the skin lesions in atopic dermatitis mice model. Methods By recombining DNA technology,the sequence of mouse SPINK5 gene was cloned into adeno?virus shuttle plasmid. Then it was transformed into HEK 293 cells with the adenoviral backbone plasmid to obtain the recombinant adenovirus. A mouse model of atopic dermatitis was established by system and local sensitization of Balb/c mice with ovalbumin . The effect of recombinant adeno?virus on the lesions of atopic dermatitis mice model was observed. Results The SPINK5 over?expressing adenovirus vector and atopic dermatitis mice model were successfully constructed. After 2 weeks of adenovirus?mediated SPINK5 gene intracutaneous injection,the redness and edema of lesions of AD model mice were obvious relieved. The pathological detection indicated that epidermal thickness and prickle cell layer ,inflammatory cell infiltration significant decreased accompanied with the model blank control. Conclusion The adenovirus?mediated SPINK5 gene had signifi?cant therapeutic effect to the atopic dermatitis mice model ,which provided a laboratory basis of application of SPINK5 gene product to therapy atopic dermatitis.

Effects of recombinant adenovirus Ad-miR-29b2c on HGC-27 cell proliferation and migration / 生物工程学报

We constructed recombinant adenoviruses expressing miR-29b2c (Ad-miR29b2c), and analyzed their effects on the proliferation and migration of HGC-27 and MGC-803 cells. miR-29b2c gene was amplified by PCR from genomic DNA and cloned into the pAdTrack-CMV vector to create the shuttle plasmid pAdT-29b2c. The recombinant plasmid was verified by restriction enzyme digestion and sequencing. The linearized shuttle vector was mixed with an adenoviral backbone plasmid (pAdEasy-1), followed by cotransformation into competent BJ5183 cells to generate the recombinant plasmid pAd-miR-29b2c. Finally, recombinant adenoviral vectors were generated by transfecting the recombinant plasmid into 293A packaging cell line. HGC-27 and MGC-803 cells were infected with the recombinant adenoviruses expressing pAd-miR-29b2c, then MTT and wound-healing assay were used to analyze the effects of pAd-miR-29b2c on the proliferation and migration of HGC-27 and MGC-803 cells. The miR-29b and miR-29c levels were significantly increased in HGC-27 cells after infected with pAd-miR-29b2c. MTT and wound-healing analysis also revealed a significant decrease in proliferation and migration of HGC-27 and MGC-803 cells compared to the control Ad-GFP-infected cells. Furthermore, western blotting results demonstrated that the protein expression level of δ-catenin was reduced in pAd-miR-29b2c transfected HGC-27 and MGC-803 cells. Taken together, the recombinant adenoviral vector was generated, and it can significantly inhibit the proliferation and migration of HGC-27 and MGC-803 cells.

Construction of BCR-ABL SH3-T79Y mutant recombinant adenovirus vectors and its promotion on apoptosis of K 562/G01 cells / 基础医学与临床

Objective To construct BCR-ABL SH3-T79Y mutant recombinant adenovirus vectors and investigate its effects on apoptosis of K562/G01 cells.Methods SH3-T79Y mutant was amplified by overlapping PCR with pMig210 as template and cloned into recombinant adenovirus vectors .After identifying , packaging and amplifying , the recombinant adenovirus vectors containing SH 3-T79Y mutant was collected .Recombinant adenovirus vectors were transferred into K562/G01 cells.Then transfection efficiency was determinated , changes of cell morphology were observed by Wright 's staining , cell apoptosis was evaluated by flow cytometry , BCR-ABL and CrkL phospho-rylation was detected by Western blot .Results The vectors were successfully constructed .Transfection efficiency was more than 80%after transferring into K562/G01 cells for 72 h;there was obvious apoptosis phenomenon , cell apoptosis significantly increased to 32.46% compared with the control groups ( P<0.05 ) , BCR-ABL and CrkL phosphorylation significantly decreased and so did the expression of BCR-ABL( P<0.05 ) .Conclusions Success-fully constructed the SH 3-T79 Y mutant recombinant adenovirus vectors and it may promote the apoptosis of K 562/G01 cells by inhibiting BCR-ABL and CrkL phosphorylation .

Constructing a recombinant adenovirus carrying human cytomegalovirus UL138 gene and evaluating the effects of UL138 gene on the functions of monocytes / 中华微生物学和免疫学杂志

Objective To construct a recombinant adenovirus carrying UL138 gene, which was re-lated to the latent infection of human cytomegalovirus, and to investigate the effects of UL138 gene on the functions of THP-1 mononuclear cells. Methods The recombinant adenovirus expressing the UL138 gene was packaged. The titer of the recombinant adenovirus was determined by calculating 50% tissue culture in-fective dose ( TCID50 ) . THP-1 mononuclear cells were infected with the recombinant adenovirus at different multiplicity of infection (MOI) and the optimal MOI was determined (100 PFU/cell) by observing the ex-pression of green fluorescent protein ( GFP ) . Changes in the expression of proinflammatory cytokines by THP-1 mononuclear cells that was induced by overexpressed UL138 were analyzed by quantitative PCR. The expression of chemokines and their receptors were measured by quantitative PCR array. Results The re-combinant adenovirus carrying the UL138 gene was successfully constructed with a titer of 1×1011 PFU/ml. The rate of THP-1 mononuclear cells that was infected with the recombinant adenovirus was 60% at the MOI of 1 ∶ 100. Results of the RT-PCR analysis and Western blot assay further confirmed that the recombinant adenovirus could infect THP-1 mononuclear cells successfully and the expression of UL138 protein increased gradually over time. The overexpressed UL138 in THP-1 mononuclear cells significantly inhibited the expres-sion of IL-18, IL-1β, IL-6, IL-8 and TNF-α as indicated by the results of quantitative PCR. Results ob-tained from the quantitative PCR array analysis showed that most of the chemokines and their receptors were downregulated in the transfected THP-1 mononuclear cells except for the chemokines of CCL17, CCL21, CCL2 and XCL2 and the receptors of CCR2, CXCR1, CXCR2, CXCR4 and CX3CR1 which were upregulat-ed. Conclusion We successfully constructed the recombinant adenovirus carrying UL138 gene which could be used to infect THP-1 mononuclear cells. Overexpressed UL138 in THP-1 mononuclear cells significantly affected the functions of THP-1 mononuclear cells.

Recombinant adenovirus containing hyper-interleukin-6 and hepatocyte growth factor enhances therapeutic efficacy on acute-on-chronic liver fai-lure in rats / 中国病理生理杂志

AIM:To determine the therapeutic efficacy of recombinant adenovirus containing hyper-interleu-kin-6 (HIL-6) and hepatocyte growth factor (HGF) (Ad-HGF-HIL-6) on acute-on-chronic liver failure (ACLF) in rats u-sing that of recombinant adenovirus HIL-6 or HGF ( Ad-HIL-6 or Ad-HGF) for comparison.METHODS:The rat model of ACLF was established and the model rats were randomly divided into model group, Ad0 group, Ad-HGF group, Ad-HIL-6 group and Ad-HGF-HIL-6 group.The sera and liver tissues were collected for biochemical, pathological and molecular bio-logical examinations.RESULTS:Compared with Ad0 group, prothrombin time ( PT) and the serum levels of alanine amin-otransferase (ALT), tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ) and high-mobility group box-1 (HMGB1) were markedly reduced in the ACLF rats treated with Ad-HGF, Ad-HIL-6 and Ad-HGF-HIL-6, and similarly, reduced he-patic damages and apoptotic activity, reduced Bax at protein level, and increased expression of Ki67 and Bcl-2 at protein levels were observed.Among them, treatment with Ad-HGF-HIL-6 showed the most significant therapeutic efficacy without obvious side effects.CONCLUSION:The therapeutic efficacy of Ad-HGF-HIL-6 is more potent than that of Ad-HGF or Ad-HIL-6 alone on ACLF rats with no significant side effects.

Construction and effect of recombinant adenovirus vector containing siRNA targeting Raf-1 on cardiomyocyte hypertrophy in neonatal rats / 中国比较医学杂志

Objective To construct an adenovirus vector expressing small interfering RNA ( siRNA) targeting to rat Raf-1 gene and identify its function in cardiomyocytes.Methods The siRNA containing DNA sequence targeting to Raf-1 and its negative control sequence were designed, synthesized, annealed and subcloned into adenoviral shuttle vector pAdTrack-CMV.The recombinant adenovirus vector pAd-siRaf-1 was obtained by homologous recombination with pAdTrack-siRaf-1 linearized by PmeI and pAdeasy-1 in bacteria BJ5183, then transfected into HEK293 cells to package the adenovirus.Cardiomyocytes were infected with the adenovirus pAd-siRaf-1, and the expressions of Raf-1 and NF-κB protein were detected by Western blotting.[ 3 H ]-leu incorporation was evaluated by scintillation.The surface area of cardiomyocytes was measured using a HJ2000 image analysis system.Results The adenovirus vectors were verified by enzyme digestion and DNA sequencing.Compared with the Ang II group, Raf-1 and NF-κB expression, the surface area and [ 3 H]-leu incorporation of cardiomyocytes were significantly decreased in cardiomyocytes infected with the adenovirus PAd-siRaf-1.Conclusions A recombinant adenovirus vector containing rat siRaf-1 gene is successfully constructed.It can effectively reduce Raf-1 and NF-κB expression and cardiomyocyte hypertrophy induced by Ang II.

Construction and identification of recombinant adenovirus vectors carrying the human transcription factor PU.1 gene / 医学研究生学报

Objective PU.1 plays a key role in innate immune function in the alveolar macrophage.This study was to con-struct and identify recombinant adenovirus vectors carrying the human transcription factor PU.1 gene. Methods The recombinant shut-tle plasmid was obtained from the PU.1 gene ( SPI1) and eukaryotic expression vector that digested by restriction enzymes and connected by T4 DNA ligase.The target fragment SPI1-IRES-EGFP was amplified by PCR.The product was cloned into the intermediate pDONR221 and then recombined with the adenovirus backbone plasmid pAd/CMV/V5-DEST to form a recombinant adenovirus vector. The recombinant adenovirus vector was linearized by PacI and then transfected into human embryonic kidney (HEK293) cells to obtain the recombinant adenovirus pAD-SPI1-IRES-EGFP, which was then propagated in HEK293 cells, filtered and purified to obtain high-con-centration adenoviruses.The adenovirus titer was determined by TCID 50 assay.The PU.1 gene expression in the HEK293 cells was con-firmed by fluorescence microscopy and real-time qPCR. Results PCR amplification, restriction digestion and sequencing analysis showed the recombinant adenovirus carried the correct PU.1 gene.The final virus titer, calculated by TCID 50, was 8 ×1011 IU/mL. Green fluorescence was observed under the fluorescence microscope. Real-time qPCR confirmed that the expression of PU.1 mRNA was increased by 2189.93 folds. Conclusion The recombinant adenovirus vector carrying the PU.1 gene was constructed and obtained successfully, which could contribute to further studies of the influence of PU.1 overex-pression on the innate defense against Aspergillus fumigatus.

Construction of recombinant adenovirus harboring fusion gene NT4p53(C22)Ant and its killing effect on HepG2 tumor cells / 西安交通大学学报(医学版)

ABSTRACT:Objective To construct a recombinant adenovirus vector harboring fusion gene NT4p53(C22)Ant and study its killing effect on HepG2 tumor cells.Methods Using molecular cloning technology,the rAVV-NT4p53(C22)Ant was produced by homologous recombination.Then we collected virus supernatant and measured its titer after it was amplified by PCR.The effect of this fusion gene on HepG2 tumor cells was evaluated by IHC, MTT assay,PI staining and flow cytometry.Results The recombinant adenovirus was successfully constructed. The p53 expression rate in rAAV-NT4p53(C22)Ant group was (44.88±2.45)%.MTT assay showed that rAAV-NT4p53(C22)Ant could strongly suppress the growth of HepG2 tumor cells.Flow cytometry showed that rAAV-NT4p53(C22)Ant could induce obvious apoptosis of HepG2 tumor cells.Conclusion The recombinant adenovirus vector encoding gene NT4p53(C22)Ant has been successfully constructed and expressed in this experiment,and it can inhibit proliferation and induce apoptosis of HepG2 tumor cells.

Construction of Adenovirus Vector Carrying Human HSP75 Gene and Expressing in C17.2 Neural Stem Cells / 中国康复理论与实践

@#Objective To construct recombinant adenovirus vector carrying the human HSP75 gene and detect its expression in C17.2 neural stem cells. Methods HSP75 gene was amplified from plasmid carrying the human HSP75 gene and inserted to the polyclonal site of adenovirus shuttle plasmid pHBAd-MCMV-GFP. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pHBAd-MCMV-HSP75-GFP and large adenovirus helper plasmid pHBAd-BHGloxΔE1,3 Cre in mediation of LipofiterTM. The recombinant adenovirus was obtained and the viral titer was examined using the method of TCID50. The transfection and expression of HSP75 was detected by fluorescence microscope, flow cytometer and Western blotting. Results Restriction digestion, sequencing analysis and PCR amplification revealed the successful construction of recombinant shuttle plasmid and recombinant adenovirus. The titer of recombinant adenovirus was 1.0×1010 PFU/ml. Western blotting indicated HSP75 gene could be expressed effectively in neural stem cells after transfection. Conclusion The recombinant adenovirus vector carrying HSP75 gene was successfully constructed and can be expressed after transfected in C17.2 neural stem cells.