Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus / 生物医学与环境科学（英文）

OBJECTIVE@#To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene.@*METHODS@#A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay.@*RESULTS@#The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses.@*CONCLUSION@#A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Research progress of intravitreal Dexamethasone implant in the treatment of non-infectious uveitic macular edema / 国际眼科杂志(Guoji Yanke Zazhi)

@#Macular edema(ME)is a typical non-specific complication of uveitis, one of the common causes of visual impairment in patients with non-infectious uveitis(NIU). The treatment of uveitis related ME is still challenging in clinic. Various agents, such as corticosteroids, anti-vascular endothelial growth factors, and immune-modulators, have been used for combating uveitis related ME. However, there is not enough evidence to support the efficacy of any of these agents. Intravitreal dexamethasone implant(IDI, Ozurdex©; Allergan Inc, Irvine, CA)is a widely administered corticosteroid for the long-term management of uveitic ME in certain cases. Recent studies have demonstrated that IDI effectively improves uveitis related ME, and this effect could be sustained for at least six months with close monitoring and retreatment, as needed. Currently, we reviewed major clinical studies about IDI in eyes with NIU and briefly overviewed their results.

Analysis of Common Herbicides in Blood by UPLC-HRMS / 法医学杂志

Objective To develop a method to screen and quantify 10 common herbicides (paraquat, diquat, glyphosate, glufosinate, cyanazine, atrazine, metazachlor, acetochlor, chlorsulfuron, and metsulfuron) in blood.Methods With acetonitrile-water solution[V (acetonitrile) ∶V (water) =3∶1]as protein precipitant, 10 common herbicides in blood were detected using ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS).Results All the 10 herbicides had good linearity in their linear range (coefficient of determination R2≥0.993), with the recovery rates 67.4％-111.9％, the relative standard deviations 1.5％-10.8％, the accuracies 85.1％-106.1％, intra-day precisions 2.7％-13.5％, and inter-day precisions 3.3％-13.3％.Conclusion This method is easy to operate with high recovery rates.It enables rapid and accurate qualitative screening and quantitative analysis of various herbicides in blood simultaneously.

Treatment of calcaneal fracture by closed reduction and minimally invasive plate fixation assisted with bidirectional distractor distraction / 中国骨伤

<p><b>OBJECTIVE</b>To investigate clinical effects of calcaneal fracture with closed reduction and minimally invasive plate fixation assisted with bidirectional distractor distraction.</p><p><b>METHODS</b>From September 2015 to October 2016, 11 male patients(13 feet) with calcaneal fractures treated with bidirectional distractor distraction assisted with minimally invasive plate fixation were retrospectively studied. They were aged from 24 to 57 years old with an average of 36.4 years old;8 feet were type IIand 5 feet were type III according to Sanders classification. Postoperative incision, fracture healing, Böhler angle, Gissane angle were observed and Maryland scoring system was used to evaluate clinical effects.</p><p><b>RESULTS</b>All fractures healed well without incision inflammation and incision disunion. All patients were followed up from 12 to 15 months with an average of 13.5 months. Böhler angle were improved from (9.6±7.3)° before operation to (20.2±4.6) ° at 1 year after operation, and had statistical meaning; Gissane angle increased from (92.7 ±8.5)° before operation to (121.7 ±7.6) ° at 1 year after operation. Maryland score at 1 year after operation was 88.79±8.25, and 11 feet got excellent results and 2 feet moderate.</p><p><b>CONCLUSIONS</b>Bidirectional distractor distraction assisted with minimally invasive plate fixation could effectively fix calcaneal fractures, reduce postoperative complications, and get satisfied results of postoperative images and functional recovery. It is one of effective methods for treating Sanders II and III calcaneal fractures.</p>

Neutralizing Antibody Titer Test of Ebola Recombinant Protein Vaccine and Gene Vector Vaccine pVR-GP-FC / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice.</p><p><b>METHODS</b>A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test.</p><p><b>RESULTS</b>Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1:105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies.</p><p><b>CONCLUSION</b>An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.</p>

A New Method for Blood NT-proBNP Determination Based on a Near-infrared Point of Care Testing Device with High Sensitivity and Wide Scope / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop a rapid, highly sensitive, and quantitative method for the detection of NT-proBNP levels based on a near-infrared point-of-care diagnostic (POCT) device with wide scope.</p><p><b>METHODS</b>The lateral flow assay (LFA) strip of NT-proBNP was first prepared to achieve rapid detection. Then, the antibody pairs for NT-proBNP were screened and labeled with the near-infrared fluorescent dye Dylight-800. The capture antibody was fixed on a nitrocellulose membrane by a scribing device. Serial dilutions of serum samples were prepared using NT-proBNP-free serum series. The prepared test strips, combined with a near-infrared POCT device, were validated by known concentrations of clinical samples. The POCT device gave the output of the ratio of the intensity of the fluorescence signal of the detection line to that of the quality control line. The relationship between the ratio value and the concentration of the specimen was plotted as a work curve. The results of 62 clinical specimens obtained from our method were compared in parallel with those obtained from the Roche E411 kit.</p><p><b>RESULTS</b>Based on the log-log plot, the new method demonstrated that there was a good linear relationship between the ratio value and NT-proBNP concentrations ranging from 20 pg/mL to 10 ng/mL. The results of the 62 clinical specimens measured by our method showed a good linear correlation with those measured by the Roche E411 kit.</p><p><b>CONCLUSION</b>The new LFA detection method of NT-proBNP levels based on the near-infrared POCT device was rapid and highly sensitive with wide scope and was thus suitable for rapid and early clinical diagnosis of cardiac impairment.</p>

Visual detection of HIV-1 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of HIV-1.</p><p><b>METHODS</b>RT-LAMP primers were designed according to conservative sequences of HIV-1 gag gene, and their sensitivity and specificity were evaluated by the established RT-LAMP protocol with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The performance of RT-LAMP on clinical samples was compared with real-time reverse transcription PCR(qRT-PCR).</p><p><b>RESULTS</b>The RT-LAMP assay showed a high specificity, and its detection limit was 1000 copies RNA per tube. The sensitivity and specificity of this method using 43 clinical samples were 94.6% and 100%, respectively,in comparison with those of qRT-PCR.</p><p><b>CONCLUSION</b>RT-LAMP assay using hydroxynaphthol blue dye does not need expensive instruments, and offer an alternative for the rapid detection of HIV-1 with the potential to be applied in field diagnosis.</p>

A novel SELEX method for screening of HIV-1 P24 antigen / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To screen aptamers that can bind P24 antigen tightly and specificly, and verify its specificity and affinity.</p><p><b>METHODS</b>Polycarbonate PCR plate was coated with P24 antigen and SELEX technology was used to screen aptamer on the PCR plate. The primary and secondary structure of these aptamers was analyzed by software. Through HRP labeled streptavidin and biotin labeled aptamers, the affinity and specificity of obtained aptamers were verified by ELISA.</p><p><b>RESULTS</b>The polycarbonate PCR plate could be coated with P24 antigen. Electrophoretic analysis showed the aptamers had been enriched. Sequence aligment analysis showed that these aptamers have consensus sequence and their apatial structure was multiple; ELISA verified that aptamers had high affinity with P24 antigen.</p><p><b>CONCLUSION</b>A simple method was established for screening aptamers that can bind HIV-1 P24 antigen specificly and tightly.</p>

Connective tissue growth factor induced differentiation of placenta mesenchymal stem cell into dermal fibroblast / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the possibility of placenta mesenchymal stem cells (PMSCs) differentiation into dermal fibroblast, and the potency of PMSCs used in cutaneous wound healing and stored as seed cells.</p><p><b>METHODS</b>Enzyme digestion method was used to obtain PMSCs, and PMSCs were amplified after culture in vitro. Flow cytometry assay, osteogenic and adipogenic differentiation were done for MSCs identification. The induction medium composed of DMEM/F12 + 50 microg/ml VC + 100 ng/ml connective tissue growth factor (CTGF) was added into the 24-well plate for 16 days induction period. Pictures were taken to record morphologic change. Immunofluorescence tests were performed to detect Vimentin, FSP-1, collagen I , collagen III, desmin and laminin expression before and after induction. At the same time osteogenic and adipogenic differentiation were used to assay the differentiation ability change after induction. The induced dermal fibroblasts were frozen in liquid nitrogen and recovery and trypan blue was used to detect cell viability.</p><p><b>RESULTS</b>After CTGF induction, PMSCs got obvious fibroblasts morphology, the protein level of Vimentin, FSP-1, collagen I, collagen III and Laminin increased, PMSCs started to express Desmin, the dermal fibroblasts specific proteins, and osteogenic and adipogenic differentiation ability was diminished. PMSCs were successfully induced into dermal fibroblasts, and these induced cells could get a high cell viability ( more than 90% ) after recovery.</p><p><b>CONCLUSIONS</b>PMSCs could be induced into dermal fibroblasts by CTGF in vitro. PMSCs have the potential application in skin wound healing, and can be used as seed cells of dermal fibroblasts.</p>

High-risk human papilloma virus DNA detection kit (cervista HPV HR) should be highly validated clinically in cervical cancer screening programs / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The aim of this study is to evaluate the clinical significance of High-risk Human Papilloma virus DNA Detection Kit (Cervista HPV HR) designed to the utilized in cervical cancer screening programs.</p><p><b>METHODS</b>The investigation for Cervista HPV HR test is designed to detect 437 residual liquid-based cytology specimens collected during routine liquid-based Pap tests at standard care vistis and to identify the presence of HR HPV. We compared Cervista HPV HR Test against standard PCR, in order to examine the performance of Cervista HPV HR Test in populations with cervical intraepithelial neoplasia grade 2+ (CIN 2, CIN 3 and Cancer, CIN 2+), and the capabilities of A5/A6, A7, A9 oligonucleotides of Cervista for predicting CIN2+.</p><p><b>RESULTS</b>The accuracy of Cervista compared to PCR with bi-directional sequencing was 88.26%. The positive percent of Cervista HPV HR Test and PCR were 38.96% and 29.08%, respectively. The sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of Cervista HPV HR Test for the detection of CIN2+ were 98.46%, 58.49%, 99.54% and 29.68%, respectively. The A9 oligonucleotides positivity percent was significantly higher in CIN2 + (odds ratio: 24.037, 95% CI: 10.086 - 57.283).</p><p><b>CONCLUSION</b>The Cervista HPV HR test can be clinically used for detecting HR HPV types during routine cervical cancer screening. A9 oligonucleotides were also strongly associated with CIN2+ diagnosis, which is improtant in cervical cancer screening for triage to colposcopy.</p>

Gemin3 inhibits cell apoptosis through suppression of p53 expression / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the role of Gemin3 in cell proliferation and its regulation pathway.</p><p><b>METHODS</b>Using co-immunoprecipitation and GST pull-down assay to determine the domain of Gemin3 and p53 binding and interaction in vitro and in vivo. To check the effect of Gemin3 on p53 by luciferase reporter assay. Stable Gemin3 knock-down cell lines were generated by lentivirus-delivered small hairpin RNA then puromycin selection. Real-time PCR was used to confirm the effect of Gemin3 level on p53 and its downstream genes, and flow cytometry was used to analyze the effect of Gemin3 on apoptosis.</p><p><b>RESULTS</b>The C-terminal of Gemin3 interacted with the DNA binding domain of p53. The p53 reporter gene, PA3M-p53 and increasing amount of GFP-Gemin3 were co-transfected into Saos-2 cells. Gemin3 repressed p53 expression at transcription level. Real-time PCR indicated that the expression of p53, p21 and Bax in Gemin3 knock-down cells was higher than that in the control cells. Western blot showed Gemin3 knock-down cells had a higher p53 espression. Flow cytometric assay showed that knock-down Gemin3 expression led to an increased cell apoptosis.</p><p><b>CONCLUSION</b>Gemin3 binds with p53 forming a complex and plays an anti-apoptotic role by repressing the p53 expression.</p>

Psoriasis complicated with venous thromboembolism: report of two cases and a literature review / 中华医学杂志(英文版)

Cases of psoriasis complicated with venous thromboembolism are rarely reported. Here, we report two cases and review the current literature on the subject. Two patients with long-standing severe psoriasis presented with chest pain, shortness of breath and breathing difficulties. The patients were diagnosed using lung ventilation-perfusion scans or computed tomographic pulmonary angiography. Anticoagulation or thrombolytic therapy was initiated, and long-term continuous anticoagulation with warfarin prevented any recurrences.

Expression and purification of avian influenza virus H5N1 NP protein, and screening interaction proteins of host cells in vitro / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express and purify H5N1 influenza virus (A/Anhui/1/2005) NP in prokaryotic system and to explore the NP-interacting proteins of human bronchial epithelial cells BEAS-2B in vitro.</p><p><b>METHODS</b>The full length H5N1 NP gene fragment was amplified by PCR, inserted into prokaryotic expression vector (pET30a) to generate NP expression plasmid pET30a-NP. After transforming pET30a-NP into E. coli (BL21), the expression of soluble NP protein was induced by IPTG. The expressed NP protein was purified by two steps with metal chelation chromatography and ion exchange chromatography. Then the total proteins of BEAS-2B cells was extracted for screening the components which have protein-protein interaction with purified NP by pull-down and LC-MS/MS methods.</p><p><b>RESULTS</b>The expression of H5N1 NP protein could be induced by IPTG in bacterial system using expression plasmid pET30a-NP. The soluble NP was purified. Twenty proteins were found by pull-down and LC-MS/MS, the further experiments may be needed to prove protein-protein interaction between them.</p><p><b>CONCLUSION</b>The soluble H5N1 NP fusion protein with high purity was obtained and twenty proteins were found which could interact with it by pull-down and LC-MS/MS.</p>

Study on the knowledge, behavior and attitude of influenza A (H1N1) among residents in Shuangqiao District, Chengde city / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To understand the the residents' knowledge, behavior and attitude of influenza A (H1N1) in Shuangqiao District of Chengde city, and provide the basis for making proper strategies of health education, prevention and control on influenza A (H1N1).</p><p><b>METHODS</b>211 residents from 1 community and 1 village of Shuangqiao District were selected to participate the questionnaire interview with multi-stage clustering sampling.</p><p><b>RESULTS</b>97.6% of the interviewed had received some kind of information on influenza A (H1N1); Total awareness rate of influenza A (H1N1) knowledge was 58.5%, which increased with the level of education and varied among diverse occupations; 48.2% of respondents conceded that their lives was affected by the influenza A ( H1N1) in some degree, and 9% of selected residents believed that there would be a severe pandemicity in this winter, while 7% of respondents didn't consider any form of preventive methods in the future; 78% of respondents expressed their wishes to be vaccinated, but 11.0% of respondents refused to received inoculation explicitly; on the issue of assessment on services provided by governments and health facilities, 93% of respondents expressed their satisfaction hierarchically. Conclusion Residents in Shuangqiao District lacked of comprehensive knowledge of influenza A (H1N1), and some specific health education should be carried out.</p>

Expression and purification of EBV-LMP2 protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain a second Epstein-Barr virus membrane protein (LMP2) in insect cells.</p><p><b>METHODS</b>The full length EBV-LMP2 gene was inserted into baculovirus expression transfer vector pFastBac HT B to obtain the recombinant baculoviruses Bac-LMP2. And generation of recombinant baculoviruses was followed by transfection of the recombinant Bac-LMP2 into insect cells, then the recombinant LMP2 protein was recognized by SDS-PAGE and western blot. The expressed LMP2 protein was purified by one step with Ni-NTA metal chelation chromatography.</p><p><b>RESULTS</b>The expressed LMP2 protein was confirmed by SDS-PAGE and western blot. The purity of purified LMP2 protein is up to 86% by HPLC analysis.</p><p><b>CONCLUSION</b>The EBV-LMP2 was expressed in insect cells, and the purified LMP2 protein was obtained.</p>

The study of multiple RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a multiplex RT-PCR-based reverse dot blot hybridization technique to detect influenza viruses.</p><p><b>METHODS</b>Obtain the HA nucleotide sequences of seasonal influenza H1N1, seasonal influenza H3N2, influenza H1N1 and human avian influenza H5N1 from GenBank. Design primers in conservative district and probes t in high variable region respectively, after analyzing the HA nucleotide sequences of influenza virus through the Vector NTI 9.0. Establish and optimize multiple RT-PCR system by comparing amplification efficiency and specificity at different primer concentrations. Establish the reverse dot hybridization system after optimizing the concentration of probes. To compare the sensitivity and specificity of this technique and the general RT-PCR Method through extracting the viral RNA of the mentioned influenza virus which are to be the reference substance.</p><p><b>RESULTS</b>Successfully establish a multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses. This technique is 100-1000 times more sensitive than gel electrophoresis method, and it has a good specificity.</p><p><b>CONCLUSION</b>Successfully established multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses.</p>

Study on immunogenicity of a recombinant adenovirus vaccine containing neuraminidase gene of H5N1 influenza virus (A/Anhui/1/2005) in mice / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate immunity of a recombinant adenovirus vaccine (rAdV) containing codon-modified neuraminidase (Mod. NA) gene of H5N1 influenza virus in BALB/c mice and to screen for appropriate dose.</p><p><b>METHODS</b>BALB/c mice were immunized with the rAdV-Mod.NA vaccine intramuscularly twice (double injection at 0 and 4th week) in three groups, low dosage (10(5) TCID50 per dose), medium dosage (10(7) TCID50 per dose) and high dosage (10(9) TCID50 per dose). The effect of humoral and cell-mediated immunity were analysed at 5th week.</p><p><b>RESULTS</b>(1) The rAdV-Mod.NA vaccine could elicit both humoral and cell-mediated robust NA specific immunity in mice by neuraminidase inhibitor assay and IFN-gamma ELISpot assay; (2) 10(7) TCID50 per dose was the appropriate dose; (3) Peptide NA(109-124): CRTFFLTQGALLNDKH and peptide NA(182-199): AVAVLKYNGIITDTIKSW were the dominant epitopes for neuraminidase-immunized BALB/c mice, which was screened out from the whole length of neuraminidase of an H5N1 virus, A/Anhui/1l/2005.</p><p><b>CONCLUSION</b>The recombinant adenovirus NA could induce specific humoral and cellular immune responses in BALB/c after immunization, which suggest rAdV-Mod.NA vaccine was a potential vaccine candidate against H5N1 influenza and worthy of further investigation.</p>

Expression and purification of HIV-1 subtype C Gp120, and its antibodies preparation / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare HIV-1 subtype C Gp120 protein and to produce its polyclonal antibodies.</p><p><b>METHODS</b>A C-terminal fragment of gp120 gene was amplified by PCR from a plasmid expressing full-length HIV-1 subtype C gp160 gene. The length of the subtype C gp120 fragment was 612 nt and it encodes 204 amino acid residues. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-30a) and recombinant pET-30a-gp120 was expressed in Escherichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine (His6) tag at the C-terminus for convenient purification. To produce subtype C Gp120-specific polyclonal antibodies, New-Zealand rabbit was immunized with the purified Gp120 protein. Serum samples were tested by enzyme-linked immunosorbent assays (ELISA) to determine the level of antibodies. And Western blotting was used to further verify whether the polyclonal antibodies could specifically recognize subtype C Gp160 protein expressed in mammalian cells.</p><p><b>RESULTS</b>HIV-1 subtype C Gp120 protein was successfully acquired and the titer of its polyclonal antibodies was 1:204 800. The polyclonal antibodies efficiently recognized Subtype C Gp160 protein expressed in COS-1 cells.</p><p><b>CONCLUSION</b>HIV-1 subtype C Gp120 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.</p>

Study on the immunogenicity of adeno-vector vaccine against H5N1 influenza A virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct adenovirus vector vaccine against H5N1 influenza virus and study on the immunogenicity.</p><p><b>METHODS</b>In this study, we amplified hemagglutinin (HA) gene sequence of H5N1 influenza virus (A/Anhui/1/2005), then constructed an adenovirus vector vaccine (Adv-HA), followed by tests in BALB/c mice for the immunogenicity with the vaccine and immunization strategies.</p><p><b>RESULTS</b>The recombinate Adv-HA vaccine could effectively induce both humoral and cellular immunity against human H5N1 influenza virus.</p><p><b>CONCLUSION</b>The Adv-HA vaccination against H5N1 influenza is a potential strategy and worthy of further investigation.</p>

Two recombinant adenovirus vaccine candidates containing neuraminidase Gene of H5N1 influenza virus (A/Anhui/1/2005) elicited effective cell-mediated immunity in mice / 病毒学报

The aim of this study is to develop the recombinant adenovirus vaccine (rAdV) candidates containing neuraminidase (NA) gene of H5N1 influenza virus and test in BALB/c mice the effect of cell-mediated immunity. In this study, two kind of NA gene (WtNA gene, the wild type; Mod. NA gene, the codon-modified type) derived from H5N1 influenza virus (A/Anhui/1/2005) were cloned and inserted respectively into plasmid of adenovirus vector, then the rAdV vaccines candidates (rAdV-WtNA and rAdV-Mod. NA) were developed and purified, followed by immunization intramuscularly (10(9) TCID50 per dose, double injection at 0 and 4th week) in BALB/c mice, the effect of cell-mediated immunity were analysed at 5th week. Results indicated that: (i) NA protein expression was detected in two rAdV vaccines candidates by Western blotting; (ii) the rAdV-Mod. NA vaccine could elicit more robust NA specific cell-mediated immunity in mice than that of rAdV-WtNA vaccine (P = 0. 016) by IFN-gamma ELIspot assay. These findings suggested rAdV-Mod. NA vaccine was a potential vaccine candidate against H5N1 influenza and worthy of further investigation.