Dengue virus envelope domain III immunization elicits predominantly cross-reactive, poorly neutralizing antibodies localized to the AB loop: implications for dengue vaccine design.

Dengue virus (DENV) is a mosquito-borne virus that causes severe health problems. An effective tetravalent dengue vaccine candidate that can provide life-long protection simultaneously against all four DENV serotypes is highly anticipated. A better understanding of the antibody response to DENV envelope protein domain III (EDIII) may offer insights into vaccine development. Here, we identified 25 DENV cross-reactive mAbs from immunization with Pichia pastoris-expressed EDIII of a single or all four serotype(s) using a prime-boost protocol, and through pepscan analysis found that 60 % of them (15/25) specifically recognized the same highly conserved linear epitope aa 309-320 of EDIII. All 15 complex-reactive mAbs exhibited significant cross-reactivity with recombinant EDIII from all DENV serotypes and also with C6/36 cells infected with DENV-1, -2, -3 and -4. However, neutralization assays indicated that the majority of these 15 mAbs were either moderately or weakly neutralizing. Through further epitope mapping by yeast surface display, two residues in the AB loop, Q316 and H317, were discovered to be critical. Three-dimensional modelling analysis suggests that this epitope is surface exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially on the surface of the mature virion. It is concluded that EDIII as an immunogen may elicit cross-reactive mAbs toward an epitope that is not exposed on the virion surface, therefore contributing inefficiently to the mAbs neutralization potency. Therefore, the prime-boost strategy of EDIII from a single serotype or four serotypes mainly elicited a poorly neutralizing, cross-reactive antibody response to the conserved AB loop of EDIII.

Evaluation and analysis of dengue virus enhancing and neutralizing activities using simple high-throughput assays.

The risk of antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is a major obstacle for the development of dengue vaccine candidates. Here, we described a novel approach for assessment of ADE by measuring DENV nonstructural protein 1 (NS1) production in culture supernatants with Fcγ receptor-expressing K562 cells in ELISA format (ELISA-ADE). Enhancing activities quantified by measurement of kinetics of NS1 production were in a good agreement with the results of the virus titration assay. In conjunction with the previously established enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well format, the observable dose-response profiles of enhancing and neutralizing activities against all four DENV serotypes were produced with two flaviviral envelope cross-reactive monoclonal antibodies and four primary DENV-1-infected human sera. The simple high-throughput ELISA-ADE assay offers advantages for quantitative measurement of infection enhancement that can potentially be applied to large-scale seroepidemiological studies of DENV infection and vaccination.

[Establishment and preliminary application of dengue virus envelope domain III IgG antibody capture enzyme-linked immuno-absorbent assay].

OBJECTIVE: To establish a highly sensitive and specific assay to detect dengue virus (DENV) envelope protein domain III (EDIII) IgG antibody, and to explore its value in the diagnosis and seroepidemiological survey of dengue. METHODS: The DENV EDIII IgG antibody capture ELISA was developed using the recombinant full-length DENV EDIII, which was prepared by Pichia yeast expression system as the capture antigen. The serum samples were collected from the same group of 35 DENV-1 patients of primary infection during disease period in 2006 and their follow-up phase in 2010; and the sensitivity of the assay was compared to that of the commercial Panbio DENV IgG ELISA. RESULTS: The sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from disease period and follow-up phase was 87% (20/23) and 94% (33/35), respectively; whereas the sensitivity of Panbio DENV IgG ELISA was 71% (25/35) and 0, respectively. The sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from both periods was similar, without statistical significance (χ(2) = 0.946, P = 0.331). For serum samples from disease period, the sensitivity of DENV EDIII IgG ELISA was comparable with that of Panbio DENV IgG ELISA (χ(2) = 1.924, P = 0.165). However, DENV EDIII IgG ELISA demonstrated a significantly higher sensitivity than Panbio DENV IgG ELISA in detecting the serum samples from follow-up phase (χ(2) = 62.432, P = 0.000). CONCLUSION: DENV EDIII IgG capture ELISA is highly sensitive in detecting IgG in the serum samples from either disease period or follow-up phase. This method might be a promising alternative for diagnosis and seroepidemiologic survey of dengue.

[To evaluate the neutralizing abilities of anti-dengue virus antibodies with nonstructural protein 1 antigen capture enzyme-linked immunosorbent assay].

OBJECTIVE: To produce neutralizing antibodies against envelope protein domain III (EDIII) of dengue virus serotype I (DENV-1) and evaluate the nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA) for identification of antibody neutralizing abilities. METHODS: Five BALB/c mice and one New Zealand Rabbit were immunized with recombinant EDIII protein of DENV-1 for the production of hybridomas and hyperimmune sera. Indirect ELISA, immunofluorescence assay (IFA) and Western Blot analyses were applied to identify specificity of antibodies. Comparing to plaque reduction neutralization test (PRNT), the new established DENV-1 specific NS1 antigen capture ELISA was used for detecting the neutralizing abilities of these antibody. RESULTS: Four strains of monoclonal antibodies (mAbs) named 1A1, 1B3, 3D3 and 9D6 and one hyperimmune serum of rabbit were obtained, all of which were approved to have neutralizing abilities to DENV-1 with the PRNT titer of 1:1024, 1:512, 1:256, 1:4096 and 1:4096. MAb 3D3 with the lowest neutralization titer in PRNT had not shown neutralizing ability to DENV-1 in NS1 antigen capture ELISA, while MAbs 1A1, 1B3 and 9D6 and the rabbit hyperimmune serum could protect the C6/36 from being infected by DENV-1 with the neutralization titer of 1:32, 1:32, 1:128 and 1:128 in this assay. CONCLUSION: NS1 antigen capture ELISA could be used to identify antibody neutralizing abilities to DENV, it was a faster and more convenient way to screen antibodies with high neutralization titer and might also be used as one of the methods to evaluate the effects of vaccines.

Structural and Functional Characterization of a Cross-Reactive Dengue Virus Neutralizing Antibody that Recognizes a Cryptic Epitope.

Understanding the molecular basis of the neutralizing antibody response to dengue virus (DENV) is an essential component in the design and development of effective vaccines and immunotherapeutics. Here we present the structure of a cross-reactive, neutralizing antibody, 3E31, in complex with domain III (DIII) of the DENV envelope (E) protein and reveal a conserved, temperature-sensitive, cryptic epitope on DIII that is not available in any of the known conformations of E on the dengue virion. We observed that 3E31 inhibits E-mediated membrane fusion, suggesting that the antibody is able to neutralize virus through binding an as-yet uncharacterized intermediate conformation of DENV E and sterically block trimer formation. Finally, we show that, unlike cross-reactive fusion peptide-specific antibodies, 3E31 does not promote antibody-dependent enhancement of infection at sub-neutralizing concentrations. Our results highlight the 3E31 epitope on the E protein DIII as a promising target for immunotherapeutics or vaccine design.

A patient with asymptomatic severe acute respiratory syndrome (SARS) and antigenemia from the 2003-2004 community outbreak of SARS in Guangzhou, China.

An asymptomatic case of severe acute respiratory syndrome (SARS) occurred early in 2004, during a community outbreak of SARS in Guangzhou, China. This was the first time that a case of asymptomatic SARS was noted in an individual with antigenemia and seroconversion. The asymptomatic case patient and the second index case patient with SARS in the 2003-2004 outbreak both worked in the same restaurant, where they served palm civets, which were found to carry SARS-associated coronaviruses. Epidemiological information and laboratory findings suggested that the findings for the patient with asymptomatic infection, together with the findings from previously reported serological analyses of handlers of wild animals and the 4 index case patients from the 2004 community outbreak, reflected a likely intermediate phase of animal-to-human transmission of infection, rather than a case of human-to-human transmission. This intermediate phase may be a critical stage for virus evolution and disease prevention.

Biosorption of uranium by lake-harvested biomass from a cyanobacterium bloom.

The aim of this work was to study some basic aspects of uranium biosorption by powdered biomass of lake-harvested cyanobacterium water-bloom, which consisted predominantly of Microcystis aeruginosa. The optimum pH for uranium biosorption was between 4.0 and 8.0. The batch sorption reached the equilibrium within 1 h. The isotherm fitted the Freundlich model well. Although the Langmuir model fitted the experiment data well at pH 3.0, 5.0 and 7.0, it did not fit at pH 9.0 and 11.0 at all. This implies that different biosorption mechanisms may be involved at different pH values. 0.1 N HCl was effective in uranium desorption. The results indicated that the naturally abundant biomass of otherwise nuisance cyanobacterium bloom exhibited good potential for application in removal of uranium from aqueous solution.

[Association between gene mutation of cytochrome P450 1A1 in exon 7 A4889G locus and susceptibility to endometriosis].

OBJECTIVE: To assess the possible association between gene mutation of cytochrome P450 1A1(CYP1A1) in exon 7 A4889G locus and the susceptibility to endometriosis (EM). METHODS: Allele specific-polymerase chain reaction method was used to analyze gene mutation in exon 7 A4889G locus of CYP1A1 in 76 patients with endometriosis and 80 healthy controls. RESULTS: The frequency of allele G on A4889G locus of CYP1A1 gene showed a significant difference between the study cohort and the control group (Chi2=7.498, P<0.01), with an odds ratio of 1.957. Statistically significant difference in the frequencies of genotypes AA, AG and GG was observed between the two groups (Chi2=6.915, P<0.05). Individuals with homozygotes for G allele were at higher risk of suffering from EM when compared against those with homozygotes for A allele, the odds ratio being 3.437 (Chi2=5.430, P<0.05). CONCLUSION: The above results suggest that gene mutation of CYP1A1 in exon 7 A4889G locus might be a genetic susceptible factor of endometriosis. The mutation allele of CYP1A1 gene appears to increase the risk of endometriosis.

[Association between glutathione S-transferase M1 gene deletion and genetic susceptibility to endometriosis].

OBJECTIVE: To evaluate the possible association of the glutathione S-transferase M1 (GSTM1) gene polymorphism with the susceptibility to endometriosis in women of Han nationality in Guangdong Province. METHODS: Polymerase chain reaction was used to identify the GSTM1 genotypes in 76 patients with endometriosis and 80 controls (surgical patients for gynecological problems other than endometriosis). RESULTS: The frequencies of the GSTM1 null genotypes in patients with endometriosis and controls were 65.8% and 46.3%, respectively, showing a significant difference between the endometriotic cohort and the control group (X(2)=6.03, P < 0.05). Individuals with GSTM1 null genotype were exposed to risks for endometriosis 2.24 times that of subjects without these genotypes OR=2.24, 95% CI=1.17-4.27 . CONCLUSION: GSTM1 gene deletion might bea risk factor for endometriosis in women of Han nationality who are native residents in Guangdong Province.

[Susceptibility to endometriosis in women of Han Nationality in Guangdong Province associated with Msp I polymorphisms of cytochrome P450 1A1 gene].

OBJECTIVE: To assess the possible association of the Msp I polymorphisms of cytochrome P4501A1(CYP1A1) gene with the susceptibility to endometriosis in women of Han Nationality in Guangdong Province. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was employed to analyze the 3 genotypes m1m1, m1m2 and m2m2 in 3'-flanking region of CYP1A1 in 76 patients with endometriosis and 80 healthy controls. RESULTS: The frequencies of genotypes m1m1, m1m2 and m2m2 were 30.3 %, 50.0 % and 19.7 % in patients with endometriosis while 42.5 %, 45.0 % and 12.5 % in the controls, respectively, showing no statistically significant difference in the frequencies of the three genotypes between the 2 groups. The frequencies of two alleles were of no significant difference between the patients and controls, either. CONCLUSION: Msp I polymorphisms of cytochrome P4501A1 in itself might not be associated with the susceptibility to endometriosis in women of Han Nationality in Guangdong Province.

[Preparation and identification of monoclonal antibodies against Penicillium marneffei].

OBJECTIVE: To prepare and identify monoclonal antibodies (mAbs) against Penicillium marneffei. METHODS: Recombinant mannoprotein1 (MP1) of Penicillium marneffei was used to immunize BALB/c mice, and anti-MP1 mAbs were obtained by means of hybridoma. Screening and identification of the mAbs were subsequently performed with indirect enzyme-linked immunosorbent assay and Western blotting, respectively. RESULTS: Four hybridomas producing antibodies against Penicillium marneffei were obtained, and the IgG isotypes of the 4 mAbs were identified as IgG1 with affinity constants (K) of 8.2x10(-9), 4.7x10(-9), 6.5x10(-9) and 2.7x10(-9), respectively. Western blotting demonstrated specific recognition of Aspergillus fumigatus MP1 by the obtained mAbs. CONCLUSION: The 4 hybridomas producing anti-MP1 mAbs with high specificity and affinity can be of significant value in the diagnosis of Penicilliosis marneffei infections.

Preparation and characterization of monoclonal antibodies against S1 domain at N-terminal residues 249 to 667 of SARS-associated coronavirus S1 protein.

OBJECTIVE: To prepare and characterize monoclonal antibodies (mAbs) against S1 protein of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). METHODS: 6-His-tagged recombinant fragment at N-terminal residues 249 to 667 of SARS-CoV S1 protein including S-protein receptor-binding domain was expressed in E.coli. The immunogenicity of this S1 domain was identified and used to immunize BALB/c mice for the production of hybridomas. The identification of the mAbs against this S1 domain was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA) and Western blotting, respectively. RESULTS: Three hybridomas producing mAbs specific to the S1 domain was obtained, with a relative molecular mass of 48,500. None of the 3 mAbs were reactive with human coronaviruses 229E and OC43. Two of the mAbs were IgG2a isotype, and the other was IgG1. CONCLUSIONS: This is the first report of mAbs produced against S-protein receptor-binding domain of SARS-CoV. The 3 S1-specific mAbs may be useful for further study of the function of the S protein and for diagnosis of SARS-CoV infection.

Rapid and efficient preparation of monoclonal antibodies against SARS-associated coronavirus nucleocapsid protein by immunizing mice.

OBJECTIVE: To develop a rapid and efficient method for preparing monoclonal antibodies (mAb) against SARS-associated coronavirus (SARS-Cov) nucleocapsid (N) protein. METHODS: BALB/c mice were injected with the recombinant N protein of SARS-Cov into the foot-pads for the immunization, and the popliteal lymph nodes were isolated 15 d later for mAb-producing hybridomas, from which the mAbs against the N protein of SARS-Cov were screened. The identification of the mAb against the N protein of SARS-Cov was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA), and Western immunoblotting. RESULTS: Four strains of hybridomas were obtained that produced the mAb specific to the N protein without detectable cross-reactivity with other pathogens. Of the 4 strains, 2 were identified as the immunoglobulin G1 (IgG1) isotype, 1 IgG2a, and the other IgG2b, with affinity constants (Ka) of 2 of the strains being 4.14 x 10(-9)M and 3.19 x 10(-9)M respectively. CONCLUSION: This is the first report on the preparation of mAb that is specific to the SARS-Cov, and the high-specificity and high-affinity mAb produced by the 4 strains of hybridomas provide a basis for further researches on the pathogenesis and early diagnosis of SARS.

[Antibody response of patients with severe acute respiratory syndrome (SARS) to nucleocapsid antigen of SARS-associated coronavirus].

OBJECTIVE: To assess serum antibody responses of patients with severe acute respiratory syndrome (SARS) to nucleocapsid (N) antigen of SARS-associated coronavirus. METHODS: The serum levels of IgM and IgG antibodies to N antigen were measured in 200 healthy blood donors and 13 SARS patients at different time points of acute and convalescent phases using indirect enzyme-linked immunosorbent assay (ELISA) with N fusion proteins of SARS-associated coronaviruses. RESULTS: The IgM positive critical value of 0.233 and IgG of 0.239 were selected as the threshold value for positive results that equals the product of 2.1 and the mean IgM and IgG levels of 200 healthy blood donors. In 13 patients with SARS, the antibody responses to N antigen were not detectable in the first week after the onset of symptoms. The IgM and IgG seroprotection rates were 83.3% and 66.7% respectively in the second week, both reaching 100% at the third week. IgM seroprotection rates was 61.5% in the second month, and 38.5% at third month. The IgG peaked one month after the onset and remained at high levels in the following 2 months. CONCLUSION: The antibody responses suggest that N protein of SARS is immunodominant and plays an important role in viral pathogenesis. This ELISA-based test for detecting anti- N antigen may be of significant value for SARS diagnosis.

Prognostic implications of microRNA-100 and its functional roles in human epithelial ovarian cancer.

Dysregulation of microRNAs (miRNAs) has been found to be associated with a variety of diseases, including epithelial ovarian cancer (EOC). Recently, miR-100 was reported to be downregulated in human ovarian carcinoma, however, the clinical significance and functional roles of miR-100 expression in human EOC are unclear. TaqMan real-time quantitative RT-PCR assay was performed to detect the expression of miR-100 in 98 EOC tissues and 15 adjacent normal epithelial tissues. The relationship between miR-100 expression and clinicopathological factors in 98 EOC patients was statistically analyzed. The effect of miR-100 expression on patient survival was determined. Finally, the role of miR-100 in EOC cell growth and its possible mechanisms were analyzed with miR-100 precursor or inhibitor-transfected cells. We showed that the level of miR-100 was significantly lower in EOC tissues compared to adjacent normal tissues. Low miR-100 expression was found to be closely correlated with advanced FIGO stage, higher serum CA125 expression level and lymph node involvement. Also, low miR-100 expression was correlated with shorter overall survival of EOC patients, and multivariate analysis showed that the status of miR-100 expression was an independent predictor of overall survival in EOC. Additionally, miR-100 could affect the growth of EOC cells by post-transcriptionally regulating polo-like kinase 1 (PLK1) expression. Together, these results suggest that low miR-100 expression may be an independent poor prognostic factor and miR-100 can function as a tumor suppressor by targeting PLK1 in human EOCs.

Enzyme-linked immunosorbent assay-format tissue culture infectious dose-50 test for titrating dengue virus.

A dengue nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA)-based tissue culture infectious dose-50 (TCID(50)) test (TCID(50)-ELISA) was developed as an alternative to the standard plaque assay for titrating dengue virus. Virus titers obtained by TCID(50)-ELISA were comparable to those obtained by the plaque assay and by the traditional TCID(50)-cytopathic effect (CPE) test (TCID(50)-CPE), with a better reproducibility and a lower coefficient of variation. Quantitative comparison of TCID(50)-ELISA and TCID(50)-CPE resulted in a correlation coefficient of 0.976. Moreover, this new method showed a wider application to C6/36, Vero E6, BHK-21, and Vero cells compared with other titration methods. In summary, the novel TCID(50)-ELISA method described here provides a more reliable and more accurate alternative compared to the plaque assay and TCID(50)-CPE for titration of dengue virus.

[Inhibitory effect of short hairpin RNA targeting survivin gene on human ectopic endometrial cells].

OBJECTIVE: To construct a lentiviral expression vector for short hairpin RNA (shRNA) of human survivin gene, and assess its gene silencing effect in human ectopic endometrial cells. METHODS: Human survivin gene shRNA sequence was designed using a software available on-line. The synthesized shRNA sequence was cloned into the pGCL-GFP vector to construct LV-survivin shRNA, which was confirmed by PCR and DNA sequence analysis. The packaging 293T cells were cotransfected with LV-survivin shRNA, pHelper 1.0 and pHelper 2.0, and the titer of the lentivirus was determined. The recombinant lentivirus was injected into human ectopic endometrial cells and the survivin mRNA expression was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in comparison with that in the non-transfected and blank vector-transfected human ectopic endometrial cells. RESULTS: PCR analysis and DNA sequencing confirmed correct insertion of the shRNA sequence into the lentiviral vector. The titer of virus after packaging was 8x10(8) U/ml. Survivin mRNA expression in human ectopic endometrial cells transfected by LV-survivin shRNA was significantly inhibited compared with those in the non-transfected and empty vector transfected human ectopic endometrial cells (P<0.01), and no significant difference was found between the latter two groups. CONCLUSION: The lentiviral shRNA vector of survivin gene constructed can effectively inhibit the expression of survivin gene in human ectopic endometrial cells in vitro. This vector provides a tool for investigating the role of survivin gene in the occurrence and progression of endometriosis and for searching new therapeutic targets.

Development of an antigen capture immunoassay based on monoclonal antibodies specific for dengue virus serotype 2 nonstructural protein 1 for early and rapid identification of dengue virus serotype 2 infections.

The dengue virus (DENV) has four distinct serotypes (DENV1, DENV2, DENV3, and DENV4) that require differentiation for effective prevention of morbid diseases. The recently developed DENV1-specific NS1 antigen capture enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibodies (MAbs) that recognize distinct epitopes on nonstructural protein 1 (NS1) of a specific DENV serotype is convenient and cost-effective, but assays have not yet been developed for DENV serotypes 2 to 4. This paper describes the development and validation of a DENV2-specific NS1 antigen capture ELISA by selection and optimization of the pair of well-characterized MAbs that recognized epitopes specific for DENV2 NS1 from a large panel of MAbs. The DENV2 NS1 ELISA displayed exclusive sensitivity with the DENV2 serotype and did not cross-react with the other three DENV serotypes. The sensitivity and specificity of the DENV2 NS1 ELISA were 83.3% (25/30) and 100% (504/504) when used to test 30 acute-phase serum samples from patients infected with DENV2 identified by virus isolation or reverse transcription-PCR serotyping and 504 serum samples from healthy individuals, respectively. The specificity of this assay was also evaluated using a panel of serum samples which were positive for DENV1, other flaviviruses, and nonflaviviruses; no cross-reactions were observed in these clinical samples. The DENV2 NS1 ELISA was eightfold more sensitive than a commercially available serotype-cross-reactive NS1 ELISA (Panbio Diagnostics, Brisbane, Australia) when the two assays were used to test the DENV2-infected cell culture supernatants in parallel. The Panbio NS1 ELISA displayed variation in sensitivity between DENV serotypes. The DENV2-specific NS1 antigen capture ELISA can be used as a tool for the rapid identification of DENV2 infections.

Well-characterized monoclonal antibodies against cell wall antigen of Aspergillus species improve immunoassay specificity and sensitivity.

The diagnosis of invasive aspergillosis (IA) based on the detection of Aspergillus galactomannan (GM) is complicated by the presence of cross-reactive GM epitopes in patient specimens. We have developed a novel and specific Aspergillus antigen-capture enzyme-linked immunosorbent assay (ELISA) by the selection of two well-characterized monoclonal antibodies from 17 candidate antibodies. The epitopes recognized by the monoclonal antibodies were present on the cell walls of the hyphae and the conidia of Aspergillus species, which were circulating or excreted as immunodominant antigens during the acute phase of IA established in the animal models. The detection of experimental Aspergillus-mediated antigenemia was suitably sensitive, and the sensitivity was comparable to that of a commercial GM detection ELISA kit (the Platelia Aspergillus assay). Moreover, the specificity of this assay was 100% when it was used to test 382 serum specimens and 120 urine specimens from healthy individuals. Cross-reactivity with other common opportunistic fungi, such as Penicillium and Candida species, and with purified GM protein derived from Aspergillus was not evident. Therefore, the chemical nature of the epitopes captured in this assay is most likely not associated with the GM structure, indicating that this newly developed Aspergillus antigen-capture ELISA is a promising tool for the diagnosis of IA without the risk of the false-positive results that are problematic with current GM antigen assays.

[Expression and identification of H5 subtype hemagglutinin of avian influenza A virus in insect cells].

OBJECTIVE: To clone and express avian influenza A virus [A/Hong Kong/482/97(H5N1)] H5 subtype hemagglutinin in baculovirus-insect cell expression system and investigate the antigenicity and bioactivity of the recombinant protein. METHODS: H5 gene of influenza A virus was amplified by PCR. The recombinant bacmid was obtained by cloning the gene to the donor plasmid of pFastBacHTB and transformed into DH10Bac competent cells. The recombinant baculovirus stock was prepared by transfecting the recombinant bacmid DNA into the insect cell line for protein expression after amplification. Immunofluorescene assay (IFA) and Western blotting were performed to identify the antigenicity of the recombinant protein, and hemagglutination assay was used to identify its bioactivity. RESULTS: The recombinant his-H5 protein was expressed in the insect cells with a relative molecular mass of 64,000, which showed erythrocyte-agglutinating activities with the red blood cells of guinea pig. Western blotting and IFA demonstrated that the recombinant his-H5 could be recognized and bound by standard anti-H5 sera. CONCLUSION: The recombinant his-H5 with a post-translation modification is successfully obtained in insect cells, which may provide a potential source for further study of the antigen's biological function and for production of the subunit vaccine or monoclonal antibodies.