Development Of Carbohydrate Microarray On Surveillance Of Host Specificity Of Avian Influenza Virus

Background: Host specificity of type A influenza virus is mediated by hemagglutinin (HA), a kind of glycoprotein on the surface of virus. Receptor of HA is oligosaccharides chains, terminal of which have sialic acid (SA). Avian Influenza Virus (AIV) mainly binds with SAα2-3 Gal on the surface of epithelial cells of gastrointestinal tract in fowl, while human influenza virus manily binds with SAα2-6 Gal of oligosaccharides chains. Many kinds of antigens and frequent variation of AIV make defending ourselves against highly pathogenic avian influenza (HPAI) more difficult. At present conventional means of detecting AIV cann't monitor and evaluate host specificity completely. So it's necessary to develop new technology to monitor antigenic variation and evaluate host specificity in AIV. It's reported in the documents, carbohydrate microarrays have been applied to study host specificity of H1,H3 virus. The glycan microarrays we intended to produce could ascertain host range of AIV quickly in terms of oligosaccharides chains recognized by virus and decide whether AIV infects humans directly, which could provide theoretical basis for precaution of HPAI.Purpose: HA is main criterion of judging new variety in subtypes of AIV. In this experiment we intended to study HA of H5, H9 subtype of AIV, which harmed our country seriously. Glycan microarrays were to be developed to monitor and assess pathogenic variation and host specificity of AIV. Infection factors of infected material or infected virus samples in laboratory also should be screened quickly by glycan microarrays. Moreover, oligosaccharides chains binding with HA specificly could be screened.Methods: First, HA gene of H5,H9 subtype were cloned and HA proteins were expressed by baculovius expression vector system. Second, the two different carbohydrate microarrays were successfully prepared based on the hydroxyl-modified surfaces and the hydrazide-modified surfaces. Besides, the two different carbohydrate microarrays were compared on property respect. Third, carbohydrate microarrays were used for researching host specificity of the expressed HA proteins.Result: Part 1 Through amplification of HA gene of H5,H9 subtype, products of 1700bp were obtained. Employing Bac-to-Bac baculovius expression vector system, the two recombinant donor plasmids named H5HA-pFastBacHTa and H9HA-pFastBacHTa were successfully constructed after they were identified by PCR, restriction endonucleases digestion and sequenced. Then the two recombinant donor plasmids were transformed into E.coli DH10Bac containing bacmid and helper to form recombinant bacmid named H5HA-Bacmid,H9HA-Bacmid. The recombinant bacmids were transfected into Sf9 cell to produce recombinant baculovius and express recombinant HA protein. The molecular weight of the expressed HA protein was 63 Kd. IIF (Indirect Immuno fluorescent) and Westernt blotting showed that, similar to natural HA protein, the expressed H5HA and H9HA recombinant protein were recognized by immune serum of AIV and possessed good biological activity.Part 2 After comparing the hydroxyl-modified slides with the hydrazide-modified slides, we obtained the following results:1 Based on the hydroxyl-modified surfaces, the minimum detectable concentration of mannose and glucose were determined to be 1 nmol/L and 10μmol/L respectively. While based on the hydrazide-modified surfaces, the minimum detectable concentration of mannose and glucose were determined to be 5μmol/L and 100nmol/L respectively.2 When the concentration of mannose immobilized on slides was 1 mmol/L, on the hydroxyl-modified surfaces the limit of detection (LOD) for ConA, the minimum concentration of detected ConA, was 0.25nmol/L. While LOD for ConA was 2.5 nmol/L on the hydrazide-modified surfaces.3 Utilization of the prepared carbohydrate microarrays for the characterization of carbohydrate-protein interaction revealed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays, that was, Mannose,Glucose and GlcNAc could bind with ConA specifically, both Lactose and Galactose could bind with ECA and Fucose could bind with LT specifically.Through comparing the hydroxyl-modified surfaces with hydrazide-modified surfaces, the results showed that LOD for ConA on the hydroxyl-modified surfaces was much lower. Therefore the hydroxyl-modified slides were used to prepare carbohydrate microarray for next research.We compared two different methods of preparing carbohydrate microarrays and identified the hydroxyl-modified slides with the lower LOD and stable combination to prepare carbohydrate microarrays. Besides, HA proteins with good biological activity were expressed in sf9 successfully. Because of time constraints, the expressed HA proeins haven't been applied for the carbohydrate microarrays. At present, we were preparing carbohydrate microarrays with 40 kinds of oligosaccharides chains to research host specificity of the expressed proteins.