Compositions that include hemagglutinin, methods of making and methods of use thereof
a technology of hemagglutinin and hemagglutinin, which is applied in the field of compositions that include hemagglutinin, methods of making and methods of use thereof, can solve the problems of ineffective prevention or treatment of illness, and achieve the effect of avoiding serious illness and death and cost effectiveness
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example 1
Design of Portions of a Naturally Occurring Hemagglutinin of Influenza A
Materials and Methods
[0361] Design of the HA1-1, 1-2 and 1-3 globular head constructs for A / Puerto Rico / 8 / 34 (H1N1). Influenza strain A / Puerto Rico / 8 / 34 (PR8) is a well-characterized mouse-adapted strain of influenza A virus. The published crystal structure of the mature PR8 HA (SEQ ID NO: 1) (Gamblin et al. 2005. Science 303:1838-1842; PDB accession number 1RU7) was used in combination with the Molecular Modeling Database to determine the boundaries for three PR8 globular head constructs. The complete list of solved crystal structures for influenza A hemagglutinin molecules can be found in the Protein Data Bank (PDB) or National Center for Biotechnology Information (NCBI) Structure website (http: / / www.ncbi.nlm.nih.gov / Structure).
[0362] The three dimensional crystal structure for the PR8 HA was viewed using the Cn3D program within the NCBI website. The trimeric HA molecule has the shape of a mushroom. The glo...
example 2
Design of Portions of a Naturally Occurring Hemagglutinin of Influenza B
Materials and Methods
[0395] Design of the HA1-1, 1-2 and 1-3 globular head constructs for B / Lee / 40. Structural considerations in the design of an influenza B HA vaccine are similar to those for influenza A, i.e., the domain boundary of the globular head of HA must be identified so that the flagellin-HA fusion protein can fold correctly or be refolded correctly to expose appropriate antigenic epitopes. Unlike influenza A, well-defined X-ray crystallographic structures are not available for influenza B HA, thus it is more difficult to unambiguously define the domain boundary of globular head. Therefore the influenza B HA model must be predicted based on bioinformatic and structural models (Tung et al. 2004. J. Gen. Virl. 85, 3249-3259). These investigators used a “knowledge-based” approach which depends on a high degree of sequence homology between the known structure from the protein data bank and the target un...
example 3
Cloning and Expression of Recombinant Flagellin-Hemagglutinin Fusion Proteins in E. coli
Materials and Methods
[0416] Cloning of HA influenza A subunits. Subunits of the HA globular head from several strains of influenza. A were cloned and expressed alone or as fusions with flagellin. We also expressed in fusion with the HA globular head domain, two proteins widely used as carrier proteins in conjugated vaccines. CRM-197 is a mutated diphtheria toxin (DTx) from Corynebacterium diphtheriae and LTB, is E. coli heat labile toxin B subunit.
[0417] These constructs were generated in one of four different methodologies:
[0418] Method #1: In this protocol, a fusion gene comprising flagellin (STF2) (SEQ ID NO: 212) and the HA subunit was codon-optimized for E. coli expression and obtained from a commercial vendor (DNA2.0 Inc., Menlo Park, Calif.) by chemical synthesis. The gene was excised with NdeI and BlpI enzymes, the insert fragment was gel purified and ligated to pET24a (Novagen, San D...
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