Browsing by Author "Chen, Shuxiong"

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    Design and engineering of self-assembling antigens towards particulate vaccines : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2019) Chen, Shuxiong
    Natural and synthetic self-assembling polymers and proteins could be bioengineered to display and/or encapsulate antigens to serve as innovative antigen carrier systems for the induction of desirable immunities. Polyhydroxyalkanoates (PHAs) are naturally occurring polyesters synthesized as cytoplasmic polyester inclusions (polyester particles) by various bacteria. The particles have been used as an antigen delivery platform by translationally fusing antigens to the particle surface-associated protein, PHA synthase. Furthermore, it has been found that protein inclusion bodies contain a large amount of correctly folded and biologically active proteins and could be engineered to perform as an antigen carrier system. Tuberculosis (TB) is a global health issue for both humans and animals. Inaccurate diagnosis and inefficacious vaccination make TB control problematic. The Mantoux tuberculin skin test gives false positive results if humans or animals are vaccinated with the Bacille Calmette-Guérin (BCG) strain or exposed to environmental mycobacteria. BCG cannot provide effective protection against TB. Subunit vaccines have great promise to protect against infectious diseases, but they are often weak immunogenically. A strategy to circumvent this problem is the use of self-assembly particulate vaccines, which could present multiple copies of antigens and serve as a depot for prolonged multivalent antigen display to induce enhanced immunogenicity. In this thesis, four specific TB diagnostic antigens — CFP10, Rv3615c, ESAT6, and Rv3020c — were displayed on polyester particles. The results showed that polyester particles displaying TB antigens specifically distinguished TB-infected from non-infected cattle. Antigen immunogenicity was dramatically enhanced after the display on polyester particles, which lowered the antigen concentration (0.1 to 3 μg dose/inoculum) required for skin tests. Mycobacterial vaccines H4 (Ag85B-TB10.4) or H28 (Ag85B-TB10.4-Rv2660c) were bioengineered to display H4/H28 on polyester particles and/or self-assemble H4/H28 into protein inclusion bodies. The results demonstrated that polyester particle-/protein inclusion body-based particulate TB vaccines increased overall immunogenicity by enhancing humoral (for example, IgG1 and IgG2c) and cellular (for example, IFNγ and IL17A) immune responses when compared to respective soluble antigens.
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    Functional protein display on the surface of biobeads produced by recombinant Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2013) Chen, Shuxiong
    Polyhydroxyalkanoic acids (PHAs) are biopolyesters produced by various bacteria. They are deposited as spherical water-insoluble cytoplasmic inclusions (beads) containing an amorphous hydrophobic polyester core and surrounded by a phospholipid monolayer and embedded proteins, including PHA synthase (PhaC), the key enzyme required for PHA bead formation. Although inactive PhaC cannot produce PHA beads, fusing inactive PhaC to green fluorescent protein (GFP) leads to GFP protein bead formation. Both PHA and protein beads could serve as a versatile platform for display of desired proteins suitable for various biotechnological and medical applications. The tuberculin skin test (TST) for diagnosing bovine tuberculosis (TB) in cattle uses the purified protein derivative (PPD) that is prepared from Mycobacterium bovis. However, some antigens in the PPD are also present in environmental mycobacteria. Therefore, the TST lacks specificity if animals are exposed to non-pathogenic environmental mycobacteria. In this study, three specific TB antigens, CFP10, ESAT6, and Rv3615c — which are present in pathogenic but absent in most non-pathogenic mycobacteria — were displayed on the surface of PHA beads. The results demonstrated that these triple antigen-displaying PHA beads can differentiate TB-infected from non-infected cattle, making this an attractive alternative to current skin test diagnostic reagents. IgG binding domains displayed on GFP protein beads have a higher IgG binding ability when compared to their counterpart displayed on PHA beads. However, it is unclear whether an enhancement of IgG binding ability due to GFP protein beads could be achieved by immobilization on other fluorescent protein (FP) beads. The results showed that other FP (including yellow, red and cyan) beads displaying IgG binding domains have an approximately 1.5–2 fold greater IgG binding ability when compared to PHA beads displaying the same binding domains. To investigate whether protein beads displaying iron-binding peptides could be magnetized while maintaining IgG binding function, an iron binding peptide was displayed. The results demonstrated that protein beads displaying both IgG and iron binding peptides can be magnetised by iron oxide and retain a strong IgG binding ability. Finally, this study revealed that different cell disruption techniques could affect the morphology and functionality of FP protein bead.