Efficient production of porcine circovirus virus-like particles using the nonconventional yeast Kluyveromyces marxianus.

Porcine circovirus type 2 (PCV2) is a ubiquitous virus with high pathogenicity closely associated with the postweaning multisystemic wasting syndrome (PMWS) and porcine circovirus diseases (PCVDs), which caused significant economic losses in the swine industry worldwide every year. The PCV2 virus-like particles (VLPs) are a powerful subunit vaccine that can elicit high immune response due to its native PCV2 virus morphology. The baculovirus expression system is the widely used platform for producing commercial PCV2 VLP vaccines, but its yield and cost limited the development of low-cost vaccines for veterinary applications. Here, we applied a nonconventional yeast Kluyveromyces marxianus to enhance the production of PCV2 VLPs. After codon optimization, the PCV2 Cap protein was expressed in K. marxianus and assemble spontaneously into VLPs. Using a chemically defined medium, we achieved approximately 1.91 g/L of PCV2 VLP antigen in a 5-L bioreactor after high cell density fermentation for 72 h. That yield greatly exceeded to recently reported PCV2 VLPs obtained by baculovirus-insect cell, Escherichia coli and Pichia pastoris. By the means of two-step chromatography, 652.8 mg of PCV2 VLP antigen was obtained from 1 L of the recombinant K. marxianus cell culture. The PCV2 VLPs induced high level of anti-PCV2 IgG antibody in mice serums and decreased the virus titers in both livers and spleens of the challenged mice. These results illustrated that K. marxianus is a powerful yeast for cost-effective production of PCV2 VLP vaccines.

Integrated application of uniform design and least-squares support vector machines to transfection optimization.

BACKGROUND: Transfection in mammalian cells based on liposome presents great challenge for biological professionals. To protect themselves from exogenous insults, mammalian cells tend to manifest poor transfection efficiency. In order to gain high efficiency, we have to optimize several conditions of transfection, such as amount of liposome, amount of plasmid, and cell density at transfection. However, this process may be time-consuming and energy-consuming. Fortunately, several mathematical methods, developed in the past decades, may facilitate the resolution of this issue. This study investigates the possibility of optimizing transfection efficiency by using a method referred to as least-squares support vector machine, which requires only a few experiments and maintains fairly high accuracy. RESULTS: A protocol consists of 15 experiments was performed according to the principle of uniform design. In this protocol, amount of liposome, amount of plasmid, and the number of seeded cells 24 h before transfection were set as independent variables and transfection efficiency was set as dependent variable. A model was deduced from independent variables and their respective dependent variable. Another protocol made up by 10 experiments was performed to test the accuracy of the model. The model manifested a high accuracy. Compared to traditional method, the integrated application of uniform design and least-squares support vector machine greatly reduced the number of required experiments. What's more, higher transfection efficiency was achieved. CONCLUSION: The integrated application of uniform design and least-squares support vector machine is a simple technique for obtaining high transfection efficiency. Using this novel method, the number of required experiments would be greatly cut down while higher efficiency would be gained. Least-squares support vector machine may be applicable to many other problems that need to be optimized.