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Manufacturing Process Study of HIV-1 DNA Vaccine and Construction, Expression and Purification of PTD-P24 Fusion Protein

Author: ChengHai
Tutor: TangXinZuo
School: Anhui Agricultural University
Course: Microbiology
Keywords: DNA vaccines Production PTD-P24 protein Building Expression
CLC: R392
Type: Master's thesis
Year: 2006
Downloads: 142
Quote: 0
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Abstract


Following the traditional vaccines and genetic engineering subunit vaccines, DNA vaccines are a new generation of vaccines, large scale preparation of plasmid DNA to the industrialization of the DNA vaccine has important significance. The first part of this paper, starting from the demand of major research projects as well as the prospects of development of the biopharmaceutical industry established HIV-1 DNA vaccine trial production process research topics. In cooperation with the upstream and downstream research, production and quality control personnel, through unremitting efforts, the more successful completion topics task. Based on the basis of the laboratory small pilot process research in GMP conditions, successfully achieve DNA amplification of the vaccine production process, to establish the clinical use of DNA vaccine preparation process with independent intellectual property rights. Stable 75 L fermentor fed-batch fermentation process, fermentation 20 hours after harvest bacterial approximately 60g wet cells / L, plasmid yield ≥ 60 mg / L,. Under the premise of ensuring high-density culture to achieve a high yield of the plasmid. Through a combination of various means of purification of plasmid DNA and optimization of the various parameters, the establishment of a stable, simple, easy-amplified DNA vaccine trial purification process. Use the LiCl precipitation spermine precipitation crude pure elimination of the means of the vast majority of impurities in the large-capacity, low-cost crude pure stage. Minimize expensive and time-consuming the chromatography process steps, simplifying the entire process on the basis of good thick pure. DNA vaccine purification process development to better implement the guiding principles of the the enhanced extraction simplify refined \Compared with foreign processes, the process step of refining process, to shorten the purification cycle, and reduce the production cost. In addition, we have also established a complete quality control system, to ensure the stability of the production process and product quality. HIV-1 DNA vaccine finished product is certified by the China Pharmaceutical and Biological Products, all test indicators are in line with the quality standards of the clinical use of DNA vaccines for China's AIDS vaccine clinical trials. Existing protein subunit vaccines A major drawback is not effective in inducing antigen-specific cellular immune response, the second part of the present subject idea is the use of protein transduction domains (protein transduction domain, PTD), the target protein is introduced into cells to explore the possibility of enhancing immune. Carried by the use of HIV-TAT protein transduction domain of protein coding sequence, the 5 'end of the HIV-1 CN54 p24 gene fusion using the PCR method of the the TAT gene sequence, build PTD-P24 fusion gene. Double digestion with Nde I and EcoR I, insert pThioHisA expression vector constructed pThioHisA-PTD-P24 plasmid. Actually at the same time build a carrier containing pThioHisA-PTD, after digestion with BamH I and EcoR I can easily insert another gene, and other research. Optimization of expression conditions in shake flasks to achieve efficient expression in Escherichia coli of soluble non-fusion protein PTD-P24, SDS-PAGE PTD-P24 protein expression levels reached up to 45% of the total bacterial protein. Western-blot experiments show that the target protein antigen. The weak cation exchange chromatographic purification PTD-P24 protein, more than 95% purity, to this purification strategy is easy to enlarge, the step is simple, high yield. Build PTD-P24 protein expression and purification work has laid a solid foundation for further pre-clinical research and pilot production.

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