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Expression and Purification of M1 Gene of H5N1

Author: ZhangLiZuo
Tutor: QiBaoMin
School: Fujian Agriculture and Forestry University
Course: Basic Veterinary Science
Keywords: Avian influenza virus M1 gene H5N1 Expression Purification
CLC: S852.65
Type: Master's thesis
Year: 2009
Downloads: 58
Quote: 0
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Abstract


Avian influenza , especially the H5N1 avian flu supplies poultry industry has caused huge economic losses , has been a serious threat to human health . Avian influenza M1 protein play an important regulatory functions in the viral replication process . In this study, a comprehensive study of the H5N1 avian influenza M1 gene expression in E. coli , and its expression product was purified . To obtain high-level expression of recombinant M1 protein , in order to further study the relationship between structure and function , the preparation of the M1 monoclonal antibody , but also to lay the foundation for further research on the H5N1 avian influenza diagnostic methods . According to the cDNA sequence of the mature peptide the GenBank H5N1 avian influenza in the M1 , combined with the characteristics of the expression vector pET-28a design , synthesis , a pair of primers . The method M1 protein cDNA fragment was amplified by PCR , and then cloned into the vector pET-28a prokaryotic expression recombinant vector pET- 28a -M1 ; using PCR, restriction enzyme digestion and DNA sequencing method were identified. The correct identification of positive recombinant plasmid was transformed to E. coli BL21 by stable expression of kanamycin resistance screening positive engineering bacteria ; to 1mMIPTG induced engineered bacteria efficient expression of recombinant proteins pET- 28a - M1 , using affinity chromatography , agarose gel gel chromatography on recombinant M1 protein was purified , and the protein content was measured by Lowry's method . Results: 1 successful build the prokaryotic expression vector pET - 28a - M1 ; 2 , the establishment of a stable transfer dye prokaryotic expression vector pET - 28a - M1 plasmid BL21 cell strains , and confirmed that M1 cDNA stably transfected cell strains obtained efficient soluble expression ; 3, high efficient expression and purification of recombinant M1 protein, a protein content of 25mg/mL . To further resolve the relationship between tertiary structure to study the structure and function of the preparation of monoclonal antibodies, the foundation of R \u0026 D commercialization of diagnostic reagents .

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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