酶工程-魏铭-Produce Enzyme by Baculovirus Expression Vector System

Why baculovirus can be used in production?
Baculovirus
• Only infect Anthropoda, Insecta specially, Lepidoptera mostly. • Taxonomy: Belongs to Baculoviridae, includes NPV and GV. • Structure:
Principles of BEVS
• polh gene 1. Non-essential;
Deletion of polh will not influence the infection, replication and transcription of virus.
2. Encode polyhedrin;
5. Electraporation;
6. Others.
2. BEVS-infected Bm/Bm cells
• One of the four most important eukaryotic expression system; (yeast, mammal cells, fungus) • Using recombined baculovirus infect insect cell/ individual with high efficiency, and express wanted protien with high level; • Developed by Meada in 1985; • First commercialized product, Feline IFN, in 1993;
Structure protein contribute the envelope of virus.
3. Promotor with high expression level;
1mg/mL, 50%-70%.
4. Easily screened
Change the appearance of colony. • • Insert foreign gene at the site of polh gene to express/produce protein. More than 1000 types of protein have been successfully expressed in this system, include medicine, cytokine, antibody, enzyme, etc. Some have been commercialized.
pH;
Moisture content// Dry the products and measure the change of weight; Microbes contamination;
UV spectrophotometry;
Molecular weight; Purity; Titer// ELISA, exam the effective components by molecular method; Bioactivity// Exam the effectivity by testing on animal. etc.
Expression of hemagglutinin-neutraminidase (HN) protein of bovine
parainfluenza virus type 3 (BPIV-3) in baculovirus expression system
• HN involves in pthogenicity of virus. • Laid a foundation of diagnosis of BPIV-3 infection and evaluation of vaccine efficacy.
• Break & Significance:
1. Bacmid behave both charecteristics of baculovirus and plasmid;
2. Complete all of the recombination process in bacteria; 3 Save time (7-9 days vs 6-9 weeks); 4. Easily screen.
Expression of hemagglutinin-neutraminidase (HN) protein of bovine
parainfluenza virus type 3 (BPIV-3) in baculovirus expression system
• HN involves in pthogenicity of virus. • Laid a foundation of diagnosis of BPIV-3 infection and evaluation of vaccine efficacy.
Procedure
• Extract total RNA of BPIV-3; • revers transcription PCR, get cDNA; • PCR, get DNA; • Construction & Identification of donor plasmid (pFastBac-HN) • Construction & Identification of shuttle plasmid (Bacmid-HN) • Transfect cells with Bacmid-HN, produce recombined virus; • Infect cells with recombined virus; • Identification of HN protein.
Procedure
• HN gene sequence from GenBank (AB770484. 1、KT071671), 1746bp;
• Design primers:
F：5′-AATGGATCCGCCACCATGGAATATTGGAAACACACAAAC-3′(BamHⅠ)， R：5′-CGGAAGCTTATTAGCTGCAGTTTTTCGGAACTTCTGTT-3′(HindⅢ）.
M13F & M13 R.
Procedure
• Extract total RNA of BPIV-3; • revers transcription PCR, get cDNA; • PCR, get DNA;
Procedure
• Extract total RNA of BPIV-3; • revers transcription PCR, get cDNA; • PCR, get DNA; • Construction & Identification of donor plasmid (pFastBac-HN)
Methods of BEVS
• Process
Methods of BEVS
• Traditional method
1. Construction of transferring vector;
2. Construction & screening of
recombined virus;
a. calcium phosphate coprecipitation method; b. Lipofecin method.
• A bioreactor may refer to any manufactured or engineered device or system that supports a biologically active environment.
Bombyx mori(Bm) bioreactor
1. Transgenic Bm
2. BEVS-infected Bm/ Bm cells
1. Transgenic Bm
• Insert foreign gene into choromosome of Bm individual (ideal: fibroin gene); • Constantly express and inherit the gene; • Method: 1. Microinjection;
Procedure
• Extract total RNA of BPIV-3; • revers transcription PCR, get cDNA; • PCR, get DNA; • Construction & Identification of donor plasmid (pFastBac-HN) • Construction & Identification of shuttle plasmid (Bacmid-HN)
Enzyme Production by Bioreactor
-How to produce enzyme using baculovirus expression vector system .
魏铭 13生物技术(英) 2013332860049 2016.04.19
Definition of Bioreactor
2. Gene gun;
3. Pression infiltration; 4. Sperm carrying;
• Disadvantage: 1. unprecise.// the site of induced gene; 2. unstable;// expression level and inheritance; 3. unpractical.
Methods of BEVS
• Developed method -Bac to Bac System