Avibacterium paragallinarum HA fusion protein, tripolymer thereof, prepared vaccine composition, preparation method and application
A technology of vaccine composition and fusion protein, which is applied in the field of pharmaceutical preparations containing antigens or antibodies, and can solve problems such as inability to fully absorb lumps at injection sites, and inability to administer whole-bacteria antigen vaccines to young broiler chickens, etc.
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Embodiment 1B
[0064] Example 1Construction of BL21(DE3)-A-GCN4-tHA expression engineering strain
[0065] 1.1 Extraction of Avian Bacillus paragallinarum type A DNA
[0066] According to the instructions of the virus DNA extraction kit, take the Avibacterium paragallinarum Serotype A Strain HN3 strain (Avibacterium paragallinarum Serotype A Strain HN3), which has been preserved in Chinese typical culture on January 20, 2015 Collection Center (CCTCC), deposit number: CCTCC NO: M2015051; deposit address: Wuhan University, Wuhan, China, published in Chinese patent application CN106267176A) pure culture, pick several colonies in 100 μL sterile double distilled water, boil for 10 minutes , ice bath for 10 minutes, centrifugation at 12000r / min for 1 minute, take the supernatant as a template for subsequent experiments.
[0067] 1.2A-tHA protein fragment gene amplification
[0068] Oligonucleotide primers were synthesized according to the conserved region sequences at the 5' and 3' ends of the t...
Embodiment 2B
[0074] Example 2 Study on protein expression characteristics of BL21(DE3)-A-GCN4-tHA expression engineering strain
[0075] 2.1A-GCN4-tHA protein expression
[0076] The correctly identified expressing engineered bacteria were streaked on LB plates (containing 50 μg / mL kanamycin) and cultured at 37°C for 14 hours. Pick a single colony, inoculate it in 3mL LB liquid medium (containing 50μg / mL kanamycin), cultivate at 37°C on a shaker (220r / min) for 14 hours, and transfer it to 100mL LB liquid medium according to 1mL / bottle (containing 50 μg / mL kanamycin) in a 250 mL conical flask, incubate for 4 hours on a shaker (37 °C, 220 r / min), cool down to 28 °C, and add isopropylthioβ with a final concentration of 0.2 mmol / L -D-galactoside (IPTG), and the incubation was continued for 5 hours. Take the culture and centrifuge at 10,000 r / min for 5 minutes at 4°C, collect the bacteria, resuspend in 10 mL of sterile saline, and sonicate in an ice-water bath for 35 minutes. 400 watts. The...
Embodiment 3B
[0083] Example 3 Construction of BL21(DE3)-A-tHA expression engineering strain and A-tHA protein expression
[0084] After electrophoresis of the PCR product of Avian Bacillus paragallinarum type A tHA protein, a DNA gel recovery kit was used to purify the obtained DNA fragment after double digestion with BamHI and XhoI, and then ligated with the pET28a plasmid treated with the same double digestion, and the ligation product Transform E. coli DH5α competent, and screen positive clones. The transformant plasmid was extracted with a plasmid extraction kit and identified by double-enzyme digestion. The correct plasmid identified by enzyme digestion was sequenced and analyzed, and the correct recombinant plasmid was named pET28a-A-tHA. The expression engineering strain BL21(DE3)-A-tHA was constructed with reference to the method in Example 1, and the correct strain identified by enzyme digestion and sequencing was stored at -70°C for future use.
[0085] The tHA protein was expre...
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