Fusion protein of equine influenza virus h3n8 subtype and its preparation method, application and vaccine
A fusion protein, H3N8 technology, applied in antiviral immunoglobulin, veterinary vaccines, biochemical equipment and methods, etc., to achieve high expression, good antigenicity, and wide application range
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Examples
preparation example Construction
[0047] The present invention also provides a method of preparing the above fusion protein, the preparation method, by expressing the gene of the fusion protein in the host, for example, but is, but is not limited to, in E. coli expression system, yeast expression system, insect expression The system, plant expression system, or mammalian expression system can, due to the protein expressed by mammalian cells, the structure and biological characteristics are closer to the natural protein, and thus the present invention preferably uses a mammalian expression system to express the fusion. The gene of the protein is more preferably using a gene of a CHO cell expression system to express the fusion protein. CHO cells are Chinese hamster ovary, and CHO cell expression systems have the following advantages:
[0048] (1) With accurate translational folding and modification function, expressed proteins are closest to natural proteins molecules in molecular structures, physicochemical proper...
Embodiment 1
[0068] Example 1: HA-NA gene design and synthesis
[0069] HA-NA gene design and synthesis: HA gene (GenBank login number KX815377.1) After optimization, the design length 810bp has sequences shown in SEQ ID NO.1; NA gene (GenBank login number MG132049.1) is optimized The design length is 900 bp and has the sequence shown in SEQ ID NO.2. The HA gene is connected to the NA gene, and the Linker sequence has a sequence shown in SEQ ID NO. The HA-NA gene is 1740 bp, which has an amino acid sequence as shown in SEQ ID NO.4 and a nucleotide sequence as indicated by SEQ ID NO. HA-NA gene was completed by Shanghai University of Furious Bio.
Embodiment 2
[0070] Example 2: PEE6.4-HA-NA recombinant plasmid construction
[0071] 2. Add Enzyme Substation: Addition of Enzyme Substances by PCR amplification at the upstream and downstream of the HA-NA gene sequence: HindII, SMAI, PCR amplification upstream primers such as SEQ ID NO.6, PCR amplification downstream The primers are shown in SEQ ID NO. 7. PEE6.4-HA-NA plasmid spectrum figure 1 Indicated.
[0072] 2.2HA-NA gene and carrier double enzyme digestion
[0073] 2.2.1 Constructing 50 μl of the reaction system, and mixed the components at 37 ° C for 2 h after mixing each component.
[0074]
[0075]
[0076] 2.2.2 Objective DNA fragment recycling: DNA gel recovery kit (purchased from Beijing Laibao Technology Co., Ltd.), recovered fragment, steps as follows:
[0077] (1) After agarose gel electrophoresis, the destination DNA strip was carefully cut from the agarose gel, and in the 1.5 ml EP tube, weigh weight.
[0078] (2) Add 3x volume sol liquid to the EP tube, 50-55 ° C water ...
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Abstract
Description
Claims
Application Information
- IPC
- C07K19/00; C12N15/62; C12N15/85; C07K16/10; G01N33/569; A61K39/145; A61P31/16
- CPC
- A61K39/12; A61K2039/552; A61P31/16; C07K14/005; C07K16/1018; C12N15/85; C12N2760/16122; C12N2760/16134
- Inventors
- 贺笋; 任立松