Fusion protein as well as preparation method and application thereof
A technology of fusion protein and G protein, applied in the field of biomedicine, can solve the problem of no protective effect and achieve good immunogenicity and good antigenicity
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Embodiment 1
[0028] Example 1: Design and screening of rabies virus G protein epitope polypeptides
[0029] Select the G protein of GenBank: MW055078.1, through the analysis and research of the protein sequence, remove 19 amino acids at the N-terminal (signal peptide sequence) and 55 amino acids at the C-terminal (transmembrane region and intracellular region, see figure 2 ), 30 epitope polypeptides were designed and synthesized (Table 1). Thirty antigenic epitope polypeptides were immunized into mice respectively, and mouse serum was collected for ELISA antibody titer detection, and the antigenic epitope polypeptides with the highest antibody titer were screened. After testing, a total of 6 epitope polypeptides have the highest antibody titers (Table 1). Therefore, these 6 epitope polypeptides (4th, 5th, 12th, 16th, 19th and 27th) were selected as focus of follow-up research.
[0030] The antibody titer was detected by a conventional ELISA method, which is briefly described as follows:...
Embodiment 2
[0033] Example 2: Design and preparation of rabies virus G protein fusion protein
[0034] The 6 antigenic epitope polypeptides screened in Example 1 were combined, and the two were connected by "AAYAAY", and an amino acid sequence (KLTNKHSPE, which can induce the production of IFN-γ) was added to the N-terminus, and the C-terminus was added. A segment of amino acid sequence (QARESKDNKIGKTENP, one of the B cell epitopes), thereby forming a complete fusion protein, the sequence of the fusion protein is:
[0035] KLTNKHSPE AAYAAY CTGVVTEAETYTNFVGYVTTTFKRK AAYAAY MAGDPRYEESLHNPYPDYHWLRTVK AAYAAY SKGSKTCGFVDERGLYKSLKGACKL AAYAAY IHDFRSDEIEHLVVEELVKKREECL AAYAAY VSFRRLSHLRKLVPGFGKAYTIFNK AAYAAY ADPSTVFKDGDEAEDFVEVHLPDVH AAYAAY QARESKDNKIGKTENPHHHHHH.
[0036] The fusion protein sequence was delivered to Nanjing GenScript Biotechnology Co., Ltd. for Escherichia coli codon optimization and whole gene synthesis; the synthesized gene was connected between the restriction ...
Embodiment 3
[0038] Example 3: Preparation and application of rabies virus fusion protein vaccine
[0039] Vaccine preparation: The fusion protein prepared in Example 2 was diluted to 50 μg / ml, and the vaccine was prepared by emulsification with ISA 201VG adjuvant (purchased from Sepec, France) according to the mass ratio of 1:1. 25μg / ml. According to the current appendix of "Chinese Veterinary Pharmacopoeia", there is no bacterial, mycoplasma and exogenous virus contamination; the viscosity is 25.8cP, <200cP; 10ml of the vaccine is added to the centrifuge tube, centrifuged at 3000r / min for 15 minutes, the bottom of the tube is Precipitation, good stability. Prepared and tested vaccines are stored at 2-8°C for later use. Referring to this vaccine preparation method, the G protein expressed in the eukaryotic system used for ELISA detection in the examples was used to prepare a vaccine for comparison.
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