Fusion proteins comprising carriers that can induce a dual immune response
a technology of fusion proteins and carriers, applied in the field of animal and human health, can solve the problems of inability to mount an effective antibody response against gnrh, weak immunogenicity, and inability to induce an effective immune response of animals, and achieve the effect of protecting vertebrate cells and inhibiting the activity of peptides
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example 1
Plasmids Expressing gD / GnRH Fusion Proteins
[0133] Construction of pQE-tmgD: The plasmid FlgD / Pots207nco(#79) (encoding a full-length gD, hereinafter “gD / Pots”) was digested with NcoI / XbaI and the resulting 1.26 kb fragment was cloned into the corresponding sites of pUC21, generating the plasmid pUC-FLgD. The complete sequence of the NcoI / XbaI fragment in the plasmid pUC-FLgD was determined on both DNA strands using Sanger fluorescent dideoxy chain termination sequencing technology. FIG. 3 shows the sequence results and characteristics. The nucleotide sequence encoding gD / Pots is included in SEQ ID NO: 16.
[0134] DNA alignment between gD / Pots and published BHV-IgD (GenBank Accession No. M59846) shows 94.7% homology with the majority of the mismatches occurring 3′ of the transmembrane domain (FIG. 4).
[0135] Amino acid alignment between gD / Pots and M59846 shows four amino acid differences, one of which is located in the signal sequence and the other three in or around the transmembra...
example 2
Expression of GnRH / gD Fusion Proteins by Transformed Bacterial Cells
[0156] All of the pQE constructs described in Example 1, above, were transformed into E. coli DH5α-F′IQ cells for expression. For induction of expression, cells were grown to an OD600 of 0.7-0.9 in a 2 liter baffled culture flask in 2xYT broth containing 100 μg / ml Ampicillin and 25 μg / ml Kanamycin, then induced with 1-2 mM IPTG and incubated for 4 hours at 37 degrees Celsius. Average OD600 readings at harvest time were 1.3. Expression of all four constructs was confirmed by Western blot analysis.
example 3
Formulation of Fusion Protein Vaccines and Immunization of Mice
[0157] Vaccine Assembly: Fusion proteins from pQE-tmgD (as a control), pQE-GnRH:gD, pQE-GnRH:gD:GnRH, and pQE-gD:GnRH were concentrated from inclusion body preparations by preparative electrophoresis on 9% polyacrylamide gels. Bands cut from SDS PAGE gels were dissolved in 25 mM Tris, pH 8.3, 192 mM glycine and 0.1% SDS (w / v). The equivalent of 10%1 g gD / mouse dose was adjuvanted with SEAM1 (Squalene Emulsion Adjuvant Metabolizable) emulsion (10 μg QuilA / 100 μl dose). Vaccine formulations were stored at 4° C. SEAM1 is 5% squalene, 0.1% Vitamin E acetate, 1% Span 85, 0.70% Tween 80, 2 mg / ml QuilA, and 400 μl / ml cholesterol.
[0158] Mice: BALB / c males were used in the study after they were 8 weeks of age (10 / group). Mice were initially housed in groups of 10, however, controls were subsequently moved to individual cages to prevent fighting.
[0159] Immunization: Mice were immunized subcutaneously with 10 μg fusion protein i...
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